MXPA94005272A - Vaccines for salmone - Google Patents

Abstract

The present invention relates to Salmonella virulence genes as well as Salmonella vaccines, useful for inducing an immune response in an organism, particularly a human organism.

Description

VACCINES FOR SALMONELLA INVENTORS: SAMUEL I. MILLER, III and JOHN J. MEKALANOS, both citizens of the United States, residing at 25 Cro ninshield Road, Brookline, Massachusetts, respectively; and 78 Fres Pond Lane, Cambridge, Massachusetts, both in the United States.

CAUSAHABIENTES: THE GENERAL HOSPITAL CORPORATION and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, a society and a charitable institution, respectively, of the United States, domiciled respectively at 55 Fruit Street, Boston, Massachusetts; and 124 Mt. Auburn Street, Cambridge, Massachusetts, both in the United States.

Summary Salmonella virulence genes are described as well as vaccines for Salmonella, useful for inducing an immune response in an organism, particularly a human organism. Background of the Invention This invention relates to vaccines. Enteric fevers and diarrheal diseases, such as typhoid fever and cholera, are the leading causes of morbidity and mortality in developing countries, Hook et al., 1980, in Harrison's Principles of Internal Medicine, 9th edition, 641 -848, McGraw Hill, New York. Traditional approaches to the development of vaccines for bacterial diseases include parenteral injection of purified components or dead organisms. These vaccines administered parenterally require a technologically advanced preparation, are relatively expensive, and often, due to the discomfort of needle-based injections, are a cause of resistance on the part of patients. Live strains of oral vaccines have several advantages over parenteral vaccines: low cost, ease of administration and simple preparation. The development of live vaccines has often been limited by a lack of understanding of the pathogenesis of the disease of interest at the molecular level. The candidate strains of live vaccines require nonreversible genetic alterations that affect the virulence of the organism, but not its induction of an immunological response. The works that define the mechanisms of Vijrío cholerae toxigenesis have made it possible to create live vaccine strains based on the suppression of the toxin genes, Mekalanos et al., 1983, Nature 306: 551, Levine et al., 1988, Infect. Iptmun. 56: 161. Recent studies have begun to define the molecular basis of the survival and virulence of the macrophage of Salmonella typhimurium, Miller et al., 1989, Proc. Nati Acad. Sci. USA 86.: 5054, incorporated herein by reference. Strains of Salmonella typhimurium with mutations in the phoP positive regulatory regimen are markedly attenuated in virulence for BALB / c mice. The phoP regution is composed of two genes present in an operon, called phoP and phoQ. The products of the phoP and phoQ genes are highly similar to other members of transcription regulators of two bacterial components that respond to environmental stimuli and control the expression of a large number of other genes. A mutation in one of these regulated genes in the phoP regulatory region, pagC, confer a virulence defect. Strains with pagC, phoP or phoQ mutations provide partial protection to subsequent attack by wild-type S. typhimurium. Salmonella species cause a spectrum of clinical diseases including enteric fevers and acute gastroenteritis, Hook et al., 1980, supra. Infections with Salmonella species are more common in immunosuppressed persons, Celum et al., 1987, J. Infect. Dis. 156: 998. S. typhi, the bacterium that causes typhoid fever, can only infect man, Hook et al., 1980, supra. The narrow specificity of S. typhi hosts has resulted in extensive use of rat infection with S. enteridi tis typhimurium as a laboratory model of typhoid fever, Carter et al., 1984, J. Exp. Med. 139: 1189. S. typhimurium infects a wider range of hosts, causing acute gastroenteritis in humans and a disease similar to typhoid fever in mice and cows. Salmonella infections are acquired by oral ingestion. Organisms, after traversing the stomach, replicate in the small intestine, Hornik et al., 1970, N. Eng. J. Med. 283: 686. Salmonella is capable of invasion through the cells of the intestinal mucosa, and S. typhi can pass through this mucosal barrier and spread via the Peyer patches to the lamina propria and regional lymph nodes. Colonization of the reticuloendothelial cells of the host then occurs after bacteremia. The ability of S. typhi to survive and replicate within the cells of the human reticuloendothelial system is essential to its pathogenesis, Hook et al., 1980, supra, Hornick et al., 1970, supra, and Carter et al., 1984, supra. Salmonella typhi immunity involves humoral and cell-mediated immunity, Murphy et al., 1987, J. Infect. Dis. 156: 1005, and can be obtained by vaccination, Edelman et al., 1986, Rev. Inf. Dis. 8_: 324. Recently, field trials with humans have shown considerable protective efficacy against S. typhi infection after intramuscular vaccination with partially purified Vi antigen, Lanata et al., 1983, Lancet 2: 441. The antibody-dependent increase in S mortality. typhi by T cells has been demonstrated in individuals who have received a live S. typhi vaccine, indicating that these antibodies may be necessary for the host to generate a cell-mediated immune response, Levine et al., 1987, J. Clin. Invest. 79_: 888. The cell-mediated immune response is important in typhoid immunity since dead vaccines that do not induce this immune response are not protective in humans, Collins et al., 1972, Infect. Immun. 41: 742. SUMMARY OF THE INVENTION The invention provides a vaccine for Salmonella that does not cause transient bacteremia. In general, the invention includes a bacterial cell, preferably a Salmonella cell, for example a S. typhi cell, S. enteri tidis typhimurium or S. cholerae-suis, whose virulence is attenuated by a first mutation in an PhoP regimen and a second mutation in a synthetic aromatic amino acid gene. As used herein, PhoP regu- lation is defined as a DNA comprising an expression unit of the virulence gene of Salmonella characterized by two regulatory genes, phoP and phoQ, and structural genes, whose expression is regulated by phoP and phqQ, for example repressed genes from the regulatory region phoP (prg) or genes activated by the regulatory region phoP (pag). Such a bacterial cell can be used as a vaccine to immunize a mammal against salmonellosis.

The Salmonella cell can be of any serotype, for example S. typhimurium, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. pylorum, S. dublin, S. heidel -berg, S. newport, S minnesota, S. inf antis, S. virchow, or S. panama. The first mutation may be a non-reversible null mutation at the PhoP / PhoQ site. Preferably, the mutation is a deletion of at least 100 nucleotides; more preferably, the mutation is a deletion of at least 500 nucleotides; even more preferably, the mutation is a deletion of at least 750 nucleotides; and most preferably, the mutation is a deletion of the nucleotides 376 to 1,322 of the PhoP / PhoQ regulatory site. The second mutation may be a non-reversible null mutation in an aroA site or a non-reversible null mutation in an aroC / aroD site, or another place involved in aromatic amino acid biosynthesis. To further attenuate the virulence of the bacterial cell of the invention, the cell can still contain another mutation, for example a deletion, in a synthetic non-aromatic amino acid gene, for example a mutation that converts the cell into auxotrophic for a non-aromatic amino acid. , for example histidine. In preferred embodiments, the bacterial cell of the invention is a cell of S. typhi with the genotype AroA, His, PhoP / PhoQ, for example TyLH445.

The invention may also include a Salmonella cell, whose virulence is attenuated by the constitutive expression of a gene under the control of a two-component regulatory system. In preferred embodiments, the constitutive expression is the result of a mutation in a component of the two-component regulatory system. In preferred embodiments, the bacterial cell includes a second mutation that attenuates virulence. In still other preferred embodiments of the vaccine, the two-component regulatory system is the phoP regulatory region, and the gene under the control of the two-component system is gene regulated by a phoP regulatory region, for example a prg gene, for example prgA, prgB, prgC, prgE or prgH, or the gene pag, for example pagC. In preferred embodiments, the constitutive expression is the result of a change or mutation, for example a deletion (preferably a nonreversible mutation) in the promoter of the regulated gene or the phoP regulatory region, eg a mutation in the gene phoQ or the phoP gene, for example the PhoPc mutation. In another aspect, the invention includes a vaccine that includes a bacterial cell that is attenuated by the reduction in the expression of a virulence gene under the control of a phoP regulatory region, for example a prg gene, for example prgA, prgB, prgC , prgE, or prgH. In preferred embodiments of the vaccine, the Salmonella cell includes a first mutation, for example a deletion that attenuates virulence, eg a mutation in a phoP regulatory region gene, eg a mutation in the phoP or phoQ gene, for example PHoPc, or a mutation in a gene regulated by the regulatory region phoP, and a second mutation that attenuates virulence, for example a mutation in a synthetic gene of aromatic amino acid, for example a gene aro, a mutation in a regulated gene for the regulatory region phoP, for example a mutation in a prg gene, for example prgA, prgB, prgC, prgE, or prgH, or place pag, for example a mutation pagC. In still other preferred embodiments, the bacterial cell includes a first mutation in a phoP regulatory region gene and a second mutation in a synthetic aromatic amino acid gene, for example a gene. In another aspect, the invention includes a vaccine, preferably a live vaccine, including a bacterial cell, whose virulence is attenuated by a mutation, for example a deletion, in a gene under the control of a two-component regulatory system. In preferred embodiments, the bacterial cell includes a virulence attenuating mutation in a second gene, for example in a synthetic aromatic amino acid gene, for example a gene. In still other preferred embodiments of the vaccine, the bacterial cell is a Salmonella cell, the two-component regulatory system is the phoP regulatory region, and the gene under its control is a prg gene, for example prgA, prgB, prgC , prgE, or prgH, or a gene pag, for example the gene pagC. In another aspect, the invention includes a vaccine, preferably a live vaccine, including a Salmonella cell, for example a S. typhi cell, S. enteri tidis typhimurium, S. cholerae-suis, including a first mutation that attenuates the virulence in an aromatic amino acid biosynthetic gene, for example a gene, and a second quen mutation attenuates virulence in a phoP regulatory region gene, for example a phoP mutation. In another aspect, the invention includes a bacterial cell, or a substantially purified preparation thereof, preferably a Salmonella cell, for example a S. typhi cell, S. enteri tidis tyhpimurium, or S. cholerae-suis, which constitutively expresses a gene under the control of a two-component regulatory system and that includes a virulence-attenuating mutation, for example a deletion, that does not result in the constitutive expression of a gene under the control of the two-component regulatory system . In preferred embodiments, the bacterial cell includes a mutation in a component of the two-component regulatory system. In preferred embodiments, the bacterial cell is a Salmonella cell that expresses a gene regulated by the phoP regulatory region constitutively (the constitutive expression preferably caused by a mutation, preferably a nonreversible mutation, for example a deletion in the region regulatory phoP, for example a mutation in the phoQ or phoP gene, for example phoPc), and which includes a virulence attenuating mutation, preferably a nonreversible mutation, for example a deletion, preferably in a synthetic aromatic amino acid gene, for example a gene ring, or in a gene regulated by the regulatory region phoP, for example a gene prg, for example prgA, prgB, prgC, prgE, or prgH or pag, for example pagC, which does not result in the expression constitutive of a gene under the control of the phoP regulatory region. In another aspect, the invention includes a bacterial cell, or a substantially purified preparation thereof, for example a Salmonella cell, for example a S. typhi cell, a S. enteri tidis typhimurium cell, or an S cell. cholerae-suis, including a virulence-attenuating mutation in a gene regulated by a two-component regulatory system. In preferred embodiments, the virulence attenuating mutation is in a gene regulated by the regulatory region phoP, for example a gene prg, for example a gene prgA, prgB, prgC, prgE, or prgH or pag, for example pagC. In preferred embodiments, the bacterial cell includes a second mutation, for example in a synthetic aromatic amino acid gene, for example a gene, in a phoP regulatory region gene, for example the phoP or phoQ genes, or in a regulated gene for the phoP regulatory region, for example a prg gene, for example a gene prgA, prgB, prgC, prgE or prgH or a gene pag, for example pagC, which attenuates virulence but does not result in the constitutive expression of a gene regulated by the phoP regulatory region. The invention also includes a living Salmonella cell, or a substantially purified preparation thereof, for example a S. typhi cell, S. enteridi tis typhimurium, or S. cholerae-suis, into which it is inserted into a gene. of virulence, for example a gene regulatory region phoP, or a gene regulated by the regulatory region phoP, for example a prg gene, for example prgA, prgB, prgC, prgE or prgH or a pag site, for example pagC, a gene encoding a heterologous protein, or a regulatory element thereof. In preferred embodiments, the living Salmonella cell carries a second mutation, for example an aro mutation, for example an aroA mutation, for example arA 'or aroA-DEL407, which attenuates virulence. In preferred embodiments, the DNA encoding a heterologous protein is under the control of an environmentally regulated promoter. In other embodiments, the Salmonella live cell further includes a DNA sequence encoding T7 polymerase under the control of an environmentally regulated promoter and a T7 transcriptionally responsive promoter, the T7 transcriptionally responsive promoter controlling the expression of the heterologous antigen. The invention also includes a vector capable of being integrated into the Salmonella chromosome, including: a first DNA sequence encoding a heterologous protein; a second DNA sequence (optl) encoding a marker, for example a selective marker, for example a gene that confers resistance to heavy metals or a gene that complements an auxotropic mutatcarried by the strain to be transformed; and a third DNA sequence, for example a gene encoded by a phoP regut for example a prg gene, for example prgA, prgB, prgC, prgE or prgH or a pag site, eg pagC, encoding a regulated gene product for the phoP regulatory regnecessary for virulence, the third DNA sequence being mutatlly inactivated. In other preferred embodiments: the first DNA sequence is arranged in the vector so as to mutatlly inactivate the third DNA sequence; the vector can not replicate in a wild type strain of Salmonella; the heterologous protein is under the control of an environmentally regulated promoter; and the vector further includes a DNA sequence encoding T7 polymerase under the control of an environmentally regulated promoter and a T7 transcriptlly responsive promoter, the T7 transcriptlly responsive promoter controlling the expressof a heterologous antigen.

In another aspect, the invention includes a method of vaccinating an animal, for example a mammal, for example a human being, against a disease caused by a bacterium, for example Salmonella, including administering a vaccine of the invention. The invention also includes a vector that includes DNA encoding the gene product of pagC; a cell transformed with the vector; a method of producing the gene product, including culturing the transformed cell and purifying the gene product of the cell or the culture medium; and a purified preparation of the gene product pagC. In another aspect, the invention includes a method of detecting the presence of Salmonella in a sample, which includes contacting the sample with pagC that encodes DNA and detecting the hybridization of the pagC that encodes DNA to the nucleic acid in the sample. The invention also includes a vector that includes DNA encoding the prgH gene product; a cell transformed with the vector; a method of producing the prgH gene product, including culturing the transformed cell and purifying the prgH gene product of the cell or culture medium; and a purified preparation of the prgH gene product. In another aspect, the invention includes a method of detecting the presence of Salmonella in a sample, including contacting the sample with prgH which encodes DNA and detecting the hybridization of prgH which encodes DNA to nucleic acid in the sample. In another aspect, the invention includes a method of attenuating the virulence of a bacterium, the bacterium including a two-component regulatory system, including causing a gene that is under the control of the two-component system to be constitutively expressed. In preferred embodiments, the bacterium is Salmonella, for example S. typhi, S. enteri tidis typhimurium, or S. cholerae-suis, and the two-component system is the phoP regulatory region. In yet another aspect, the invention includes a substantially pure DNA that includes the sequence given in the sequence identified with No. 5 or a fragment thereof. The invention also includes a substantially pure DNA that includes a sequence encoding pagD, for example nucleotides 91 to 354 of the sequence identified with No. 5 (oreading frame (DAG) and its degenerate variants to encode a product. with essentially the sequence of amino acids given in the sequence identified with No. 6, as well as ORF for pD and its non-coding 5 'region (nucleotides 4 to 814 of the sequence identified with No. 15) containing the pagD promoter. The DNA in the region between ORF pagC and ORF pagD (nucleotides 4 to 814 of the sequence identified with No. 5), DNA that includes the pagC promoter (nucleotides 562 to 814 of the sequence identified with No. 15), and DNA that includes the pagD promoter alone (nucleotides 4 to 776 of the sequence identified with No. 15) are also within the claimed invention. The invention also includes a substantially pure DNA that includes a sequence encoding envE, for example nucleotides 1,114 to 1,650 of the sequence identified with No. 5 (ORF, envE) and its degenerate variants that encode a product with essentially the sequence of amino acids given in the sequence identified with No. 7. Another aspect of the invention includes a substantially pure DNA that includes a sequence encoding msgA, for example nucleotides 1,825 to 2,064 of the sequence identified with No. 5 (ORF). msgA) and its degenerate variants encoding a product with essentially the amino acid sequence given in the sequence identified with No. 8, as well as ORG msgA with its 5 'non-coding region, nucleotides 1,510 to 1,824 of the sequence identified with No. 5 containing the msgA promoter. Also within the invention is a substantially pure DNA comprising the msgA promoter alone (nucleotides 1,510 to 1,760 of the sequence identified with No. 5). In yet another aspect, the invention includes a substantially pure DNA that includes a sequence encoding envF, for example nucleotides 2,554 to 3,294 of the sequence identified with No. 5 (ORF envF) and its degenerate variants that encode a product with essentially the amino acid sequence given in the sequence identified with No. 9, as well as ORF envF with its 5 'non-coding region, nucleotides 2,304 to 2,553 of the sequence identified with No. 5 containing the envF promoter. Also within the invention is a substantially pure DNA that includes the sequence given in the sequence identified with No. 10 or a fragment thereof. The invention also includes a substantially pure DNA that includes a sequence encoding prgH, for example nucleotides 688 to 1866 of the sequence identified with No. 10 (ORF prgH) and, its degenerate variants encoding a product with essentially the sequence of amino acids given in the sequence identified with No. 11, as well as the ORF prgH with its promoter region (nucleotides 1 to 689 of the sequence identified with No. 10). The invention also includes a substantially pure DNA that includes a sequence encoding prgl, for example nucleotides 1,981 to 2,133 of the sequence identified with No. 10 (ORF prgl) and its degenerate variants that encode a product with essentially the amino acid sequence given in the sequence identified with No. 12, as well as ORF prgl with its promoter region (nucleotides 1 to 689 of the sequence identified with No. 10). In another aspect, the invention includes a substantially pure DNA that includes a sequence encoding prgJ, for example nucleotides 2.152 to 2.457 of the sequence identified with No. 10 (ORF prgJ) and its degenerate variants encoding a product with essentially the sequence of amino acids given in the sequence identified with No. 13, as well as ORF prgJ and its promoter region (nucleotides 1 to 689 of the sequence identified with No. 10). In still another aspect, the invention includes a substantially pure DNA that includes a sequence encoding prgK, for example nucleotides 2,456 to 3,212 of the sequence identified with No. 10 (ORG prgK) and its degenerate variants that encode a product with essentially the sequence of amino acids given in the sequence identified with No. 14, as well as ORF prgK with its promoter region (nucleotides 1 to 689 of the sequence identified with No. 10). The invention also encompasses a bacterial cell whose virulence is attenuated by a mutation, for example a deletion, in one or more genes selected from the group consisting of pagD, pagE, pagF, pagG, pagH, pagl, pagJ, pagK, pagL, pagM , pagN, pagP, envE, and envF. Also included is a bacterial cell that is attenuated by a mutation, for example a deletion, in one or more genes selected from the group consisting of pagC, pagD, pagJ, pagK, pagM and msgA. A bacterial cell, whose virulence is attenuated by a mutation, for example a deletion, in one or more genes selected from the group consisting of prgH, prgl, prgJ and prgK, is also within the claimed invention. "Two-component regulatory system," as used herein, refers to a bacterial regulatory system that controls the expression of multiple proteins in response to environmental signals. The two components referred to in the term are a sensor, which for example can detect an environmental parameter and in response to it promote activation, for example by promoting phosphorylation, of the second component, the activator. The activator affects the expression of genes under the control of the two-component system. A two-component system may include, for example, a histidine protein kinase and a phosphorylated response regulator, as seen in both gram-positive and gram-negative bacteria. In E. coli, for example, ten kinases and eleven response regulators have been identified. They control the chemotaxis, nitrogen regulation, phosphate regulation, osmo-regulation, sporulation and many other cellular functions, Stock et al., 1989, Microbiol. Rev. 53: 450-490, incorporated herein by reference. A two-component system also controls the virulence of tumor formation of the Agrobacterium tumef sciens plant, Leroux et al., EMBO J. 6: 849-856, incorporated herein by reference. Similar virulence regulators are implicated in the virulence of Bortedella pertussis, Arico et al., 1989, Proc. Nati Acad. Sci. USA 86: 6671-6675, incorporated herein by reference, and Shigella flexneri, Bernardini et al., 1990, J. Bact. 172: 6274-6281, incorporated herein by reference. Environmentally regulated, as used herein, refers to an expression pattern where the expression of a gene in a cell depends on the levels of some characteristic or component of the environment in which the cell resides. Examples include promoters in biosynthetic pathways that are activated or deactivated by the level of a specific component or components, for example iron, promoters that respond to temperature, or promoters that are more actively expressed in specific cell compartments, for example in macrophages or vacuoles . As used herein, a vaccine is a preparation that includes materials that evoke a desired biological response, for example an immune response, in combination with a suitable carrier. The vaccine may include living organisms, in which case it is usually administered orally, or dead organisms or components thereof, in which case it is usually administered parenterally. The cells used for the preferensia invention vaccine are alive and thus are able to colonize the intestines of the inoculated animal. As used herein, a mutation is any sambio (in somparasion are the appropriate parental sense) in the DNA sesuensia of an organism. These sambios may arise, for example, spontaneously, by chemical, energétisa (X-rays) or other forms of mutagenesis, by genetic engineering, or as a result of mating or other forms of genetic information inter-assembly. Mutations include, for example, base changes, deletions, insertions, inversions, translocations or duplications. A mutation attenuates virulence if, as a result of the mutation, the level of virulence of the mutant cell is reduced in comparison to the level in a cell of the parental strain, as measured by (a) a significant reduction (e.g. at least 50%) of the virulence in the mutant strain in comparison to the parental strain, or (b) a significant redussion (eg, at least 50%) in the amount of the polypeptide identified as the virulence pheromone in the know mutant in somparation with the parental strain. A nonreversible mutation, as used herein, is a mutation that can not be reversed by a single base pair change, for example deletion or insertion mutations and mutations that involve more than one lesion, for example a mutation deposited by mutations in two separate points. The phoP regulatory region, as used herein, is a two-component regulatory system that controls the expression of the pag and prg genes. Includes the place phoP and the place phoQ. Genes regulated by the phoP regulatory region, as used herein, refer to genes such as the genes pag and prg. pag, somo is used in the present, refers to a gene that is positively regulated by the phoP regulatory region. prg, as used herein, refers to a gene that is negatively regulated by the phoP regulatory region. A synthetic aromatic amino acid gene, as used herein, is a gene that codifies an enzyme that satalizes a step in the synthesis of an aromatic amino acid. Examples of such genes in Salmonella are aroA, aroC and aroD. Mutations in these genes can attenuate virulence without a total loss of immunogenicity. Abnormal expressions, as used herein, means expression that is greater or less than sight in the wild type. Heterologous protein, as used herein, is a protein that in the wild type is not expressed, or is expressed from a different sromosomal site, for example a heterologous protein is a sodifised by a gene that has been inserted into a second gene . Virulene gene, as used herein, is a gene whose inactivation results in a Salmonella cell with less virulence than that of a similar Salmonella cell in which the gene is not inactivated. Examples include the genes phoP, pagC, prgH. A marker, as used herein, is a gene product whose presence is readily determined, for example a gene product that confers resistance to a heavy metal or a gene produsto that allows or inhibits sresylation under a given set of condiions. Purifidated preparation, as used herein, is a preparation, for example of a protein, which is purified from proteins, lipids and other materials are assorted. The preparation is preferensia purifisada at least 2-10 veses. "Sonic expression," as used herein, refers to a gene expression that is modulated or regulated to a lesser degree than the expression of the same gene in an appropriate control strain, for example a parental or wild-type strain. For example, if a gene is normally repressed under a first set of conditions and derepressed under a second set of conditions, constitutive expression would be expression at the same level, for example the repressed level, the derepressed level, or an intermediate level, regardless of the sondisiones Partial constitutive expression is included within the definition of constitutive expression and the difference between two levels of expression is reduced in somparasion is what is seen in an appropriate control strain, for example a wild type or parental type. A substantially purified preparation of a bacterial cell is a cell preparation where the contaminating cells without the desired mutant genotype make up less than 10%, preferably less than 1%, and are the least preferable to less than 0.1% of the total number of cells in the cell. the preparation. The invention allows the attenuation of virulence of bacteria and vaccines that include bacteria, especially vaccines that include living bacteria, by mutations in two-component regulatory systems and / or in genes regulated by these systems. The vasunas of the invention are highly attenuated in virulence but retain the immunogenicity, so that they are both safe and effective. The vectors of the invention allow the rapid construction of strains containing DNA encoding heterologous proteins, for example antigens. The DNA that sodifices heterologous proteins is chromosomally integrated, and thus stable, unlike plasmid systems that depend on antibiotic resistance or other selection pressure for stability. Living Salmonella cells from the invention in which the expression of the heterologous protein is under the control of an environmentally responsive promoter do not express the heterologous protein at times when such expression would be undesirable, for example during culture, vaccine preparation or storage, to the viability of the cells, but when they are administered to humans or animals, they express great sanctities of protein. This is desirable because the high expression of mushas heterologous proteins in Salmonella can be asosified with bacterial toxicity. The use of only an integrated copy of the DNA that sodifies the heterologous protein also contributes to the minimal expression of the heterologous protein at times when expression is not desired. In forms of realization where a virulence gene, for example in gene pagC or the gene prgH, they are the site of the integration for the DNA that sodifises the heterologous protein, it attenuates the virulence of the organism. A "substantially pure DNA", as used herein, refers to a nucleic acid sequence, segment or fragment, which has been purified from the sequences that flank it in a natural state, for example a DNA that has been removed of the sequences that are normally adjacent to the fragment, for example the sequencies adjacent to the fragment in the genome where they naturally occur. The term also applies to DNA that has been substantially purified from other somponents naturally associated with DNA, for example DNA that has been purified from proteins that naturally accompany it in a cell. Other aspects and advantages of the invention will be apparent from the following description of preferred embodiments and claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the drawings will be described. Drawings Figure 1 is a graph of the survival of Salmonella strains within macrophages. Figure 2 is a map of the restriction endonuclease sites of the pagC site. Figure 3 is a map of the DNA sequencing of the pagC region (sesuensia identified with No. 1). Figure 4 is a map of the location of prg? inside the place hil. The fleshas induce the targeting direction of the neomycin promoter of the Tn5B50 insertions within the hil site and the transsripssion diression of the prgH.1: - fusion protein. TnphoA. The restriction endonuclease sites are represented by B, BamHl; H, HindIII; X, Xhol; S, Sacl; V, .EcoRV. Figure 5 is a DNA sesuensia of the prgH gene (pIBOl plasmid) (sequence identified with No. 3). Figure 6 is a bar graph showing a comparison of the sensitivity of wild-type sepals (ATCC 14028), PhoP-null mutant (CS015) and pag: - mutants. TnphoA to defensin NP-1. The y-axis represents the defensin mortality index (DKI), which represents dead basterias due to exposure to NP-1. The DKI is defined as the logarithmic function of the relasion of control bacteria to surviving basterias incubated with NP-1 [DKI = log (CFU without NP-1 / CFU with NP-1)]. The individual bars represent the mean and standard error of five separate experiments. The x axis indicates the mutated allele. It was determined that the average DKI for one of the tested strains was not different from that of Salmonella wild type (P <0.05). In contrast, the phoP mutant was considerably different (P <0.0001). Figure 7 is a diagram showing a partial physical map of the restriction endonuclease sites of the chromosomal region pagC. The lethal doses at 50% (LDS0) of mice for sepals are transposon insertions in page, envE, msgA and pagC, are shown above sada gene. The horizontal arrows show the transshipping direction. The vertical fleshas denote TnphoA insertions and the hollow triangle denotes a Mud insertion. "Below the chromosomal map is a representation of the DNA insert in plasmid pCAA9, which was mutagenized are TnphoA and MudJ". The designations are letters are: A, Accl; B, Clal; E, ECoRI; H, Hpal; P, Ps i; and V, EcoRV. Figure 8 is a DNA sesuensia of the sorptive region above pagC and the tradussion of sada ORF. The Hpal and Clal sites at the beginning and at the end of the region are unavailable. The potential regions of Shine-Delgarno are underlined and the stem loop structures (Rho-independent potential terminators) are indexed with a line below and above the sequence. The knowledge of flesha denote the location of the representative transposon insertion within each gene. Horizontal arrows in the promoter regions pagD and msgA mark the transsriptional inisium sites, and the asterisks mark the -10 and -35 sequences. The consensus lipid binding site in EnvF is enserrado in sorshetes. The ORF pagD begins in the nusleotide 91 and ends in the nusleotide 354 of the identified sesuensia are No. 5; the ORF sends somienza in the nusleotide 1,114 and ends in the nusleotide 1,650 of the sesuensia identified with the No. 5; the ORG msgA begins in the nusleotide 1,825 and ends in the nusleotide 2,064 of the identified sesuensia are No. 5; and the ORF envF inisia in the nusleotide 2,554 and ends in the nusleotide 3,294 of the sesuensia identified with the No. 5. Figure 9 is a sequencing of the DNA that contains the prgH, prgl, prgJ and prgK genes. The start codon (ATG) of each gene is underlined, and the end codon is indicated with an asterisk. The ORF prgH starts at nucleotide 688 and ends at 1866 of the sequence identified with No. 10; the ORF prgl begins at nucleotide 1891 and ends at nusleotide 2,133 of sesuensia identified with No. 10; the ORF prgJ is in the nusleotide 2,152 and ends in the nusleotide 2,457 of the identified sesuensia are No. 10; and the ORF prgK is in the nusleotid 2,454 and ends in the nusleotide 3,212 of the identified sesuensia are No. 10. Figure 10 is a linear graph showing the rates of sresimiento of the parental separator of Salmonella (AroA-) and the know of vaccine (AroA-, PhoP-). Figure 11 is a bar graph showing the defensin sensitivity of mouse vasuna sp. (S. typhimurium). Figure 12 is a bar graph showing the PhoP astivation as measured by the LacZ activity using the PagB: LasZ log merge combination. Figure 13 is a bar graph showing the defensin sensitivity of the S. Typhi vaccine strain TyLH445 compared to the parental strain AroA. "Figure 14A is a graph showing the relative expression of the sonstitutive expression (610 and 617) and expression regulated by phoP (pagC and pagD) of AP fusion proteins. Figure 14B is a graph showing the immunological response to lipopolysaccharide (LPS) Figure 14C is a graph showing the immunological response to the model heterologous antigen, AP Figure 15 is a sequencing of DNA containing the intergenic region pagC-pagD The translation initiation site of pagC (ATG in the opposite DNA strand) is underlined (nusleotides 1-3 of the sense identified with No. 15). The tradussion initiation site of pagC (nucleotide 562) ) is indicated by an arrow pointing to the left.The transgriptional start site of pagD (ATG) is underlined (nusleotides 815-817 of sequencing identified with No. 15) .The transcriptional start site pagD is indicated are a arrow pointing to the right (nucleotide 776) Deposit of Strains Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Misroorganisms for the Purpose of Patent Procedure, a the deposit of the following materials in the American Type Culture Collection (ATCC) of Rockville, Maryland, United States, regardless of whether Mexico is not yet part of the internal treaty. The foregoing is manifested by virtue of the fact that, to date, as far as the applicants know, regulations regulating the deposit of samples of misroorganisms and / or genetic material nor deposit institutions recognized and authorized by the General Directorate of Technological Development of the Secretary of Commerce and Industrial Development of the Mexican federal government. The transferee, The General Hospital Corporation, states that the ATCC is a depository institution that allows to achieve the permanence of the deposit and an easy access to it by the public once a patent is granted. All restrictions on the availability to the public of the material deposited in such terms will be irrevocably eliminated upon granting the patent. The material will be available during the time the patent application is being processed by the person appointed by the United States Patent and Trademark Commissioner. The deposited material will be conserved with all the necessary care to keep it viable and uncontaminated for a period of at least fifty years after the most recent request for the supply of a sample of the deposited plasmid, and in any case for a period of at least thirty (30) years after the date of deposit or for the duration of the patent, whichever is greater. The session ratifies its obligation to replace the deposit if the deposit institution is unable to provide a sample if it is solved due to the condition of the deposit. The PhoPc CS022 strain (described below) has been deposited at the American Type Culture Collestion (Roskville, Maryland, United States) and has received the designation ATCC 55130. The pIBOl plasmid, which contains the prgH gene, was deposited on July 9. 1993 at the American Type Culture Collection (Rockville, Maryland, United States), and received the designation ATCC 75496. Does the PhoP Regulatory Expression Attenuate the Virulence of Salmonella? Survival within Macrophages The constitutive allele phoP (PhoPc), pho-24, results in derepression of the sites pag. Using mutagenesis of S. typhimurium LT-2 with diethyl sulfate, Ames et al. Isolated the strain pho-24 TA2367 (all the known, the materials and the methods referred to in this section are described below), which contained a phoP mutation that gave as a result the constitutive production of acid phosphatase in rich medium, Kier et al., 1979, J. Bacteriol. 138: 155, incorporated herein by reference. This acid phosphatase regulated by phoP is sodifised by the gene phoN, a site pag, Kier et al., 1979, supra, Miller et al., 1989, supra. To analyze whether the pho-24 allele insensitive to the expression of other pag sites, the effect of the pho-24 allele on the expression of other pag sites recently identified as transcriptional fusion proteins (eg pagA and pagB) and tradussion was determined. (eg, pagC) that require phoP and phoQ for expression, Miller et al., 1989, supra. The fusion genes of the pag gene, isogenic with the exception of the pho-24 allele, were constructed and tested for fusion protein activity. The PhoPc derivatives of the strains pagA:: MU dJ and pagB:: Mu dJ processed 480 and 490 U, respectively, of β-galastosidase in medium riso, an insertion of 9 to 10 veses on the values for the fusion defects are a place phoP wild type, see Table 1. Gene fusion pagC: -. TnphoA produced 350 U of AP, an increase of three to four times over the product in CS119, which is isogenic except for the pho-24 mutation, Miller et al., 1989, supra. These results are a nine-step insight into the astivity of acid phosphatase in CS022 on the introduction of the pho-24 allele. Therefore, these assays available for the expression of the gene show that the pho-24 mutation causes constitutive expression of the pag sites other than phoN.

