Papers by Herb Schellhorn
Environmental Microbiome, Jan 14, 2024
Background Fecal bacterial densities are proxy indicators of beach water quality, and beach posti... more Background Fecal bacterial densities are proxy indicators of beach water quality, and beach posting decisions are made based on Beach Action Value (BAV) exceedances for a beach. However, these traditional beach monitoring methods do not reflect the full extent of microbial water quality changes associated with BAV exceedances at recreational beaches (including harmful cyanobacteria). This proof of concept study evaluates the potential of metagenomics for comprehensively assessing bacterial community changes associated with BAV exceedances compared to nonexceedances for two urban beaches and their adjacent river water sources. Compared to non-exceedance samples, BAV exceedance samples exhibited higher alpha diversity (diversity within the sample) that could be further differentiated into separate clusters (Beta-diversity). For Beach A, Cyanobacterial sequences (resolved as Microcystis and Pseudanabaena at genus level) were significantly more abundant in BAV non-exceedance samples. qPCR validation supported the Cyanobacterial abundance results from metagenomic analysis and also identified saxitoxin genes in 50% of the non-exceedance samples. Microcystis sp and saxitoxin gene sequences were more abundant on non-exceedance beach days (when fecal indicator data indicated the beach should be open for water recreational purposes). For BAV exceedance days, Fibrobacteres, Pseudomonas, Acinetobacter, and Clostridium sequences were significantly more abundant (and positively correlated with fecal indicator densities) for Beach A. For Beach B, Spirochaetes (resolved as Leptospira on genus level) Burkholderia and Vibrio sequences were significantly more abundant in BAV exceedance samples. Similar bacterial diversity and abundance trends were observed for river water sources compared to their associated beaches. Antibiotic Resistance Genes (ARGs) were also consistently detected at both beaches. However, we did not observe a significant difference or correlation in ARGs abundance between BAV exceedance and non-exceedance samples. This study provides a more comprehensive analysis of bacterial community changes associated with BAV exceedances for recreational freshwater beaches. While there were increases in bacterial diversity and some taxa of potential human health concern associated with increased fecal indicator densities and BAV exceedances (e.g. Pseudomonas), metagenomics analyses also identified other taxa of potential human health concern (e.g. Microcystis) associated with lower fecal indicator densities and BAV non-exceedances days. This study can help develop more targeted beach monitoring strategies and beach-specific risk management approaches.
Editorial: Role of transcription factors and sigma factors in bacterial stress physiology
Frontiers in Microbiology
Journal of Great Lakes Research, Feb 1, 2019
Environmental water monitoring is an important responsibility of municipal governments. In this s... more Environmental water monitoring is an important responsibility of municipal governments. In this study, we partnered with several municipalities in an extensive sampling program to investigate the effects of spatiotemporal and environmental factors on bacterial diversity in a complex watershed ecosystem containing specific environments including creeks, a river, canals, stormwater outfalls and freshwater lakes of the Niagara Peninsula. Samples were collected using standard municipal protocols and bacterial DNA extracted from these samples was sequenced using high-throughput DNA sequencing targeting the V3-V4 regions of the 16S rRNA gene. Average taxonomic richness and alpha diversity differed significantly between samples collected from lakes and creeks (P b 0.05), and between lakes and stormwater outfalls (P b 0.05). Beta diversity also differed significantly (P b 0.0001) between habitats suggesting that each of these habitats harbours distinct bacterial groups. Among the environmental factors examined, dissolved oxygen (DO) level was strongly associated (P b 0.001) with bacterial diversity. Using a Bayesian source tracking method, the proportional contribution of creeks, river, canals and stormwater outfall habitats in shaping lake bacterial community structure was quantified. Sequences associated with genera known to contain pathogens as well as fecal indicator bacteria were found in every habitat. This study demonstrates that DNA sequence analysis can augment traditional methods of watershed monitoring and management by providing additional information on bacteria of interest to water quality policy makers. Future work may integrate taxonomic and functional analyses to obtain a greater understanding of pathogen survival, nutrient cycling and microbial interactions in freshwater ecosystems.
