DE19916176A1 - New essential bacterial genes and their proteins - Google Patents

Abstract

The invention relates to nucleic acids and to protein sequences derived from said nucleic acids which possess functions that are essential to the survival of Escherichia coli. The invention also relates to vectors and host cells that have been transformed with these vectors for expressing essential proteins of the type described above and to proteins produced with these vectors. Finally, the invention also relates to the use of the proteins produced with these vectors in screening methods for identifying antibacterial substances and to the antibacterial substances found with these proteins.

Description

In the past, new and innovative antibiotics were mainly modified and modified Optimization of known classes of substances such as B. β-lactams (penicillin Cephalosporins I-V) and testing their effect on microorganisms in so-called MHK testing researched and developed.

At the same time, the search for new active principles has been assisted in recent years of biochemically, microbiologically well-characterized targets (points of attack) established. However, the selection of targets was based on these methods empirical procedure, which makes finding new points of attack very expensive designed.

Knowledge of complete genome sequences of pro- and Eukaryotes in connection with methods of bioinformatics and molecular biology the possibility of systematic and rational selection of antibacterial attack Points.

So, by analyzing different bacterial genomes, a large number of genes were found unknown function discovered.

Unknown proteins from the gram-negative model organism Escherichia coli (F.R. Blattner et al., Science 277: 1453-1462, 1997) usually have names that start with "Y": B. YQGF. However, some proteins have unknown functions names deviating from this system because the genes coding for them in the Neighboring functionally characterized genes are: z. B. KDTB (T. Clementz and C.R.H. Raetz, J. Biol. Chem. 266: 9687-9696, 1991; T. Clementz, J. Bacteriol. 174: 7750-7756, 1992).  

Against the background of the discovery of anti-infectives, genes represent unknown Function and proteins derived from them represent new potential targets, if can be shown that they are essential for the survival of microorganisms (F. Argoni et al., Nature Biotechnology, 16: 851, 1998; D. T. Moir et al., Antimicrob. Agents chemother. 43: 439-446, 1999; US 5821076; B. J. Akerly et al., Proc. Natl. Acad. Si. USA 95: 8927-8932, 1998).

To analyze the essentiality of such gene sequences for an organism Knockout technologies are used. Knockout strategies are based on the Exchange of the intact (wild-type) gene sequence information in the chromosome of a Bacterium against a gene sequence destroyed by insertion or deletion. From the Ability to only with an intact complementary copy in the cell (on one Plasmid or a second artificial copy in the chromosome) survivable such a gene sequence thus qualifies the coding gene product as essential for this cell.

Knockout attempts are considered recombination events of homologous DNA Sequences performed. For this purpose, vectors are used that are controllable may lose the ability to replicate the plasmid, as in the case of temperature sensitive replicons (pKO3, pMAK700 or pSC101) or vectors used in cannot replicate the desired target cell (E. coli): e.g. B. Gram positive Plasmids (pC194, pT181) in Gram-negative bacteria (E. coli). Integration vectors such as pMAK700 carry sequences for the temperature sensitive Replicon additionally markers such as antibiotic resistance (e.g. chloramphenicol) that can be used for selection for plasmid-carrying cells.

The invention relates to genes with previously unknown function, the ver to show spread in the phylogenetically diverse bacterial realm and in particular to select such genes that have no or only a very distant one Possess homology in eukaryotes, and for these genes their essentiality for the To prove the viability of microorganisms.  

Another requirement for the genes to be found is through a suitable choice of Search strategy to select only those whose derived proteins are based on their the expected physicochemical properties suggest water solubility, so that they can be used advantageously in cell-free assays.

From this analysis, gene sequences can be selected that are based on conventional not chosen as a point of attack for antibiotic drug binding would have been.

For this purpose, the gene products of said genes are expressed in the context of the invention and cleaned. The new proteins thus obtained are used in detection assays used by agonists and antagonists of these proteins. Beyond that also over- or under-expression mutants for the corresponding genes in assays used to find agonists and antagonists.

The present invention relates to essential genes which are the proteins from the group Encode YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB, YJEE, KDTB, and genes from other microorganisms that have the corresponding orthologic gene products encode, as well as genes as indicated above, for which it can be shown that their controllable switch-off leads to growth inhibition of the host organism, and genes as indicated above from Escherichia coli which encode gene products which are 95-100% identical, and for which it can be shown that their controllable Elimination leads to growth inhibition of the host organism, as well as genes such as from other microorganisms which encode orthologic gene products, 42-100% identical or cons. to the corresponding gene product from E. coli fourth exchanged amino acids, and for which it can be shown that their controllable switch-off leads to growth inhibition of the host organism, and Genes that are part of their nucleic acid sequence from at least one gene from the Group of the genes indicated contain all or part, as well as the Gene products of the above genes.  