Table 1 Basterian Strains and Properties Enzymatic Activity Source or Strain Genotype (U) a Reference 10428 Wild type 180 (A) ATCC; Miller et al., 1989, supra TA2367 phs-24 1,925 (A) Kier et al., 1974, supra.

CSO03 DeltapJioP DeltapurB < 10 (A) Miller et al., 1989, supra CS022 pho-24 1,750 (A) This work CS023 pho-24 phoN2 25 (A) This work zxx:: 6251Tnl0d-Cam CSO 12 pagrAl:: MU dJ 45 (B) Miller et al., 1989, supra CS013 pag l -. -. Mu dJ 120 (B) Miller et al., 1989, supra CS119 pagCl:: TnphoA phoN2 85 (C) Miller et al., 1989, supra zxx-. : 6251Tnl0d-Cam SC024 pagAl :: Mu dJ pho-24 450 (B) This work SC025 PasrBl:: u dJ pho -24 980 (B) This work SCO 26 pa = rCl:: Tt? PhoApho- 4phoN2 385 (B) This job zxx:: 6251Tnl0d-Cam CS015 phoP102:: Tnl0d-Cam < 10 (A) Miller et al., 1989, supra TT13208 phoPl 05:: TnlOd < 10 (A) - b aA, acid phosphatase; B, β-galactosidase; C, alkaline phosphatase (AP) b Gift from Ning Zhu and John Roth.

Identifications of Protein Species that are Repressed As Well as Activated in the PhoPc Mutant Strain Whole-cell proteins of the CS022 isolate were analyzed to estimate the number of protein thickenes that can be potensively regulated by the PhoP regution. Notably, the analysis by proteins of the proteins produced by the sepals is the PhoPc phenotype, although some protein species were reduced in terms of expression when many presumably gene products were fully induced by the pho-24 mutation. The reduced proteins in the PhoPc strain can represent gene products that are repressed by the PhoP regulator. The genes encoding the proteins reduced by the pho-24 allele are designated prg sites for repressed phoP genes. The protein somparation of wild-type mutant strains, PhoP "and PhoPc shows that cresting in LB medium at 37 ° C represents repression modes for gene products and derepression sonsions for prg gene products. To estimate the total number of potency-regulated phoP gene products, the total cell proteins of wild-type and PhoPc mutant strains grown in LB were analyzed by two-dimensional gel electrophoresis, at least 40 thickeners underwent greater fluttering in expression in response to mutation pho-24 Virulence Defects of PhoPc Strain Notably, strains are the only mutation pho-24 were attenuated in a marsated manner for virulence in mice (Table 2) .The number of PhoPc organisms (2 x 105) that killed 50% of the BALB / s mice clogged (LD50) by the intraperitoneal route (ip) was close to the number of PhoP bacteria "(6 x 105), Miller et al., 1989, supra. The PhoPc strains had a comparable growth to wild type organisms in rich and minimal media. The PhoPc mutants were also tested for alterations in lipopolysaccharide, which may explain the observed virulence defect. The CS022 had normal sensitivity to phage P22, reastivity group B normal to the antigen to 0 antigen, and a lipopolysaccharide profile identical to that of the parental strain, as determined by polyacrylamide gel electrophoresis and color marking.

Table 2 Virulence and Protective Efficacy of Salmonella Strains PhoPc and PhoP "Number of Number of Survivors / Total Survivors After Wild Type Attack Dose Initial Dose / Total Immunization 5 x 107 5 x 105 5 x 104 5 x 103 Organisms PhoPc 15 13/13 5/5 4/5 50 4/4 4/4 1. 5 x 102 11/11 4/4 3/3 5 x 102 16/16 4/4 1. 5 X 103 5/5 3/3 2/2 5 x 103 4/4 4/4 1. 5 x 104 5/5 3/3 2/2 5 x 10"19/23 4/4 1. 5 x 10 = 5/5 3/3 2/2 5 X 105 1/4 1/1 x 10e 0/6 3 x 109 (*) 5/5 5/5 3 x 1010 (*) 5/5 5/5 1.5 x 1011 (*) 5/5 5/5 PhoP Organisms "6 x 103 36 / 36 0/12 0/12 0/12 6 x 104 36/36 0/12 0/12 3/12 6 x 105 19/36 0/12 0/12 3/12 x 1010 (*) 7/7 3/7 (*) The organisms were administered orally, In all other experiments, the organisms were administered by i.p.

As strain TA2367 pho-24 was constructed by chemical mutagenesis and may have another linked mutation responsible for its virulence defect, PhoP revertants were isolated to determine if the pho-24 allele was responsible for the attenuation of virulence observed. The revertants of the PhoPc phenotype, identified by the normal levels of acid phosphatase in a laughing medium, were isolated among the resurfaced basterias of the livers of the infested mice, namely CS022. Six separate phenotypic revertants, designated CS122 through CS128, were found to be fully virulent (LD50 of less than 20 organisms for BALB / c mice). The place responsible for the reversion phenotype was mapped in the six revertants tested for virulence by contransduction of bacteriophage P22 and had linkage characteristics consistent with the phoP site (more than 90% binding to purB). These data indicate that these reversal mutations are not extragenic suppressors but that they are true intragenic or revertant suppressors of the pho-24 mutation. In this way, the virulence defect of the PhoPc mutants is probably the result of a single reversible mutation in the phoP site and not the result of a second non-relasioned mutation acquired during mutagenesis. Reversion Frequency of the PhoPc Phenotype The reversion frequency of the PhoPc mutation in vivo in mice was investigated to determine if the reversion could reduce the LD50 value of this strain. The presence of the revertants of strain CS022 was tested by administering 106, 104 and 102 attack organisms to each of the eight animals by i.p. On day 7, three animals that received PhoPc organisms died. That day, the livers and spleens of all animals were cultured and homogenized in saline. After appropriate dissolution, 10% of the tissue was placed on LB plates containing the chromogenic phosphatase substrate XP. The revertants were identified by their blue solonias plus slaras in somparasión are PhoPc basterias and were confirmed by suantitative assays of acid phosphatase. An estimate of 107, 105 and 103 organisms per organ were recovered from the animals by one of the three respec- tive doses. The revertants were identified only at the highest dose and comprised 0.5 to 1%, or 105 organisms per organ, at the time of death. It is feasible that the revertants are able to sompetir more efestively for sresimiento in these organs that are have mastrófagos, because the CS022 strain is deficient in survival within macrophages (see below). However, the revertants were not identified if less than 105 organisms were administered at the questioning dose, suggesting that the reversion fresuensia should be approximately 10".5 The reversion rate of the PhoPc phenotype for CS022 basterias sultivadas in LB is hesho 6 x 10"4 suando is salified by the same phenotypes of solonia. The percentage of revertants of animal death resistances suggests that in vivo pressure is applied to revertants of the PhoPc phenotype and implies that the observed virulence defect can be much greater quantitatively for a strain with a non-reversible PhoPc mutation. The PhoPc strain is Deficient in Survival within Macrophages Due to the importance for Salmonella virulence of survival within macrophages, Fields et al., 1986, Proc. Nati Acad. Sci. USA 83: 5189, incorporated herein by reference, PhoPc bacteria were tested for this property. Strain CS022 was defective in the ability to grow and persist in masophagi in somparasión with wild type organisms (figure 1). In Figure 1, the supervi-vensia of the CS022 (PhoPc) sepa (triangles) in cultured macrophages is compared to that of wild type S. typhimurium ATCC 10428 (isolates). The experiment shown is a representative one. The difference between the two strains at 4 and 24 hours is considerable (P <; 0.05). PhoP bacteria "have a defect in survival in maarophages suitably similar to that of PhoPc bacteria but consistently survive better two or three times in side-side experiments." Increased resuper- sion of organisms that revert to the PhoPc phenotype in rich mouse organs The content of macrophages is consistent with the reduced survival in masrophages of PhoPc mutants in vi tro.

Use of PhoP Strain as a Live Vaccine It has been previously reported that PhoP isolates are useful as live viruses to protect mouse typhoid, Miller et al., 1989, supra.The immunogenicity of PhoPc suando is used as live attenuated vasunas in The mice were somparada are those of PhoP ". This was confirmed by simultaneous determination of the survival, after dosages of graded questioning are known wild type ATCC 10428, in previously immunized mice are dosed doses of the two live vasunas, CS015 phoP:: Tnl0d-Cam and CS022 pho -24, so we are a saline solution. The results obtained (Table 2) suggest the following conclusions: (i) small doses i.p. of the PhoPc strain (eg, 15 organisms) effectively protect mice from doses as large as 5 x 105 bacteria (a challenge dose representing more than 104 ip.D.50), (ii) large doses of orally given PhoPc organisms completely protect mice of an oral challenge consisting of 5 x 107 wild-type bacteria (more than 200 LD50s wild-type oral), and (iii) in comparison, a large dose of. PhoP organisms "(5 x 105) does not provide similar protection.The reversal of the PhoPc mutation in vivo makes up a very small part of the analysis of the use of these somo somas insults, since revertants of the CS022 (ie desir, wild type cells). However, it was not possible to identify revertants by examining 10% of the spleen and liver tissue available from those mice that had 104 or less organisms.Strains, Materials and Methods The knowledge, materials and methods used in the work of PhoP regution described above were the following: Strain 14028 of the American Type Culture Collestion (ATCC), a mild virulent strain of S. typhimurium, was the parental strain for all virulence studies.The strain TT13208 was a gift from Nang Zhu and John Roth The TA2367 strain was a generous gift from Gigi Stortz and Bruce Ames, Kier et al., 1979, supra.The p22HT intterpharyophagous was used in transdussional struces for to construct isogenic strains except for mutations of phoP sites, Davis et al., 1980, Advanced Bacterial Genetics, p. 78, 87, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, incorporated herein by reference. Luria broth was used as a rich medium, and the minimum medium was M9, Davis et al., 1980, supra. The chromogenic phosphatase substrate of 5-bromo-4-sloro-3-indolyl phosphate (XP) was used to assess the production of aceid and AP in solid media. Derivatives of S. typhimurium ATCC 10428 with the pho-24 mutation were constructed by the use of strain TA2367 as a donor of the purB gene in a P22 transductional cross with the DeltaphoP DeltaphoP strain CS003, Miller et al., 1989, supra. Then colonies were selected regarding the ability to grow in minimal medium. A transductant designated CS022 (phenotype PhoPc) that synthesized 1,750 U of acid phosphatase in medium rhyme (a nine-vein inset over the wild-type level in medium riso) was used in further studies. Strains CS022 and CS023, phoN2 zxx:: 6251Tnl0d-Cam and negative acid phosphatase derivative of CS022 were constructed, containing fusions of the gene for transducer crosses P22 of basteriófago, using selection of TnphoA- or resistance to encoded kanamycin by Mu dJ. Strains were verified for intact gene fusion by demonstrating the appropriate loss of fusion protein activity upon introduction of a phoP105 allele:: Tnl0d or phoP102:: Tnl0d-Cam. Assays of acid phosphatase, AP, and β-galactosidase were carried out as previously distilled, Miller et al., 1989, supra, and are reported in units, as defined by Miller, 1972, Experiments in Molecular Genetics, p. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, insorporated in the present by referensia. In studies of virulence and vaccination of mice, they were washed and diluted in saline solution normal basterias sultiva-das overnight in Luria broth. The wild-type parental strain of CS022 (ATCC 10428) was used for all challenge studies of live vaccines. This strain has a lethal dose (LD50) for adult BALB / s mice of less than 20 subara organisms are administered by intraperitoneal (i.p.) and 5 x 104 when administered orally in NaHCO3. The mice were purchased from Charles River Breeding Laboratories, Inc. (Unlimited, Massachusetts, United States) and were 5 to 6 weeks old at the time of initial questioning. All inoculations i.p. were carried out as previously described, Miller et al., 1989, supra. Oral challenge experiments were carried out with bacteria cultured in LB broth and consented by sentrifugation. The basterias were resuspended in 0.1 M NaHCO3 to neutralize the stomatal acids, and administered as a 0.5 ml bolus under ether anesthesia. The colonies accounts were carried out to accurately access the number of organisms administered. All the challenge experiments were carried out one month after inoculation i.p. and six weeks after the oral questioning. Inosules of questioning were administered by the same route as vaccinations. The care of all the animals was under the institutional guidelines, as established for the animal committees of the Massachusetts General Hospital and Harvard Medical School. It was led to taste protein electrophoresis as follows. Gel protein electrophoresis, one-dimensional, was carried out by the method of Laemmli, 1970, Nature 227: 680, incorporated herein by reference, in whole cell protein extracts of the stationary phase cells cultured during the night in Luria's broth. The gels were fixed and marked with Coomassie bright blue R250 in 10% acetic acid and 10% methanol. Two-dimensional gel protein electrophoresis was carried out by the method of O'Farrell, 1975, ". Biol. Chem. 250: 4007, incorporated herein by reference, on the same whole cell extracts. 1.5% ampholynes, pH 3.5 to 10 (LKB Instruments, Baltimore, Maryland, United States) was taken to flavor for 9,600 V hour (700 V for 13 h 45 min.) The final pH gradient of the tube gel extended the pH from 4.1 to 8.1, measured by means of a superfisial pH sensor (BioRad Laboratories, Richmond, California, United States) and asethylated cytoster markers, single-stranded (Calbioshem-Behring, La Jolla, California, United States) Adyasente tube The gels in piece were dizzy are silver, Merril et al, 1984, Methods Enzymol 104: 441, insorporated in the present by referensia.In the experiments of survival in masrophages were carried out experiments as described above, Mille r et al., 1989, supra, by the method of Buchmeier et al., 1989, Infect. Immun. 5? : 1, incorporated herein by reference, modified from the method of Lissner et al., 1983, J. Immuno 1. 131: 3006, incorporated herein by reference. Stationary phase cells were opsonized for 30 minutes in normal mouse serum before exposure to bone marrow-derived macrophages, cultured, harvested from BALB / c mice. One hour after infection, gentamicin sulfate (8 μg / ml) was added to kill the extracellular bacteria. All points in time were made in triplicate and repeated on three separate occasions. PhoPc Mutant Strains Are More Effective as Live Vaccines The PhoP0 mutant S. typhimurium is very effective when used as a living vessel in the typhoid fever of mice and is superior to the PhoPs basteria. "So many poses 15 PhoPc basterias protect mice against 105 LD50 (lethal doses at 50%) of wild-type organisms by the intraperitoneal route (Table 3) .This suggests that the produtos of the gene, pag are important antigens for immunity against the typhoid of mice. Preliminary results have documented that the antigens recognized by the serum of sphygmomanometers of typhoid resonored some of the prodrugs of the phoP-regulated gene of S. typhi. If the target antigens are only expressed within the host, then the dead ones sultivated only in half-laughs may not induce an immunological response against these proteins. The use of different preparations of killed vaccines of S. typhimurium containing different mutations in the phoP regution was evaluated. As can be seen in Table 3, no preparation of dead cells (even those containing mixtures of PhoP bacteria "and PhoPc) is as effective as those of living bacteria.This suggests that there are other properties of live vaccines that increase immunogenicity or important antigens not regulated by PhoP are not in these preparations.The only protein observed in any of the animals studied was the lowest dose of challenge for those immunized are PhoPc bacteria.This also suggests that the genes activated by phoP are important antigens promoters. Table 3 Salmonella are Regulatory Mutations phoP Used as a Vaccine Dead Vaccination Dose Questioning Organisms Type Wild Strain Phenotype 6 x 103 6 x 105 None (3) (5) ATCC10428 Wild type (8) (9) CS015 PhoP- (10 ) (13) CS022 PhoPc 2/7 (*) (14) CS022 / CS015 PhoPVPhoP0 (8) (13) CS015 = phoP102:: TnlOd-Cam CS022 = phs-24 BALB / c mice were i immunized twice, seven days apart, with 5 x 10 bacteria removed with formalin. Three weeks after the second vaccination, the mice were challenged with wild-type organisms at the two doses indicated. The numbers in parentheses indicate that there were no survivors after questioning and the average number of days until death. (*) List of survivors to questioned number. phoPc indicates unregulated expression, constitutive of genes activated by phsP and lack of expression of genes repressed by phoP. phoP- indicates a lack of expression of the genes activated by phoP and the expression of genes repressed by phoP.

Double Regulatory Mutative strains phoP Recent efforts by Stocker, Levine and their co-workers have focused on the use of auxotropic mutant strains in amino acid and purine amino acids as live vaccines, Hosei th et al., 1981, Nature 291 : 238, incorporated herein by reference, Stocker, 1988, Vaccine 6: 141, incorporated herein by reference, and Levine et al., 1987, J. Clin. Invest. 79: 888, incorporated herein by reference. The purine mutations were found to be too attenuating of immunogenicity, possibly because no purine is available to the organism within a mammalian host, Sigwart et al., 1989, Infect. Immun. 57_: 1858, incorporated herein by reference. Because auxotropic mutations can be somplemented by means of homologous resombination events, they are wild-type sopias donated from environmental organisms or by acquiring the necessary metabolite within the host, it would be prudent for living organisms to have a second attenuating mutation in a different mechanism. virulence (that is, not just a second mutation in the same metabolic path). Additionally, in mice the aroA mutants have some residual virulence. Several strains with aroA mutations combined with phoP regution mutations were investigated for virulence attenuation and immunogenicity. Table 4 demonstrates that a PhoP or PhoPc mutation additionally attenuates the aroA mutant S. typhimurium by at least 100-fold and that, at least at high levels of vaccine organisms, immunogenicity is retained. Both pgPc and phoPc are any mutations alone, therefore phoP regution mutations can increase the safety of preparations of live aroA vaccines Table 4A Additional Attenuation of aroA Mutants by Regulatory Mutations PhoP Survivors of Varied Numbers of Mutant Organisms of Salmonella (*) Strain Phenotype 10s 107 108 109 1010 (**) CS004 aroA- 6/6 1/6 0/6 0/6 6/6 SL3261 aroAdel His "6/6 1/6 0/6 0/6 6/6 CS322 aroA- PhoPc 6/6 6/6 6/6 1/6 6/6 CS323 S13261 PhoPc 6/6 6/6 6/6 2/6 6/6 CS315 aroA- PhoP "6/6 5 / 6 6/6 2/6 6/6 CS316 SL3261 PhoP "6/6 6/6 6/6 1/6 6/6 CS027 page" PhoPc 6/6 4/6 0/6 0/6 6/6 Table 4B Eficasia Protester of Salmonella with Regulatory Mutations aroA / phoP Survivors of Dose of Questioning of Wild Type Organisms (*) Strain Phenotype Inoculum 5 x 5 X 107 CS004 aroA '10s 4/4 5/5 S 3261 aroAdel His "106 4/4 4/5 CS322 aroA "PhoPc 10s 5/5 CS323 S 3261 PhoP c 106 5/5 CS322 aroA" PhoPc 107 5/5 CS323 S 3261 PhoPc 107 5/5 CS322 aroA ~ PhoPc 108 5/5 CS323 SL3261 PhoPc 108 5/5 CS315 aroA 'PhoPc 5/5 CS316 S 3261 PhoPc 10a 5/5 (*) Ratio of survivors to number of mice questioned. (**) Indicates oral inoculation; all other experiments were inoculation i.p.

CS004 = aroA554:: rnl0. SL3261 = aroADEL407 hisG46. CS322 = aroA554:: Tnl0 pho-24. CS323 = ar-oADEL407 pho-24. CS315 = aroA554:: Tnl0 phoP102:: TnlOd-Cam. CS316 = aroADEL407 hisG46 phoP102:: TnlOd-Cam. CS026 = pagCl:: TnphoA pho-24 phoN2 zxx:: 6251TN10d-Cam.