Journal of Bacteriology, Jul 1, 1992
Chromosomal transcriptional and translational lacZ fusions to the katE (structural gene for the H... more Chromosomal transcriptional and translational lacZ fusions to the katE (structural gene for the HPII hydroperoxidase) and katF (putative sigma factor required for katE expression) genes ofEscherichia coli were isolated, and the regulation of these fusions was used to identify factors that control the expression of these two important antioxidant factors. While katE was found to be regulated primarily at the level of transcription (since induction patterns were similar for both transcriptional and translational fusions), katF expression was a function of both transcriptional and translational signals. The katE gene was induced 57-fold as cells entered the stationary phase, while katF was induced 23-fold. katF induction was coincident with katE induction and occurred at the onset of the stationary growth phase. Expression of both katE and katF could be induced by resuspending uninduced exponential-phase cells in spent culture supernatant recovered from stationary-phase cells. The component of stationary-phase culture supernatant responsible for induction of the katF regulon appeared to be acetate, since expression of both katE and katF fusions was induced when exponential-phase cells were exposed to this weak acid. Other weak acids, including propionate and benzoate, were also found to be effective inducers of expression of both katF and katE. Induction of katE and katF fusions was unaffected in merodiploid strains containing both mutant and wild-ype alleles, indicating that expression of both genes is independent of the wild-type gene product. Examination of catalase zymograms prepared from cells exposed to various levels of acetate revealed that both HPI and HPII catalases are induced by this weak acid, suggesting that there is a common link in the regulation of these two enzymes.
Faculty Opinions recommendation of The H-NS-like protein StpA represses the RpoS (sigma 38) regulon during exponential growth of Salmonella Typhimurium
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Dec 14, 2009
Journal of Bacteriology, 1997
Escherichia coli produces an inducible set of proteins that protect the cell from exogenous perox... more Escherichia coli produces an inducible set of proteins that protect the cell from exogenous peroxide stress. A subset of these genes is induced by hydrogen peroxide and is controlled at the transcriptional level by the OxyR protein. To identify additional genes involved in protection from hydrogen peroxide, a library of random transcriptional fusions of placMu53 was screened for hydrogen peroxide sensitivity and 27 such mutants were identified. These fusions were transduced into nonlysogenic strains to ensure that the phenotypes observed were the result of a single mutation. The mutants were grouped into three classes based on the expression of the lacZ fusion during growth in oxyR ؉ and ⌬oxyR backgrounds. The expression of the lacZ fusion in 8 mutants was independent of OxyR, 10 mutants required OxyR for expression, and 6 mutants showed reduced levels of expression in the presence of OxyR. OxyR dependence varied from 2-to 50-fold in these mutants. The OxyR-dependent phenotype was complemented by a plasmid-borne copy of oxyR gene in all mutants. Three mutants exhibited dual regulation by OxyR and RpoS. We sequenced the fusion junctions of several of these mutants and identified the genetic loci responsible for the hydrogen peroxide-sensitive (hps) phenotype. In this study, we report the identification of several genes that require OxyR for expression, including hemF (encoding coproporphyrinogen III oxidase), rcsC (encoding a sensor-regulator protein of capsular polysaccharide synthesis genes), and an open reading frame, f497, that is similar to arylsulfatase-encoding genes.