The scope of the invention further includes vectors that include one or more of the contain above genes, as well as transformed microorganisms, which one contain or more of the above genes, and the use of construct ter, recombinant microorganisms in which one of the above genes can be expressed in an adjustable manner to find substances that are in contact with said Bind gene products, as well as the use of the gene products of the above Genes in assays for finding substances that bind to said gene products, and the use of said gene products in assays, said assays based on are based on the principle of affinity selection, and the use of said gene products of YQGF in assays, the assay being a flavin mononucleotide or a Flavinadenine dinucleotide used as a substrate, and an indicator system that the Indicates the redox state of the protein and the use of said gene products from YJEE in assays where the assay uses an ATP / GTP as a substrate, as well as the use of said KDTB gene products in assays, the assay being a Nucleoside derivative used as a substrate and its phosphorylation and / or Dehydrogenation is measured, as well as the use of said gene products from YGBP in assays, the assay being a nucleoside diphosphate derivative as a substrate used and its implementation is measured, and the use of said YGBB gene products in assays, the key step of the assay being the hydrolytic cleavage of the substrate, as well as the use of said Genpro YCHB products in assays, the key step of the assay being either a Phosphorylation or dehydrogenation is, as well as the use of said YHBC gene products in assays measuring DNA binding, and the use of said YGGJ gene products in assays, the Key step of the assay is phosphorylation, as well as use said gene products or the use of parts of said gene products for up Finding or producing antibodies or other proteins that bind to be said to bind gene products. The scope of the invention also includes use Use of substances that bind said gene products for the manufacture of medicines agents, as well as the use of substances that bind said gene products for  Production of antimicrobial drugs, as well as their use said gene products for finding substances linked to these gene products bind, as well as the production and use of antisense constructs, ge targets the above genes. Procedures for cleaning the said gene products using antibodies to the scope of the invention.

The substances found using the methods listed above, which bind to the gene products mentioned above, and which are derived from these substances Manufactured medicines that can be used to treat infections caused by Pathogens in humans and animals can be used.

This preferably includes diseases caused by bacterial pathogens chosen from the families of Bacillaceae, Bacteroidaceae, Chlamydiales, Entero bakteriaceae, Micrococcaceae, Mycobakteriaceae, Neisseriaceae, Pasteurellaceae, Pseudomonadaceae, Rickettsiaceae, Spirillaceae, Spirochaetaceae, Streptococcaceae and Vibrionaceae.

The treatment of diseases caused by a or several bacterial pathogens selected from the genera Porphyromonas gingivalis, Chlamydia trachomatis, Escherichia coli, Yersinia pestis, Staphylococcus, aureus, Mycobacterium tuberculosis, Neisseria gonnorhoeae, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae, Pseudomonas aeruginosa, Rickettsia prowazekii, Campylobacter jejuni, Helicobacter pylori, Borrelia burgdorferi, Treponema pallidum, Enterococcus faecalis, Streptoccus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Vibrio cholerae, Bacillus subtilis can be caused.

The substances found with the methods listed above, which bind the above gene products, and those made from these substances Medicines can be used in humans and animals to treat diseases and especially used to treat infections affecting the blood; the  Cardiovascular system; the central nervous system and its appendages; the Bones, the bone marrow; the muscles and fascia, the teeth; the joints and their appendages and cavities; the internal organs, their cavities, inner and outer skins and appendages; the gastrointestinal tract and its Cavities, inner and outer skins and appendages; the skin, skin on hanging structures and soft parts and their appendages; the genitourinary system and his appendages; localized and / or generalized inflammation and / or general bacteremia and sepsis, as in the context of general Er disease, trauma, polytrauma, and / or after surgery can; opportunistic infections and sepsis also related to others Diseases such as blood disorders, viral diseases, consuming er diseases and / or antineoplastic and / or immunosuppressive therapy, affect.

For example, diseases can be treated that after infection with Porphyromonas gingivalis in the area of the neck, nose, ear, head and neck, in the Oral cavity wound area after surgical and / or dental treatment lung, and / or after bites of humans and animals; Escherichia coli in the area of the Genitourinary system including urinary tract, bladder, kidney, Renal pelvis, urosepsis and sepsis after surgery and others Dream; Staphylococcus aureus as a causative agent of inflammatory diseases of the Airways and nasopharynx, lungs, bones, skin, the Skin appendages and soft tissues, of the heart and heart valves, as well as the pathogen caused toxic diseases such as hemolysis of the blood Epidermolysis of the skin, enterocolitis of the gastrointestinal tract, the toxic Shock syndrome; Mycobacterium tuberculosis as tuberculosis and / or tuber culoid diseases of the lungs, skin, and other organs and organ systems; Neisseria gonnorhoeae and the resulting clinical picture of gonorrhea; Neisseria meningitidis as a causative agent in acute purulent meningitis; Bordetella pertussis as causative agent of whooping cough; Haemophilus influenzae as a causative agent in meningitis, Epiglottitis, osteomyelitis, pneumonia, septicemia, laryngotracheitis, pharyngitis,  Sinusitis, otitis media, septic arthritis, acute endocarditis, cellulitis; Pseudo monas aeruginosa and resulting inflammation of wounds, des Genitourinary tract, the lining of the heart, the respiratory tract, the gastrointestinal tractes; Rickettsia prowazekii and resulting typhus; Chlamydia trachomatis and resulting conjunctivitis granulosa trachomatosa des Eye; Yersinia pestis and the resulting plague disease; Campylobacter jejuni and resulting inflammatory diseases of the gastrointestinal tract such as enteritis, colitis, proctitis and ulcer disease; Helicobacter pylori and resulting inflammatory chronic diseases of the gastrointestinal tract such as gastritis and duodenal ulcer, as well as ulcer disease; Borrelia burgdorferi and resulting Lyme disease and erythema migrans disease; Treponema pallidum and the resulting clinical picture of syphilis; Enterococcus faecalis and resulting disease of the urinary tract, the urogenital system, the Cardiac membranes and wound infection; Streptoccus mutans and resulting Endocarditis and / or inflammation of the heart valves; Streptococcus pneumoniae and resulting inflammatory diseases of the upper and lower respiratory tract such as pneumonia, otitis media, sinusitis, as well as meningitis, peritonitis, and Ent ignitions of the inner lining of the heart and the pericardium, as well as caries; Streptococcus pyogenic and resulting inflammatory diseases of the upper respira tion tracts such as streptococcal pharyngitis, scarlet fever, otitis media, and pyoderma and skin erysipelas and sepsis; Vibrio cholerae and the resulting Clinical picture of cholera; Bacillus subtilis and the resulting sick picture of oppurtunistic infections, disseminated spread and secondary infections in the context of other bacterial infections such as otitis, mastoiditis, Urinary tract infection, bacteremia, meningitis, endocarditis occur.