PhoP Regulatory Mutations of Salmonella typhi The phoP regution is conserved at least parsially in S. typhi DNA hybridization studies, thus transdussional sperm from basteriophagous P22 have shown that the genes phoP, phoQ and pagC appear highly guarded between mutations of S. typhi and S. typhimurium in these genes in S. typhi. Live Vaccines for Salmonella as Delivery Systems for Heterologous Antigens The vestor used in the delivery system of vasuna is a derivative of pJM703.1 dessrito in Miller et al., 1988, J. Bact. 170: 2575, insorporated in the present by referensia. This vector is a derivative R6K with a suppression in the pir gene. The R6K derivatives require that they replicate the protein product of the pir gene. E. coli that are present in the pyr gene present as a basteriophagous lambda profago can support the replisation of this vector. Cells that do not contain the pir gene will not support replication of the vector as a plasmid. This vector also contains the mob region of RP4, the sual will allow the mobilization to other gram negative basterias by mating from strains of E. coli such as SM101 lambda pir, which can provide the function of mobilization in trans. The pagC region is shown in Figures 2 and 3. Figure 2 shows the sites of restriction endonuclease of the place pagC. The heavy bar indicates the coding sequence pagC. The TnphoA insert is indicated by an inverted triangle. The direction of transcription is indicated by the arrow and is from left to right. The numbers do not specify the location of the endonuclease sites, in number of base pairs, in relation to the start codon of the predicted pagC tradustion, the positive numbers indicating the place downstream of the inisio codon and the negative numbers indicting the place sorriente Above the start Sodon. A is Accl, B is Bgll, C is Clal, D is Dral, E is EcoRI, H is Hpal, N is NruI, P is PstI, S is SspI, T is Stul, U is PvuII, V is EcoRV, and II It's Blgil. Figure 3 shows the sequencing of DNA (identified sesuensia are number 1) and the translation of pagC:: TnphoA. The highly stressed sesuensia indicates a potent ribosomal binding site. The underlining of the senses and the double indisances sesuensias in the main primordial materials were constructed in a somplementary way to these nucleotides for the extension of the primordial material of the RNA analysis. The asterisk indicates the approximate start of transcription. The flesha indicates the decision of the transcription. The sesuensias in suadros indisan a region that can work in ligature of polymerase and resonosimiento. The inverted triangle is the site of the insertion junction TnphoA sequenced. The arrow indicates a potential site for secuensia sensilla rupture. Three kilobases of DNA containing the pagC gene (from the 5 'nucleotides of the restriction endonuclease site PstI 1,500 at the start of the pagC translation to the restriction endonuclease site EcoRI 1,585 nucleotides of the translation termination pagC) were inserted into the derivative pJM703.1 discussed above. The pagC sequence of the Clal restriction endonuclease site was deleted (490 nusleotides) and replaced with a synthetic oligonucleotide poly-linker that creates unique restriction endonuclease sites. DNA encoding one or more heterologous proteins, for example an antigen, can be inserted into this site. This creates a vestor that allows the insertion of multiple foreign genes into the DNA background. The vestor can be mobilized towards Salmonella by mating or any other delivery system, for example heat shock, bacteriophage transduction or electroporation. As it can not replicate, the vector can only be inserted into Salmonella by specific recombination. On site are the homologous DNA on both sides of the pagC gene. This will break down and inactivate the native place and replace it with broken page DNA, worn in the locker room. Such resombination events can be identified by exchange of markers and selective media if the foreign DNA inserted in the pagC site confers a growth advantage. The insertion of genes for resistance to antibiotics for selection is less desirable since this would allow an increase in resistance to antibiotics in the natural population of the basteria. Genes that resist resistances to substances other than antibiotics, for example heavy metals or arsenide (for resistance to mercury, see Nucifora et al., 1989, J. Bact. 171: 4241-4247, herein incorporated by referensia), can be used. to identify transformants. Alternatively, it can be carried out by using a Salmonella receptor strain carrying an auxotropic mutation in a metabolic tray and a vestige carrying DNA that is somatically the auxotropic mutation. Many prototypes of live Salmonella vaccines contain mutations in histidine or purine trajectories, so that complements of these metabolic auxotropies can be used to train members. (Specifically it has been shown that purine mutations are too attenuated for use in humans.) Additional evidence of marker exchange can be documented by loss of ampicillin resistance (carried in the plasmid backbone) or by hybridization analysis . A useful gene for selection can be cloned by complementation of a vaccine strain with a metabolic auxotropy. Specific examples include the cloning of DNA encoding both purB and phoP by complementation of a suppressed strain for function of both genes. Libraries of Salmonella genes have been constructed in a pLAFR cosmid vector (Frindberg et al., 1984, Anal. Biochem. 137. -266-26, incorporated herein by reference) by methods known to thematists in the art. The pLAFR sásmidos are plasmids of a wide range of hosts that can be mobilized to Salmonella from E. coli. A whole bank of such strains can be mobilized towards Salmonella vaccine strains and selected for complementation of an auxotropic defect (for example, in the case of purB culture in media without adenine). The DNA that is able to complement this defect is then identified and can be cloned into the antigen delivery vector. As discussed above, heterologous genes can be inserted into the poly-linker that is inserted into the pagC sequence of the vector. The heterologous genes can be under the control of any of the numerous environmentally regulated promoter systems that can be expressed in the host and eliminated in the laboratory. Because the expression of foreign proteins, especially membrane proteins (as they are the most important antigens), is frequently toxic to bacteria, the use of environmentally regulated promoters that would be expressed in mammalian tissues at high levels but which can be cultured in the laboratory without expression of heterologous antigens would be very desirable. Additionally, the high expression of antigens in host tissues can result in increased attenuation of the organism by deviation of the metabolism fuel of the organism to the synthesis of heterologous proteins. If foreign antigens are expressed specifically in host phagocytic cells, this can increase the immune response to these proteins because these cells are responsible for processing antigens. Promoter systems that may be useful include those nutrient-regulated promoter systems for which it has been shown that a thickened nutrient is not available to basterias in mammalian hosts. Puras, Sigwart et al., 1989, Infect. I mun. 57_: 1858 and iron, Finkelstein et al., 1983, Rev. Infect. Dis. 5_: S759, for example, are not available within the host. Promoters that are regulated by iron, such as the promoter of the aerobastine gene, as well as promoters of biosynthetic genes in purine trajectories, are thus excellent candidates for testing as promoters that can be eliminated are found in high sonsentrasions of these nutrients. Other useful environmentally regulated promoters of Salmonella include promoters for genes that encode proteins that are expressed specifically within masrophages, for example the DanK and GroEL proteins, which are increased by high temperature culture, as well as some phoP gene products, Buchmeier et al., 1990, Science 248: 730, insorporated in the present by referensia. Therefore, it would be expected that promoters such as pagC 5 'that control sequences and promoters better characterized for heat shock genes, for example GroEL and DnaK, would be expected to astively mirror within the masrophage. The macrophage is the site of antigen processing and the expression of heat shock genes in macrophages and the extensive conservation of heat shock genes in nature may explain the immunodomain of these proteins. A consensus heat shock promoter sequence is used and can be used in the vectors (Cowling et al., 1985, Proc. Nati Acad. Sci. USA 82.:2679, incorporated herein by reference). The vectors can include an environmentally regulated T7 polymerase amplifisation system to express heterologous proteins. For example, the T7 polymerase gene (standed by Stan Tabor and Charles Rishardson, Current Protocols in Molecular Biology, editors Ausubel et al., 1989 (page 3.5, 1.2), John Wiley and Sons, insorporated in this by referensia) the sontrol of a promoter regulated by iron can be included in vestores before dessritos. The promoter of the E. coli aerobastine gene has been inserted. It is the CATTTCTCATTGATAATGAGAATCATTATTGACATAATTGTTATTATTTTACG (Identified sequence is No. 2), Delorenzo et al., J. Bact. 169: 262, insorporated herein by reference, in front of the T7 polymerase gene and demonstrated regulation by iron of the gene product. This version of the vector will also include one or more heterologous antigens under the control of the T7 polymerase promoters. It is well known that RNA can be synthesized from promoters of purified synthetic T7 and T7 oligonucleotide in vi tro. When the body is under iron, T7 polymerase will be synthesized and the high expression of genes with T7 promoters will be facilitated. PagC Fusion Proteins in S. Typhimurium The expression of heterologous antigens within macrophages under the control of promoters regulated by phoP can be used as an effective method both to attenuate Salmonellae and to improve the immunogenicity of foreign antigens. As discussed above, the expression of pagC is induced in the antigen processing cell, i.e. a masrophage. In this way, the expression of a heterologous antigen under the control of the pagC promoter is also difficult to be inducible in macrophages. To evaluate the immunological response to a heterologous antigen expressed under the control of inducible pag promoters, mice were inosulated with basterias that expressed the AP antigen, under the control of the regulatory sequences pagC or pagD. Pag-AP fusion proteins were produced in these bacteria from a single chromosomal copy of the gene encoding AP. Bacteria were generated using two methods: mutagenesis of TnphoA and genetic engineering techniques using a suicide vector, both having been previously described. As mice, mice were inosulated are basterias expressing an AP fusion protein under the control of the sonstitutive promoters. The consititutive promoter was completely independent of regulation by genes in the PhoP regimen. Two such bacterial strains, strains 610 and 617, were constructed using the methods described above. The expression of AP in strain 610 was moderate, while the expression of AP in strain 617 was high (see Figure 14C). These strains were injected intraperitoneally in BABL / c mice. Serum samples were taken three weeks after inosulation. Normal mouse serum (MNS) was used as control. Standard ELISA assays were used to test the sera for the presensia of AP-specific antibodies. The sera were also tested for LPS-thickened antibodies using S. typhimurium LPS. Antibodies directed to LPS were detested in all tested murine sera, but only those strains in which AP was expressed as a Pag fusion protein from a single copy of chromosomal gene spawned an immunological response against the model heterologous antigen, AP (see figures 14A and 14B). Despite the approximately 10-fold greater constitutive expression of the AP fusion protein in strain 617, only a minimal immunological response to this antigen was observed after immunization with strain 617. In contrast, an intense response was observed in inoculated mice with strains that expressed the Pag-AP fusion protein. These data indicate that the phoP- regulation resulting from the in vivo induction of protein expression within <of macrophages increases the immunogenicity of heterologous antigens expressed under the control of the pag promoters. Any promoter that directs the inducible expression, specific for a cell, of a protein in macrophages or other antigen-presenting cells, eg, pags described herein, can be used to increase the immunogenicity of an antigen expressed in Salmonella. The Gene yagC and the Gene Product pagC Strains, Materials and Methods The following methods, strains and materials were used in the cloning of pagC and in the analysis of the gene and its gene product. The rich means were Luria's balance (LB) and the minimum means were M9, Davis and collaborators, 1980, supra. The sonstrussion of strain CS119 pagCl: -. TnphoA phoN2 zxx: -. 6251 TtilOd-Cam of S. typhimurium was previously described, Miller et al., 1989, supra. Strain 10428 of S. typhimurium from the American Type Culture Collection (ATCC) included CS018, which is isogenic to CS119, except for phoP105:: Tnl 0d, Miller et al., 1989, supra, CS022 pho-24, Miller et al, 1990, J. Bacteriol. 172. -2485-2490, herein incorporated by referensia, and CS015 phoP102:: Tnl0d-cam, Miller et al., 1989, supra. Other wild-type strains used for the preparation of the chromosomal DNA included S. typhimurium LT2 (ATCC 15277), S. typhimurium Ql and S. drypool (Dr. J. Peterson, U. of Texas Medisal Branch, Galveston, Texas, United States), and Salmonella typhi Ty2 (Dr. Caroline Hardegree, FDA). The pLAFR sásmidos were mobilized from E. coli to S. typhimurium using the MM294 separator of E. coli, containing pRK2013, Friedman et al., 1982, Gene 18-289-296, incorporated herein by reference. PA activity was evaluated on solid media using the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (XP). Assays of AP were carried out as previously described, Brickman et al., 1975, J. Mol. Biol. 96: 307-316, incorporated herein by reference, and reported in units as defined by Miller, 1972, supra, pp. 352 -355. Uni-dimensional gel electrophoresis of the protein was carried out by the method of Laemmli, 1970, Na ture 227: 680 -685, insorporated in the present by referensia, and hybridization using anti-body antibody to PA as previously disengaged, Peterson et al. , 1988, Infect. Immun. 56_: 2822 -2829, insorporated in the present by referensia. Whole cell protein extracts were prepared from cultures saturated in LB at 37 ° C with aeration, boiling the cells in SOS-pagE sample buffer, Laemmli, 1970, supra. Two-dimensional gel electrophoresis was carried out by the method of O'Farrell, 1975, J. Biol. Chem. 250. - 007, incorporated herein by referensia. The proteins in the 10% polyasilamid piece gels were visualized by marsado are silver, Merril et al., 1984, Methods in Enzymology 104: 441. incorporated herein by reference. Chromosomal DNA was prepared by the method of Mekalanos, 1983, Cell 3_5.-253 -263, incorporated herein by referensia. The DNA, fractionated by size in agarose gels, was transferred to nitroselulose (for hybridization) by the method of Southern, 1975, J. Mol. Biol. 913: 503-517, insorporated in the present by referensia. DNA probes for Southern hybridization analysis were radio-mated by the primordial random material method, Frinberg et al., 1984, 'supra. Plasmid DNA was transformed into E. coli and Salmonella by slurry of salsium and thermal shock, Mekalanos, 1983, supra, or by electrophoresis using a Genepulser apparatus (BioRad, Richmond, California, United States), as recommended by the manufacturer, Dower and collaborators, 1988, Nucí. Acids Res. 16: 6127-6145, insorporated in the present by referensia. DNA sequencing was carried out by the dideoxy sadena termination method of Sanger et al., 1977, Proc. Nati Acad. Sci. USA 74: 5463-5467, incorporated herein by referensia, modified for use are Sequenase (registered arsa) (U.S. Bioshemisal, Cleveland, Ohio, United States). Oligonucleotides were synthesized in an Applied Biosystems machine and used as primordial materials for sequencing reactions and extension of primordial RNA material. Specific primordial materials, unique for the two ends of TnphoA, one of the suals corresponds to the sequencing of sodifisation of AP and the other to the sesuensia IS50 deresha, were used to detect the junctions of the transposon insertion. Construstion of a cosmid gene bank of S. typhimurium in pLAFR3 and evaluation for slides containing wild-type pagC DNA were carried out as follows. DNA from strain ATCC 10428 of S. typhimurium was partially digested using the restriction endonuclease Sau3A and then selected by size in a density gradient of 10 to 40% sucrose. T4 DNA ligase was used to ligate chromosomal DNA of size from 20 to 30 kilobases to the cosmid vector pLAFR3, a derivative of pLAFR1, Friedman et al., 1982, Gene 18_: 289-296, incorporated herein by reference, which was digested with the restriction endonuclease BamEI. Cosmid DNA was packaged and trans-infested in the E. coli DH5-alpha separator using extratases purchased from Stratagene, La Jolla, California, United States. Solonies were evaluated by hybridization analysis. The analysis of proteins produced from sloned DNA by transsripsion / tradussion assays in vi tro was carried out as follows. These assays were carried out in cell-free extracts (Amersham, Arlington Heights, Illinois, United States) and using the dessritas condisions by the manufacturer. The resulting radio-dizziness proteins were analyzed by means of SDS -pagE. RNA was purified from early Salmonella sultans and stationary phase by the hot phenol method, Case et al., 1988, Gene 7_2. - 219-236, incorporated herein by reference, and run on agarose-formaldehyde gels for hybridization analysis, Thomas, 1980, Proc. Nati Acad. Sci. USA 77_: 5201, incorporated herein by reference. Analysis of extension of primordial RNA material was carried out as previously described, Miller et al., 1986, Nuc. Acids Res. 1? : 7341-7360, incorporated herein by reference, using AMV reverse transcriptase (Promega, Madison, issonsin, United States) and primordial synthesized oligonucleotide materials complementary to nucleotides 335-350 and 550-565 of the pagC site. Identification of an 18 kDa protein missing in a pagC mutant of S. typhimurium The mutant strain of pagC CS119 was analyzed by two-dimensional protein electroporesis to detest protein thickeners that might be absent as a result of the Tnp? OA insertion. Only one species of missing protein was observed, approximately 18 kD and pI-8.0, when the strains, isogenic except for their transposon insertions, were subjected to this analysis. This 18 kDa thickener was also missing in similar analyzes of Salmonella isolates are the phoP and phoQ mutations. Although the bi-dimensional analysis of gel proteins may not detest subtle protein expression in CS119, this suggests that only a larger protein species was absent as a result of the insertion of pagC:: TnphoA. Additional examination of the bi-dimensional gel analysis revealed a new protein threesome of around 45 kDa which is fastible to be the pagC-AP fusion protein. The pagC-AP fusion protein was also analyzed by Western-type analysis using anti-sera to AP and was shown to be similar in size to the native AP (45 kDa) and not expressed in PhoP-S. typhimurium. Pacr C Insertion Cloning:: T.sp.phoA Sromosomal DNA was prepared from CS119 of S. typhimurium and a thick physical map of the restriction endonuclease sites in the pagC: - fusion region was determined. TnphoA using a DNA fragment of TnphoA as a probe, in hybridization analysis. This work indicated that ecoRV restriction endonuclease digestion resulted in a single DNA fragment that includes the pagC:: TnphoA insertion in addition to several kilobases of flanking DNA. Chromosomal DNA of strain CS119 was digested with EcoRV (blunt end) and ligated into the bacterial plasmid vector pUC19 (New England Biolabs) which had been digested with the restriction endonuclease Smal (blunt end). This DNA was electroporated into the DH5 algae (BRL) of E. coli and colonies were placed on the LB agar containing the antibiotics sanamisine (TnphoA sodified) and ampicillin (pUC19 encoded). A single resistance to ampicillin and kanamycin containing a plasmid designated pSMIOO was selected for further studies. A radio-labeled DNA probe from pSMIOO was constructed and used in a Southern hybridization analysis of strain CS119 and its wild-type parental strain ATCC 10428 to prove that the pagC: fusion was cloned. - TnphoA. The probe contained sequencias immediately adjacent to the transposon at the opposite end of the AP gene [DNA fragment generated by Hpal endonuclease that included 186 bases of IS50 to the deresha of the transposon and 1, 278 DNA bases of Salmonella (figure 2). As expected, the probe derived from pSMIOO was hybridized to a 12-12 kb Accl endonuclease digested DNA fragment of the strain containing the transposon insert, CS119. This was approximately 7.7 kb (size of TnphoA) greater than the Accl fragment of 3.9 kb present in the wild-type strain that hybridizes to the probe. In addition, a derivative of the plasmid pSMIOO, pSMIOl (which did not allow expression of the pagC-PhoA gene fusion from the lac promoter), was transformed into strains of Salmonella phoP- (strain CS015) and phoN- (strain CS019) and Cloned AP activity was found to be phoP dependent for expression. Therefore, it was concluded that the slung DNA was fusing the pagC: - fusion. TnphoA. The presence of the pagC gene was also demonstrated in other strains of S. typhimurium, as well as in S. typhi and S. drypool. All of the Salmonella isolates examined showed strong hybridization similar to EcoRV of 8.0 kb and an Accl restriction endonuclease fragment of 3.8 kb suggesting that pagC is a gene of virulence common to the Salmonella species. The gene pagC probe from nucleotides -46 (with 1 somo the first base of methionine) to 802 (site PstI to Blgll site) could not hybridize sruzadamente to the DNA of Ci trobacter freundii, Shigella flexneri, Shigella sonnei, Shigella dysenterial, Escherichia coli, Vibrio cholerae, Vibrio vulnificus, Yersenia whole eolithic, and Klebsiella penumonia. DNA Cloning of Wild Type and Its Complementation of the Virulence Defect of a Mutant Pacr C of S. t? Phimuriu The same restriction endonuclease fragment before dessr was used to evaluate a pool of semiside genes of the wild type strain ATCC 10428. A single slone, designated pWP061, was holding 18 kilobases of AD? of S. typhimurium and strongly hybridized to the pagC AD probe ?. It was found that? WP06l contained AD? of Salmonella identical to that of pSMIOO when analyzed by restriction endonuclease analysis and hybridization studies of AD ?. Probes derived from pWP061 were also used in hybridization analysis are AD? of S. typhimurium wild type and CS119. Identical patterns of hybridization were observed to those observed are pSMIOO. We also mobilized pWP061 to have CS119, a mutant pagC strain. The resulting strain had wild type virulence for BALB / c mice (an LD50 less than 20 organisms when administered by i.p. inyection). Therefore, the AD? cloned somplementa the virulence defesto of a mutant pagC. Whenever it was ensoned that a wild type smashed DNA from the place pagC somplemented the virulence defect of a mutant pagC strain of S. typhimurium,. it was concluded that the pagC protein is a membrane protein (see below) of 188 amino acids (18 kDa), essential for survival within macrophages and virulence of S. typhimurium. Physical Mapping of Restriction Endonuclease Sites, DNA Sequencing, and Product Determination of Gene pacrC Restriction endonuclease analysis of the plasmids pSMIOO and pWP061 was carried out to obtain a physical map of the place pagC, and in the case of pSMIOO, to determine the transcriptional digestion (figure 2). Sub-slonas of AD? were generated and the fusion bonds TnphoA were sesuensiadas, so are the AD? of Salmonella extending from the Hpal site, 828 nusleotides 5 'to the phoA fusion junction, to the ScoRI site of 1,032 3' nusleotides to the TnphoA insertion (figures 2 and 3). The mareso sorresto de lestura of the sesuensia of AD? It was dedusido of the required to synthesize a fusion of gene ast astivo. The amino acid sessedia of this open reading space was predicted to sodify a protein of 188 amino acids with a pI + 8.2 predesido. These data were found to be the two-dimensional analysis of polyasrilamide gel of CS119 in the sual, an 18 kDa protein of approximately pI + 8.0 was absent. No other open reading frames were embedded, predicted by sodipizing larger peptides of 30 amino acids. The deduene amino acid sesuensia of the open-latitudinal lattice of 188 amino acids is a methionine initiation sodon at 33 amino acids from the fusion of pagC and AP (Figure 3). This pagC contribution of 33 amino acids to the fusion protein was consistent with the size observed in Western analysis and contains a hydrophobic N-terminal region, identified by the method of Kyle et al., 1982, J. Mol. Biol. 157.-105-132, incorporated herein by reference, which is a typical Basterian signal sesuensia, Von Heinje, 1985, J. Mol. Biol. 184: 99-105, incorporated herein by reference. Specifically, aminoaside 2 is positively serged lysine, followed by a hydrophobic domain and aminoaside 24 is a negatively-harvested aspartate residue. A sonsense cleavage site for this leader peptide is presumed to be an alanine residue in the amino acid 23, Von Heinje, 1984, J. Mol. Biol. 173: 243-251, insorporated herein by reference. DNA sesuensia also revealed a typical ribosomal ligation site, Shine et al., 1974, Proc. Nati Acad. Sci. USA 71: 1342-1346, incorporated herein by reference, at 6-2 nucleotides 5 'to the predicted start of the translation (figure 3) (nucleotides 717-723). This suggests that the open reading frame, in fact, was translated and additionally supports the assumption that this was the deduced amino acid sequence of the pagC protein interrupted by the TnphoA insert (figure 3). In Vitro Synthesis of Proteins by the Place pacrC Clonado To detect if other proteins were encoded by pagC and to determine the approximate size of the gene prodigal pagC, a coupled in vitro transcription / translation analysis was carried out. A 5.3 kilobase I? CoRI fragment from pWP061 was inserted into pUC19 so that the pagC gene can not be expressed from the lac promoter. This plasmid was used in a coupled transcription / translation assay in vi tro. A single protein of approximately 22 kilodaltons was synthesized by the cell-free system. The size was compatible with that of the pagC protein precursor containing its leader peptide. These data also support the conclusion that only the single product of gene pagC was identified. Identification of RNA Coded by pagC An RNA of approximately 1,100 nucleotides is encoded by pagC. The pagC gene is highly expressed by cells with a phoP constitutive phenotype of astigmentation pagr, in somparasión they are phenotype wild type and constitutive phoP of pag activation, in somparasión are the wild type and phoP 'basterias. In these hybridization experiments, only pagC is detected in wild-type cells in rich media during stationary culture. This result, considered with the previous works, Miller et al., 1989, supra; Miller et al., 1990, supra, demonstrates that pagC is regulated transsripsio-nally by the prodrugs of the phoP gene and is only expressed during the early logarithmic phase of culture in rich media by cells with a phoP constitutive phenotype. The size of the pagC transscript is approximately 500 nusleotides more than the need to sodipize the 188 amino acid protein. Analysis of the extension of primordial material of Salmonella RNA using primordial oligonucleotide materials for the sessensia pagC was carried out to determine the approximate site of the transsripsión and to determine if these nucleotides can be transcribed 5 'or 3' to the pagC gene product of 188 amino acids. Analysis of extension of primordial material with an oligonucleotide predicted to be somplementary to the 550-565 nusleotides of pagC, 150 nusleotides 5 'to the predoside insonium, resulted in a produsto of primordial material extension of approximately 300 nusleotides. Therefore, a primordial material more sorriente above, supplementary to the nusleotides 335-350 of pagC was used and was used in a similar analysis. A produsto extension of primordial material of 180 nusleotides was observed as a thickened to the primordial material. This is consistent with the transcription that originates in the nusleotide 170 (figure 3). Upstream of the predesido transsripsional inisium, in nucleotides 153-160, a clásiso site of RNA polymerase ligation was observed with the TATAAT sequence in -12 nucleotides as well as the TAATAT sequencing in -10 nusleotides. No somatic sorrespondensias were observed for the consensus RNA polymerase recognition site (TTGACA) 15-21 nucleotides upstream of the -10 region. AT-39 (126-131) nucleotides (TTGGAA), -38 (127-132) nucleotides (TTGTGG), and -25 (135-140) nucleotides (TTGATT) are sequences that have sorrespondensias are the most freshly protected nusleotides of this sesuensia Based on the above results, it was predicted that the transsripsion would end up close to the translation stop codon of the 188 amino acid protein (nucleotide 1,295, Figure 3). From hesho, a stem loop configuration was engineered at nucleotides 1,309-1,330 which can function as a transcriptional terminator. This was consistent with the lack of evidence of open lestura frames downstream of the 188 amino acid protein and the lack of synthesis of another transsripsion / tradsion using the pagC slonado DNA. This also suggests that the insertion pagC: -. TnphoA inastivates the synthesis of a single protein. PacrC Similarity with Ail and Lom A computer analysis of protein similarity using the protein sequence base of the "Protein Identification Resource" of the National Biomedical Research Foundation, George et al., 1986, Nucleic Acids Res. 14: 11-15, incorporated herein by reference, was carried out to identify other proteins that had similarity to pagC in an attempt to find clues about the molecular function of this protein. Notably, pagC was found to be similar to a bacteriophage lambda protein, Lom, which has been located on the outer membrane in minicell cell analysis, Court et al., 1983, Lambda II, Hendrix, R. W. et al., Editors, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY), pp. 251-277, incorporated herein by reference, and was shown to be expressed by lambda lysogens from E. coli, Barondess et al., 1990, Nautre 346: 871-874, incorporated herein by reference. Recently, the sequencing of dehydrated amino acids from the Y. enterocollar slit gene product was determined and ensonded to be also similar to Lom, Miller et al., 1990, J. Bacteriol. 172: 1062-1069. Therefore, an alignment of proteins family sesuensia was carried out using a software algorithm that establishes families of protein sesuensias and sonsense sesuensias., Smi th et al., 1990, Proc. Nati Acad. Sci. USA 87: 118-122, incorporated herein by reference. The formation of this family is indicated by the internal values of similarity database between these proteins: PagC and Lom (107.8), PagC and Ail 104.7), and Ail and Lom (89.8). These same proteins were searched against 314 control sequences in the database and the mean values and ranges were 39.3 (7.3-52.9) pagC, 37.4 (7.3-52.9) Ail, and 42.1 (7.0-61.9) Lom. The similarity values for this family of proteins are all greater than standard deviations of 3.5 over the highest value obtained by similarity to the 314 random sequencing. No other similarities or other family members were found in the database. The regions of similarity are located not only in the transmembrane domains of the leader peptide but throughout the entire protein. Mutant Strains for Attenuation in Virulence Salmonella typhimurium strains of the invention with a pagC mutation were attenuated in virulence at least 1,000 times. In addition to pagC, other pagr genes described herein may be useful in the development of live vaccines for Salmonella. Mutations in the activated phoP genes can be used to construct live, attenuated Salmonella vaccines. By constructing multivalent Salmonella vector vaccines, activated PhoP-promoters can increase immunogenicity by targeting. expression of foreign proteins to macrophages that present antigens. Identification of Newly Activated phoP Genes To further analyze the role of activated phoP genes in bacterial virulence, a bank of strains was generated with fusions of active phoA gene by mutagenesis of TnphoA. Strain CS019 was the parental strain for mutagenesis of TnphoA because it has wild type bacterial virulence and carries the phoN2 allele, which results in minimal phosphatase activity in the background. Strains are fusions of asthous gene phoA were identified by colony blue phenotype after culture in agar containing XP. Such strains were evaluated for reduced activity of fusion protein in the acquisition of the phoP12 allele that results in the null PhoP phenotype. 2,064 strains expressing AP were isolated and the colonies were purified from 240 independent matings. Strains are AP astivity were isolated at a frequency of 0.8% from the total set of kanamycin resistant bacteria (containing TnphoA). A total of 54 inserts pag: -. TnphoA candidates were isolated from the bank of strains expressing AP, and 49 of these were determined to have a more than six-fold reduction in AP activity in the absence of functional phoP / phoQ. Therefore, approximately 2% of the colonies expressing AP were identified as gene fusions of pag-phoA gene. Identification of Thirteen Unique Places Three methods were used to determine if the 49 TnphoA inserts defined pari passages. First, physical maps of the restriction endonuclease sites EcóRI and HindIII 5 'were defined to the TnphoA insertions. Secondly, we carried out analysis of enlases to transposon insertions strongly linked to pag-sonoside sites. Third, the strains that were determined to be unique by the above methods were evaluated on the basis of a bank of strains with transposon insertions of known chromosomal locus. Hybridization analyzes showed that 13 of the 49 strains had restriction endonuclease sites unique 5 'to the TnphoA insert. The numbers of sepals with physical maps similar to 5 'to the TnphoA insertion varied from 1 to 7. One of the 13 physical maps was similar to the one expected for an insertion in pagC and was observed in seven of the isolated strains as if they were inserting pag:: TnphoA candidates. The analysis of these seven areas indicated that only three of them were pagC insertions: -. TnphoA, because the analysis of hybridization with a fragment of pagC somo probe and analysis of links to transposon insertions highly linked to pagC did not mean that these insertions were not in PagC. Another of the merges pag:: phoA, denoted pagP, had the endonusleasa physical map of 5 'restriction that would be expected for phoN. However, it was determined that this insertion was not within phoN by linkage analysis and hybridization. A transductional cross was carried out between wild type bacteria and CS1247 strain containing pagP -. : TnphoA and zxx: -. 6215Tnl 0d-cam. These transductants were linked in sanamisine, ensuring the inheritance of pagP:: TnphoA that encodes kanamycin resistance. These colonies were then evaluated for resistance to chloramphenol, which would be unacceptable for zxx: 6215Tnl0d-cam to pagP. No link was found that indicated that pagP was not linked to phoN. Hybridization using a porsion of phoN somo probe was also carried to sabo in CS1247 and indicated that this strain was a wild-type phoN site. 13 pag places were defined and designated pagD-P. To define the PhoP regulation of the 13 fusion proteins pag:: TnphoA, the AP astivity was tested in isogenic strains, except for the phoP site. The AP activity was assayed during bacterial culture in medium riso in the phases of logarithmic and stationary sultivo (Table 13). The dependensia of the place phoP intasto for expression sompleta permanesió esstante for the different stages of cultivation; however, the relative amount of AP expression was increased by limiting the sultivo. The difference in expression of gene fusions varied from six to 48 suas were compared isogenic strains with a wild type and place phoP null. Of the five pag sites previously identified, only phoN, pagC and pagA have sromosomal places sonosidos. Linkage analysis of the 13 newly identified pag sites was carried out using strains containing transposon inserts linked to pagC (AK3233 and AK3140), and pagA (AK3255). It was found that three pag:: nphoA inserts were linked to AK3140, the sual is a region near pagC at 24-25 minutes on the chromosome. These were designated page, page and page. pagD:: nphoA was also similarly linked to the transposon insertion of AK3233 (83%) and AK3140 (33%), as previously reported for pagC. The TnphsA insertion of this strain has been further defined and is transcribed divergently from pagC. pagE and pagF exhibited different links to the insertions of AK3233 and AK3140 that pagC and pagD, suggesting a considerably different chromosomal location. The insert pagE:: TnphoA is linked 39% to the transposon insert of AK3233 and 99.1% linked to that of AK3140, while pagF: -. TnphoA is linked 31% to the insert of AK3140 but not to that of AK3233. These different lines in adtion to the physical maps of the restriction endonuclease sites 5 'to the TnphoA insertion indicated that these were new places pag. Therefore, three new pag sites were found in the 25-minute region, one of which is strongly linked to the previously defined pagC. Binding analysis was then carried out using a group of Tnl0deltal6deltal7 inserts defined at random in the ten strains with TnphoA insertions with no known place. Of these ten alleles pag:: TnphoA, only two showed links to the insertion bank Tnl0deltal6deltal7. The inset pagG:: -TnphoA was shown to have 97% binding to the transposon insert of AK3258 located at approximately 30 minutes. The insertion pag:. - TnphoA, designated pagH, exhibited 23% of the inscription of AK3091. The link to the transposon insert of AK3091 was similar to the link previously shown for prgE (26%). Therefore, this chromosomal region contains PhoP genes both activated and repressed. This Tndeltal6deltal7 insertion was analyzed using pulse sampo gradient of sromosomal DNA pulse from AK3091 digested with the restriction endonuclease Xbal and Blnl. These data indicate that the transposon insertion of AK3091 was located in the region of 20-25 minutes and that pagH and prgE are located in this region of the chromosome. Strains with Inserts pag.TnphoA Have Wild Type Sensitivity to Defensin Rabbit NP-1 Strains of S. typhimurium with null mutations in the phoP operon have increased sensitivity to a variety of cationic antimicrobial peptides, including defensins, magainins, and protamine. Defensins are a family of mammalian peptides present in neutrophil granules, lung macrophages, and intestinal Paneth cells. Resistance to these peptides may contribute to bacterial virulence and the ability to colonize mucosal surfaces. Strains with insers pag: -. TnphoA were tested on highly sensitive rabbit NP-1 defensin sensitivity. None of the strains are a single insertion: TnphoA demonstrated an increased sensitivity to the NP-1 defensin (see figure 6). Thus, despite the demonstrated sensitivity of the null PhoP mutants to the rabbit NP-1 defensin, no mutations defined in places were associated with defensin sensitivity. Four strains with inserts pag; zTnphoA Show Marked Attenuation of Virulence in Mice To further define whether these new pag sites are a tribute to virulence in mice, the 13 sepals are transposon insertions pag were evaluated in vivo. The mice were injected intraperitoneally with approximately 100 organisms. Four strains with transposon insertions in pagD, pagJ, pagK and pagM show attenuated virce. The mice ingested with these strains all survived and showed no signs of systemic infections, such as hepatos-plenomegaly and piloeressión due to fever. These smooth sepals were subjected to additional virce tests by intraperitoneal injection of multiple doses of organisms in a total of ten mice in two separate osasions. The mean LD50 was determined from these subsessive injections and is listed in Table 14. One of these is the insertion of the Pd:: TnphoA, has an LD50 10,000 veses greater than S. typhimurium wild type. The other three isolates were also attenuated in a dizzy manner as a result of virce in mice LD50 values higher than 1,000 to 10,000 veses of wild-type organisms. These data indicated that the places regulated by PhoP, paG, pagJ, pagK and pagM, suando mutated, resulted in attenuation of bacterial virce. Strains Pag:: TnphoA Attenuated in Virce in Mice Have Reduced Survival Within Macrophages As Salmonella PhoP mutant is deficient in supervi-vensia within masrófagos, the strains that are mutated in the pag genes that had attenuated virce in mice were tested for reduced viability within masrophages. As shown in Table 14, all of the pag mutant isolates demonstrated remarkably reduced survival within masrophages. The redrosed intraselular survival of the pag mutants was not observed until the predicted time would be maximally expressed pag. Four sepals are mutations in the places pagC, paGD, pagJ, pagK and pagM were ensontraron to be attenuated in virce in mice and survival within masrófagos. Knows are mutations in these sinuses had all variable degrees of virce attenuation. The strains are a mutation in pagJ they had a virce defect comparable to that observed for pagC mutants (more than 1,000 x the LD50 of the wild type organisms). Insertion pagD: -. TnphoA resulted in the greatest attenuation of virce, in somparasión they are that of a null PhoP mutation (more than 10,000 x the LD50 of the wild type organisms). Mutants pagK and pagM had virce attenuation that was intermediate between the pagJ and pagD mutants. The assumed effect of the deletion of pagC, pagD, pagJ, pagK and pagM, if it is additive and similar to the observed attenuation are inserts TnphoA, can be larger than that observed by suppression of phoP alone. The observation that many of these genes are somewhat expressed in the stationary phase in the absence of PhoP suggests that functional Pag proteins can be produced in vivo in the absence of PhoP. A virginity gene pagM is expressed significantly in the absence of PhoP, although it may still require PhoP / PhoQ for indussion within phagosomes of masrophages. These data suggest that the suppression of gene products may lead to greater attenuation of virce than the suppression of regulatory proteins. Salmonella Envelope Proteins as Virce Factors: Defensin Sensitivity Based on the methods used to identify pag sites, ie translational gene fusions to phoA, and the observation that pagC gene fusions produce AP, it has been discovered now that many pagr sodify bacterial envelope proteins. They have not been found to be mutations pag sensitive that confer sensitivity to defensins or other cationic peptides. The data suggest that alteration of the basterian envelope is the result of sambio in the synthesis of the whole envelope protein aggregate mediated by PhoP / PhoQ may be important for the virce of S. typhimurium. Defensins are small satined peptides, amphipathic, of approximately 30-35 amino acids in length, whose antimicrobial action implues penetration and membrane disruption, possibly forming selective anionic channels.