Regulators of Oxidative Stress Response Genes inEscherichia Coliand Their Conservation in Bacteria
John Wiley & Sons, Inc. eBooks, Aug 12, 2016
Journal of Bacteriology, Dec 15, 2004
RpoS is a conserved alternative sigma factor that regulates the expression of many stress respons... more RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging. RpoS, an alternative sigma factor, controls a large regulon that is specifically expressed when the cell is nutrient deprived or is subjected to external stress, such as osmotic shock (32). Genes that are dependent on RpoS have been identified in different contexts from many studies of regulation in various gram-negative bacteria (38). From these studies it is clear that the regulon encodes many proteins that help bacteria adapt to adverse conditions, including those that are involved in nutrient scavenging, DNA repair, protein turnover, and protection from external environmental insult (31). Characterization of the RpoS regulon has relied on several experimental approaches, including two-dimensional gel electrophoresis (50), identification of genes that are induced by RpoS-related stimuli such as carbon starvation (85), and by identifying gene fusions that depend on RpoS for expression (66). Use of bacterial cDNA microarrays or macroarrays, while potentially useful in identifying additional members of the RpoS regulon, have been employed in only a few studies of Escherichia coli and have not yet been used to directly assess RpoS dependence of genes in isogenic wild-type and rpoS mutant strains. In a previous study (66), our lab introduced an RpoS-null allele into a bank of random operon-lacZ fusions to facilitate the identification of RpoS-dependent genes based solely on RpoS requirement. This resulted in the identification of several new highly RpoS-dependent genes in E. coli (66), and similar approaches have been employed with Salmonella spp. where other RpoS-dependent genes have been identified (37). Lac gene fusions are useful measures of gene expression, because the product, -galactosidase, is stable and easily assayed (16). This makes lacZ fusions particularly suitable for the study of genes that are expressed in stationary phase, where the stability of the RNA and protein products of the affected gene is not known. In this study, we report the completion of the identification of reporter fusions identified in a previous genetic screen of lacZ-expressing fusion mutants. In addition, we examined the expression of the RpoS-dependent operon fusions in rich and minimal media and classified the RpoS-dependent genes according to their function. MATERIALS AND METHODS Growth conditions. Expression assays were performed using derivatives of E. coli K-12 (Table 1). Cultures were grown overnight from single colony isolates in M9 minimal medium or Luria-Bertani (LB) medium containing appropriate antibiotics. The rich medium used was LB broth (53). Cell growth was monitored by measuring optical density at 600 nm (OD 600) (Novospec II; Pharmacia LKB, Cambridge, United Kingdom). For -galactosidase expression studies, the cultures were maintained in early exponential phase (OD 600 Ͻ 0.2) for at least eight generations prior to the start of each experiment. Bacterial cultures were grown at 37°C and shaken at 200 rpm, sampled, and assayed for -galactosidase activity. Chemicals and media. Chemicals were supplied by Fisher Scientific, Ltd.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Dec 14, 2012
Genetic exchange is common among bacteria, but its effect on population diversity during ecologic... more Genetic exchange is common among bacteria, but its effect on population diversity during ecological differentiation remains controversial. A fundamental question is whether advantageous mutations lead to selection of clonal genomes or, as in sexual eukaryotes, sweep through populations on their own. Here we show that in two recently diverged populations of ocean bacteria, ecological differentiation has occurred akin to a sexual mechanism: a few genome regions have swept through subpopulations in a habitat specific manner, accompanied by gradual separation of gene pools as evidenced by increased habitat-specificity of the most recent recombinations. These findings reconcile previous, seemingly contradictory empirical observations of the genetic structure of bacterial populations, and point to a more unified process of differentiation in bacteria and sexual eukaryotes than previously imagined. How adaptive mutations spread through bacterial populations and trigger ecological differentiation has remained controversial. While it is agreed that the key factor is the balance between recombination and positive selection, theory and observations remain seemingly at odds. On the one hand, evidence for genes spreading through populations independently via recombination ('gene-specific sweeps') is found in observations of environment-specific genes (1) and alleles (2), and reduced diversity at single loci amidst high genomewide polymorphism (3, 4). On the other hand, mathematical modeling suggests that empirically observed rates of homologous recombination should not be high enough to unlink a gene, which is under even moderate selection, from the rest of the genome (5, 6). Importantly, this recombination/selection balance, expressed most saliently by the ecotype theory, leads to a prediction that is actually observed but that is at odds with gene-specific
Faculty Opinions recommendation of Rate of environmental change determines stress response specificity
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Apr 17, 2013
The conserved stationary phase regulator, RpoS is required or the expression of many paralogous genes in Escherichia coli
Abstracts of the General Meeting of the American Society for Microbiology, May 14, 1999
Journal of Bacteriology, 1988
Journal of Bacteriology, Oct 1, 1991
Epidemiology and Ecology of Emerging Viruses in Two Freshwater Lakes of the Northern Hemisphere
Changing patterns of waterborne viral diseases across the globe has been the focus of many studie... more Changing patterns of waterborne viral diseases across the globe has been the focus of many studies in recent years. Despite the recent increase in recreational water-related diseases, studies performed in the Great Lakes region have primarily focused on the identification of fecal indicator microorganisms or select pathogenic viruses. Although these studies provide useful information in determining sources of fecal contamination of aquatic environments, they do not provide a comprehensive profile of viral species inhabiting this region. Understanding the dynamics and the diversity of total viral communities is essential in assessing the impact of these communities on water quality and the risk they pose to public health. Therefore in this chapter, we have summarized the current understanding of viral community structures of the lower Great Lakes area, particularly of Lake Ontario and Lake Erie, the epidemiology and ecology of pathogens, their sources, and the public health implicati...