Bioinformatic analysis of different bacterial genomes:
The amino acid sequences of the 4289 proteins from Escherichia coli K12 MG1655 with the Genbank / EMBL entry U00096 were compared with the sequences of the complete protein sets from the Gram-positive model organism Bacillus subtilis (Genbank / EMBL accession no. AL009126) and from the Gram-negative pathogen Haemophilus influenzae ( Genbank / EMBL accession no. L42023) as well as with all other amino acid sequences of the public database (Genpept, Swiss-Prot).

Comparative analyzes were carried out with the FASTA program (FASTA vers. 2; Pearson and Lipman 1988).

All E. coli proteins with significant [expected value E (N) <10 ^-4 ] sequence homology to proteins from H. influenzae and B. subtilis were selected.

Sequences which have a homology of an expected value E (N) <10 ^-4 to yeast proteins (HW Mewes et al., Nature, suppl. Vol. 387: 9, 1997), as well as all proteins with known function or with homologies to known proteins from other organisms were excluded.

Furthermore, proteins from E. coli were excluded, for which homologous protein sequences occur within the species of E. coli [expected value E (N) <10 ^-4 ].

In addition, sequences with more than one predicted trans membrane domain [program: "Peptide Structure", Wisconsin Sequence Analysis Package (Vers. 9, Genetics Computer Group, Madison WI, USA)] excluded.

Results of the bioinformatic analysis of different bacterial genomes:
27 protein sequences were selected by way of example from the sequences of potential targets thus obtained:

KDTB, SMPB, YBAB, YBAD, YBAK, YBAX, YBEA, YBEY, YCHB, YFGB, YGAG, YGBB, YGBP, YGGJ, YGGV, YHBC, YHBJ, YHBY, YHIN, YIBK, YIDA, YIGZ, YJEE, YJEQ, YQGF, YRAL, YRFI.  

"Knockout" construction and essentiality tests using temperature-sensitive Plasmids material and methods 1. Bacterial strains and plasmids

Table 1

E. coli strains used

Table 2

Plasmids used

Culture media used LB (Luria Bertani) medium

10 g tryptone, 5 g yeast extract, 10 g NaCl on 11 H ₂

O.

M9 minimal medium

6 g Na ₂

HPO ₄

.3 g KH ₂

PO ₄

, 0.5 g NaCl, 1 g NH ₄

Cl to 11 H ₂

O.

Table 3

Antibiotic concentrations in the media used

Molecular biological techniques Basic molecular biological techniques

Basic molecular biological techniques such as plasmid isolation, cutting with Restriction endonucleases, modification of DNA (dephosphorylation, filling reactions, ligation), production of competent E. coli cells and transformation have been carried out with common regulations known to the person skilled in the art, such as they z. See, for example, J. Sambrook et al., Molecular Cloning, and Ausubel et al., Current Protocols in Molecular Biology.

Polymerase chain reaction (PCR)

All PCR primers used had calculated melting temperatures of 58 ° C to 62 ° C.  

The following DNA polymerases were used for cloning purposes: Pwo, Taq (Boehringer, Mannheim, Germany), Akku-Taq- (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and Pfu-DNA polymerase (Stratagene, Heidelberg, Germany).

Genes or their flanking regions were made from 100 ng of chromosomal DNA amplified from E. coli W3110 F in a reaction volume of 25 to 100 ul. The Primer concentrations were 1 µM and dNTP concentrations each 250 µM. Twenty-five to thirty reaction cycles of 30-60 s at 94 ° C, 30-60 s at 48-55 ° C and 1-3 min at 72 ° C with a final 5-minute Step at 72 ° C [ref .: PCR Protocols: a Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990)].

Assembly PCRs

Amplified 5 'and 3' flanking gene regions were fused by 1 µl of the two previous PCR approaches as a template for a second PCR were mixed with the distally located primers. This PCR was among the conditions described above.

Diagnostic colony PCRs

Diagnostic colony PCRs were performed using a mixture of Taq DNA Polymerase (Boehringer) and Thermo-Sequenase (Amersham) performed.

One colony at a time was resuspended in 50 µl H ₂ O. 5 μl of this was used for the PCR, which was carried out in a 50 μl volume under the conditions described above.

Overexpression of essential proteins in E. coli

Various vector systems are used to overexpress essential proteins in E. coli used: pBAD / His (Invitrogen), pQE vectors (Quiagen).  

PBAD / His plasmids (pUC vector derivatives) constructed for regulated, dose dependent expression and purification for recombinant proteins in E. coli were used:
The vector system pBAD / His contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein. The maximum expression of the soluble, recombinant protein is achieved by targeted activation of the araBAD promoter with arabinose. The purification of the proteins was carried out using affinity chromatography on nickel-NTA agarose.

To purify the fusion proteins, the cells containing the pBAD construct must first be grown to an optical density (OD _600nm ) of 0.3 to 0.6. The targeted protein production is then started with arabinose [0.002-0.2% (w / v)] and incubated for a further 2-3 h. After the incubation has ended, the cells are harvested by centrifugation and the cell pellet is frozen. Cell disruption and purification procedure were carried out according to "The QIAexpressionist, A handbook for high-level expression and purification of 6xHis tagged proteins, Quiagen, Hilden, Germany 1997". The cleaning steps were checked with polyacrylamide gels (and then stained with Coomassie blue).