Although the defensins are extensively enscribed in neutrophils and Paneth cells, these or other relasionated moliosulas do not contribute to the non-oxidative mortality of phagocytosed basterias by macrophages. Although it is still possible for a single unidentified page to encode a protein responsible for defensin resistance, it appears more plentiful than an assumed hypothesis of expression of several envelope proteins sodified per page may result in resins resistance to defensins. A sambium added in a large number of basterian envelope proteins can alter membrane twill, electride potential, or lipid content, such that the interaction of defensins with bacterial membranes can change. Identification of Transcriptional Units Linked to PagC To identify genes upstream of pagC, E. coli carrying the plasmid pWPL17 containing 2.8 kb of 5 'DNA to pagC (Table 15 and Figure 7) was mutagenized with the transposons MudJ and TnphoA, and identified know are AP or β-galactosidase astivity in chromogenic substrates. In addition, as part of an effort to identify additional PhoP genes, the random mutagenesis of the Salmonella sromosome was carried out by TnphoA, and the APs were evaluated for TnphoA inserts bound to Tnl0deltal6deltal7 of the strain AK3233. it is linked 75% to pagC. Several sepals that are plasmids are gene fusions generated TnphoA or MudJ astivas were identified. In addition, two assays were identified that contained active chromosomal TnphoA inserts linked closely to pagC. Physical maps of the restriction endonuclease sites surrounding the transposon insertions in strains with fusions of lacZ gene and phoA chromosomal or active plasmid were carried out to determine the ratio of the transposon insertions to pagC. This analysis revealed that several regions of the DNA were transcribed oppositely to pagC (figure 7). Several inserts of TnphoA that resulted in active phoA gene fusions were identified. These data indicated that genes linked to pagC encoded membrane or sesretate proteins. Genes Linked to PacrC Encode Four Novel Proteins To analyze the genes defined by transposon insertions, the DNA sesuensia of this region was determined (Figure 8). DNA was thinned by stretching this region; 4 kb of DNA between the Hp 737 bp site upstream of the start codon of pagC to a Clal site upstream were sequenced. The sequencing of DNA from the fusion junctions of all the gene fusions TnphoA and MudJ was also determined. Based on these data, the mareso sorresto de lestura de sada gene was determined. The data from DNA sesuensia revealed that the ORFs predesid to be transsritos and tradusidos are base in the data derived from the insertions TnphoA and MudJ. All ORFs revealed tipotic sites of ribosomal ligation 6 to 11 bases from the predicted onset of transduction. The translation of the ORF immediately upstream and transcribed oppositely to PagrC, indicates that a sorta envelope protein of 87 amino acids (without prosing) is sodified. It is followed by a second ORF (envE) that encodes an envelope protein of 178 amino acids (unprocessed). This ORF is followed by a estrustura that can function as a Rho-independent transcriptional terminator (see figure 8). The third ORF, msgA (super survival gene in macrophages), encodes a small protein similar in size to that of the first gene produsto (79 aminoasides) and is followed by a strusture that can function as a Rho-independent transsripsional terminator (see figure). 8). The DNA prediction sesuensia that this protein is composed of several residues loaded with a large number of negatively charged amino acids with a large number of negatively charged amino acids residing in the term carboxy. The predicted protein product does not contain a structure that resembles a signal sesuensia at its amino terminus or any hydrophobic stretch; therefore, it is unpleasant that the third ORF encodes an envelope protein. The final ORF (envF) encodes an envelope protein of 278 amino acids (unprocessed). A computer search of known protein motifs revealed that EnvF contains a consensus prokaryotic membrane lipid binding site and, therefore, it is feasible to be a lipoprotein (see figure 8 for consensus site location).

The predesid proteins, produced by pAG, envE and envF, contain a typical structure of sesuensia of bacterial signal. In addition, the hydrophobic profiles confirmed the hydrophobic nature of the amino termini of these proteins. The EnvE and EnvF proteins also contain hydrophobic stretches that can function as membrane scanning domains. The G + C content of the genes in this region is: paGC, 43.4%; pagD, 42.1%; envE, 45.9%; msgA, 46.8%; and envF, 40.5%, which is considerably lower than the average G + C content of S. typhimurium (52%). A complete search of the database with the protein predesid sesuensias of these four ORFs did not show significant similarities. Strains containing three distinctive TnphoA inserts and one MudJ insertion, one of which was characterized in one of the soft genes, were selected for additional treatment. A Gene PagD, Transcribed Opposite to PagC, is Regulated Positively by PhoP / PhoQ Representative strains are transposon inserts were examined to evaluate whether transsritos genes opposed to pagC were insremented in synthesis in the PhoP presensia. To determine whether these genes were regulated PhoP, it was necessary to resign inserts of plasmids in the Salmonella sromosome. When replacing the wild-type gene are the gene that are transposon insertion, P22HTinü lysates made in these strains were transduced to a known PhoP suppressed (PhoP ") and the levels of AP or β-galactosidase were monitored. of gene generated by transposon showed a significant increase in astivity between PhoP and WT funds, while the other insersions did not show regulation by PhoP (Table 16) This place of pagD is adjacent to and transcribed divergently from pag C. The transposon insertion representative in envF it was unable to recombine in the sromosome, possibly due to an insufficient sanctity of homologous DNA downstream of the transposon.In order to examine the possibility of PhoP regulation of the envF gene, an upstream region of this gene through and including the phoA gene of the transposon TnphoA was cloned as a Pvul (blunt end) -Xhlo fragment of 3 kb at the EcoRV-SalI sites of the stor suisida pGP704. This slone was paired in the Salmonella strain CS019, and rescinders resistant to ampicillin were selected (creating a strain designated envF:: pGPP2). A phoP105: mutation:: TnlOd-Tet was transduced into this strain to create an isogenic pair that only differs in the ability to produce a functional PhoP protein. As shown in Table 16, the introduction of phoP105:: TnlOd-Tet had no effect on the AP levels of these two tests, demonstrating that envG is not an astigated PhoP gene. Transposon Insertions in Genes Linked by PagC Mitigate Virulence and Cause Reduced Survival Within Macrophages As the transposon insertions in pagC significantly replenish the LD50 of S. typhimurium in BALB / c mice, strains containing transposon inserts linked to pagC were evaluated in terms of virulence attenuation in mice. As shown in figure 7, although the transposon insertion in envE had no effect on the virulence of the sepa, a TnphoA insertion in pagD and the insertion MudJ 1.8 kb downstream in msgA attenuate the virulence of S. typhimurium in more than 300 veses in somparasión are wild type organisms (LD50 <20 organisms). These data suggest that these two sites are esensial for virulence. To examine the survival layers of those strains that have a virulence deft, S. typhimurium containing inserts in pagD or msgA was used to infest macrophages derived from the bone marrow. The results, shown in Table 15, demonstrate a survival defect of masrophages for these two samples. The survival defect is greater for the insertion of pagD (MSI = 0.002) in somparasión are the msgA insertion (MSI = 0.01), and both defests are equal to or greater than those of the PhoP know "(MSI = 0.01) .The transposon insertions in this gene may not be resorbed in the sromosome.Thus, it was necessary to demonstrate that the virulence and the defons of Survival of msgA macrophages is not due to a polar effect of the MudJ insertion on the transcription of envF, therefore, pGPP2 was recognized in the msgA:: MudJ and the AP activity of this strain was compared to that of CS019. Recombinant pGPP2 These data (shown in Table 16) demonstrate that transcription of the envF gene is not affected by the insertion of msgA:: MudJ and is transcribed from its own promoter, however, it is possible that under different conditions At the same time, other promoters may be astivated that can single out msgA and envF over the same transsripsion.Determination of the mscrA Transcription Start Sites and pagD The 5 'regions of these genes were examined s to define the transsripsion start sites of msgA and pagD. Somplementary oligonusleotides at the 5 'end of the ORF sada from the upstream sorptive region were used in an analysis of extension of primordial material. The results of this analysis revealed that the transsripsión of pagD somienza 39 bases sorriente upstream of the translation start. The predicted regions -10 (TTCCAT) and -35 (TTGAAT) were ensontradas to be similar to the sessenias of sonsenso sonocidas for the promoters of E. coli. PagD transcription was detected only in Salmonella PhoP RNA and not in Salmonella PhoP RNA. "It was found that the start of transcription of msgA starts 58 bases upstream of the beginning of the translation and contains the predicted sequences -10 (CAAAAC) and -35 (TTACGT) .These regions do not do well to the consensus sequences -10 and -35; however, the cDNA of this transsripsion was easily detected using various primordial materials in the primordial RNA material extensions of both PhoP "c and PHoP" and to produce an abundant RNA. Distribution of genes pagD and msgA in Enterobacteriaceae and in two organisms with G + C content The G + C content of the chromosomal region pagC is much lower than the average G + C content of Salmonella. The gene encoding the acid phosphatase regulated by PhoP of S. typhimurium (phoN) also has a low G + C content (39%), and DNA homologous to phoN was found only in two organisms with low G + C of several genera submitted testing. The DNAs of several members of Enterobacteriaceae and two organisms with low G + C were examined for similarity to pagD and msgA by hybridization. PCR fragments highly thickened to ORF sada were dizzy and used as probes. This analysis showed hybridization to an elevated astringency to all the Salmonella thickets examined as well as Shigella sonnei, Shigella flexneri, Klebsiella pneumoniae and Ci trobacter freundii. Hybridization was not seen in the organisms are under G + C Morganella morganii or Providencia stuartii. Identical patterns of hybridization were seen to be specific probes for both genes, indicating that these genes are also linked in organisms other than Salmonella. A Cluster of Regulated Virulence Genes for Survival of Salmonella typhimurium Within Macrophages - d9 - Four upstream and transsritos genes opposed to the pagC gene of Salmonella typhimurium have now been identified. Three genes (pagD, envE and envF) are predesigned to be envelope proteins that are based on the isolation of active TnphoA insertions at these sites and the presence of a typical signal sesuence at the amino terminus of each protein. None of the four proteins has significant homology with any protein in the database. Only the gene immediately upstream of pagC and transcribed oppositely (pagD) was determined to be regulated PhoP. Transposon insertions in this gene greatly attenuate virulence and the body's ability to survive within murine macrophages. It has been shown that the transcription of several pags (including pagC) is induced by suing Salmonella is within the phagosome of the masrophage. In adisión, analysis of proteins produced by Salmonella after the infestation of cell lines derived from macrophages indicates that the pag produtos are induced and that pagC may be among the most abundant products indusidos when masrophage infesion occurs. As it is required for the survival of masrophages, it is possible that the transsripsion of this gene is also induced within the phagosomes of masrophages. The pagD protein is small (87 amino acids, unprocessed), and has no strong hydrophobic domains; therefore, it is feasible that it is a periplasmic or secreted protein.