Science Progress, 2019
Conventional microbiological water monitoring uses culture-dependent techniques to screen indicat... more Conventional microbiological water monitoring uses culture-dependent techniques to screen indicator microbial species such as Escherichia coli and fecal coliforms. With high-throughput, second-generation sequencing technologies becoming less expensive, water quality monitoring programs can now leverage the massively parallel nature of second-generation sequencing technologies for batch sample processing to simultaneously obtain compositional and functional information of culturable and as yet uncultured microbial organisms. This review provides an introduction to the technical capabilities and considerations necessary for the use of second-generation sequencing technologies, specifically 16S rDNA amplicon and whole-metagenome sequencing, to investigate the composition and functional potential of microbiomes found in water and wastewater systems.
Journal of Water and Health, 2019
Residents in rural communities across Canada collect potable water from aquifers. Fecal contamina... more Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has been under the longest active boil water advisory in Canada due to FIB contamination in groundwater wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also...
Water Research, 2017
Recreational waters and adjacent beach sands harbor complex microbial communities which may conta... more Recreational waters and adjacent beach sands harbor complex microbial communities which may contain human pathogens that cannot be detected by conventional methods. Here, we investigate the diversity of bacterial populations inhabiting four freshwater beaches of the Great Lakes region using shotgun metagenomic sequencing approach. Our analysis suggests that average taxonomic richness and alpha diversity are significantly higher (P < 0.001) in beach sands compared to the corresponding water environments. Compared to the water environments, beach sands harbored taxa from a more diverse range of phyla, including a higher proportion of sequences from unclassified phyla. Unique phyla were also identified in sand which included species from Aquificae, Candidatus Microgenomates, Latescibacteria, and Candidatus Aminicenantes. Sequences originating from pathogens were detected in both sand and water, with some pathogens enriched in both environments. Both lakes exhibited similar community composition suggesting that geographic location did not appear to have any major impact on bacterial diversity. These findings reveal the diversity of bacterial communities of freshwater beaches and highlight the importance of monitoring pathogens in recreational beaches, especially in the sand environment of these beaches.
Validation of qPCR method for enterococci quantification at Toronto beaches: Application for rapid recreational water monitoring
Journal of Great Lakes Research
Canadian Journal of Public Health
Objectives We evaluated the potential impacts from using a rapid same-day quantitative polymerase... more Objectives We evaluated the potential impacts from using a rapid same-day quantitative polymerase chain reaction (qPCR) monitoring method for beach posting outcomes at two Toronto beaches. Methods In total, 228 water samples were collected at Marie Curtis Park East and Sunnyside Beaches over the 2021 summer season. Water samples were processed using the USEPA 1609.1 Enterococcus qPCR-based method. Escherichia coli (E. coli) culture data and daily beach posting decisions were obtained from Toronto Public Health. Results No significant correlation was observed between previous-day and same-day (retrospective) E. coli enumeration results at any Sunnyside Beach transect, and only relatively low (R = 0.41–0.56) or no significant correlation was observed at sampling transects for Marie Curtis Park East Beach. Comparing our same-day Enterococcus qPCR data to Toronto’s 2-day E. coli geometric mean beach posting decisions, we noted the need for additional postings for 1 (2%) and 3 (8%) misse...
Water
The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering diges... more The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering digester design and by manipulating environmental variables that affect microbial community composition. Community composition can potentially be assessed using high-throughput DNA sequencing, but the two most common methods of community DNA sequencing (16S and shotgun sequencing) generally yield different taxonomic identification profiles and can perform differently according to the sampled environment. To evaluate the use of these two approaches in monitoring OWTS operation, we conducted a comparative parallel analysis using both 16S rDNA and shotgun sequencing in a controlled field study. Results indicate that when examining microorganisms above 0.1% relative abundance, 16S and shotgun sequencing produced similar results in terms of individual sample species richness and between-sample community similarity. However, shotgun sequencing provided comparatively higher taxonomic richness for th...
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Papers by Herb Schellhorn