The vector system pQE contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein. The maximum expression of the soluble, recombinant Protein is activated by targeted activation of the phage and T5 promoter system Induction achieved with IPTG. The purification of the proteins was carried out with the help of Affinity chromatography performed on nickel NTA agarose.  

Cell culture and purification of fusion proteins from cells that construct pQE included, according to the regulation from "The QIAexpressionist, A handbook for high-level expression and purification of 6xHis-tagged proteins, Quiagen, Hilden, Germany 1997 ".

Cloning strategy Knockout plasmids

To test the essentiality of the open reading frames of the selected genes, "Knockout" plasmids were used, the temperature-sensitive replica have origin of pSC101. That means: These plasmids can be at 30 ° C reproduce independently in E. coli, but not at 42-44 ° C. Areas of DNA, which are homologous to the E. coli chromosome can be cloned into such plasmids these plasmids at 42-44 ° C with the help of homologous recombination in the targeted Integrate investigating gene locus of the E. coli chromosome. The subsequent one Excision of the plasmid at 30 ° C can be exchanged for the wild-type sequence lead the sequence originally cloned in the plasmid.

The flanking areas of the examined genes in the range of 400-800 each bp were cloned into pMAK705 and pKO3, respectively. Instead of the gene under investigation either a tetracycline or a kanamycin resistance cassette in each integrated plasmids. Two ways were used for the cloning strategy steps.

Integration of the resistance cassette into the gene

The gene of interest including its flanking regions (400-800 bp) amplified by means of PCR from chromosomal E. coli W3110-F DNA and into a Cloning vector cloned (pCR blunt). With the help of restriction digests and at closing ligations became most of the coding region of the gene of interest replaced by an antibiotic cassette, or the gene was replaced by such a cassette is interrupted (tetracycline or kanamycin resistance cassette).  

Then the cassette with the original flanking the gene Sequences cloned into the "knockout" plasmid pMAK705 or pKO3.

Cloning of "in-frame" deletions with integration of a resistance cassette

DNA regions with lengths of 500 to 800 bp, the S 'and 3' end of the surrounding each gene were identified in two separate PCR reactions chromosomal E. coli W3110-F DNA amplified. The necessary primers were selected so that the ends of the PCR products that are the 5 'and 3' ends of the gene also covered a 21 bp "tag" with one or two restriction sites owned. Accordingly, the two PCR products contained two 21bp long mutually complementary extensions. These were used to both Products in a second PCR step using the outside primer assemble.

Via restriction sites at the 5 'ends of the primers on the outside were incorporated, the fusion PCR product could be cloned into pKO3. The Fusion PCR product contained the flanking areas (400-800 bp each) as well exactly 18 nucleotides of the 5 'end and 36 nucleotides of the 3' end of the examined Gene, with the 5 'and 3' ends of the gene interrupted by a 21 bp "tag" were. In this way, a so-called "in-frame" deletion of the gene generated. A restriction site was inserted into the 21 bp "tag" Kanamycin resistance cassette integrated.

Temperature "shift" experiments (integration and excision)

E. coli JM101 and E. were used to integrate pMAK705 and pKO3 constructs. coli MC 1061 transformed with the plasmids according to standard protocols and at 43- Incubated 44 ° C on LB agar plates with chloramphenicol.

The 43/44 ° C clones were used to enable excision of the plasmids (Cointegrate) then inoculated to 30 ° C-LB. Depending on the vector system (pMAK705 or pKO3) different paths were followed.  

Temperature "shifts" with pMAK705

43/44 ° C clones were inoculated in 100 ml of LB chloramphenicol liquid medium and incubated at 30 ° C for 16 h. After two passages in fresh medium New incubations at 30 ° for 16 h each were made from 1 ml of the last grown Culture the plasmids isolated. The prepared plasmids were restricted digest was examined for the presence of the wild-type copy of the gene in the plasmid. The culture in which positive plasmids were found were based on LB chlorine isolated amphenicol plates at 30 ° C.

A number of colonies were picked and used for plasmid preparation dressed. The prepared plasmids were restricted to the Existence of the wild-type copy of the gene in the plasmid was examined. Positive clones were checked by PCR for the presence of the antibiotic resistance cassette in the corresponding chromosomal locus verified. In this way, a Chromosomal knockout for the gene under investigation in E. coli generated at the same time early complementation by a functional gene copy on the plasmid pMAK705.

Temperature "shifts" with pKO3

The cointegrates of the pKO3 derivatives in E. coli MC1061 were cut out at simultaneous elimination of the pKO3 plasmid from the cell accordingly written method [A. J. Link et al., J. Bacteriol. 179: 6228-6237 (1997)]. Here three 43/44 ° C colonies were diluted in Luria-Bertani (LB) medium and opened LB-5% (w / v) sucrose-kanamycin plates plated and incubated overnight beer. Subsequently, 50 to 100 of the resulting clones per integration clone again on LB-sucrose-kanamycin and LB-chloramphenicol plates inoculated ("replica plating"). Chloramphenicol sensitive, but kanamycin resistant colonies were finally analyzed by PCR using the gene flanking primers were used. Based on the size of the PCR product whether the respective clone is replaced by a kanamycin resistance cassette  of the wild type gene. In this way, a chromosomal knockout for the gene under investigation was generated in E. coli without functional complementation.

Evidence of essentiality

The essentiality of the genes was shown in different ways:
Proof of essentiality by means of temperature shifts with the pMAK705 vector system:
Aliquots were taken from 5 ml of overnight LB-chloramphenicol cultures of the respective complemented knockout strains obtained at the end of the pMAK705 experiments. These were set to a uniform OD ₆₀₀ of 0.1. Dilutions thereof were plated out on LB plates with or without chloramphenicol and incubated at 30 ° C. and 43/44 ° C. overnight.