It was found that the transposon insertions in the msgA gene have an effect on virulence in mice and the survival of macrophages. It is possible that this gene can also be induced within phagosomes of assidulated masrophages because there are other genes necessary for the survival of macrophages. If this gene is induced by the environment of the macrofuge, its expression (as well as other genes necessary for the survival of masrophages) can be controlled by a regulatory system separate from the PhoP / PhoQ system. These pagC-linked genes do not stop to form an operon. Because none of the sorriente genes downstream of pgD is regulated PhoP, transsritos will not be from the pagD promoter. The presensia of a potential transsripsion terminator at the end of the envE gene makes it infeasible that msgA is so-transsrit are envE. The data suggest that the insert msgA:: MudJ is not polar in envF, which makes it very feasible for envF to have its own promoter. Additionally, a potential transsripsion terminator following msgA as well as an intergenic region of 493 bp makes it unlikely that these genes are co-transsritos. The primordial material extension analysis of these genes confirms that all four genes are transcribed from their own promoter. The other two genes examined in this study, envE and envF, are predicted to produce membrane proteins that are in regions of potency membrane scanning. The prodrug of gene envF is feasible to be a lipoprotein based on the presensia of a consensus lipid binding site and is likely to play a role in the virulence of Salmonella. The low G + C content of the genes in the pagC region suggests that they may have been acquired by horizontal transmission. The Southern analysis of organisms with low G + C probed are the genes msgA or pagD did not show homology, but this does not eliminate the possibility that they were acquired from another organism are low G + C content. There is also the possibility that these genes reside in a mobile gene element acquired from another source. The msgA and pagD probes hybridized in identical patterns are some members of Enterobacteriaceae other than Salmonella. However, it has been shown that the pagC gene is unique to the Salmonella thicket. This may indicate that the products of the genes upstream of pagC do not form a complex with PagC or that their functions do not require PagC interassión. Alternatively, because there are proteins that have homology are PagC in other members of Enterobacteriaceae (in the absence of any DNA homology), a PagC homology can bind to msgA and pagD in other species that were not detected by the hybridization experiments. of DNA. Promoter Region of pagC / pagD: Expression of Heterologous Proteins pagC and pagD are transcribed divergently and both are PhoP activated. Other regulated genes, divergently transcribed, are conosides in the material (Beck and collaborators, 1988, Microbiol, Rev. 52: 318-326), for example the Klebsiella pneumoniae pulA-malX region (Chapon et al., 1985, J. Bacteriol 164: 639-645). The transcription of most of such genes requires accessory proteins, such as CAP, in addition to the regulator to activate transsripsión. These two genes are transsritos divergently, and their promoters are willing back are back. There is a 134 bp region between the sites of transcription of these genes, which is similar to the intergenic region between pagC and pagD. The pulA-malK promoter region is predestined to have two MalT ligation sites (the regulatory protein of this system), one for each gene. Other activated MalT genes require the CAP protein for expression, but the pulA and malX genes do not, possibly due to their high consallation of the MalT regulator. As between the sites of the transcription of pagC and pagD (the sequels -35 predesidas) are only 137 bp (the nusleotides 562 to 776 of the sesuensia identified are No. 15), it is feasible that there is only one PhoP binding site in the intergenic region, and that the ligation of one or more phosphorylated PhoP molecules positively regulates both genes. This intergenic region pagC / pagD which contains the divergent promoters can be used to construct vestores to express two heterologous proteins, one in each direction. Prg genes As previously discussed, the phoP / phoQ (PhoPc phenotype) mutations increase the expression of pags and repress the synthesis of approximately 20 proteins sodifised by repressed phoP genes (prgs). The PhoPc basterias are attenuated in virulence in mice, suggesting that prgs are virulence genes. Through the use of the transposon, TnphoA, fifty unlinked prg sites were identified. In general, the conditions of the means (hunger) that activate the expression pag repress the expression prg. It was shown that a prg, prgH place contributed to the virulence in mice both by oral route and interaperito-neal. It was found that both PrgH and S. typhimurium PhoPc mutant had defects in induction of endocytosis by the epithelial cells. The identification and mutation of such virulence genes will be useful in the development of vaccines. Nucleotide sequence of the prsH, pral, praJ and prcrK genes The sequence identified with the? O. 10 represents the nucleotide sequence of a 5,100 bp H ± ndIII fragment containing the hyperinvasive hil site. Four ORFs that sodifish suatro prg genes are located within this AD? (see figure 9). The ATG starter is underlined; the asterisks indicate the positions of the stop serums prgH, prgl, prgJ and prgK. These prg sites are required for basterian invasion of epithelial cells, full virulence in mice, and transepithelial migration of neutrophils. A bacterium attenuated by a mutation in one or more of these places can be used to vaccinate individuals with infection by the wild type pathogen. Strains, Materials and Methods All the bacterial strains used in the facesterisation of the prg genes are listed in Table 5. Table 5 Strain Reference or Genotype or Description Relevant Source S. typhimurium Derivates 14028s 14028s Wild type ATCC CSO02 phoP12 This work CS003 DeltapiíofÜeltapurB This work CSO 12 pagAl :: Mu dJ This work CSO 13 pagBl :: Mu dJ This work CS119 pagCl :: TnphoA phoN2 zxx :: 6251 TnlOd-Cm This work CS015 pho -102 :. -TnlO d-Cm This job CS019 phoN2 zxx:: 6251Tnl0d-Cm This job CS022 pho-24 This job CS023 pho-24 phoN2 zxx:: 6251Tnl0d-Cm This job CS030 phoN2 zxx:: 6251Tnl0d-Cm phoP12 This job AD154 phoP12 purB1744 : -TnlO Gift from E. Eisenstadt CS031 pho-24 purB1744:.-TnlO This work IB001 phoN2 zxx: 6251Tnl0d-Cm deltaphoPdeltapurB This work IBOO 2 CS030 with prgAl :: nphoA This work IB003 1B002 with pho-24 purB1744:. -TnlO This work IB004 IB002 with phoP12 purB1744: Tnl0 This work IB005 CS019 with prgAl :: TnphoA This work IB006 CS015 with prgAl :: TnphoA This work IB007 CS030 with prgBl :: TnphoA This work IB008 IB007 with pho-24 purB1744:; -.TnlO This work IB009 IB007 with phoP12 purB1744 :; : TnlO This work IB010 CS019 with prgBl :: TnphoA This work IB011 CS015 with prgBl :: nphoA This work IB012 CS030 with prgB2 :: TnphoA This work IB013 IB012 with pho-24 purB1744: TnlO This work IB014 IB012 with phoP12 purB1744:: TnlO This work IB015 CS019 with prgB2:: TnphoA This work IB016 CS015 with prgB2 :: nphoA This work IB017 CS030 with prgCl:: TnphoA This work IB018 IB017 with pho-24 purB1744:: Tnl0 This work IB019 JB017 with phoP12 purB1744 :. -TnlO This work IB020 CS 019 with prgCl:: TnphoA This work IB021 CS015 with prgCl :: TnphoA This work IB022 CS030 with prgEl :: TnphoA This work IB023 IB022 with pho-24 purB1744:. -TnlO This work IB024 1B022 with phoP12 purB1744: TnlO This work IB025 CS019 with prgEl :: TnphoA This work IB026 CS015 with prgEl. -TnphoA This work IB027 CS030 with prgE2 :: TnphoA This work IB028 JB027 with pho-24 purB1744:: TnlO This work IB029 JB027 with phoP12 purB1744:. -TnlO This work IB030 CS019 with prgE2: TnphoA This work IB031 CS015 with prgE2: TnphoA This work IB032 CS030 with prgE3: TnphoA This work IB033 IB032 with pho -24 purBl 744:: Tnl O This work IB034 IB032 with phoP12 purBl 744:: TnlO This work IB035 CS019 with prgE3:: TnphoA This work IB036 CS015 with prgE3:; TnphoA This work IB037 IB001 with prgHl:: TnphoA This work IB038 IB037 with pho- 24 purB1744 :. -TnlO This work IB039 IB037 with phoP12 purBl 744:: Tnl O This work IB040 CS019 with prgHl:: TnphoA This work IB041 CSO 15 with prgHl:: TnphoA This work IB042 TnSB50-38 O in IB 040 This work IB043 pwKSH5 in IB040 East work IB044 pwKSH5 in CS022 This work CS032 oxyA AlO 49:: Mu dl- 8 supDlO This work CS033 OXIC1048:: Mu dl- 8 supDlO This work CS034 oxiE4:: Mu dl del ta nadAlOO This work Other derivatives of S. typhimurium A 3011 Collection of inserts Tnl0deltal6delta 17 randomly spaced (19) TT520 srl -202:: Tnl 0 (41) TT2979 srl-211:: Tn5 (41) TN3061 zcf-845:: Tnl0 dcp-1 zhg-1635:. -TnlOdCm (41) SH7782 ompD:: Tn5 (41) 4115 invA :: cat (13) EE517 deltahil-517 (Tn5B50-380) Rega]. Or C. Lee JF897 Oxial 049:: Mu di -8 SupDl 0 (2) JF896 OXIC 1048:: Mu dl-8 supDlO (2) JF739 oxyE4:: Mu di deltanadAlOO (2) S. enteritidis CDC5 isolated wild type clinical (45) SM7 STr ^ smb (45) E. coli SM10 (pRT291) contains the plasmid pRT291 (TnphoA) (49) derived from pRK290 that selects TCr and Kmr MM294 (pPHIJI) contains the plasmid Gm pPHIJI, which is (49) incompatible with pRK290 W42 (p KSH5) contains the plasmid pWKSH5, a derivative of (51) pSClOl which contains an i-zi-dlll fragment of 5.1 kb of hil DNA including prgH V. Bajaj and C. Lee (19) Kukral et al., Journal of Bacteriology, 169: 1787-1793, 1987 (41) Sanderson et al., Microbiological Reviews, 52: 485-532, 1988 (13) Gal ny collaborators, Infection and Immunity, 59. -3116 -3121, 1990 (2) Aliabadi et al., Journal of Bacteriology, 165: 780-786, 1986 (45) Stone et al., Journal of Bacteriology, 174: 3945-3952, 1992 Bacteria were cultured as follows: Luria-Bertani broth (LB) was used as a rich medium. Antibiotics were used at the following concentrations in culture media or agar: 100 μg / ml ampicillin (Ap), 25 μg / ml cloramfenisol (Cm), 30 μ / ml gentami-sine (Gm), 45 μm / ml sanamycin (Km) ), and tetracislina 25 μ / ml (Ts). The sromogenous substrate 5-bromo-4-sloro-4-indolyl-phosphate (p-toluidine salt) (X) was used to detest the asthma of phosphatase in agar at a final sonsension of 40 μ / ml. P-Nitrophenyl phosphate (p-NPP) was used as a substrate for the quantitative measurement of AP activity. The media were buffered to various pH ranges by 1 M sodium sitrate. E medium (Vogel-Bonner minimum) was prepared as described by Davis et al., 1980, Advanced Bacterial Genetics: A Manual for Genetic Engineering, Cold Spring Harbor Laboratory , Cold Spring Harbor, NY. Nitrogen, carbon and phosphate free media (NCP) were prepared as dessibed by Kier et al., 1977, J. Bacteriol. 130: 399, insorporated herein by reference. This means of famine was supplemented with 0.04% (weight / volume) of glucose somo source of sarbono, 10 mM NH4C1 somo source of nitrogen, and 1 mM NaH2P04.H20 somo source of phosphate. The carbon concentration is logarithm of one less than the stripping by Kier et al., Supra. The AP activity of isogenic strains except for mutations in the phoP site was measured in cultures from a single colony inoculum under various oxygen tensions with or without agitation, at 37 ° C. Anaerobic cultures were sultivated in an anaerobic sámara (Coy Laboratories Produsts, Ins.) Are a mixture - 37 - 80% N2, 10% 02, and 10% C02 at 37 ° C. For regulation of acid, aliquots of logarithmic media were removed to measure the inessial pH and AP astivity. Sodium citrate 1M (pH> 6.0) or acidic 1M sym trum (pH 4.7) were added to equivalent amounts of culture at a final 50 mM concentration of sitrate. The sultives were performed aerobically for two hours at 37 ° C and then the pH and AP measurements were taken. AP astivity was measured as previously dissorted (Michaelis et al, 1983, J. Bacteriol 154: 366-376, herein incorporated by referensia). The AP units were calculated by means of the following formula: units =. { OD420 / [time (minutes) x volume x OD60]} x 1,000, as defined by Miller for β-galactosidase (Miller et al., 1972, Experiment in Molecular Genetics, pp. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Standard bacterial genetic techniques were used to study the prg sites. Transduction mediated by the bacteriophage P22HTint was carried out in accordance with methods described in the material. The mutagenesis of TnphoA was carried out using a large host range plasmid (pRT29l) to deliver TnphoA (Taylor et al., 1989, J. Bacteriol 171: 1870, incorporated herein by reference). Transpositions of TnphoA into Salmonella DNA were identified by the use of the incompatibility plasmid pPHIJI (Taylor et al., Supra). Evaluation of genes repressed by phoP was carried out using CS031, the donor strain of the pho-24 allele. CS031 was constructed by a P22 bacteriophage transduction cross between strains AD154 and CS022, which contain the purB:: TnlO allele and the pho-24 allele, respectively. The binding of pho-24 and purB:: TnlO was 70%, similar to the binding of purB to other phoP alleles. Therefore, when transdussional tastes of the P22 basterfagogo were taken between CS031 and the isolates that are gene fusions to phoA, the sepals can be evaluated with respect to the loss of the astivity of the fusion protein when acquiring tetrasislin resistensia. The initial evaluation involved detesting the loss of AP astivity in approximately 70% of the solonias that acquired tetrasislin resistensia., because it is presumed that they were alleging the pho-24 allele. In addition, strips were made using AD154 which contains the same allele purB:: TnlO linked to a null phoP allele, phoP12. The plasmid DNA was transformed into the strain LB5010 of S. typhimurium by means of the calsium chloride and thermal shock method (Maclachlan et al., 1985, J. Bacteriol. 161: 442). Isolation of strains with TnphoA insertions in phoP genes repressed mutations in the phoP site (PhoPc phenotype) that give a result of unregulated expression of pags in an unregulated form also considerably attenuate the virulence of S. typhimurium and survival in macrophages. The virulence defect of PhoPc strains can be explained by their reduced expression of approximately 20 polypeptides encoded by repressed phoP genes (prgs). A PhoP'PhoN strain "(IBOOl) was constructed by means of a P22 transductional cross between CS019 and CS003, whereby IBOOl are TnphoA (so that the background acid phosphatase, encoded by phoN, did not interfere with the measurement of the Fusion protein activity when altered to the phoP site) and 1,800 individual blue colonies with asthma of PhoA fusion protein were isolated on XP-containing LB agar plasmas.These colonies were the result of 18 separate matings with approximately 20 sets on one sada. sepans were subjected to redussion tests of fusion protein activity when acquiring the pho-24 allele (CS031), which resulted in a PhoPc phenotype, so that AP assays were carried out in isogenic strains, except for the phoP site. The phenotype PhoPc was confirmed in these strains by means of preparation of extracts of complete cell protein and SDS-PAGE analysis.All strains are a phenotype PhoP demonstrates The distinctive, expected pattern of expression of proteins in PhoPc isolates is called thickened proteins of repressed proteins. Osho strains were identified with gene fusions to phoP genes repressed. As shown in Table 6, the synthesis of most of the prg:: TnphoA fusion proteins was completely repressed by the pho-24 allele. Although two sites had complete repression of the activity of the fusion protein, others only showed partial repression. The expression of pags in PhoPc strains is 5 to 10 times lower than that observed after the bacteria are phagocytosed by macrophages, suggesting that the degree of repression of some prg sites may be higher when pags are maximally activated within phagosomes of assidulated masrophages. Lower values for prgB-phoA fusions in sepals are a wild type phoP site (Table 7B) compared to PhoP strains "(Table 7) may represent some degree of suppression in the presence of PhoP Table 6 PhoP allele" PhoPc repression (fold) prgAl:: TnphoA 29 7 4 prgBl:: TnphoA 137 27 5 prgB2 -. ': TnphoA 77 19 4 prgCl:: TnphoA 14 1 14 prgEl:: TnphoA 21 5 4 prgE2:: TnphoA 34 6 6 prgE3:: TnphoA 25 6 4 prgHl:: TnphoA 92 2 46 In Table 6, a comparison is made of the effect of mutations in the phoP site on the activity of the Prg-PhoA fusion protein. PhoP "indicates that the strain tested contains the allele phoP12 (CS030), and PhoP ° indicates that the assay tested is the allele pho-24 (CS031) .The values were salted from sultivos in stationary phase.The numbers denote the values representative samples of experiments carried out in three separate osasions and represent astivity in units of AP, as previously defined Table 7A Middle Average Starvation Strain Riso IB010 prgBl:: TnphoA 21 26 IB040 prgHl:: TnphoA 7 181 CS119 pagCl: -. TnphoA 1263 102 Table 7B Anaerobic Microaerophilic Aerobic Allele Strain IB010 prgBl:: TnphoA 33 777 1521 IB040 prgHl:: nphoA 142 85 41 CS119 pagCl:: TnphoA 431 173 81 Table 7C Strain Allele pH 4.5 pH 7.0 IB010 prgBl:: TnphoA 332 26 IB040 prgHl:: TnphoA 8 18 CS119 pagCl:: TnphoA 145 27 Table 7 shows the effects of environmental conditions on the regulation of places prg. Table 7A shows the effect of famine on the expression of prg and pag. The starvation medium (N "C" P ") (17) was 0.4% gummy, 10 mM NH4C1, and 1 mM NaH2P04.H20 The astivity of the fusion protein for the starvation medium was measured after 48 hours of sultivo (OD600 = 0.5), while in medium riso (LB) was measured in late logarithmic sultivo (OD600 = 1.0). * All sultives were aerobically sultivated.Table 7B shows the effect of oxygen tension on the expression of activated phoP and repressed phoP genes Expression in rich medium is compared under aerobic conditions in stationary phase (OD600 > 1.4), microaerophilic (OD600 = 0.8), and anaerobic striated aredisiones are 80% of N2, 10% of 02, and 10% C02 (ODS00 = 0.6) after 24 hours of culture Table 7C shows the effect of pH on the expression of the fusion protein activity of prg and pag sites. Expression was measured from growing cultivated cultures. logarithmic (OD600 = 0.5) in LB medium buffered at various pHs c on sodium citrate. All numbers represent activity in units of AP, as defined above. Chromosomal Location of the Locations prg; : TnphoA Prgr:: TnphoA linkage analysis was performed in a pool of strains with Tnl0deltal6deltal7 inserts randomly spaced to determine sromosomal locations and if the prg:: TnphoA alleles were unlinked sites. The inserts prg: -. -TnphoA were in cinso different groups of enlase. Three alleles, prgEl -3:: TnphoA were linked identically to the insertion Tnl0deltal6deltal7 of ADK3091 (26%) and two other alleles, prgBl -2: -. TnphoA were linked similarly to the Tnl0deltal6deltal7 insertion of AK3190 (94%), AK3249 (89%) and AK3186 (50%). Another allele, PrgHl:: TnphoA was found to be linked 37% to the insertion Tnl0deltal6deltal7 of the AK3304 sepa. The other two prg alleles did not prove to be in the test sampled. The sromosomal DNA of these two sepals was analyzed by Southern hybridization analysis using a portion of TnphoA as a probe, and a physical map of the sites located adjacent to the TnphoA insertion was determined. These alleles, prgA and prgC, had different restriction endonuclease sites surrounding the TnphoA insertions. In addition, the repression of the prgA and prgC fusion protein activity in strains are pho-24 mutation was different; PrgC was completely repressed, while prgA was only partially repressed, indicating that these places are different. In this way, unlinked sites were identified that sodify envelope proteins repressed in the PhoPc phenotype. Although three prg sites that were linked to the transposon inserts were identified, none of the insetions Tnl0deltal6deltal7 had a sonoside map location. The physical map site of two of these transposon insertions, AK3249 and AK3304, was analyzed using digestion, Xbal restriction endonuclease, and pulse sampo gel electrophoresis (PFGE). Since Tnldeldel6deltal7 is a single Xbal site, these inserions Tnl0deltal6deltal7 can be assigned to a Xbal fragment specific to the sonoside map site (Liu et al, 1992, J. Bacteriol 174: 16622). AK3249 was assigned to 28-32 minutes, while AK3304 was assigned to either end of the 58-70 minute fragment. It was carried to additional P22 transdussión to sonoside receptors in those regions. The insertion Tnl0deltal6deltal7 of the AK3249 and prgBl: -. TnphoA ensontró not be linked are the insertion TnlO of the know TN3061 (6% bound to dcp), which has a transposon insertion to 28 minutes, or to the ompD:: Tn5 insert of the SH7782 strain at 32 minutes. It was found that prgHl:: TnphoA was very weakly bound to the insertion srll202:: TnlO of the TT520 separator (<; 0.1%) at 59 minutes. These data indicate that the prgs are unlinked in the chromosome of Salmonella, and the function of PhoP / PhoQ as global regulators is consistent. The sromosomal location of TnphoA insertions in repressed phoP genes (prg:: TnphoA) was determined by binding analysis to a bank of strains with Tnl0deltal6del-tal7 insertions (Kukral et al., 1987, J. Bacteriol. 169: 1787, incorporated in the present by reference). Cells are TnphoA inserts were scattered on agar-LB plates containing 10 μg / ml tetracycline and 40 μg / ml XP. Then P22 lysates cultured in strains with Tnl0deltal6del-tal7 inserts were found on the plates with a multi-tip inoculator. After inoculation during the night, the plates were checked for binding, looking for mixed blue and white colonies. Binding was confirmed and quantified leading to individual transdusional tastes between the strains that are Tnl0deltal6deltal7 and the strain with the TnphoA insertion. After selection of resistance to tetracislin sodifidated by Tnl0deltal6deltal7, strains were classified by loss of blue color and resistance to cannamisin encoded by TnphoA. Some TnphoA isolates were found to be linked to the Tnl0deltal6deltal7 strains without location sonoside on the map. Two of these insertions Tnl0deltal6deltal7 were physically mapped using PFGE following digestion with restriction endonuclease Xbal. Based on physical mapping, we determined whether the analysis of binding to other transposon inserts was necessary for the transduction of bacteriophage P22. Chromosomal DNA was prepared as described by Mekalanos, 1983, Cell 3_5: 253, incorporated herein by reference, using proteinase K instead of pronase. The purification of the plasmid DNA was carried out by sonnentional methods. Respiratory endonuclease digestion was performed according to the manufacturer's recommendations (New England Biolabs). The DNA, fractionated in size in agarose gels, was transferred to Genescreen Plus membranes (New England Nuslar / DuPont, Boston, Massashusetts, United States) for hybridization by the Southern method, well known in the art. DNA probes were purified from agarose gels by the songelation-ensogimiento method (Tautz et al., 1983, Anal. Biochem. 132: 14) and radio-dizzy are [32P] dCTP by the primary material method. eatorio (Feinberg et al., 1983, Anal. Biochem. 132: 6). Gene Cloning from TnphoA Fusions The gene coding prgH has been squelched using the methods outlined below. The plasmid, pIBOl, containing the prgH gene, was deposited at the American Type Culture Collection, July 9, 1993 (Rockville, Maryland, United States), and received the designation ATCC 75496. Figure 5 shows the partial DNA sequence of prgH (sequence identified with No. 3). Figure 9 shows the location and sequence of the entire prgH gene. Genes described herein that have been identified by TnphoA insertion can be slunched using methods disclosed in the art (Beattie et al., 1990, J. Bacteriol 172: 6997). For example, sromosomal DNA from sada know how to fuse a prg: - gene fusion. TnphoA is digested are a restriction enzyme such as BahHl, which cuts at a single site in TnphoA, maintaining the fusion junction, phoA sequences and the neo gene. Similarly, a plasmid such as pUC19 is digested with the same enzyme. Dromosomal and plasmid digested DNA are ligated overnight at 15 ° C and transformed into competent E. coli. The transformations are placed in plasmas on LB-agar containing ampisiline and sanamisine to select the gene of pUC19 and the neo gene of TnphoA. The chromosomal DNA containing the gene fusion prg:: TnphoA can then be sequenced using convensional methodology, as described above, such as the Sequenase (registered trademark) equipment (United States Biochemical). Universal primordial material (United States Biochemisal), corresponding to the DNA sesuensias in the plasmid or the primordial material TnphoA (5 '- AA-TATCGCCCTGAGCA-3') (sesuensia identifisada are No. 4) corresponding to bases 71 to 86 TnphoA can be used as a primary material. To clone the wild-type gene, a chromosomal DNA fragment flanking TnphoA sequences can be used to evaluate a cosmid gene bank of wild-type Salmonella strain ATCC 10428 using the previously dessritos methods to slang wild-type pagC. Environmental Regulation of Prg Sites As PhoP / PhoQ are regulators that respond environmentally, the effects of different sultivo sondisiones on the expression prg- were tested. : TnphoA. The rate of yield of strains with insertions prg: -. TnphoA was comparable to that of wild type organisms under all the sondisiones. The expression of all the places prg was maximum in the phase of late logarithmic sresimiento suando the basterias were sultivadas in medium riso (LB). An example of this is the somparation of the expression values of prgH: -. TnphoA in Table 7A (rich medium and stationary growth) and Table 7C (pH 7.0, logarithmic phase). Since the expression of the places pag was maximum in famine (which only attains OD600 = 0.5 maximum) and the stationary phase of sultivo, this was consistent with a respectable relation between the expression of pags and prgs. Additional analysis of expression of prg sites under famine sonsions confirmed this resiprosous recession (Table 7A). The expression of prgH was repressed (Table 7A) and other prgs were minimally afflicted under starvation scenarios, in contrast to the expression induction pag when the bacteria were subjected to starvation (Table 7A). Due to its role in baster-mediated endocytosis (BME), the effect of oxygen tension in rich medium on the expression of pag and prg was also tested (Table 7B). Different but not suitable regulation of places pag and prg was sung in the sultivo to different tensions of oxygen. Although the pagA and pagB sites were minimally afflicted at different oxygen stresses, the place of virulence pagC was about 5 times repressed suando sultivan anaerobically bacteria in comparison are aerobic sultivo (Table 7B). Feasibility was also noted in the expression of prg sites in response to sultive conditions in oxygen absences. One place, Prg, was suppressed three times in anaerobic sultivo, while another place, PrgB, was indicted sasi 50 veses suando was sultivó anaerobically (Table 7B). Other prg sites had minimal expression in fusion protein expression as a result of different oxygen tensions in the culture medium. Low pH conditions also had a variable effect on prg expression (Table 7C). The expression of the activity of the pagC fusion protein was induced under rough condi tions, as is known. When bacteria were sultivated in the logarithmic medium, no significant induction of the relative repression of prgH was observed in media with low pH, while the expression of prgB was induced by exposure of bacteria at low pH (Table 7C). Thus, the sites expressed maximally under various environmental conditions can all be repressed by the PhoPc phenotype. The sensitivity to acids was tested by the method of Foster et al., 1990, J. Bacteriol. 172: 771, incorporated herein by reference. Strains were cultured aerobically in E medium and 0.4% gumose at 37 ° C at OD600 of 0.5. The pH of the bacterial culture was reduced to about 3.3 by the addition of 1 M hydrochlorohydric acid. An aliquot (t0) was immediately taken, the rest of the culture was added at 37 ° C, followed by aliquots removed at 40 minutes ( T40) and 80 minutes (t80). The pH of the crops remained 3.3. The aliquots were diluted 1:10 in cold PBS, washed and resuspended in normal saline solution before placing in serial disolusions in series for units of formations of solonias. prcrH is a Virulence Site for S. typhimurium As the PhoPc phenotype resulted in virulence attenuation and suppressed the synthesis of approximately 20 proteins, the virulence of the sepals are simple mutations in prg sites was tested (Table 8). Strains with insertions prg:. - TnphoA were evaluated for virulence defects by intraperitoneal injection of approximately 150 organisms in BALB / s mice. They also performed sontroles are wild type basterias. A considerably longer time course of disease progression was noted that a mutant prg in somparasión are wild type basterias. Mice that were injected intraperitoneally with strains containing the prgHl:: TnphoA insert developed signs of typhoid fever, such as a "depressible" phenotype (fever and piloerection) and hepatosplenomegaly in approximately 10-14 days, compared with approximately 24 hours for wild-type bacteria. Despite the prolonged time of disease development, all mice eventually died. The progression of the disease of the mice that were injected are other known insistences prg:: TnphoA showed a similar pattern of the disease to the wild type basterias. Table 8 Intraperitoneal Injection LD50 14028s Wild type <; 10 IB040 prgHl 5. 6 X 101 CSO15 phoP-102 6. 7 X 105 IB041 prgH phoP-102 1. 2 X 107 Oral Inoculation 14028s Wild type 6. 5 X 104 IB040 prgHl 6. 5 X 105 Table 8 shows the effect of the prgHl:: - TnphoA mutation on the Salmonella virulens in mice. The sepals were isogenic and administered by intraperitoneal injection and oral inoculation in 35-day-old BALB / c mice. The number of animals used at bastherian solutions sersan at LD50 for the sada allele is listed in parentheses. The LD50 determinations were repeated in three separate osases. Additional tests of the LD50 of sepals containing mutations prgH were carried to sabo. It was determined that the prgH mutants have an LD50 of about 60 organisms, in somparasion they are a value of < 10 for wild type basterias. Due to the difficulty of delivering organisms to small doses in mice, a known strain is a mutation in both prgH and phoP. The PrgH'PhoP strain had an increase of more than 10 fold in LD50 compared to CS015, an isogenic PhoP strain (Table 8). The combined effect of the two mutations, additionally documented that the prgHl:: -TnphoA mutation attenuated the virulence of S. typhimurium and it was not known that the mutations "that affected two phases of the gene expression regulated by PhoP / PhoQ were additive in their efesto Virulenesia were also tested for their antiviral properties when administered by the oral route, and a 10-fold reduction in virulence (increase in LD50) was observed (Table 8). Efficacy of sepals are inserts for the musosal barrier was tested by competition experiments with wild-type basterias.For the first 72 hours after oral inoculation with mutant bacteria, prgHl - mutants were not recovered. - TnphoA of the bloodstream of mice, in somatosis with control experiments in which the organisms were routinely isolated from the blood of mice inoculated with wild type bacteria. Other strains with prg mutations were also tested for virulence defects by the oral route, but no significant change in virulence was observed. Virulence studies in mice were carried out as follows. Bacteria were grown aerobically at 37 ° C to stationary phase, washed with LB and diluted in normal saline. 35-day-old BALB / s female mice (16-18 g) were purchased from Charles River Breeding Laboratories, Ins. (Wilmington, Massashusetts, United States). Baseline samples diluted in saline solution were injected by intraperitoneal route are an inosulo of 0.1 to 0.15 ml. The basterias were administered orally as a 0.5 ml bolus to mice on an empty stomach for two hours, via a 2-inch, 18-gauge, stainless steel oral feeding needle (Harvard Apparatus, Inc., of South Natick, Massachusetts, United States) under light anesthesia with 2-bromo-2-chloro-1,1,1-trifluoroethane (Halothane). The number of organisms administered per plaquedcation was quantified by cfu / ml in LB-agar. LD50 values in mice were determined by conventional methods (Reed and Muench, 1938, Amer. J. Hygiene 22: 493). The LD50 determinations were repeated on three separate occasions. Competent assays were performed after bacteria were orally administered to mice, as noted above. Bacteremia was determined on days 1-4 from bleeding in the tail or intracardiac punctures with 50 μl of blood analyzed immediately and then cultured in LB broth at 37 ° C overnight. Spleen and bowel biopsies were performed on days 1-6, the organs were homogenized in 3 ml of 0.9% sodium sloride. Samples and sultives were analyzed in series dilutions. S. typhimurium was confirmed by the carateristic growth (black colonies) on the enteric agar-Hektoen (Difso Laboratories) and by the agglutination test in masroscopic objective holders with rabbit serum of Salmonella group B (antigens 4)., 5, 12) (Fisher Scientific). Mutations in Oxygen-Induced Genes That Do Not Affect Virulence in Mice It was shown that places prgH and pagC both were repressed by anaerobic culture and required for full virulence, thus suggesting that a sambio from anaerobic to aerobic sondisions can serve as a general signal of induction of virulence genes. Strains with mutations in oxygen-inducible sites (Aliabadi et al., 1986, J. Bacteriol. 165: 780) were constructed. Derivatives of ATCC 14028s are mutations oxiA, oxyC and oxyE were heshos (called CS032, CS033, CS034, respectively). These know were so virulent as wild-type basterias. Although these gene fusions can still marsar operons by sustaining virulence genes, these data suggest that these sites are not sensential for full virulence and that oxygen induction is not always correlated is the virulence function. PrcrH Mutants Have Normal Survival Within Macrophages As the PhoP ° phenotype resulted in a defect in basterian survival in masrophages, evidence was made of the effect of this mutation on protein synthesis encoded by prgH. A strain with the prgHl:: TnphoA insertion was subjected to intraselular survival tests in bone marrow-derived masdrugs of BALB / s mice and J774.2 cells, a sellar line derived from masrophages. There was no evidence of intraselular survival. Also tests were made of a know are an insertion prgBl:. - TnphoA and it was found that it did not have a survival defect within macrophages. Assays were carried out to determine bacterial survival within macrophages, as described by Buchmeier et al., 1989, Infect. Immun. 57_: 1, incorporated herein by reference. Bacteria grown at stationary phase were opsonized for 30 minutes in normal mouse serum before exposure to bone marrow-derived, cultured macrophages, coseshados of BALB / s mice. One hour after the infestation, 10 μg / ml gentamicin was added to kill the extracellular bacteria. All points in time (1, 4 and 24 hours) were made in triplicate and repeated on three separate occasions. Cultured bone marrow macrophages were coseshados of BALB / s mice acquired from Charles River Breeding Laboratories. The J774.2 masrophages were sultivated in Dulbesso minimum esensial medium are 10% fetal bovine serum (DMEM / FBS 10%). Insertions prg:: TnphoA Do not Suppress PhoP Mutations of PhoP Mutants Several phenotypes of phoP mutants, including sensitivity to defensins and ásidos, as well as virulence attenuation in mice, were subjected to suppression tests by the addition of a pr :: TnphoA mutation. To test the phoP mutation layering to suppress the synthesis of prg prodrugs, isogenic phoP mutants except for prg:: TnphoA mutations were constructed and tested for virulence in mice, where deletion would imply an instement in virulence, or reduced sensitivity to acids and defensin. The inserts prg: -. TnphoA had no effect on the virulence phenotypes of PhoP basterias. "These results indicate that the mutations prg:: TnphoA tested did not suppress the PhoP phenotype null somo mutasiones sensillas Mutants PrgH and PhoPc Are Defective in Bacterial-mediated Endocytosis by Cultured Epithelial Cells BME strains of S. typhimurium prg: - TnphoA and PhoPc were tested The following observations (described here) suggest that prg genes may be involved in bacterial-mediated admission by eukaryotic cells: it was shown that prgHl :: TnphoA was located at 59 'in the bacterial sromosome, a place where other genes agglomerate other essential genes for the invasion; it was shown that the prgH mutants were defective in sompetensia are wild-type organisms that reach the bloodstream of mice the first 72 hours after oral ingestion; and the expression of a place prg, prgB, was induced dramatically under the anaerobic sis- reeity of sultivo. The known mutations prgH and pho-24 had a considerable reduction (p value <0.01) in their ability to induce admission by polarized epithelial cells of Madin-Darby canine kidneys (MDCK) in comparison are wild type basterias. Other prg strains with TnphoA insertions do not demonstrate a statistically significant defect in BME from epithelial cells (Table 9). The adherence of defective strains in BME was not affected by the insertion prgH:: TnphoA suando is determined by sfu / ml asosiada to cells before administration of gentamisin (Table 9) and by microscopy. To test bacterial adherence and basterias admission by epithelial cells, bacterial strains were cultured at 37 ° C without agitation (misroaerophilic) at a final density of approximately 2 x 108 units forming solonias (sfu) / ml.

Tests were carried out by seeding 105 MDCK / savity cells in tissue samples from 24-multisavities tissues. The cells were incubated overnight at 37 ° C in an atmosphere of 5% C02 / 95% air in DMEM / 10% FBS without antibiotics until > 80% confluent. Adhesion and invasion assays were carried out according to the protocol of Lee and Falkow, 1990, Proc. Nati Acad. Sci. USA 82: 4304, incorporated herein by reference. Table 9 Strain Genotype Adhesion Invasion 14028 Wild type 4.2% 3. .8% SM7 Strr smb 0. .6% * CS119 pagCl:: TnphoA 1. .9% IB005 prgAl:: TnphoA 7., 6% IB010 prgBl:: TnphoA 2. .9% IB020 prgCl:: TnphoA 1. .5% IB025 prgEl:: TnphoA 1. .9% IB040 prgHl -. : TnphoA 5.7% 0.,% 1 * CS022 pho -24 1.9% 0. 6% * IB043 pWKSH5 in IB040 17.5% * IB044 PWKSH5 in CS022 0. 09% * In Table 9, the effect of prg:: TnphoA insertions on Salmonella-mediated endocytosis by MDCK epithelial cells is shown. The microaerófllámente sultivated basterian sepals were evaluated for changes in adherence and invasion. Adherence was determined as the percentage of bacteria adhered to the cells after sentrifugation and 30 minutes of insubassion at 4 ° C / total number of basterias added to each cavity. The invasion was determined as the percentage of bacteria that had invaded after insubassion of two hours are gentamisin / total number of basterias added to sada savity. There was no difference between S. typhimurium wild type and S. enteri tidis CDC5 wild type with respect to adhesion and fresuensia of the invasion. The asterisso (*) represents statistical signifisansia by variance analysis of the invasion data performed in triplicate in comparison with wild type (p value <0.01). The MDCK sonfluent monolayers were washed three times with PBS, then 0.9 ml of cold DMEM / 10% FBS was added to each well. The basterias were washed in LB and resuspended in an equivalent volume of DMEM / 10% FBS. Approximately 5 x 107 basterias per savity were added. The plasmas were rotated at 500 rpm at 4 ° C for 10 minutes, then incubated at 4 ° C for 30 minutes. The adherent basterias were recovered by washing the plates three times are buffered saline solution are phosphate (PBS), using the epithelial cells in 0.5 ml of 1% Triton-X-100 / PBS, and forming plasmas for sfu / ml on agar-LB . A morphological determination of adherence was carried out by marsing bacterially-infected epithelial cell monosapas cultured overnight on sub-sections for 7 minutes in 1 μg / ml of 6-diamidino-2-phenylinindole (DAPI). These marsated subsides are DAPI were examined by both fluorosurveyed microscopy and phase contrast using a Letiz Laborlux 12 microscope. Invasion of baster-mediated endosytosis (BME) was evaluated by allowing the bacteria to adhere as described above. Plates containing basterias and epithelial cells were incubated for two hours at 37 ° C in an atmosphere of 5% C02 / 95% air. Each savity was washed three times are PBS to remove the non-asosiada basterias are the cells. 10% DMEM / FBS were added, and 10 μg / ml of gentamisin were added to kill the extracellular basterias. After 90 minutes of incubation, the cell monolayers were washed three times with PBS and the viable intracellular bacteria were released by vigorous shaking in pipettes with 0.5 ml of Triton-X-100 / PBS. A mutant of Salmonella enteri tidis and a wild type, clinical, invasive isolate of S. enteri tidis were used as controls for BME. Viable bacteria were quantified by plating for cfu / ml on LB-agar medium. All assays were performed in triplicate and repeated at least three times. MDCK epithelial cells were used between passages 40-58 to maximize bacterial adhesion and invasion. The epithelial cell lines were cultured in DMEM / 10% FBS and 1% penicillin / streptomycin solution at 37 ° C in an atmosphere of 5% C02. To test bacterial sensitivity to defensin, defensin NP-1 was purified from rabbit peripheral neutrophils, according to methods disclosed in the art (Selsted et al., 1985, J. Biol. Chem. 260: 4579; Selsted et al., 1984, Infect. Immun. £ 5: 655). Typically, 105 basterias in 0.5% tryptone in a volume of 100 μl were exposed to 50-100 μg defensin / ml at 37 ° C for 2 hours. The reassurances were stopped by diluting the 0.9% NaCl reassessment. Appropriate disolusions were placed in plasmas to determine the cfu / ml of the surviving bacteria. Tests were carried out in duplicate, at least two times for each strain. Appropriate assays with sensitive (PhoP ") and resistant (wild-type) sepals were carried out on somoles .. Mapping of pr jH The location of prgH relative to other sites of invasion at 59 minutes was determined using binding analysis Transduction binding analysis P22 indicated that the Tnl0deltal6deltal7 of strain AK3304 had similar binding to inVa (40%) and prgH (37%), however, invA was not linked to sorbital.The insertion was found to be prgHl: - .TnphoA was linked (99.6% ) to the transposon insertion of EE517, a strain with an 8.5 kilobase deletion adjacent to the Tn5B50 - 378 insert, a physical map of the restriction endonuclease sites surrounding the TnphoA insert of strain IB037 was made (FIG. ), revealing that there were no similarities with the respecific endonuclease map of the invA-E region.Plasmids containing the inv-hil DNA were then used as probes. in analysis of Southern hybridization of sromosomal DNA of basterias ATCC 10428s wild type and IB040 containing the insertion prgHl. - .- TnphoA. When a plasmid that contains other invasion sites highly linked to invA-E (invH, invF and part of invG) was used as a probe, no differences were observed in the pattern of hybridization between wild-type basterias and the IB040 isolate, indicating that prgH It was not located within the inv region. However, when a plasmid containing a region of 5 kb immediately downstream of the insert Tn5B50-380 of hil was used as a probe, the prgHl:: TnphoA insert was shown to be located within this region. By using the conoside map of restriction of the hil place (Lee et al., 1992, Proc. Nati. Acad. Sci. USA 8_9: 1847) and the sonoside sites of TnphoA restriction endonuclease, the physical map of this area and the ratio of prgHl:. - nphoA inside her (figure 4). The insertion prgHl:. - TnphoA was oriented so that the transcriptional direction of the phoA fusion protein was opposite that of the Tn5B50 insertions that confer the hil phenotype and contain a constitutive neomisin promoter that is transcribed from the transposon (figure 4). Although it was found that prgH was located within the hil place, this gene is unique in that it is transcribed oppositely and unlike any other genes identified within the hil place, prgH is regulated by the phoP regution. As it was possible that a protein whose expression is alternating by the insertion Tn5B50-380 could alter the expression of prgH, we know how to separate both inserts and the astivity of prgH-phoA fusion protein under different environmental sondisions. When basterias were subjected to famine or anaerobically sultivated, depression of the fusion protein activity was observed. The Table 11 shows the effect of insertion Tn5B50-380 on the expression of the activity of the prgH fusion protein. Table 11 Strain Allele Famine LB (aerobiso) LB (anaerobium) IB040 prgHl: -. TnphoA 5 152 41 IB042 Tn5B50-380 46 248 227 prgHl:: TnphoA These data demonstrate that the insertion Tn5B50-380 increased the expression of prgH, even though the prgH: transcript was opposite to that of Tn5B50-380 encoded by the neomisin promoter. . Famine (repression sondisiones for prgs) indicates that the bacteria were aerobically cultured for 48 hours in a famine medium (N "C" P ") containing 0.04% glucose, 10 mM NH4C1, and 1 mM NaH2P04.H20. LB (aerobic) indicates that the basterias were sultivadas in balance of Luria-Bertani (medium riso) until late logarithmic sresimiento (conditions of non-repression) (OD600 > 1.0). LB (anaerobic) indicates that the basterias were sultivated under strict anaerobic conditions for 24 hours (OD600 = 0.6). All numbers represent activity in units of AP, as described above.

To avoid the possibility that the BME defect of the prgH mutant was an artifact of the prodrug PhoA fusion protein, complementation analysis was carried out with a plasmid (pWKSH5) containing a 5.1 kb HindIII fragment that included the hil and prgH sites . The plasmid was crossed in mutant bacteria PrgH (IB040) and PhoPc (CS022) to create separations IB043 and IB044, respectively. The BME phenotype of the PrgH mutant was similar to the wild type with the same plasmid insert. The BME phenotype of the PhoPc mutant was not supplemented by this plasmid. These results indicate that a gene product altered in synthesis as a result of the prgH:: TnphoA insert was necessary for BME. Using a strain with a place mutation PhoP / PhoQ that simulates the environmental activation of pags in a sonorous way (PhoP phenotype) were defined cinso phoP repressed unison sites, which sodifisan envelope proteins. It was shown that the repressed phoP genes were widely spased in the sromosome and the expression of the prg site was repressed under famine conditions, when pag sites were induced (Table 2).