The clones form a significantly reduced number of colonies on LB chloramphenicol plates at 43/44 ° C compared to the number of colonies on LB +/- chlorine amphenicol at 30 ° C, since the majority of the clones at 43/44 ° C the pMAK705 construct loses and can no longer develop resistance to chloramphenicol. The number of colonies is reduced by at least a factor of 10 ² . Under these conditions, the clones are not considered to be viable.

Criterion for chromosomal knockout in an essential gene

Clones which, after the colony count on LB without chloramphenicol at 43/44 ° C, formed at least 10 ² fewer colonies than at 30 ° C on LB (with or without chloramphenicol) and at most 10 times more colonies than on LB plates with chloramphenicol 43/44 ° C, had a chromosomal knockout in an essential gene according to our criteria. The clones were not viable if they lost the plasmid at 43/44 ° C on LB without chloramphenicol (measured in "number of viable colonies").

On the other hand, all the clones had LB at 43/44 ° C without chloramphenicol as many (+/- factor 10) colonies formed as on LB (with and without chlorine  amphenicol) at 30 ° C, a chromosomal knockout in a non-essential Gene. Removal of the plasmid with the complementary functional copy from the E. coli cells on LB without chloramphenicol at 43/44 ° C does not lead to a decrease in the viability of the clones (measured in "number of survivors capable colonies ").

Proof of essentiality by means of knockout generation only in the presence of a complementary gene copy (pKO3 system)

The clones generated with the pKO3 vector system, which are chloramphenicol sensitive and a kanamycin resistance cassette instead of the wild-type gene held a chromosomal knockout in a non-essential gene. A plasmid-coded complementation no longer existed.

If all colonies remained chloramphenicol resistant, no knockout in the Chromosome are generated with simultaneous loss of the plasmid.

A knock was attempted to further verify the essentiality of these genes out the corresponding genes in the chromosome with simultaneous functional complexes generate mentation.

For this the E. coli strain MG1655 was used, which in contrast to MC1061 has a full araBAD locus. First the araBAD Genes from E. coli MG1655 through the functional copy of each to below seeking gene replaced. The plasmid pAL761 was used for this. After trans formation of the pAL761 derivatives and subsequent integration and excision steps was the replacement of the araBAD genes with the functional copy of a gene detected by means of PCR on the clones that were not on M9 minimal medium 0.2% (w / v) arabinose could grow.

Positive clones were subsequently identified with the pKO3 derivative of the corresponding Gene transformed. The subsequent cointegration and excision was as above  described performed with the exception that the integration and excision step was carried out in the presence of 0.2% (w / v) arabinose.

The genotype of the. Was identified among the chloramphenicol-sensitive colonies by PCR deleted chromosomal gene locus diagnosed with kanamycin cassette. Showed the clones obtained had a growth dependence on arabinose (i.e. no single Colony formation on LB agar plates without arabinose in contrast to growth on LB agar plates with arabinose), the corresponding genes were considered essential.

Proof of essentiality with the help of "in-frame" deletions (pKO3 system)

In some cases, the genes could not be replaced by the corresponding ones Broken or deleted copies with antibiotic resistance cassettes also in Do not create the presence of a complementation. This can be caused by the Cassettes may be polar effects that cause expression essential genes located downstream of the gene under investigation are impaired becomes. For this reason, the "in-frame" deletions described above plasmids of the corresponding genes without the kanamycin cassette for the following Experiments used.

"In-frame" deletion clones were transformed into E. coli MC 1061. Integration, Excision and elimination of pKO3 and the subsequent "replica plating" were carried out as described above with the modification that no longer applied Kanamycin was selected. The chloramphenicol sensitive colonies should statistically speaking, 50% of the gene deletion carries. If under 50 using PCR colonies tested, no clone carrying the deletion, further experiments followed to confirm the essentiality of the gene under investigation.

For this purpose a deletion of the gene in the chromosome was carried out with simultaneous comple mentation, as described above, and the growth dependence of Arabinose shown.  

Proof of essentiality in complemented knockouts without arabinose-dependent growth

On the complemented knockout strains that are not dependent on arabinose Growth, an attempt was made to find the functional copy of the gene in araBAD Replace locus with a Kanamycin cassette. For this purpose, the corresponding Strains transformed with pAL767, a pKO3 derivative with kanamycin resistance cassette and araBAD-flanking regions.

After integration, excision and elimination of the pAL767 100% of the resulting clones (200 clones considered) both kanamycin and Chloramphenicol-resistant, it was shown that pAL767 is still in the chromosome integrated template. So it was not possible to get the intact copy of the gene from the Remove araBAD locus. This finding underlines the essentiality of the gene in question. Could be in the kanamycin-resistant and chloramphenicol-sensitive cloning via PCR the successful exchange of the functional gene copy against the kanamycin resistance cassette was shown the gene is not essential.  

Results of proof of essentiality

Table 4

Genes, the essentiality of which has been assessed using the above methods

The 27 genes examined therefore became eight essential genes identified. With the protein sequences derived from these genes, a Homology search against nucleotide sequences completely and incompletely ent key bacterial genomes using the TBLASTN program.

With this method, by choosing a threshold value, the expected value P (N), all significant homologous protein sequences can be distinguished from sequences that match at random. Using a stringent threshold value (expected value P (N) <10 ^-4 ), gene products with a significant sequence homology could be found in a large number of disease-relevant bacteria.

From the homologues of a bacterial species found in this way were considered to be similar most selected those who have the maximum number of identical and / or  conserved exchanged amino acids compared to the corresponding E. coli Have protein (Table 5). Such proteins are called orthologic proteins (Gene products) referred to, as the expert knows that they have the same function exercise like in Escherichia coli.

The orthologic proteins listed in Table 5 have at least 21% identical or 42% identical or conserved amino acids exchanged, d. H. 21% identity / 42% similarity.  

Table 5

Distribution of the orthologic proteins of the identified essential E. coli gene products in pathogenic bacteria. Results of a TBLASTN search with the protein sequences found to be essential from E. coli against nucleotide sequences of fully or incompletely decoded bacterial genomes, such as those stored in the publicly accessible genome database of the National Center for Bio technology Information (NCBI): criterion for significant homology is an expected value P (N) <10 ^-4 . The identity and similarity of the orthologists in comparison to the corresponding E. coli protein are given as% identity /% similarity. Fully sequenced genomes are marked with (*). Missing homology in incompletely decoded genomes is marked with a (?).

The following functions can be predicted on the basis of sequence motifs in the amino acid sequence of the eight essential gene products and / or their spatial structure to be expected from the amino acid sequence:
YJEE is likely to be an ATP or GTP binding protein. The protein may have ATPase / GTPase or ATP / GTP synthase activity.
KDTB is probably a sugar and / or nucleoside and / or nucleoside derivative binding protein with phosphorylation or dehydrogenase activity.
YQGF may be a flavin derivative binding protein. It can bind flavomononucleotide (FMN) or flavin adenine dinucleotide (FAD) and transfer electrons to another substrate / protein.
YHBC is a putative DNA-binding protein with a modulatory / regulatory function.
YGGJ is a possible phosphotransferase.
YGBP is probably a nucleoside diphophate derivative phosphorylase.
YCHB is a putative kinase or putative oxidoreductase.
YGBB is a putative hydrolase.

Examples Example I to YJEE Construction of the integration vector with the gene sequence for yjeE

The integration plasmid pMAK705 (5.5 kb) is possible via homologous To integrate recombination into the chromosome of E. coli JM101. The plasmid contains in addition to a temperature sensitive origin of replication from pSC101 a chloramphenicol resistance gene for selection of the plasmid in the host organism.

The coding sequence for yjeE and 420 base pairs (bp) upstream and 413 bp downstream of the coding sequence for yjeE were amplified by PCR (Cycler Perkin Elmer 480). 100 ng of chromosomal DNA from E. coli W3110 F were in 25 cycles with 45 sec. at 94 ° C, 45 sec. at 48 ° C and 3 min. at 72 ° C and one subsequent incubation at 72 ° C for 5 minutes with Pfu DNA polymerase in Reaction volume of 100 ul amplified. Primers were used as amplification primers A (5'-CCT GCT GGC AAT CAA TCC CGA TA-3 ') and primer B (5'-AGG CGG TGG CGG CAC ATCGGC GTT-3 ') used. The amplification product (1482 bp) was transformed into the vector after cleaning with Qiaex (Qiagen, Hilden, Germany) pCR-blunt using the "pCR-blunt cloning kit" (Invitrogen Corporation, Carlsbad, CA). In the restriction site BpmI from A tetracycline resistance cassette was cloned into pCR-blunt / yjee. The Tetracycline cassette was made from plasmid pBR322 as 1400 bp EcoRI / AvaI Fragment isolated and blunt ends were made with Klenow polymerase.

The resulting plasmid pCR-blunt / yjeE :: tet was with KpnI / XbaI restriction digested enzymes. A 2.8 kb DNA band was isolated and in the with KpnI / XbaI pretreated vector pMAK705 (host organism E. coli JM101) cloned.  

The resulting construct (pMAK705yjee) was used for the integration experiments used.

Production and analysis of the yjeE knockout

Cells with the plasmid pMAK705yjee were overnight in LB medium at 30 ° C grown and the next day on LB test plates with chloramphenicol plated and incubated for 16 h at 43 ° C. Integration of the plasmid into the chromosome of E. coli JM101 was selected by selecting chloramphenicol-resistant colonies that could grow at 43 ° C. After identifying the integration (at 43 ° C) the cells were incubated in 100 ml LB medium at 30 ° C for 16 h. This Cells were transferred twice into fresh medium and continued for 16 h each Incubated at 30 ° C.

After this series of incubations, plasmid rapid preparations of 1 ml each were used of the last incubation approaches with Qiaprep (Qiagen, Hilden, Germany). The prepared plasmids were checked for their presence by restriction digestion the wild-type copy of the gene in the plasmid was examined. Cells from the approaches that contained positive clones (yjeE gene from the wild-type chromosome on the Plasmid) were isolated on LB plates with chloramphenicol at 30 ° C.

The plasmid DNA of the isolated clones was again targeted for the intact yjeE gene tested the chromosome and identified positive colonies. The integration of the copy of yjeE in the chromosome inactivated by insertion of the tetracycline cassette was tested via PCR.

The clones checked in this way could now focus on the essentiality of the target for each Survival of the E. coli cell can be used. The cells were placed on LB test plates incubated with and without chloramphenicol at 30 ° and 43 ° C.  