). Table 10 Environment pags prgs half rich hunger 02 aerobic -pagC aerobiso-prgH anaerobic -prgB pH 3.3-5.5 3.3-5-5 -prgB > 6.0 -prgH mammalian epithelial macrophage cells It was shown that prgH lies between two Tn5B50 inserts that confer the hil phenotype. As the deletion mutants in this region have also been shown to have BME defects, and the BME defect of the prgH mutants can be supplemented with a plasmid containing this site, it is possible that a protein not synthesized as a result of the prgHl: insertion. - TnphoA promote BME (figure 4). Contrary to the expectation that genes essential to the hil phenotype would be induced under microaerophilic conditions similar to those found for expression of prgB, the expression of prgH was maximal during aerobic sultivo and the insertion Tn5B50-380, which results in a hil phenotype, derepressed the expression of prgH. In addition, the transcription direction predicted by the insertion prgHl: -. TnphoA is opposite to that of the neomycin promoter encoded by Tn5B50-380 associated with the hil phenotype, suggesting that a regulatory protein interrupted by or transcribed from the Tn5B50-380 insert affects the expression of prgH. In view of the observation that pWKSH5, a plasmid containing prgH (hil), does not somplement PhoPc basterias for BME, it is possible that other invasion genes may also be regulated by PhoP / PhoQ. If prgH were expressed from pWKSH5, despite the presence of the pho-24 mutation, this would suggest that other genes repressed as part of the PhoPc phenotype would be necessary for BME. The identification and characterization of prgH has shown that PhoP / PhoQ oppositely regulates factors necessary for bacteria to enter or survive within mammalian cells., documenting further the importance of gene regulation for bacterial virulence. The identification of prg sites can be used to study the regulation of bacterial genes after infection of mammalian cells. Understanding the regulation of virulence genes, such as prgH, can also be used with attenuated pathogenic bacteria for the development of new living vasunas for typhoid fever. Role of Prg genes in Virulence The prg place, prgH, was found to contribute to virulence in mice by administering S. typhimurium both orally and intraperitoneally. It was further found that the PrgH mutants as well as PhoPc were defestuous in basal cell-mediated admission by epithelial cells, suggesting that an inability to cross the epithelial barriers may contribute to the observed virulence attenuation. Sompetensia studies in mice after oral ingestion of basterias additionally supported that the prgH mutants were deficient in transsitosis through the epithelial barrier of the intestine. Therefore, at least two phases of protein expression regulated by PhoP / PhoQ essential for bacterial virulence have been defined. In one phase, prg expression promotes baseline mediated endosytosis by epithelial cells (Table 10), whereas in another phase, pag expression promotes survival within macrophages. Systemic pathogens, such as Salmonella, can produce more complex and varied environments than those that can be found by musosal pathogens. The achievement of intermediate states of expression of pag and prg may be essential for virulence at a certain stage of the infectious cycle. Consistent with this concept was the lack of uniformity observed in the expression of pags and prgs in culture at different oxygen tensions and pH sonsions. These data may also indicate that not all the regulation of pags and prgs is mediated directly through PhoP and PhoQ. Given the PhoP function we are a transshipping regulator, it is not possible that it represses prg places at the transshipping level. The approach of defining genes repressed by the pho-24 mutation has led to the discovery of at least one site of virulence, prgH, the sual can be mutated to attenuate the basteria for vaccine purposes. Attenuation of Bacterial Virulence by Constitutive Expression of Two-Component Regulatory Systems The virulence of a bacterium can be attenuated by inducing a mutation that results in gene expression under the control of a two-component regulatory system or by inducing a mutation that inactivates a gene under the control of two-component systems. A balance between the expression of genes under the control of the system of two somponents is necessary for full virulence, for example between the expression of genes pag and prg, and possibly between genes regulated by the system of two components and other genes. Mutations that break this balance, for example mutations that affect the expression of a gene under the control of the two somatic system, or a mutation that ingests a gene under the control of the two-component system, for example the gene pag, reduce the virulence. The mutations in two-component regulators can be identified by the use of a separator that contains a gene-logger fusion to a gene regulated by the two-component system. Such gene fusions in the most typical cases include sodifisation of DNA for the lacZ or AP gene fused to a gene under the control of a two-somersystem. Strains that are fusions that are highly expressed (in somparasión are wild type or parental sepals) in an unregulated form, is desir sonstitutiva, can be detected by increased color on substrates cromogénisos for enzymes. To detest sonorous mutations, a cloned virulence regulator can be mutagenized, for example by passage through a defective E. coli strain in DNA repair or by chemmis mutagenesis. The mutated DNA for the regulator would then be transferred to the strain containing the gene fusion and constitutive mutations identified by the high expression of gene fusion (blue color in the case of lacZ fusion cultured in medium containing X-gal). Constitutive mutations in a component of a two-component regulatory system can also be made by in vitro mutagenesis after other constitutive mutations have been sequenced and identified a change of specific amino acids responsible for the constitutive phenotype. Putting several amino acid changes that all result in a constitutive PhoP phenotype would result in a reduced rate of reversion by base spontaneous changes. A constitutive mutation may also be constructed by deletion of the amino terminal portion of the phospho-asepting regulator which is the phospho-aseptan-te domain, for example suppression of sesuensia that sodifisan amino-terminal aminoasides to amino acid 119 in the phoP gene or deletion. of similar phospho-aseptic sesuensias in genes from other regulatory systems of two somponents. This may result in a sambium in conformation similar to that induced by phosphorylation and result in attenuated DNA ligation and transcriptional activation. Virulence attenuation; Deletion in the PhoP / PhoQ Regiment As discussed above, the PhoP regimen is essential for the full virulence of Salmonella. This reguion is composed of two genes, PhoP and PhoQ, located in an operon, and more than 40 genes that regulate positively and negatively (pags and prgs, respectively). Mutants of S. typhimurium PhoP nulls have been shown to be markedly attenuated and also effective vaccine strains when studied in the BALB / c mouse model of typhoid fever. This phenotype is feasibly the result of multiple activated phoP virulence genes, since transposon insertions in multiple different activated phoP genes have been shown independently to reduce the virulence of S. typhimurium. Mutants of S. typhimurium devoid of genes for the aromatic aminoasides (aroA null or aroC / aroD null mutants) are also strongly attenuated in the mouse model. However, tests of aroC / aroD mutants in humans have shown that although these knowns are immunogenic, bacteremia and side effects, such as fever, have been noted in doses as low as 105 to 107 organisms administered as a single oral dose (J Clin Invest. 90: 412-420). It has now been ensontrado that a great suppression in a global regulator of Salmonella virulence, is desir the operon PhoP / PhoQ, reduse significantly the virulensia of the basteria. This mutation, the result of a deletion of 1 kb of DNA within the phoP / phoQ site, was inessially hesha in S. typhimurium and subsequently transferred via homologous resomycin to S. typhi. In order to confer an even greater margin of safety in the construction of this vasuna, it was created in a background of suppressed layer of genes esensial for the amino acid aromatides and carrying the mutation histidine G46, a mutation that converts the organism into auxotrope for histidine. The resulting strain, S. typhi TyLH445, offers several advantages over existing candidates for vaccines, mainly immunogenicity without transient bacteremia. Use The Salmonella cells of the invention are useful as sources of immunological protection against diseases, for example typhoid fever and related diseases, in an animal, for example a mammal, for example a human being, in particular as the basis of a vaccine. living cells capable of colonizing the intestine of the inoculated animal and causing an intense immune response. Doses and appropriate administration condi tions of such a live, attenuated vaccine are discussed in Holem et al., Acute Enteric Infections in Children, New Prospects for Treatment and Prevention (1981) Elsevier / North-Holland Biomedical Press, chapter 26, p. 443 et seq. (Levine et al.), Insorporated in this by reference and in the examples that appear later. Advantages An advantage of the invention is that the bacterial cells are attenuated as a result of one or more mutations, namely the phoP / phoQ operon, which afflict a virulence trajectory. Another advantage is that the bacterial cells have mutations in two distinctly different attenuating genes, namely the path of synthesis of aromatic amino acids (Aro), and in an important operon for the virulence of Salmonella (PhoP / Q). As a result, the bacterium appears to be extremely attenuated; doses as high as 1 x 109 sfu seem to be very safe. Other vaccines that are in development, such as CVD 908, have ossured some systemic symptoms, for example fever or basteremia, at such low doses as 1 x 107 sfu. In addition to the phoP / phoQ deletion and the AroA mutation, the basterian cells of the invention may also have a histidine mutation to attenuate virulence, although the absence of the histidine mutation may improve immunogenicity. The Basterian cells of the invention are the most promising candidates for vaccines to date, because they are strongly immunogenic and safe., that is, extremely attenuated. Example 1 - Construction of a Vaccine Strain The basterian cells of the invention were made by deleting approximately 1 kb of DNA in the phoP / phoQ regution. Suppressed phoP / phoQ suicide vectors were constructed using methods conosidos in the matter. A DNA fragment containing the phoP / phoQ site was obtained by PCR using wild-type S. typhimurium chromosomal DNA as a template. Primordial PCR materials flanking the phoP / phoQ site were constructed containing sites of recognition of terminal restriction enzyme, eg recognition site for EsoRI, to facilitate subsequent cloning. Following amplification, the PCR product was digested with EsoRI and slit in the EsoRI site in the poly-linker of a high sopor vestor. The plasmid 'containing the phoP / phoQ DNA fragment was named pLH356. Analysis of sesuensias and restriction mapping of the phoP / phoQ site revealed four Hpal sites within the site; no Hpal sites were found in the vector. To create an internal deletion within the phoP / phoQ site, the pLH356 DNA was cut to completion with Hpal, and religated, to give an internal suppression of nucleotides 376-1,322 (pLH418). This suppression was confirmed by restriction digestion of the plasmid. A DNA fragment containing the internally suppressed phoP / phoQ site was cut from pLH418 using the Sasl / Sphl restriction sites within the vestigative linker region. This fragment was slugged in sompatible sites in the plasmid CVD442, which carries the sacB gene to allow positive selssion for allelic intersambio. The resulting suiside vector was called pLH423. Plasmid pLH423 was transformed into E. coli lambda pir SY327, and subsequently into E. coli lambda pir SM10 (know LH425). The strain E. coli LH425 was paired with the strain S. typhimurium CS019. Simple recombinants carrying plasmid sequences integrated into the S. typhimurium chromosome were selected by plating on agar containing ampicillin and chloramphenicol (strain LH428). It was confirmed that these strains were resistant to ampicillin and sensitive to sucrose, that is, they died in 20% sasarose plates without containing NaCl when they were added at 30 ° C. These data confirm the integration of the plasmid sesuensias in the Salmonella chromosome. A bacteriophage P22 lysate was hesho of the LH428 strain; the phage particles were concentrated 20 times by centrifugation at high volatility and transduced in the S. typhi 522Ty2 domain (one known is a deletion in the aroA gene, and the G646 mutation that renders the organotropic organism to histidine). Recombinant organisms of simple S. typhi were selected by arrangement in LB plates supplemented with aromatic amino acids, cystine, histidine, and ampicillin (strain LH435). Strain LH453 was cultured with aromatic amino acids, systine and histidine (but without ampisiline) until the semi-log-rich phase of sultivo. Disolusions in series were arranged in plasas are LB and 20% sasarose, without NaCl, and in LB plasmas without NaCl. The number of bacteria that grew on the plates without sucrose was greater than the number that occurred in plasmas supplemented with sucrose by a logarithmic fastor of three. These data suggest that many colonies lost sequencies plasmids by blowing the sacB gene. Multiple colonies of the sucrose selection were collected and confirmed to be sensitive to ampicillin and resistant to sucrose. Sromosomal DNA from approximately 10 solonias was purified and subjected to Southern analysis, using the 2.3 kb fragment of the phoP / phoQ wild type somo probe. Southern analysis revealed the loss of two Hpal sites and one Xmnl site by being within the deleted 1 kb fragment of phoP / phoQ in several strains. One of these strains was designated TyLH445. Example 2 - In Vitro Evaluation of TyLH445 TyLH445 was extensively in-terrored using conventional slinoidal misrobiological tests. The nutritional requirements of TyLH445 were evaluated. TyLH445 did not concentrate on M-9 plasmas unless they are somp1ementaran are a mixture of aromatic amino acids, systine (S. typhi grows best with cystine), and histidine. These data confirmed that TyLH445 was AroA-, His-. It was shown that TyLH445 is agglutinated with polyclonal serum against Salmonella and polyclonal serum against the S. typhi Vi antigen. The agglutination group D was found variable, perhaps due to the excess of the Vi antigen. It was also found that TyLH445 was indole negative (as all Salmonellae are) and that it produced very high hydrogen sulfide (as S. typhi mushos has). Biochemical tests using both the VITEK system and the identification system of BBL crystal enthalpy organisms were also carried out. These data indicated that strain TyLH445 was S. typhi. The culture characteristics of TyLH445 were also evaluated. It was found that TyLH445 grew just as rapidly as its parental strain, 522Ty2 (place phoP / phoQ intact). In vivo growth was measured in Luria broth supplemented with aromatic amino acids / histidine / cystine at 37 ° C. The crescent curves of the parental and vasuna separations were essentially identical (see figure 10). Standardized test methods were used to determine sensitivity to antibiotics. TyLH445 and the parental strain, 522Ty2, were found to be sensitive to ampicillin, trimethoprimus sulfamethoxazole, ciprofloxacin, aminoglycosides and sephalosporins of tersera generation. No differences were noted in the zone sizes between the parental and vaccine strains, suggesting that no other mechanism of resistance to antibiotics, for example modifisation of antibiotic transport systems, or modification of the sellable wall of the basteria, were afflicted by the introduction of the phoP / phoQ site mutated in S. Typhi The phoP / phoQ Hpal deletion mutants were subjected to defensin sensitivity tests, a phenotype of the null PhoP mutants. They took to tas trials of sensitivity to defensin somo follows.

Liquid cultures of seeds to be tested were grown overnight. The cultures were then diluted 1: 200 and cultured at an optimum density (OD600) of about 0.2, after which the cells were diluted to a concentration of approximately 1 x 105 organisms per 0.05 ml. Two reassumptions were carried out for each strain: (1) vehicle alone (0.01% of asastosis in sterilized water), and (2) defensin NP-1 solution (70 μg / ml in acetic acid at 0.01%). An equal volume of basterian suspension in tryptone was added and the test tubes were incubated on a roller at 37 ° C for two hours. The final volume in each reactive tube was 0.1 ml, making the final defensin consentration of 35 μg / ml. The defensin is inactivated by the high salt and protein sonsentrasion present in the basterian culture medium, for example LB balance. In this way, defensin activity was stopped by adding 900 μl of Luria broth to each tube. Serial solutions of each tube were placed in plates and the cfu / ml was determined for both the control tube and the treatment tube for each strain. The results were expressed as logarithm of dead basterias for sada strain. Typically, 1.0-1.5 log of wild type bacteria were killed. The null PhoP mutants generally exhibit 2-4 log of deaths. As the strains are lower sresence rates, which are less susceptible to death by defensin, the rate of sada sresistance known to be tested in the defensin sensitivity assay was measured. Strains are similar sresimiento rates were somparadas in the assay of defensin sensitivity. Hpal suppression was evaluated in both a background of S. typhimurium and in the background of S. typhi. In both backgrounds, the deletion mutation conferred sensitivity to rabbit NP-1 defensin at a consentration of 35 μg / ml. See Figures 11 and 13. The difference between deleted PhoP + and Hpal null PhoP mutants was less pronounced in the S. typhi strain, an effect that may reflect the lower crescent rate of the less resistant S. typhi strain in S. typhi. somparasión are the know of S. typhimurium that lacks additional auxotropies. The state of phoP astivation in cells is the suppression of Hp phoP / phoQ was tested using a lacZ gene logger fused to a gene phoP astivado B (pagB). As the efisiensia of the transduction using P22 in S. typhi is low, these studies were carried out in S. typhimurium instead of S. typhi. It was shown that phoP activation was from 40 to 60 units of Miller (Miller et al., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352-355) in the presence of a phoP site / phoQ intact, and barely detectable in strains with Hpal suppression (3 cfu, see figure 12). EXAMPLE 3 - In Vivo Evaluation of the S. typhimurium Suppressed Hpal strain As the strains of S. typhi are not pathogenic for mice, the Hpal suppression mutation phoP / phoQ was evaluated in both S. typhimurium wild type somo aroA. Female BALB / s mice were injected intraperitoneally with various solutions of S. typhimurium LH430, a wild-type S. typhimurium that carries Hpal suppression. The LD50 of this strain was determined to be between 8.2 x 105 and 8.2 x 10 s. (All mice that received 8.2 x 105 sfu survived, and all those that received 8.2 x 106 cfu died.) These data are available. The LD50 data obtained are sepals carrying transposon inserts in the phoP / phoQ site. The immunogenicity of Hpal suppression phoP / phoQ was evaluated in S. typhimurium aroA:: tet (LH481), a strain comparable to the strain for human vaccine. Mice were inoculated intraperitoneally with 2.3 x 105 and 2.3 x 106 cfu of LH481 (four mice per vaccine dose), and challenged 30 days later with 30 times the LD50 of wild-type organisms. All the mice, except one, survived. The mouse that died was from the group that received the lowest dose of vaccine. No animal that received the higher dose of vaccine became ill. Example 4 - Human Studies Phase I The vaccine strain was administered to human volunteers at doses of 1 x 105 to 1 x 1010 sfu / per single oral dose. Two volunteers received each dose; three volunteers were given a dose of 1 x 108 cfu / ml. Volunteers were evaluated at various points in time after administration of the vaccine. Safety To detect the presence of the vaccine strain in the patients' blood, cultures of Bactec blood were sampled in duplicate on days 4, 6, 8, 10 and 12 after taking the vaccine. No basteremia was detected in any of the volunteers. Trese adult volunteers have received increasingly simple oral doses of this new attenuated vaccine against typhoid fever. No individual has had side effects of any kind. Scarcely, there have been no fevers, no gastrointestinal symptoms, or sonic symptoms. Volunteers have undergone serial blood tests in a program that has been loaned after receiving the oral vasuna, taking two sets of Blood sultives Bastes on days 4, 6, 8, 10 and 12 after resveraging the vasuna, and no positive blood cultures have been observed. The volunteers have been reviewed up to two months after receiving the vasuna, and no late symptoms have been reported. Colonization Tests were carried out on samples of excrement related to the presence of the vasuna TyLH455 using methods known in the art. The primary excrement was evaluated for the presence of the vaccine strain in culture dishes. In some cases, it was necessary to enrich the exsurement samples for the vasuna sampler by supplying the excrement during the night with the BBL Selenite F broth, supplemented with Aro / His / Cis-tina in order to detest the basteria. This medium is somewhat inhibitory of E. coli but promotes Salmonella sresimiento. Volunteers have been colonized for various periods of time from 1-6 days after receiving the vasuna. At the highest doses (109 or 1010), the volunteers had positive primary cultures in the first 1-3 days after the vaccination, while at lower doses, they were positive only for the organism of vasuna enriched broth cultures. (selective medium for Salmonella that inhibits other enterics). No volunteer studied so far has had prolonged use of the vaccine organism after two months of follow-up. Table 17 Dose Number Colonization 105 2 No 106 2 2/2 for 1-2 days 107 2 1/2 for 3 days 108 3 1/3 for 6 days 109 2 2/2 for 4-6 days; both had positive primary plasmas on day 1 1010 ** 2 2/2 for 3-6 days; both had positive primary plates on days 1 and 2 * Measured by the tests of somplet cells and LPS ELISAS and Widal vs. flagellar antigen. The sera analyzed at 1:40 and higher dilutions in all tests. ** One of these volunteers received an amplification dose of 1010 organisms, given one month after primary inoculation (pending serology). Immunogenicity The induction of an immunological response to the vaccine strain was measured by conventional ELISA assays. Sera were collected from volunteers 0, 7, 14, 21 and 28 days after receiving a single oral dose of the vaccine. The ELISA assays were carried out using complete bacteria TyLH445 and S. typhi LPS (Sigma, St. Louis, Missouri, United States) as antigens. The serum of day 0 of each volunteer was used as an internal negative control. Serum-free sera from the previously infested strains are wild type S. typhi (mostly from Mexico) were used as positive serums. Several volunteers have measured their serosonversion 21 days after resuscitation of the vasuna, as measured by ELISAs by detesting IgG directed against complete vaccine organisms or against S. typhi LPS. Sera taken from patients before administration of the vaccine (pre-immune sera) were tested and the data used to establish a baseline. Sera from patients taken at various points in time after vaccination were considered positive if the test results were 0.2 OD ELISA units greater than the pre-immune serum. Other Forms of Realization Other forms of realization, for example strains of Salmonella containing only one deletion in the phoP / phoQ regulatory site to attenuate virulence, and strains that, in addition to a mutated phoP mutation or genetic alteration, also contain an attenuating mutation in another gene, for example the gene sya (adenylate sislase) or the srp gene (adenylate sislase receptor), are also included in the claims.

TABLE 12. Bacterial Strains Strain Genotype Source S. typhimurium 14082s Wild type ATCC CS019 phoN2 zxx :: 625 \ Tnl0d-Cm 25 CSO 15 phoP-102 :: Tnl0d-Cm 25 AD154 phoP12 purB1744 :: Tn \ 0 3 TT13208 phoP105 :: Tnl0d 26 CS585 pagDlv.TnphoA phoN2 zxx :: 6215Tn / 0d-Cm This study CS586 pagDlr.TnphoA phoP105: Tnl0dphoN2 zx? ::: 6215Tn / 0d-Cm This study CS619 pagE l: \ Tnpho A phoN2 zrc :: 6215Tn / 0d-Cm This study CS620 pagElr.TnphoA phoP105 :: Tnl0dphoN2 zra ::: 6215Tn / 0d-Cm This study CS1599 pagFlr.TnphoA phoN2 zxx :: 6215Tn / 0d-Cm This study CS1600 pagFlv.TnphoA phoP105 :: Tn! 0dphoN2 zxx :: 6215Tn / 0d-Cm This study CS334 pagGlr.TnphoA phoN2 zxx :: 6215Tn70d-Cm This study CS335 pagGl :: TnphoAphoP105: Tal0dphoN2 zxx :: 6215Tnl0d-Cm This study CS1488 pagH 1 r.TnphoA phoN2 zrc :: 6215Tn / 0d-Cm This study CS1489 pagHlr.TnphoA phoP105 :: Tnl0dphoN2 zxx :: 62 \ 5Tnl0d-C This study CS2054 pagIl :: TaphoAphoN2 zx; - :: 6215Tn./í?d-Cm This study CS2055 pagIl :: TnphoAphoP105 Tnl0dphoN2 zxx :: 6215Tn / 0d-Cm This study CS1074 pagJlr.TnphoA phoN2 z x :: 6215Tn7 (? D-Cm This study CS1075 page] r.TnphoA phoP 105 Tnl0d phoN2 zxx :: 6215Tn / 0d-Cm This study CS767 pagKl r.TnphoA phoN2 zxx :: 6215Tn / 0d-Cm This study CS768 pagK 1 -r.TnphoA phoP105 :: Tnl0dphoN2 zxx :: 62 \ 5Tnl0d-Cm This study CS993 pagLlr.TnphoA phoN2 zxx :: 6215Tn / £ 'd-Cm This study CS994 pagLlr.TnphoA phoP105: Tnl0dphoN2 zxx :: 62l5Tn! 0d-Cm This study CS1845 pagMl .TnphoA phoN2 zxx 62 \ 5Tnl0d-Cm This study CS1846 pagMlr.TnphoA phoP 105 :: Tnl0d phoN22x :: 6215Tn / 0d-Cm This study CS728 pagNlr.TnphoA phoN2 za ::: 6215Tn / 0d-Cm This study CS729 pagNl :: TnphoAphoP105 :: Tnl0dphoN2 zxx: 62 \ 5Tnl0d-Cm This study CS1194 page 1"TnphoA phoN2 zx - :: 6215Tn / 0d-Cm This study CS1195 pagOlr.TnphoA phoPl 05r.ini 0dphoN2 zxx: 6215Tnl0d-Cm This study CS1247 pagP l: TnphoA phoN2 zxx :: 6215Tn / (? D-Cm This study CS1248 pagP l: \ TnphoA phoP105 :: Tnl0dphoN2 z:? :: 6215Tn / 0d-Cm This study AK301 1-3314 Collection of inserts TnlO? 16? 17 randomly spaced 18 E. coli SM10 (pRT291) Contains the plasmid? Rt291 (TnphoA) derived from pRK290 which selects Tetr and Kmr MM294 (pPHUI) Contains plasmid pPHUI Gmr, which is incompatible with pRK290 37 Miller et al., 1989, Proc. Nati Acad. Sci. USA, 86: 5054-58 3 Behlau et al., 1993, J. Bacteriol., 175: 4475-84 26 Miller et al., 1990, J. Bacteriol., 172: 2485-90 18 Lehrer et al. , 1991, Cell, 64: 229-30 37 Taylor et al., 1989, J. Bacteriol., 171: 1870-78 TABLE 13. Comparison of activity p.phoA in wild-type strains and null phoP foci.

Activity (AP units) to Allele Logarithmic growth Stationary growth Number of times PhoP + PhoP "PhoP + PhoP" reduction "pagDl :: TnphoA 32 2 79 9 16 pagEl" TnphoA 96 2 108 3 48 pagFl :: TnphoA 89 4 276 10 22 pgGl "TnphoA 35 1 65 6 35 pagHl" TnphoA 35 5 38 6 7 pagIl :: TnphoA 12 2 24 8 6 pagJl :: TnphoA 123 8 944 88 15 pagKl "TnphoA 30 3 123 26 10 pagLl :: TnphoA 7 1 35 4 7 pagMl :: TnphoA 92 11 439 130 pagNl :: TnphoA 23 1 58 2 23 pag01 :: TnphoA 31 2 54 12 16 pagPl :: TnphoA 38 1 27 3 38 a AP activity values are presented in units defined by Miller for β-galactosidase (24). The values are representative of experiments (performed in duplicate) that were repeated on three separate occasions. PhoP + denotes the insertion pag :: TtsphoA in strain CSO 19 containing a wild-type phoP focus. PhoP "denotes an isogenic strain carrying the phoPl allele 05:: Tnl0." The values of the number of fold reduction in an enzymatic activity represent the decrease in AP activity in the acquisition of the null phoP105 allele. These were calculated from the cultures of the logarithmic growth phase and rounded to the nearest whole number.

TABLE 14. The effects of gene fusions: phoA on virulence in Salmonella mice.

Strain Genotype LD50a MSI Reference 14028s Wild type < 20 6.13 25 CS015 phoP102 :: TnlO-Cam 7.0xl05 0.40 25 CS585 pagDlv.Tx? HoA 4.0xl05 0.01 15 CS1074 pagJl :: TnphoA 4.0x103 0.56 This study CS767 pagKl :: TnphoA 9.0x104 0.04 This study CS1845 pagMl: Z •? VfhoA 3.0xl04 0.09 This study a The lethal dose at 50% was determined by intraperitoneal injection of ten mice by dilution using the method of Reed and Muench (31). The Macrophage Survival Index (MSI) was determined by dividing the mean CFU of Salmonella recovered from macrophage cultures (carried out in triplicate) 24 hours after the addition of gentamicin by the average CFU of Salmonella recovered from macrophages one hour after the addition of gentamicin.

MUIer et al., 1989, Proc. Nati Acad. Sci. USA, 86: 5054-58 15 Kier et al., 1979, J. Bacteriol., 138: 155-61 TABLE 15. Plasmids, Strains, and Relavant Properties Strains of S. tvphimurium Genotypes / Relevant Information MSIa Source "ATCC 14028 Wild type 3.90 ATCC CS019 phoN2 zxx :: 6251Tn / 0d-Cm (31) CS585 CSO 19, pagD:: TnphoA 0.002 ( 4) CS586 CS585, TJ / ZOPI 05:: TnI 0d-Tet (4) JSG205 ATCC14028, msgAv.Mudi 0.01 This job JSG225 JSG205, phoPl 05:: Tn7 OdTet This job CS811 CS019, envE ::? R? HoA This job CS812 CS8l l, phoPl0 :: Tnl0d-? Et This work CS100 ATCCl4028, phoPl05 ::? Nl0d-tet 0.01 Derived from TT13208 JSG232 JSG205, envF :: pG ?? 2 This job JSG234 CSO 19, envF :: pG ?? 2 This job JSG235 JSG234, phoPl05:: Tni 0d-Tet This job JSG244 JSG205, phoPl05:: Tni Od-Tet This job CS099 ATCC14028; zj.x3024 :: Tn70? 16? 17po / -2 (amber Whitfield pole) This work Other Salmonella Ty2 Vi positive FDA Salmonella paratyphi 'A ATCC9150 ATCC Salmonella paratyphi: B ATCC 13428 ATCC Salmonella enteriditis Clinical isolated VRI E strains coli SMlO? zV thi-l thr-l leuBβ supE44 tonAll / ac71rec_4 :: RP4-2-Tc :: Mu DH5a F-0 80dlacZAMl5A (lacZYA-argF) m 69endA 1 recAlhsdR 17deoRthi-lsupE44? -gyrA96relAl Other Enterobacteriaceae Yersinia enterocolitica Clinical isolated Bacteriological laboratory MGH Vibrio cholerae Clinical isolate Peruvian epidemic Campylobacter fetus Clinical isolate MGH bacteriological laboratory Citrobater freundii Clinical isolate MGH bacteriological laboratory Klebsiella pneumoniae Clinical isolate MGH bacteriological laboratory Shigella flexner i Clinical isolate MGH bacteriological laboratory Shigella sonnei Clinical isolate MGH bacteriological laboratory Morganella morganii Clinical isolate MGH Providencia bacteriological laboratory stuartii Clinical isolated MGH bacteriological laboratory Plasmids pWPL17 pBR322 containing a 2.8 Kb fragment of pWP061 This work pCAA9 pWPL17 containing a TnphoA insert in envF This work pGP704 Suicide vector j!? ZV-dependent (34) pGPP2 pGP704 containing the cloned fusion of the gene envF :: phoA East work pWP061 Clone of cosmid containing the pagC region (36) a MSI (macrophage survival index) is calculated by dividing the number of surviving organisms in the 24 hours after infection by the number of organisms associated with cells present after infection for 30 minutes. b MGH, Massachusetts General Hospital; ATCC, American Type Culture Collection; FDA, Food and Drug Administration; VRI, Virus Research Institute. 31 Miller et al., 1989, Proc. Nati Acad. Sci. USA, 86: 5054-58 4 Belden et al., 1989, Infect. Immun., 57: 1-7 34 Miller et al., 1988, J. Bacteriol., 170: 2575-83 36 Pulkkinen et al., 1991, J. Bacteriol., 173: 86-93 TABLE 16. Fusion activity of the alkaline phosphatase and ß-galactosidase genes Strain Relevant genotype Gene fusion activity JSG205 msgA: MudJ 461 (B) JSG244 phoPl05 :: Tnl0d-Tet 415 (B) msgA :: Mudl JSG226 envEvZ? VfhoA 50 (A) JSG229 phoPl05 ::? Nl0d-? Qt 60 ( A) envE :: TnphoA JSG204 paGD: -ZYrfhoA 76 (A) JSG225 phoPl05 :: Tnl0d-? Et 9 (A) pagD :: TnphoA JSG234 envF :: pG ?? 2 16 (A) JSG235 phoPl05 ::? Nl0d- TQt 19 (A) envF :: pG? ~ P2 JSG232 msgA :: MudJ 10 (A) envF :: pGFP2 a (AP) (alkaline phosphatase) or (B) ß-gal (ß-galactosidase) SEQUENCE NUMBER: 1 GTTAACCACT CTTAATAATA ATGGGTTTTA TAGCGAAATA CACTTTTTTA TCGCGTGTTC 60 AATATTTGCG TTAGTTATTA TTTTTTTGGA ATGTAAATTC TCTCTAAACA CAGGTGATAT 120 TTATGTTGGA ATTGTGGTGT TGATTCTATT CTTATAATAT AACAAGAAAT GTTGTAACTG 180 ATAGATATAT TAAAAGATTA AATCGGAGGG GGAATAAAGC GTGCTAAGCA TCATCGTGAA 240 TATGATTACA GCGCCTGCGA TGGCATATAA CCGTATTGCG GATGGAGCGT CACGTGAGGA 300 CTGTGAAGCA CAATGCGATA TGTTCTGATT ATATGGCGAG TTTGCTTAAT GACATGTTTT 360 TAGCCGAACG GTGTCAAGTT TCTTAATGTG GTTGTGAGAT TTTCTCTTTA AATATCAAAA 420 TGTTGCATGG GTGATTTGTT GTTCTATAGT GGCTAAAGAC TTTATGGTTT CTGTTAAATA 480 TATATGCGTG AGAAAAATTA GCATTCAAAT CTATAAAAGT TAGATGACAT TGTAGAACCG 540 GTTACCTAAA TGAGCGATAG AGTGCTTCGG TAGTAAAAAT ATCTTTCAGG AAGTAAACAC 600 ATCAGGAGCG ATAGCGGTGA ATTATTCGTG GTTTTGTCGA TTCGGCATAG TGGCGATAAC 660 TGAATGCCGG ATCGGTACTG CAGGTGTTTA AACACACCGT AAATAATAAG TAGTATTAAG 720 GAGTTGTT 728 ATG AAA AAT ATT ATT TTA TCC ACT TTA GTT ATT ACT AT AGC GTT TTG 776 Met Lys Asn lie lie Leu Ser Thr Leu Val lie Thr Thr Ser Val Leu 5 10 15 GTT GTA AAT GTT GCA CAG GCC GAT ACT AAC GCC TTT TCC GTG GGG TAT 824 Val Val Asn Val Wing Gln Wing Asp Thr Asn Wing Phe Ser Val Gly Tyr 20 25 30 GCA CGG TAT GCA CAA AGT AAA GTT CAG GAT TTC AAA AAT ATC CGA GGG 872 Wing Arg Tyr Wing Gln Ser Lys Val Gln Aep Phe Lys Asn lie Arg Gly 35 40 45 GTA AAT GTG AAA TAC CGT TAT GAG GAT GAC TCT CCG GTA AGT TTT ATT 920 Val Asn Val Lys Tyr Arg Tyr Glu Asp Asp Ser Pro Val Ser Phe lie 50 55 - 60 TCC TCG CTA AGT TAC TTA TAT GGA GAC AGA CAG GCT TCC GGG TCT GTT 968 Ser Ser Leu Ser Tyr Leu Tyr Gly Asp Arg Gln Wing Ser Gly Ser Val 65 70 75 80 GAG CCT GAA GGT ATT CAT TAC CAT GAC AAG TTT GAG GTG AAG TAC GGT 1016 Glu Pro Glu Gly lie His Tyr His Asp Lys Phe Glu Val Lys Try Gly 85 90 95 TCT TTA ATG GTT GGG CCA GCC TAT CGA TTG TCT GAC AAT TTT TCG TTA 1064 Ser Leu Met Val Gly Pro Wing Tyr Arg Leu Ser Asp Asn Phe Ser Leu 10 0 105 110 TAC GCG CTG GCG GGT GTC GGC ACG GTA AAG GCG AC TTT AAA GAA CAT 1112 Tyr Ala Leu Ala Gly Val Gly Thr Val Lys Ala Thr Phe Lys Glu His 115 120 125 TCC ACT CAG GAT GGC GAT TCT TTT TCT AAC AAA ATT TCC TCA AGG AAA 1160 Ser Thr Gln Asp Gly Asp Ser Phe Ser Asn Lys lie Ser Arg Lys 130 135 140 ACG GGA TTT GCC TGG GGC GCG GGT GTA CAG ATG AAT CCG CTG GAG AAT 1208 Thr Gly Phe Wing Trp Gly Wing Gly Val Gln Met Asn Pro Leu Glu Asn 145 150 155 160 ATC GTC GTC GAT GTT GGG TAT GAA GGA AGC AAC ATC TCC TCT AA AAA 1256 lie Val Val Asp Val Gly Tyr Glu Gly Ser Asn lie Ser Thr Lys 165 170 175 ATA AAC GGC TTC AAC GTC GGG GTT GGA TAC CGT TTC TGA AAAGC 1300 lie Asn Gly Phe Asn Val Gly Val Gly Tyr Arg Phe 180 • 185 ATAAGCTATG CGGAAGGTTC GCCTTCCGCA CCGCCAGTCA ATAAAACAGG GCTTCTTTAC 1360 CAGTGACACG TACCTGCCTG TCTTTTCTCT CTTCGTCATA CTCTCTTCGT CATAGTGACG 1420 CTGTACATAA CATCTCACTA GCATAAGCAC AGATAAAGGA TTGTGGTAAG CAATCAAGGT 1480 TGCTCAGGTA GGTGATAAGC AGGAAGGAAA ATCTGGTGTA AATAACGCCA GATCTCACAA 1540 GATTCACTCT GAAAAATTTT CCTGGAATTA ATCACAATGT CATCAAGATT TTGTGACCGC 1600 CTTCGCATAT TGTACCTGCC GCTGAACGAC TACTGAAAAG TAGCAAGGTA TGTATTTTAT 1660 CCAGGAGAGC ACCTTTTTTG CGCCTGGCAG AAGTCCCCAG CCGCCACTAG CTCAGCTGGA 1720 TAGAGCATCA ACCTCCTAAG TTGATGGTGC GAGGTTCGAG GCCTCGGTGG CGGTCCAATG 1780 TGGTTATCGT ATAATGTTAT TACCTCAGTG TCAGGCTGAT GATGTGGGTT CGACTCCCAC 1840 TGACCACTTC AGTTTTGAAT AAGTATTGTC TCGCAACCCT GTTACAGAAT AATTTCATTT 1900 ATTACGTGAC AAGATAGTCA TTTATAAAAA ATGCACAAAA ATGTTATTGT CTTTTATTAC 1960 TTGTGAGTTG TAGATTTTTC TTATGCGGTG AATCCCCCTT TGCGGCGGGG CGTCCAGTCA 2020 AATAGTTAAT GTTCCTCGCG AACCATATTG ACTGTGGTA -GGTTCACCGG GAGGCACCCG 2080 GCACCGCAAT TTTTTATAAA ATGAAATTCA CACCCTATGG TTCAGAGCGG TGTCTTTTTA 2140 CATCAGGTGG GCAAGCATAA TGCAGGTTAA CTTGAAAGAT ACGATCAATA GCAGAAACCA 2200 GTGATTTCGT TTATGGCCTG GGGATTTAAC CGCGCCAGAG CGTATGCAAG ACCCTGGCGC 2260 GGTTGGCCGG TGATCGTTCA ATAGTGCGAA TATGAATGGT TACCAGCCGC CTGCGAATTC 2320 SEQUENCE NUMBER: 2 CATTTCTCAT TGATAATGAG AATCATTATT GACATAATTG TTATTATTTT ACG 53 SEQUENCE NUMBER: 3 GAGCGCATTA TCAGATAAAT TGATTTATTT CTCACTTTCA TTCTATTTTC ATCAGGAATC 60 CCTGTGTCCT GTGCGGTAAT CTGCTGCTAT CGAGAACGAC AGACATCGCT AACAGTATAT 120 ATGGAAACAT CAAAAGAGAA GACGATAACA AGCCCAGGGC CATACATAGT TCGATTACTT 180 AACAGCTCAC TGAACGGCTG TGAGTTTCCA TTGCTGACAG GCCGAACACT CTTTGTGGTA 240 GGTCAGAGTG A? GCGCTCAC TGCTTCAGGT CAACTCCCTG ATATACCTGC CGATAGCTTT 300 TTTATCCCGC TGGACCATGG CGGAGTAAAT TTTGAAATCC AGGTGGATAC GGATGCGACC 360 GAAATTATAC TCCATGAGCT GAAAGAAGGA AATTCTGAAT CTCGTTCGGT GCAATTAAAT 420 ACGCCAATAC AGGTCGGTGA ATTGCTTATC CTGATTCGCC CGGAAAGCGA GCCGTGGGTG 480 CCCGAGCAGC CTGAGAAGTT AGAAACGTCT GCAAAAAAGA-ACGAGCCGCG TTTTAAAAAC 540 GGAATTGTAG CAGCACTGGC CGGGTTTTTT ATATTGGGAA TTGGGACTGT GGGGACGTTA 600 TGGATACTTA ACTCGCCGCA GCGGCAGGCC CGAGAGCTCG ATTCGTTATT GGGGCAGGAG 660 AAGGAGCGTT TTCAGGTGTT GCCAGGCC 688 NIJMERO SEQUENCE: 4 AATATCGCCC TGAGCA 16 SEQUENCE NUMBER: 5 GGTTAACTCT TCGTTGAATA AAAAATGTCA ATGACGTTCC ATAATTCAGG AGATGAACTT 60 CACAAGTCAT TATATATAAC AGGAGGTGCT ATGAAACATC ATGCTTTTAT GCTTTGGTCA 120 TTACTTATTT TTTCATTCCA TGTTTTGGCC AGTTCAGGCC ATTGTTCTGG TTTACAACAG 180 GCATCATGGG ATATTTTTAT CTACGATTTT GGTAGTAAAA CCCCGCAACC ACCTACAAAT 240 ACTGATAAAA AGCAAGCCAG GCAGATTAGT TCACCGTCCT GCCCGACGAC AAAACCCATG 300 ATGTCCGCAC CAGTCAATGA CGCCAGGAAA GGGAATACTT TCTCCAGAAC ATAATGTTAT 360 TTATCTACAA TGGTGCCGAC GACTACTTTT AGCCACCCGG AAATCTTGAT TGCCATCAAA 420 TATAGCTGGC ATTATTTTTC CTGACGTGTA TAGTGCGCCT .CGTTATCCCC ATTAAGGAAT 480 TTGTTTGTCT CGTAAAATGA CAGGAATTGT CAAAACCTTT GATTGTAAGA GCGGTAAAGG 540 TCTCATCACC CCCTCCGATG ACGCAAAGAT GTTCAGGTCC ACATTTCAGC ATGTCGCCAA 600 CACGAAACAG AAGCGCTTAT CCCCGGTATA CGCGTTGAGT TTTATCGTAT TAATGGCCTC 660 CGCGGACCTA CCGCCGCCAA CGTTTATCTT TCATAATTCG TCACCCGGCA TTTTTCAGAA 720 AAATTTAGCG AGTACGTCTA CCTCCGCAGC CTGCTATGAG GCTTTGCCTG AAAGGCTGCA 780 GAATGTTTTC AGTGGCGAAA ATCTAAAAGA TTTATTTTGC TAATCAGTCC TGTGACCTCT 840 TTTATCATAT ATCGGGTGCC CCCCCTTCTC ACTTTGTTTA ACGTGAAGAA ATGTACAGCC 900 GTTTTTCACT GTGATAGCAT CTAATATTGC AAAAGTATTT AACGCTATAT ACCCATTGTC 960 ACAGGAGTGG CTGCGTGCGA GCTGAGCTAT TTAACCGAAG TATTTATGTG ATCATTGGAA 1020 TTATCTCTAT TGCCGCTCAA TGCTACGTCA TATTCAGTGG GTATAAATCG CCAATATAGT 1080 TGTAACGCTA TTTATTTTTA GGGTAATAAT TGAATGACTT TGCTTTCAGG AAAAACCACA 11 0 CTGGTTCTCT GCCTCTCCTC TATTTTATGT GGATGTACGA CGAACGGCTT ACCCACACCT 1200 TATAGTATTA ATTTGTCGTT CCCGGTCATT ACACAAAACC AGATTAATTC CGGTGGTTAT 1260 TACATAAATG ACGCGGAACA AATTCGGACA ACTGATGGTC TGTGCCTTGA TGCAGGCCCA 1320 GATCAACAGA ATCGTTTGAC GCTGCGGGAG TGTAAGCATG TGCAATCTCA GCTTTTCTCA 1380 TTTCACCGAG ACAGAATCAC GCAGGGTGAG AAATGTCTGG ATGCCGCAGA CAAGGTACAA 1440 AAGAAGGCAC ACCAATCATT CTTTATTCAT GCACGGGTAA TGATAACCAG CGCTGGCTCA 1500 CTGATCATAA CAAAATTAAG GGGAAACAGA GCCGAAAATG CCTGGGCACA AATAGCATTA 1560 TTGTCAGAAA AGGCGACCCT GTTGTGTTGG CCGATTGCGA TTTTAGTCGC GCCCTGGAAT 1620 TTACCATCAG GTAGCAGGAC ACCGCTGTGA AGAGAGTGCC GCTAACCTCT TGACACGACA 1680 ACAGGTTAGC GACCTTTACT TCCACGTGCG ATCAATTTAC TTTACGTCCG CAACGTCAGG 1740 ATGACAAAAC GGCGGCTAAA CCTTGACACC AGTTATATAC CCAGCTTAAA TACTGGTCAT 1800 CCAACCAGTA AAAAGGAAAT GGCGATGTTC GTCGAACTCG TTTATGACAA GCGAAATGTT 1860 GAAGGTTTGC CAGGCGCACG CGAAATCATC CTCAATGAAC TCACAAAACG CGTACATCAA 1920 CTTTTTTCCCG ATGCGCAAGT GAAAGTTAAG CCAATGCAGG CGAACGCATT AAACAGTGAC 1980 TGTACAAAAA CCGAGAAAGA ACGGCTGCAC CGTATGCTGG AAGAGATGTT TGAAGAGGCT 2040 GATATGTGGC TGGTCGCCGA ATAACGTCCC CTCCTGCGAA AGCCAACATG TCCGATCGAA 2100 AACAGCGCCC TGAGGCGCTG TCTGTGACGA TATAACGCAA ACGCTACCAC TCAGAACATG 2160 TTGTTGTTGA TACCTCAGAC CGGTATGTGG AACCGACATT. CATCGCTTCA CTGGCCTGTC 2220 GGTATGAGTA GCCCTTATCA ACAATCAGCT GTGCGCATTC CAGCCTGAAA TCTGAAAGTA 2280 CGTTTGGTTT TGTTGTTTAT TAAGAGCCTA TCCCATTAGA CTCTTTTATT CGCCAAACTG 2340 GCTTTAACGA TTACGCCTAC TGGGATAGGT TCTAAACTTA TCATCAATAC GTAAAATACC 2400 TATTTACGAA CAAAAAGTAA CAGGTAAAAA TCCGAAATAA AACCAGCATA ACTAAAACTT 2460 ACTGCAGATA TGCACACGCA TTATTACTAT GTTTCCAGGA TAGTCTCGAC CAGTCAAGAC 2520 TATCTATTTT ATATAAAAAG GGAAATACTT CACATGAATA AAATACATGT TACATATAAA 2580 AATCTCTTAC TTCCGATTAC CTTCATCGCG GCAACTCTAA TTAGCGCCTG TGATAACGAT 2640 AAAGATGCCA TGGCGGAAGC TGAAAAAAAT CAAGAGAAAT ACATGCAAAA AATCCAGCAA 2700 AAAGAGCACC AGCAATCAAT GTTCTTTTAC GACAAAGCCG AAATGCAAAA AGCTATTGCC 2760 AATATCAACG CAAAAGGTGG AGCCAATCTT GCGATTATTG AAGTCCGTTT CTTCAAGGGC 2820 GGGTATTCAT TCATTCGACA AAGCGTTAAC ACCCCTGCTA ÁAGTAGAGGT GTTTAAATTT 2880 AACAACGGCT ACTGGGGGGG ACCTTCGCCT GTCAATTTAA CCATCTTTGG CACTATAACA 2940 GAGGAGCAAA AACAAGAAGC ACTAAAAGAG GCTTTATTCA AATTCGACTC GATCAATTTC 3000 AGCATTATAC CAGAGCGTAT TCAGGAAACA ATTAAACGCG CTAACGCCAG TGGCATCATT 3060 TCCGTTACGG AAGATAGCGA TATCGTTGTA CGAGCAGAGA TAGCTCATAA TGGCGAATTC 3120 GTCTATGACA TTACCATCAC TGCTAAAAAT ACAGCACGTG CGGTAATGAC CTTAAATAAG 3180 GATGGTTCTA TTGCCGGATA TGAGATCAAA GAACCTTTCG CCCCAAAAAA AGAAGCCGAA 3240 AAAGCACAGC AACTTGTTGA ACAATCGAGA AAAGACATTG AAAGTCCAGC GTAAAAAAGC 3300 AGCTGGAAAG ATGAACGAAA TACAGCAGAC ATTTAAAAAT AGCAGGCGAT ACAAACATTG 3360 ATAAAAATTA TAGCGCGAAA GAGCGCGTGC CAGGTACTAA GGCACTGCTT GAAGACAGCG 3420 AATCGCTATT TCATTCTCTG ACACTGTAAT TTTTCGTACT CAAGATGTTT ATTTATTGAG 3480 TCTTTTTGTGG ATAACCAGGT GAAGTTATGT GACGCCAGGA ATCTATTCCA GCGGGCGTAC 3540 TTGTTGGAGC CAGTGTGAAG CCGGGCAGCG CGCAGAAACC GGAGCGTATA CGTTGTACGT 3600 AAGAATTTCG AGCACTGCCC GACCTAAAAA TGATGAATAA AATAGATATT TTAAAGAGGT 3660 AATATGAAGA ATTTTTTCAA AATAATTACT GATTTCATCG CGGATATTTC CCTTGATCTA 3720 TTTGCTATAT TTTTATGCAT GTTATTCGTA TACAAAACAG GACCATCAAT TGGTGTGATA 3780 TCATTTTTTA TTGCATTAAT TATTTATATC ATTCTTCATT TTTTTTTACT CATTTCTTGA 3840 AAAAATCATA AAAAAAATAT TCAAATAAGT ATTTAAAATT ATTGTTTTGT GGTACAAATT 3900 CAGCGCAATA AAACAGAGCA ACTAAAAAAA ATTAGGCGTA .GCGAAGTGGA AAAGGACTGT 3960 CATGTACTGG ACCGTGAGCT GGTCGGGAGA GCAATGTACG GGAAAGAGCG AAATACTGTC 4020 ATTGATATGA GCAGGAATAT CGAT 4044 SEQUENCE NUMBER: 6 Met Lys His His Wing Phe Met Leu Trp Ser Leu Leu lie Phe Ser Phe 1 5 10 15 His Val Leu Wing Ser Ser Gly His Cys Ser Gly Leu Gln Gln Wing Ser 20 25 30 Trp Asp lie Phe lie Tyr Asp Phe Gly Ser Lys Thr Pro Gln Pro Pro 35 40 45 Thr Asn Thr Asp Lys Lys Gln Wing Arg Gln lie Ser Ser Pro Cys 50 55 60 Pro Thr Thr Lys Pro Met Met Ser Wing Pro Val Asn Asp Wing Arg Lys 65 70 75 80 Gly Asn Thr Phe Ser Arg Thr 85 SEQUENCE NUMBER: 7 Met Thr Leu Leu Ser Gly Lys Thr Thr Leu Val Leu Cys Leu Ser Ser 1 5 10 15 lie Leu Cys Gly Cys Thr Thr Asn Gly Leu Pro Thr Pro Tyr Ser He 20 25 30 Asn Leu Ser Phe Pro Val He Thr Gln Asn Gln He Asn Ser Gly Gly 35 40 45 Tyr Tyr He Asn Asp Wing Glu Gln He Arg Thr Thr Asp Gly Leu Cys 50 55 60 Leu Asp Wing Gly Pro Asp Gln Gln Asn Arg Leu Thr Leu Arg Glu Cys 65 70 75 80 Lys His Val Gln Ser Gln Leu Phe Ser Phe His Arg Asp Arg He Thr 85 90 95 Gln Gly Glu Lys Cys Leu Aep Wing Wing Asp Lys Val Gln Lys Lys Wing 100 105 110 His Gln Ser Phe Phe He His Wing Arg Val Met He Thr Ser Wing Gly 115 120 125 Ser Leu He He Thr Lys Leu Arg Gly Asn Arg Ala Glu Asn Wing Trp 130 135 140 Wing Gln He Wing Leu Leu Ser Glu Lys Wing Thr Leu Leu Cys Trp Pro 145 150 155 160 He Ala He Leu Val Ala Pro Trp Asn Leu Pro Ser Gly Ser Arg Thr 165 170 175 Pro Leu SEQUENCE NUMBER: 8 Met Phe Val Glu Leu Val Tyr Aep Lys Arg Asn Val Glu Gly Leu Pro 1 5 10 15 Gly Ala Arg Glu He He Leu Asn Glu Leu Thr Lys Arg Val His Gln 20 25 30 Leu Phe Pro Asp Ala Gln Val Lys Val Lys Pro Met Gln Wing Asn Wing 35 40 45 Leu Asn Ser Asp Cys Thr Lys Thr Glu Lys Glu Arg Leu His Arg Met 50 55 60 Leu Glu Glu Met Phe Glu Glu Ala Asp Met Trp Leu Val Ala Glu 65 70 75 SEQUENCE NUMBER: 9 Met Asn Lys He His Val Thr Tyr Lys Asn Leu Leu Leu Pro He Thr 1 5 10 15 Phe He Wing Wing Thr Leu He Wing Wing Cys Asp Asn Asp Lys Asp Wing 20 25 30 Met Wing Glu Wing Glu Lys Asn Gln Glu Lys Tyr Met Gln Lys He Gln 35 40 45 Gln Lys Glu His Gln Gln Ser Met Phe Phe Tyr Aep Lys Ala Glu Met 50 55 60 Gln Lys Ala He Ala Asn He Asn Ala Lys Gly Gly Ala Asn Leu Ala 65 70 75 80 He He Glu Val Arg Phe Phe Lys Gly Gly Tyr Ser Phe He Arg Gln 85 90 95 Ser Val Asn Thr Pro Ala Lys Val Glu Val Phe Lys Phe Asn Asn Gly 100 105 110 Tyr Trp Gly Gly Pro Ser Pro Val Asn Leu Thr He Phe Gly Thr He 115 120 125 Thr Glu Glu Gln Lys Gln Glu Ala Leu Lys Glu Ala Leu Phe Lys Phe 130 135 140 Asp Ser He Asn Phe Ser He He Pro Pro Glu Arg He Gln Glu Thr He 145 150 155 160 Lys Arg Wing Asn Wing Ser Gly He He Ser Val Thr Glu Asp Ser Asp 165 170 175 He Val Val Arg Wing Glu He Wing His Aen Gly Glu Phe Val Tyr Asp 180 185 •! • 190 He Thr He Thr Wing Lys Asn Thr Wing Arg Wing Val Met Thr Leu Asn 195 200 205 Lys Asp Gly Ser He Wing Gly Tyr Glu He Lys Glu Pro Phe Wing Pro 210 215 220 Lys Lys Glu Wing Glu Lys Wing Gln Gln Leu Val Glu Gln Ser Arg Lys 225 230 235 240 Asp He Glu Ser Pro Wing 245 SEQUENCE NUMBER: 10 TTTTGGTTTG CTGCCGTTTG GGATAACTGC ATAGAGAGCG GCCAAGTCGC TTGCGGTCGG 60 TATCTCGAGT ATATCGAAAT CCATGTGGCC ATTGACCTCT TCAAGCGCTC ACGTTAACTA 120 CCTGCTCTTT TTTGAGCACC AACATCCCAG GTTCGTCACA GTAAATCGTA TCGTGATTAT 180 TGCTAATCGT CAGTTTACCG CTCCGAAAGC AAACTAAAGT GAAACTGCTT ACATAAAGAT 240 TTTTGATGGT AACCTGCTGA GTCTGACTTT TAATTTGCTG CCGGGTATTT GTCAAAAGTG 300 ATTTTAATTT CTGTAAGTTA TCTGCGGCAG GACGCTGATG ACTATTACTT ACAAAGGTTA 360 CATTTTCCAT ATTATCCCTT TGTTGAACTT ATTTTAATGT TCCTTACTGG TATCCTACTG 420 AAAAAATCTG AGTTGTAAAT GCTCTTTATT AGCGTGTGTT GGCAATGGTC TGATTGTTAC 480 ACCAAAAGAA CCCAAATTTG GGTAATTTAT CTACAGTAGT TTAAGCCCCA ATGGGGATGA 540 TGGTTCTTTT AATATGTGTT GAGACGCATT ATACAGAATA AATTGATTTT ATTTCTCACT 600 TTTCATTCTA TTTTCATCAG GAATCCCTGT GTCCTGTGCG GTAATCTGCT GCTATCGAGG 660 AACGACAGAC ATCGCTAACA GTATATATGG AAACATCAAA AGAGAAGACG ATAACAAGCT 720 TTCCAGGGCC ATACATAGTT CGATTACTTA ACAGCTCAC. GAACGGCTGT GAGTTTCCAT 780 TGGGCCTGAC AGGCCGAACA CTCTTTGTGG TAGGTCAGAG TGATGCGCTC ACTGCTTCAG 840 GTCAATGTGA TAGCTCCCTG ATATACCTGC CGATAGCTTT TTTATCCCGC TGGACCATGG 900 CGGAGTAAAT TTTAGGGAAA TCCAGGTGGA TACGGATGCG ACCGAAATTA TACTCCATGA 960 GCTGAAAGAA GGAAATTATG TCTGAATCTC GTTCGGTGCA ATTAAATACG CCAATACAGG 1020 TCGGTGAATT GCTTATCCTG TGATTCGCCC GGAAAGCGAG CCGTGGGTGC CCGAGCAGCC 1080 TGAGAAGTTA GAAACGTCTG CATAAAAAAG AACGAGCCGC GTTTTAAAAA CGGAATTGTA 1140 GCAGCACTGG CCGGGTTTTT TATAGAAAGT TGGGAATTGG GACTGTGGGG ACGTTATGGA 1200 TACTTAACTC GCCGCAGCGG CAGGCCGCAG GTGTAAGAGC TCGATTCGTT ATTGGGGCAG 1260 GAGAAGGAGC GTTTTCAGGT GTTGCCAGGC CGGGACGGAA AATGCTCTAT GTCGCTGCGC 1320 AAAATGAAAG AGATACGTTG TGGGCTCGTC AGGTTTTAAA TAGCGAGGGG CGATTATGAT 1380 AAAAATGCGC GAGTGATTAA CGAAAACGAA GAAAATAAGC GTAGAATCTC TATCTGGCTG 1440 GATACCTATT ATCCGCAGCT GGCTTATTAT CGGATTCATT TCGATTAGAG CCGCGTAAAC 1500 CCGTTTTCTG GCTAAGCCGC CAGCGAAACA CGATGAGCAA GAAAGAGTCT CGAGGTGTTA 1560 AGTCAAAAGC TGAGAGCGCT AATGCCTTAC GCGGATTCGG TTAACATCAA ACGTTGATGG 1620 ACGATGTTAC CGCAGCAGGC CAGGCGGAAG CGGGGCTAAA ACAGCAGGCG TTAAGAAGAT 1680 TACCTTATTC CCGCAGGAAT CATAAGGGGG GCGTAACGTT TGTTATTCAG GGGGCGCTCG 1740 GTGAGATGAT GTAGAAATAC TCAGAGCCCG TCAATTTGTC GATAGCTATT ACCGCACATG 1800 GGGAATGGGA CGCTATGTGC AGTTTGCGAT CGAATTAAAA GATGACTGGC TCAAGGGGCG 1860 CTCATTTGAG CAGTACGGGG CGGAAGGTTA TATCAAAATG AGCCCAGGCC ATTGGTATTT 1920 CCCAAGCCCA GAGGGCTTTA ATTTAACGTA AATAAGGAAG TCATTATGGC AACACCTTGG 1980 TCAGGCTATC TGGATATGGA CGTCTCAGCA AAATTTGATA CGGGCGTTGA TAATCTACAA 2040 ACGCAGGTAA CAGAGGCGAT GTTACTGGAT AAATTAGCAG CAAAACCCTC CGATCCGGCG 2100 CTACTGGCGG CGTATCAGAG TAAGAAAAAC TCTCGGAATA TAACTTGTAC CGTAACGCGC 2160 AATCGAACAC GGTAAAAGTC TTTAAGGATA TGATTGATGC TGCCATTATT CAGAACTTCC 2220 GTTAATCAGT TATAAGGTGG ATTATGTCGA TTAAGCAACT ATTGTCCCTG AGAATGCCGT 2280 TATAGGGCAG GCGGTCAATA TCAGGTCTAT GGAAATAGAA CGGACATTGT CTCGCTGGAT 2340 GACCGGCTAC TCCAGGCTTT TTCTGGTTCG GCGATTGCCT AGAAACGGCT GTGGATAAAC 2400 AGACGATTAC CAACAGGATT GAGGACCCTA ATCTGGTGAC GGATTATTTC CTAAAGAGCT 2460 GGCTATTTCG CAAGAGATGA TTTCAGATTA TAACCTGTAT .GTTTCTATGA GGTCAGTACC 2520 CTTACTCGTA AAGGAGTCGG GGCTGTTGAA ACGCTATTAC GCTCATGATT CTTGGATGTC 2580 GATATCTATA TACTTTTCTG CTGGTAATGA CCCTTGCCGG CTGTAAGGAT AAGGATCTTA 2640 GCTTTTAAAA GGACTGGACC AGGAACAGGC TAATGAGGTC ATTGCCGTTC TGCAAATGCA 2700 CAGAAATATA GAGGCGAATA AAATTGATAG CGGAAAATTG GGCTATAGCA TTACCGTTGC 2760 TGAGCAGGTA CTGATTTTAC CGCTGCGGTG TACTGGATTA AAACTTATCA GCTTCCTCCC 2820 CGGCCACGGG TAATTGGAAA TAGCGCAGAT GTTCCCGGCG GATTCGCTGG TATCGTCTCC 2880 GCGAGCTGAA AAGGAAAACC AGGTTATATT CGGCTATTGA ACAGCGACTG GAACAGTCAT 29 0 TACAGACGAT GGAGGGCGAT GTGCTCTCCG CCAGGGTCCA TATTAGTTAT GATATTGATG 3000 CTGGTGAAAA TGGCCGCCCG CAAGGCAAAA CCTGTTCATC TGTCGGCATT AGCCGTATAT 3060 GAACGAGGTT CGCCGCTTGC GCATCAAGAA GATCAGCGAT ATCAAGCGTT TCTTAAAGAA 3120 TAGTTTTGCC GATGTGGATT ATGACAACAA TTTCTGTTGT GTTGTCAGAA CGTTCTGATG 3180 CCCAATTACA GGCTCCCGGC ACACCAGTAA AAGTAACGTA ATTCTTTTGC AACCAGTTGG 3240 ATTGTTTTGA TTATTTTGTT ATCCGTGATG TCAGATACAG GCTTTGGCGT CTGGTATTAC 3300 AAAAACCATT ATGCCCGCAA TAAGAAAGGC ATAACGGGGA GTACTGATGA TAAGGCGAAA 3360 TCGTCAAATG AATAGGCAGC CATTACCCAT TATCTGGCAA AGAATCATTT TTGATCCGTT 3420 ATCGTATATC CATCCTCAGC GGTTGCAGAT AGCGCCGGAA ATGATTGTCA GACCGCGCCA 3480 CGCGAAATGA GTTAATACTG GCGGCATGGC GGCGGCTTAA GAACGGAGAA AAGGAGTGTA. 3540 TTCAAAACTC ACTGACGCAG CTGTGGCTGC TCAGTGGCGC CGACTGCCGC AAGTAGCGTA 3600 TTTACTAAAC TGAGAGCCGA TCTGGCAAGG CAGGGAGCCT TGCTTGGCCT AGCCGGATTG 3660 GGCGAAATGA GTTAATACTG GCGGCATGGC GGCTTGCCAT 3700 SEQUENCE NUMBER: 11 Met Glu Thr Ser Lys Glu Lys Thr He Thr Ser Pro Gly Pro Tyr He 1 5 10 15 Val Arg Leu Leu Asn Be Ser Leu Asn Gly Cys Glu Phe Pro Leu Leu 20 25 30 Thr Gly Arg Thr Leu Phe Val Val Gly Gln Ser Asp Ala Leu Thr Ala 35 40 45 Ser Gly Gln Leu Pro Asp He Pro Wing Asp Ser Phe Phe He Pro Leu 50 55 60 Asp His Gly Gly Val Asn Phe Glu He Gln Val Asp Thr Asp Ala Thr 65 70 75 80 Glu He He Leu His Glu Leu Lys Glu Gly Aen Ser Glu Ser Arg Ser 85 90 95 Val Gln Leu Asn Thr Pro He Gln Val Gly Glu Leu Leu He Leu He 100 105 110 Arg Pro Glu Ser Glu Pro Trp Val Pro Glu Gln Pro Glu Lys Leu Glu 115 120 125 Thr Ser Ala Lys Lys Asn Glu Pro Arg Phe Lys Asn Gly He Val Wing 130 135 140 Wing Leu Wing Gly Phe Phe He Leu Gly He Gly Thr Val Gly Thr Leu 145 150 155 160 Trp He Leu Asn Ser Pro Gln Arg Gln Ala Wing Glu Leu Asp Ser Leu 165 170 175 Leu Gly Gln Glu Lys Glu Arg Phe Gln Val Leu Pro Gly Arg Asp Lys 180 185 190 Met Leu Tyr Val Wing Wing Gln Asn Glu Arg Asp Thr Leu Trp Wing Arg 195 200 205 Gln Val Leu Wing Arg Gly Asp Tyr Asp Lys Asn Wing Arg Val He Asn 210 215 220 Glu Asn Glu Glu Asn Lys Arg He Ser He Trp Leu Asp Thr Tyr Tyr 225 230 235 240 Pro Gln Leu Ala Tyr Tyr Arg He His Phe Asp Glu Pro Arg Lys Pro 245 250 255 Val Phe Trp Leu Ser Arg Gln Arg Asn Thr Met Ser Lys Lys Glu Leu 260 265 270 Glu Val Leu Ser Gln Lys Leu Arg Ala Leu Met Pro Tyr Wing Asp Ser 275 280 285 Val Asn He Thr 'Leu Met Asp Aep Val Thr Wing Wing Gly Gln Wing Glu 290 295 300 Wing Gly Leu Lys Gln Gln Wing Leu Pro Tyr Being Arg Arg Asn His Lys 305 310 315 320 Gly Gly Val Thr Phe Val lie Gln Gly Ala Leu Aep Asp Val Glu He 325 330 335 Leu Arg Wing Arg Gln Phe Val Asp Ser Tyr Tyr Arg Thr Trp Gly Gly 340 345 350 Arg Tyr Val Gln Phe Wing He Glu Leu Lys Asp Asp • Trp Leu Lys Gly 355 360 365 Arg Ser Phe Gln Tyr Gly Wing Glu Gly Tyr He Lys Met Ser Pro Gly 370 375 380 His Trp Tyr Phe Pro Ser Pro Leu 385 390 SEQUENCE NUMBER: 12 Met Wing Thr Pro Trp Ser Gly Tyr Leu Aep Asp Val Ser Wing Lys Phe 1 5 10 15 Asp Thr Gly Val Asp Asn Leu Gln Thr Gln Val Thr Glu Wing Leu Asp 20 25 30 Lys Leu Wing Wing Lys Pro Ser Asp Pro Wing Leu Leu Wing Wing Tyr Gln 35 40 45 Ser Lys Leu Ser Glu Tyr Asn Leu Tyr Arg Asn Wing Gln Ser Asn Thr 50 55 60 Val Lys Val Phe Lys Asp He Asp Wing Wing He He Gln Asn Phe Arg 65 70 75 80 SEQUENCE NTJMERO: 13 Met Ser He Ala Thr He Val Pro Glu Aen Ala Val He Gly Glp Ala 1 5 10 15 Val Asn He Arg Ser Met Glu Thr Asp He Val Ser Leu Asp Asp Arg 20 25 30 Leu Leu Gln Wing Phe Ser Gly Be Wing He Wing Thr Wing Val Asp Lys 35 40 45 Gln Thr He Thr Asn Arg He Glu Asp Pro Asn Leu Val Thr Asp Pro 50 55 60 Lys Glu Leu Wing He Ser Gln Glu Met He Ser Asp Tyr Asn Leu Tyr 65 70 75 80 Val Ser Met Val Ser Thr Leu Thr Arg Lys Gly Val Gly Wing Val Glu 85 90 95 Thr Leu Leu Arg Ser 100 SEQUENCE NUMBER: 14 Met He Arg Arg Tyr Leu Tyr Thr Phe Leu Leu Val Met Thr Leu Ala 1 5 10 15 Gly Cys Lys Asp Lys Asp Leu Leu Lye Gly Leu Aep Gln Glu Gln Wing 20 25 30 Asn Glu Val He Wing Val Leu Gln Met His Asn He Glu Wing Asn Lys 35 40 45 He Asp Ser Gly Lys Leu Gly Tyr Ser He Thr Val Wing Glu Pro Aep 50 '55 60 Phe Thr Wing Wing Val Tyr Trp He Lys Thr Tyr Glp Leu Pro Pro Arg 65 70 75 80 Pro Arg Val Glu He Wing Gln Met Phe Pro Wing Asp Ser Leu Val Ser 85 90 95 Ser Pro Arg Wing Glu Lys Wing Arg Leu Tyr Be Wing He Glu Gln Arg 100 105 110 Leu Glu Gln Ser Leu Gln Thr Met Glu Gly Val Leu Ser Wing Arg Val 115 120 125 His He Ser Tyr Asp He Asp Wing Gly Glu Asn Gly Arg Pro Pro Lys 130 135 140 Pro Val His Leu Ser Ala Leu Ala Val Tyr Glu Arg Gly Ser Pro Leu 145 150 155 160 Wing HIL Gln He Ser Aep He Lys Arg Phe Leu Lys Asn Ser Phe Wing 165 170 175 Asp Val Asp Tyr Asp Asn He Ser Val Val Leu Ser Glu Arg Ser Asp 180 185 190 Wing Gln Leu Gln Wing Pro Gly Thr Pro Val Lys Arg Asn Ser Phe Wing 195 200 205 Thr Ser Trp He Val Leu He He Leu Leu Ser Val Met Ser Wing Gly 210 215 220 Phe Gly Val Trp Tyr Tyr Lys Asn His Tyr Wing Arg Asn Lys Lys Gly 225 230 235 240 He Thr Wing Asp Asp Lys Wing Lys Ser Ser Asn Glu 245 250 SEQUENCE NUMBER: 15 CATAACAACT CCTTAATACT ACTTATTATT TACGGTGTGT TTAAACACCT GCAGTACCGA 60 TCCGGCATTC AGTTATCGCC ACTATGCCGA ATCGACAAAA CCACGAATAA TTCACCGCTA 120 TCGCTCCTGA TGTGTTTACT TCCTGAAAGA TATTTTTACT .ACCGAAGCAC TCTATCGCTC 180 ATTTAGGTAA CCGGTTCTAC AATGTCATCT AACTTTTATA GATTTGAATG CTAATTTTTC 240 TCACGCATAT ATATTTAACA GAAACCATAA AGTGTTTAGC CACTATAGAA CAACAAATCA 300 CCCATGCAAC ATTTTGATAT TTAAAGAGAA AATCTCACAA CCACATTAAG AAACTTGACA 360 CCGTTCGGCT AAAAAACATG TCATTAAGCA AACTCGCCAT ATAATCAGAA CATATCGCAT 420 TGTGCTTCAC AGTCCTCACG TGACGCTCCA TCCGCAATAC GGTTATATGC CATCGCAGGC 480 GCTGTAATCA TATTCACGAT GATGCTTAGC ACGCTTTATT CCCGCTCCGA TTTAATCTTT 540 TAATATATCT ATCAGTTACA ACATTTCTTG TTATATTATA AGAATAGAAT CAACACCACA 600 ATTCCAACAT AAATATCACC TGTGTTTAGA GAGAATTTAC ATTCCAAAAA AATAATAACT 660 AACGCAAATA TTGAACACGC GATAAAAAAG TCTATTTCGC TATAAAACCC ATTATTATTA 720 AGAGTGGTTA ACTCTTCGTT GAATAAAAAA TGTCAATGAC GTTCCATAAT TCAGGAGATG 780 AACTTCACAA GTCATTATAT ATAACAGGAG GTGCTATG 818