Table 6

Incubation cell count +/- chloramphenicol at 30 ° C / 43 ° C

As can be seen from the table, the cells can grow at 30 ° C. both with and without chloramphenicol. At 43 ° C, the growth with chlorine amphenicol as well as without chloramphenicol is drastically reduced, ie by a factor of 10 ⁴ . This clearly shows the need for the presence of the plasmid with the complementing sequence of yjeE for the growth of the E. coli cell at 43 ° C. without chloramphenicol and thus the essentiality of yjeE for the survival of these cells.

example Proof of essentiality for ygbP Cloning of "in-frame" deletions with or without integration of a Resistance cassette

DNA regions surrounding the 5 'and 3' ends of the ygbP gene were separated into two separate PCR reactions from 100 ng chromosomal E. coli W3110-F-DNA in a reaction volume of 25 ul amplified. The primer concentrations were 1 µM and dNTP concentrations were 250 µM each. Five and five twenty reaction cycles of 30 s at 94 ° C, 30 s at 52 ° C and 1-2 min at 72 ° C were performed with a final 5 minute step at 72 ° C. The PCR product that is primed with YGBP1A (5'- cgcggatccCCACATGGTCACTGCCTGG-3 ') and YGBP1B (5'- cccatccactaaactgcagctCAAATGAGTGGTTGCCATGTT-3 ') was made to holds 18 bp of the 5 'end of ygbP and 776 bp of the chromosomal region upstream from ygbP. The PCR product that is primed with YGBP2A (5'- agctgcagtttagtggatgggTACCTCACCCGAACCATCC-3 ') and YGBP2B (5'-  cgcggatccACCTGGCAGCCTTCCAGTTG-3 ') was produced, comprises 36 bp of the 3 'end of ygbP and 807 bp of the chromosomal region downstream of ygbP.

The inner primers YGBP1B and YGBP2A additionally contained at their 5 'ends 21mer "tags" that were complementary to each other (small letters of the above sequences mentioned). These were used to make both PCR products in one second PCR step using the external primers YGBP1A and YGBP2B to assemble. For this purpose, 1 ul of the two previous PCR-An mixed as a template for a second PCR with YGBP1A and YGBP2B. This PCR was carried out under the conditions described above. The PCR Product of the expected size was analyzed by agarose gel electrophoresis QIAEX (QIAGEN, Hilden, Germany) cleaned up.

Via the BamHI interface, which is at the 5 'ends of the outer primers YGBP1A and YGBP2B were built in (small letters of those mentioned above Sequences), the fusion PCR product could be cut into the BamHI dephosphorylated pKO3 can be cloned. In this way the plasmid pAL759 generated, which contained the "in-frame" deletion of ygbP. About the PstI The 21-bp "tag" (5'-ctgcag-3 ') cleaved the kanamycin resistance Cassette cloned into pAL759, obtained from pUC4K by PstI digestion has been. The resulting construct was named pAL759a.

Temperature "shifts" with pAL759a

E. coli MC 1061 was transformed with the plasmid pAL759a and opened at 44 ° C LB agar plates incubated with chloramphenicol.

Three of the 44 ° C. clones obtained (cointegrates) were diluted in LB medium, plated on LB-5% (w / v) sucrose-kanamycin plates and overnight incubated. 100 of the resulting sucrose Kanamycin clones again on LB sucrose kanamycin and LB  Chloramphenicol plates inoculated ("replica plating") and at 30 ° C overnight incubated. 100% of the kanamycin-resistant clones were also chlorine amphenicol resistant. An exchange of ygbP for a kanamycin cassette could not be carried out successfully.

Temperature "shifts" with pAL759

E. coli MC1061 was transformed with the plasmid pAL759 and at 44 ° C. on LB- Incubated agar plates with chloramphenicol.

Three of the 44 ° C. clones obtained (cointegrates) were diluted in LB medium, plated on LB-5% (w / v) sucrose plates and incubated overnight. 100 of the resulting sucrose clones were reopened per integration clone LB-sucrose and LB-chloramphenicol plates inoculated ("replica Plating ") and incubated at 30 ° C overnight. 50 chloramphenicol-sensitive each Colonies were finally analyzed by PCR using YGBP1A and YGBP2B were used as primers. Based on the size of the PCR product it was possible to show that none of the clones tested had the deletion of ygbP (PCR product with ygbP deletion: 1637 bp). The wild-type gene locus of ygbP are amplified (2294 bp). An exchange of ygbP for a deletion could not be carried out successfully.

Generation of a ygbP deletion in the presence of a chromosomal complement tion

The next step was to try to delete ygbP in the chromosome to produce simultaneous functional complementation. The E. coli Strain MG1655 used, which in contrast to MC1061 is a complete one araBAD locus. First, the araBAD genes from E. coli MG1655 were cut replaced the functional copy of ygbP. The plasmid pAL763 was used for this.  

The plasmid pAL763 was generated by using chromosomal E. coli W3110-F DNA with the primers YGBPR (5'-atcgctagcATCAGCCCGGGAATTAACATG-3 ') and YGBPT (5'-atcctcgagAATTCGCATTATGTATTCTCCTG-3 ') the whole YGBP gene including its ribosomal binding site (18 bp upstream from 5 'end) and including 8 bp downstream of the 3' end of ygbP amplified has been. The primers contain the restriction sites for at their 5 'termini XhoI (YGBPT) and NheI (YGBPR). The PCR product was via the interfaces cloned into the vector pAL761. After transformation of E. coli MG1655 with pAL763 and the following integration and excision steps (see above) became the Exchange of the araBAD genes for the functional copy of ygbP using PCR the clones detected that are not on M9 minimal medium with 0.2% (w / v) Arabinose could grow. The diagnostic PCR was carried out with the primers YGBPR (see above) and ARA2B (atcgcggccgcAAAGCCGTGCTCGCGC). The Primer ARA2B binds in the 3 'region 865 bp downstream of the araD gene, so that together with the primer YGBPR for positive clones a 1655 bp product originated. Positive clones were subsequently identified with the "in-frame" Deletion plasmid pAL759 transformed. The subsequent cointegration and Excision was performed as described above, except that the Integration and excision step in the presence of 0.2% (w / v) arabinose was carried out. By means of PCR was among the chloramphenicol sensitive Colonies of the deleted chromosomal locus genotype were diagnosed. The ygbP deletion clones containing an arabinose-regulated ygbP copy in araBAD Contained locus showed a dependence on the growth of arabinose. On LB- No single colonies could be formed in agar plates without arabinose, whereby Single colonies on LB agar plates with arabinose were obtained. The ygbP gene is therefore essential.  