Claims (11)

1. NOVELTY OF THE INVENTION CLAIMS 1. A vaccine comprising a bacterial cell whose virulence is attenuated by the constitutive expression of a gene under the control of a two-component regulatory system.
2. 2. The vaccine of claim 1, wherein said constitutive expression is the result of a mutation in a component of said two-component regulatory system.
3. 3. The vaccine of claim 1, wherein said bacterial cell comprises a second mutation that attenuates virulence.
4. 4. The vaccine of claim 1, wherein said bacterial cell is a Salmonella cell, said two-component regulatory system is the phoP regulatory region, and said gene is a gene regulated by the phoP regulatory region.
5. 5. The vaccine of claim 4, wherein said constitutive expression is the result of a mutation.
6. 6. The vaccine of claim 5, wherein said mutation is in the phoP regulatory region.
7. 7 The vaccine of claim 6, wherein said mutation is in the phoP gene.
8. 8. The vaccine of claim 6, wherein said mutation is in the phoQ gene.
9. 9. The vaccine of claim 6, wherein said mutation is a phoPc mutation.
10. 10. The vaccine of claim 6, wherein said mutation is a nonreversible mutation. The vaccine of claim 4, wherein said constitutive expression is the result of a change in the promoter of said regulated gene. The vaccine of claim 4, wherein said gene is a prg gene. 13. A vaccine comprising a Salmonella cell that is attenuated by the reduced expression of a virulence gene regulated by the phoP regulatory region. The vaccine of claim 13, wherein said reduction in expression is the result of a mutation. 15. The vaccine of claim 14, wherein said mutation is in the prgH gene. 16. The vaccine of claim 14, wherein said mutation is in the prgA, prgB, prgC or prgE genes. 17. The vaccine of claim 4, wherein said gene is a pag gene. 18. The vaccine of claim 17, wherein said place pagC is the place. 19. The vaccine of claim 4, further characterized in that the Salmonella cell comprises a first mutation that attenuates virulence and a second mutation that attenuates virulence. The vaccine of claim 19, wherein said first mutation is in a gene of the phoP regulatory region. 21. The vaccine of claim 20, wherein said first mutation is in the phoP gene. 22. The vaccine of claim 20, wherein said first mutation is in the phoQ gene. 23. The vaccine of claim 20, wherein said first mutation is a phoP0 mutation. The vaccine of claim 19, wherein said first mutation is in a gene regulated by the phoP regulatory region. 25. The vaccine of claim 19, wherein said second mutation is a mutation in a synthetic aromatic amino acid gene. 26. The vaccine of claim 25, wherein said second mutation is an aro mutation. 27. The vaccine of claim 19, wherein said second mutation is in a gene regulated by the phoP regulatory region. 28. The vaccine of claim 23, wherein said second mutation is in a prg. 29 The vaccine of claim 13, further characterized in that said Salmonella cell comprises two mutant genes, a first mutant gene that attenuates virulence and a second mutant gene that attenuates virulence. 30. The vaccine of claim 29, wherein said second gene is in a prg. 31. The vaccine of claim 30, wherein said gene is prgH. 32. The vaccine of claim 30, wherein said gene is prgA, prgB, prgC or prgE. 33. The vaccine of claim 27, wherein said second mutation is in a pag. 34. The vaccine of claim 27, wherein said second mutation is a pagC mutation. 35. The vaccine of claim 4, wherein said Salmonella is of the species S. typhi. 36. The vaccine of claim 4, where said Salmonella is of the species S. enteriditis and of the typhimurium strain. 37. The vaccine of claim 4, wherein said Salmonella is of the species S. cholerae-suis. 38. The vaccine of claim 4, wherein said vaccine is a live vaccine. 39. A vaccine comprising a bacterial cell whose virulence is attenuated by a mutation in a gene under the control of a two-component regulatory system. 40. The vaccine of claim 39, further characterized in that said bacterial cell comprises a mutation that attenuates virulence in a second gene. 41. The vaccine of claim 39, wherein said bacterial cell is a Salmonella cell and said two-component regulatory system is in the phoP regulatory region. 42. The vaccine of claim 41, wherein said gene is a prg gene. 43. The vaccine of claim 41, wherein said gene is prgH. 44. The vaccine of claim 41, wherein said gene is prgA, prgB, prgC or prgE. 45. The vaccine of claim 41, wherein said gene is a gene pag. 46. The vaccine of claim 45, wherein said gene is pagC. 47. The vaccine of claim 41, wherein said bacterial cell further comprises a mutation in a second gene, said mutation attenuating the virulence of said bacterial cell. 48. The vaccine of claim 47, wherein said second gene is a biosynthetic aromatic amino acid gene. 49. The vaccine of claim 48, wherein said second gene is a gene. 50. A vaccine comprising a Salmonella cell comprising a first mutation that attenuates virulence in an aromatic amino acid biosynthetic gene and a second mutation that attenuates virulence in a gene of the phoP regulatory region. 51. The vaccine of claim 50, wherein said first mutation is in a gene. 52. The vaccine of claim 51, wherein said second mutation is a phoP mutation. "53. A bacterial cell that constitutively expresses a gene under the control of a two-component regulatory system and that comprises a mutation that attenuates virulence which does not result in the constitutive expression of a gene under the control of said two-component regulatory system 54. The bacterial cell of claim 53, further comprising a mutation in a component of said two-component regulatory system. bacterial cell of claim 53, wherein said cell is a Salmonella cell that expresses a gene regulated by the phoP regulatory region constitutively and that comprises a mutation that attenuates virulence that does not result in the constitutive expression of a gene under the control of the phoP regulatory region 56. The bacterial cell of claim 55, wherein said expression cons The titre is caused by a mutation in the phoP regulatory region. 57. The bacterial cell of claim 55, wherein said constitutive expression is caused by a mutation in the phoP gene. 58. The bacterial cell of claim 55, wherein said constitutive expression is caused by a mutation in the phoQ gene. 59. The bacterial cell of claim 56, wherein said mutation is a phoP ° mutation. 60. The bacterial cell of claim 56, wherein said mutation is a deletion. 61. The bacterial cell of claim 55, further characterized in that said mutation attenuating virulence is in a synthetic aromatic amino acid gene. 62. The bacterial cell of claim 61, wherein said mutation attenuating virulence is an aro mutation. 63. The bacterial cell of claim 55, wherein said mutation attenuating virulence is in a gene of the phoP regulatory region. 64. The bacterial cell of claim 63, wherein said mutation that attenuates virulence is the phoP gene. 65 The bacterial cell of claim 63, wherein said mutation attenuating virulence is in the phoQ gene. 66. The bacterial cell of claim 55, wherein said mutation that attenuates virulence is in a prg. 67. The bacterial cell of claim 66, wherein said mutation attenuating virulence is in the prgH gene. 68. The bacterial cell of claim 66, wherein said mutation attenuating virulence is in the prgA, prgB, prgC or prgE gene. 69 The bacterial cell of claim 55, wherein said virulence attenuating mutation is in a pag. 70. The bacterial cell of claim 55, wherein said mutation attenuating virulence is a pagC mutation. 71. The bacterial cell of claim 55, wherein said cell is of the species S. typhi. 72. The bacterial cell of claim 55, wherein said cell is of the species S. enteridi tis and of the strain typhimurium. 73. The bacterial cell of claim 55, wherein said Salmonella cell is of the species S. cholerae-suis. 74. A bacterial cell comprising a mutation that attenuates virulence in a gene regulated by a phoP regulatory region. 75. The bacterial cell of claim 74, wherein said bacterial cell is a Salmonella cell and said mutation that attenuates virulence is in a gene regulated by the phoP regulatory region. 76. The bacterial cell of claim 75, wherein said gene is a prg gene. eleven . The bacterial cell of claim 76, wherein said gene is the prgH gene. 78. The bacterial cell of claim 76, wherein said gene is the gene prgA, prgB, prgC or prgE. 79. The bacterial cell of claim 75, wherein said gene is a pag. 80. The bacterial cell of claim 79, wherein said gene is pagC. 81. The bacterial cell of claim 74, further comprising a second mutation that attenuates virulence but that does not result in the constitutive expression of a gene regulated by the phoP regulatory region. 82. The bacterial cell of claim 81, wherein said second mutation is a synthetic aromatic amino acid gene. • 83. The bacterial cell of claim 82, wherein said second mutation is an aro mutation. 84. The bacterial cell of claim 81, wherein said second mutation is in a gene of the phoP regulatory region. 85. The bacterial cell of claim 84, wherein said second mutation is in the phoP site. 86. The bacterial cell of claim 84, wherein said second mutation is in the phoQ site. 87. The bacterial cell of claim 81, wherein said second mutation is in a gene regulated by the phoP regulatory region. 88. The bacterial cell of claim 87, wherein said second mutation is in a pag. 89. The bacterial cell of claim 75, wherein said cell is of the species S. typhi. 90. The bacterial cell of claim 75, wherein said cell is of the species S. enteridi tis and of the strain typhimuri um. 91. The bacterial cell of claim 75, wherein said cell is of the species S. cholerae-suis. 92. A living Salmonella cell in which a gene encoding a heterologous protein, or a regulatory element, of said heterologous protein gene is inserted into a virulence gene. 93. The Salmonella live cell of claim 92, wherein said virulence gene is in the phoP regulatory region. 94. The Salmonella live cell of claim 92, wherein said virulence gene is a regulated gene in the phoP regulatory region. 95. The living Salmonella cell of the claim 94, wherein said virulence gene is a prg gene. 96. The living Salmonella cell of the claim 95, where said virulence gene is the prgH gene. 97. The Salmonella live cell of claim 95, wherein said virulence gene is the prgA, prgB, prgC or prgE gene. 98. The Salmonella live cell of claim 94, wherein said virulence gene is a pag. 99. The Salmonella living cell of claim 98, wherein said pag gene is pagC. 100. The Salmonella live cell of claim 92, wherein said Salmonella cell carries a second mutation that attenuates virulence. 101. The Salmonella live cell of claim 100, wherein said second mutation is an aro mutation. 102. The Salmonella live cell of claim 92, wherein said DNA encoding a heterologous protein is under the control of an environmentally regulated promoter. 103. The Salmonella live cell of claim 92, wherein said Salmonella cell is of the species S. typhi. 104. The Salmonella live cell of claim 92, further comprising a DNA sequence encoding T7 polymerase under the control of an environmentally regulated promoter and a T7 transcriptionally responsive promoter, said T7 transcriptionally responsive promoter controlling the expression of said heterologous antigen. 105. A vector capable of being integrated into the Salmonella chromosome, comprising: a first DNA sequence encoding a heterologous protein, a second DNA sequence encoding a marker, and a third DNA sequence encoding a regulated gene product in the phoP regulatory region necessary for virulence, said third DNA sequence being mutationally inactivated. 106. The vector of claim 105, wherein said gene regulated by the phoP regulatory region is a prg. 107. The vector of claim 106, wherein said gene is prgH. 108. The vector of claim 106, wherein said gene is prgA, prgB, prgC, or prgE. 109. The vector of claim 105, wherein said gene regulated by the phoP regulatory region is a pag site. 110. The vector of claim 109, wherein said place is pagC. 111. The vector of claim 105, wherein said first DNA sequence is arranged in said vector so as to mutationally inactivate said third DNA sequence. 112. The vector of claim 105, wherein said vector can not replicate in a wild-type Salmonella strain. 113. The vector of claim 105, wherein said first DNA sequence encoding a heterologous protein is under the control of an environmentally regulated promoter. 114. The vector of claim 105, further comprising a DNA sequence encoding T7 polymerase under the control of an environmentally regulated promoter and a T7 transcriptionally responsive promoter, said T7 transcriptionally responsive promoter controlling the expression of said first DNA sequence encoding a heterologous protein. 115. A method of vaccinating an animal against a disease caused by a bacterium, comprising administering the vaccine of claim 1. 116. The method of claim 115, wherein said bacterium is Salmonella and said vaccine is the vaccine of claim 4. 117. A method of vaccinating an animal against a disease caused by a bacterium, comprising administering the vaccine of claim 39. 118. The method of claim 115, wherein said bacterium is Salmonella and said vaccine is the vaccine of claim 41. 119. A method of vaccinating an animal against an illness caused by Salmonella, which comprises administering the vaccine of claim 50. 120. A vector comprising DNA encoding the gene product of pagC. 121. A cell comprising the vector of claim 120. 122. A method of producing the gene product of pagC, which comprises culturing the cell of claim 121 and purifying the gene product of said cell or said culture medium. . 123. A purified preparation of the gene product pagC. 124. A method of detecting the presence of Salmonella in a sample, which comprises contacting said sample with DNA encoding pagC and detecting the hybridization of said DNA encoding pagC to nucleic acid in said sample. 125. A vector comprising DNA encoding the product of gene prgH. 126. A cell comprising the vector of claim 125. 127. A method of producing the prgH gene product, which comprises culturing the cell of claim 126 and purifying the prgH gene product of said cell or said culture medium. 128. A purified preparation of the prgH gene product. 129. A method of detecting the presence of Salmonella in a sample, comprising contacting said sample with DNA encoding prgH and detecting the hybridization of said DNA encoding prgH to nucleic acid in said sample. 130. A method of attenuating the virulence of a bacterium, said bacterium comprising a two-component regulatory system, comprising making a gene under the control of said two-component system be constitutively expressed. 131. The method of claim 124, wherein said bacterium is Salmonella and said two-component system is the phoP regulatory region. 132. A bacterial cell whose virulence is attenuated by a first mutation in a PhoP regution and a second mutation in a synthetic aromatic amino acid gene. 133. The bacterial cell of claim 132, wherein said bacterial cell is a Salmonella cell. 134. The Salmonella cell of claim 133, wherein said Salmonella cell is a Salmonella typhimurium cell. 135. The Salmonella cell of claim 133, wherein said Salmonella cell is a Salmonella enteridi tis. 136. The Salmonella cell of claim 135, wherein said Salmonella cell is a Salmonella pyl orum cell. 137. The Salmonella cell of claim 135, wherein said Salmonella cell is a Salmonella cell for typhi A. 138. The Salmonella cell of claim 135, wherein said Salmonella cell is a Salmonella cell for typhi B. 139. The Salmonella cell of claim 133, wherein said Salmonella cell is a Salmonella cholerasuis cell. 140. The Salmonella cell of claim 133, wherein said Salmonella cell is a Salmonella typhi cell. 141. The bacterial cell of claim 133, wherein said first mutation comprises a nonreversible null mutation in the PhoP / PhoQ site. 142. The bacterial cell of claim 141, wherein said mutation comprises a deletion of at least 100 nucleotides. 143. The bacterial cell of claim 142, wherein said mutation comprises a deletion of at least 500 nucleotides. 144. The bacterial cell of claim 143, wherein said mutation comprises a deletion of at least 750 nucleotides. 145. The bacterial cell of claim 144, wherein said mutation comprises a deletion of nucleotides 376 to 1,322 of said PhoP / PhoQ site. 146. The bacterial cell of claim 141, wherein said second mutation comprises a null, nonreversible mutation at an AroA site. 147. The bacterial cell of claim 141, wherein said second mutation comprises a non-reversible null mutation at an AroC / AroD site. 148. The bacterial cell of claim 146, further comprising a mutation in a synthetic non-aromatic amino acid gene, wherein said mutation makes said auxotropic cell for said non-aromatic amino acid. 149. The bacterial cell of claim 148, wherein said amino acid is histidine. 150. The bacterial cell of claim 149, wherein said S. typhi has the genotype AroA ", PhoP / PhoQ". 151. The bacterial cell of claim 150, wherein said S. typhi is TyH445. 152. The bacterial cell of claim 134, wherein said first mutation comprises a null, nonreversible mutation in the PhoP / PhoQ site. 153. The bacterial cell of claim 152, wherein said mutation comprises a deletion of nucleotides 376-1,322 of said PhoP / PhoQ site. 154. The bacterial cell of claim 152, wherein said second mutation comprises a null, nonreversible mutation at an AroA site. 155. The bacterial cell of claim 154, further comprising a mutation in a synthetic non-aromatic amino acid gene, wherein said mutation makes said auxotropic cell for said non-aromatic amino acid. 156. A vaccine comprising the bacterial cell of claim 132. 157. A substantially pure DNA, comprising a sequence encoding pagD. 158. The DNA of claim 157, wherein said sequence comprises nucleotides 91 to 354 of the sequence identified with number 5. 159. The DNA of claim 158, further comprising nucleotides 4 to 814 of the sequence identified with the number 15. 160. A substantially pure DNA, comprising nucleotides 4 to 814 of the sequence identified with number 15. 161. The DNA of claim 160, wherein said DNA sequence comprises nucleotides 562 to 814 of the sequence identified with number 15. 162. The DNA of claim 160, wherein said DNA sequence comprises nucleotides 4 to 776 of the sequence identified with number 15. 163. The DNA of claim 158, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with the number 6. 164. A substantially pure DNA, comprising a coding sequence. ica envE. 165. The DNA of claim 164, wherein said sequence comprises nucleotides 1,114 to 1,650 of the sequence identified with number 5. 166. The DNA of claim 165, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with the number 7. 167. A substantially pure DNA, comprising a sequence encoding msgA. 168. The DNA of claim 167, wherein said sequence comprises nucleotides 1,825 to 2,064 of the sequence identified with number 5. 169. The DNA of claim 168, further comprising the nucleotides 1,510 to 1,824 of the sequence identified with the number 5. 170. Substantially pure DNA, comprising nucleotides 1,510 to 1,760 of the sequence identified with No. 5,171. The DNA of claim 168, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the sequence of amino acids given in the sequence identified with the number 8. 172. A substantially pure DNA, comprising a sequence encoding envF. 173. The DNA of claim 172, wherein said sequence comprises nucleotides 2,554 to 3,294 of the sequence identified with number 5,174. The DNA of claim 173, further comprising nucleotides 2,304 to 2,553 of the sequence identified with the number 5. The DNA of claim 173, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with the number 9. 176. A substantially pure DNA, comprising the sequence given in the sequence identified with the number 5 or a fragment thereof. 177. A substantially pure DNA, comprising the sequence given in the sequence identified with the number 10 or a fragment thereof. 178. A substantially pure DNA, comprising a sequence encoding prgH. 179. The DNA of claim 178, wherein said sequence comprises nucleotides 688 to 1866 of the sequence identified with number 10. 180. The DNA of claim 179, further comprising nucleotides 1 to 689 of the sequence identified with the number 10. The DNA of claim 179, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with the number 11. 182. A substantially pure DNA, which comprises a sequence encoding prgl. 183. The DNA of claim 182, further comprising nucleotides 1.891 to 2.133 of the sequence identified with number 10. 184. The DNA of claim 183, further comprising nucleotides 1 to 689 of the sequence identified with number 10. 185. The DNA of claim 183, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with number 12. 186. A substantially pure DNA, comprising a sequence encoding prgJ. 187. The DNA of claim 186, further comprising nucleotides 2.152 to 2.457 of the sequence identified with number 10. 188. The DNA of claim 187, further comprising nucleotides 1 to 689 of the sequence identified with number 10. 189. The DNA of claim 187, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with the number 13. 190. A substantially pure DNA, comprising a sequence encoding prgK. 191. The DNA of claim 190, further comprising nucleotides 2,456 to 3,212 of the sequence identified with the numeral 10. 192. The DNA of claim 191, further comprising nucleotides 1 to 689 of the sequence identified with numeral 10. 193. The DNA of claim 191, and its degenerate variants, wherein said sequence encodes a product that essentially comprises the amino acid sequence given in the sequence identified with number 14. 194. A bacterial cell, whose virulence is attenuated by a mutation in one or more genes selected from the group consisting of pagD, pagE, pagF, pagG, pagH, pagl, pagJ, pagK, pagL, pagM, pagN, pagP, envE and envF. 195. A bacterial cell whose virulence is attenuated by a mutation in one or more genes selected from the group consisting of pagC, pagD, pagJ, pagK, pagM and msgA. 196. A bacterial cell, whose virulence is attenuated by a mutation in one or more genes selected from the group consisting of prgH, prgl, prgJ, and prgK. IN WITNESS WHEREOVER, I sign the above, in this city- of Mexico, D.F., on the 11th day of July 1994. By THE GENERAL HOSPITAL CORPORATION and PRESIDENT AND FELLOWS OF HARVARD COLLEGE