Example 3 Production and purification of recombinant YJEE protein

The expression and purification of YJEE protein is possible with the pBAD / His Vector system (Invitrogen) has been carried out. The sequence of yjeE was made up 100 ng of chromosomal DNA from E. coli W3110F for 1 min at 94 ° C, 1 min. at 52 ° C, 3 min at 72 ° C in 25 cycles with Pfu DNA polymerase in a 100 µl reaction mixture amplified. YJEE-START (5'-TGA AGA TCT AAT CGA GTA ATT CCG CTC CCT GAT-3 ') and YJEE-STOP (5'-TCA GAA TTC TTA ACC GGC TAA ACG CGC CAG CAA CAA TC-3 ') used. The 5 'end of YJEE START contains the sequence for the restriction enzyme BglII. The 5 'end of YJEE-STOP contains the sequence for EcoRI. The PCR approach was carried out in one Agarose gel separated and a band cut out at 450 bp. Subsequently the gang was isolated with Qiaex (Qiagen, Hilden, Germany) and with the Restriction enzymes BglII and EcoRI were incubated for 3 hours at 37 ° C.

The DNA fragment treated in this way was pre-treated with BglII / EcoRI Vector pBAD / HisB cloned [host organism: E. coli strain TOP10 (Invitrogen)]. Successful cloning was obtained by plasmid isolation Transformants and restriction shown with EcoRI. The clone with the plasmid pBAD / HisB-YjeE30 has been used for expression and purification.

The cultivation and purification of YjeE from E. coli TOP 10 with plasmid pBAD / HisB-YjeE30 was carried out according to the protocol for the purification of histidine-labeled proteins on Ni-NTA agarose (The QIAexpressionist, A handbook for high-level expression and purification of 6xHis-tagged proteins, Quiagen, Hilden, Germany 1997). The induction of the YjeE expression was started with 0.02% arabinose (w / v) at an optical density of the culture of OD ₆₀₀ = 0.5. Various concentrations of imidazole (50, 100, 150 and 200 mM) were used to elute the bound protein. The highest yield of protein was obtained at 150 mM. The purification was checked with polyacrylamide gel separation and subsequent Coomassie staining. 10 mg protein with a degree of purity <90% could be isolated from 11 culture medium (LB medium).

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SEQUENCE LOG

Claims (25)

1. Essential genes which are the proteins from the group YQGF, YHBC, YGGJ, Encode YGBP, YCHB, YGBB, YJEE, KDTB, and genes from others Microorganisms that encode the corresponding orthologic gene products. 2. Genes according to claim 1, for which it can be shown that their controllable Elimination leads to growth inhibition of the host organism. 3. Genes according to claim 1 from Escherichia coli which encode gene products which are 95-100% identical, and for which it can be shown that their controllable switch-off leads to growth inhibition of the host organism. 4. Genes according to claim 1 from other microorganisms, the ortholog Encode gene products that correspond to the corresponding gene product from E. coli 42- Have 100% identical or conserved exchanged amino acids, and for which it can be shown that their controllable switch-off for Inhibition of growth of the host organism leads. 5. Genes that are part of their nucleic acid sequence at least one gene partially or fully contained according to claims 1-4. 6. gene products of the genes according to claims 1-5. 7. Vectors containing one or more of the genes according to claims 1-5. 8. Transformed microorganisms containing one or more of the genes according to claims 1-5.   9. Use of constructed, recombinant microorganisms in which one the genes according to claims 1-5 can be expressed in a regulatable manner finding substances that bind to said gene products. 10. Use of the gene products according to claim 6 in detection assays of substances that bind to said gene products. 11. Use of the gene products according to claim 6 in assays, wherein said Assays are based on the principle of affinity selection. 12. Use of the gene products according to claim 6 of YQGF in assays, wherein the assay is a flavin mononucleotide or a flavin adenine dinucleotide used as a substrate, and an indicator system that the redox state of Indicates protein. 13. Use of the gene products according to claim 6 of YJEE in assays, wherein the assay uses an ATP / GTP as a substrate. 14. Use of the gene products according to claim 6 of KDTB in assays, wherein the assay uses a nucleoside derivative as a substrate and its Phosphorylation and / or dehydrogenation is measured. 15. Use of the gene products according to claim 6 of YGBP in assays, wherein the assay uses a nucleoside diphosphate derivative as the substrate and its implementation is measured. 16. Use of the gene products according to claim 6 of YGBB in assays, the key step of the assay being hydrolytic cleavage of the Substrate.   17. Use of the gene products according to claim 6 of YCHB in assays, the key step of the assay being either phosphorylation or is dehydrogenation. 18. Use of the gene products according to claim 6 of YHBC in assays, binding to DNA being measured. 19. Use of the gene products according to claim 6 of YGGJ in assays, the key step of the assay being phosphorylation. 20. Use of the gene products or parts of the gene products according to Claim 6 for the detection or production of antibodies or other proteins that bind to the gene products from claim 6. 21. Use of substances that bind the gene products according to claim 6 for the manufacture of pharmaceuticals. 22. Use of substances that the gene products according to claim 6 bind, for the production of antimicrobial drugs. 23. Use of the gene products according to claim 6 for finding Substances that bind to these gene products. 24. Antisense constructs directed against the genes according to claims 1-5. 25. A method for purifying the gene products according to claim 6 using Antibodies.