Search

Abstract Background RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. Results The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (twofold, P < 0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Conclusion Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains.

Abstract Background Though RpoS is important for survival of pathogenic Escherichia coli in natural environments, polymorphism in the rpoS gene is common. However, the causes of this polymorphism and consequential physiological effects on gene expression in pathogenic strains are not fully understood. Results In this study, we found that growth on non-preferred carbon sources can efficiently select for loss of RpoS in seven of ten representative verocytotoxin-producing E. coli (VTEC) strains. Mutants (Suc++) forming large colonies on succinate were isolated at a frequency of 10-8 mutants per cell plated. Strain O157:H7 EDL933 yielded mainly mutants (about 90%) that were impaired in catalase expression, suggesting the loss of RpoS function. As expected, inactivating mutations in rpoS sequence were identified in these mutants. Expression of two pathogenicity-related phenotypes, cell adherence and RDAR (red dry and rough) morphotype, were also attenuated, indicating positive control by RpoS. For the other Suc++ mutants (10%) that were catalase positive, no mutation in rpoS was detected. Conclusion The selection for loss of RpoS on poor carbon sources is also operant in most pathogenic strains, and thus is likely responsible for the occurrence of rpoS polymorphisms among E. coli isolates.

Abstract Background Arginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression. Results In this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 μM L-arginine), a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited), levels of expression were only one third of those observed in ΔargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in ΔargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS. Conclusion The results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

Background: Recreational water activities at beaches are popular among Canadians. However, these activities can increase the risk of recreational water illnesses (RWI) among beachgoers. Few studies have been conducted in Canada to determine the risk of these illnesses. This protocol describes the methodology for a study to determine the risk and burden of RWI due to exposure to fecal pollution at beaches in Canada., Methods: This study will use a mixed-methods approach, consisting of a prospective cohort study of beachgoers with embedded qualitative research. The cohort study involves recruiting and enrolling participants at public beaches across Canada, ascertaining their water and sand contact exposure status, then following-up after seven days to determine the incidence of acute RWI outcomes. We will test beach water samples each recruitment day for culture-based E. coli, enterococci using rapid molecular methods, and microbial source tracking biomarkers. The study started in 2023 and will continue to 2025 at beaches in British Columbia, Manitoba, Ontario, and Nova Scotia. The target enrollment is 5000 beachgoers. Multilevel logistic regression models will be fitted to examine the relationships between water and sand contact and RWI among beachgoers. We will also examine differences in risks by beachgoer age, gender, and beach location and the influence of fecal indicator bacteria and other water quality parameters on these relationships. Sensitivity analyses will be conducted to examine the impact of various alternative exposure and outcome definitions on these associations. The qualitative research phase will include focus groups with beachgoers and key informant interviews to provide additional contextual insights into the study findings. The study will use an integrated knowledge translation approach., Discussion: Initial implementation of the study at two Toronto, Ontario, beaches in 2023 confirmed that recruitment is feasible and that a high completion rate (80%) can be achieved for the follow-up survey. While recall bias could be a concern for the self-reported RWI outcomes, we will examine the impact of this bias in a negative control analysis. Study findings will inform future recreational water quality guidelines, policies, and risk communication strategies in Canada., (© 2024. The Author(s).)

Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix ). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring., (© 2024 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Competing Interests: OL is an inventor on patents related to vaccine adjuvants and human in vitro systems that model responses to vaccines and immunomodulatory agents. OL is a co-founder of Ovax Inc and consultant to GlaxoSmithKline GSK and Hillevax. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be constructed as a potential conflict of interest.

CrAssphage or crAss-like phage ranks as the most abundant phage in the human gut and is present in human feces-contaminated environments. Due to its high human specificity and sensitivity, crAssphage is a potentially robust source tracking indicator that can distinguish human fecal contamination from agricultural or wildlife sources. Its suitability in the Great Lakes area, one of the world's most important water systems, has not been well tested. In this study, we tested a qPCR-based quantification method using two crAssphage marker genes (ORF18-mod and CPQ&#95;064) at Toronto recreational beaches along with their adjacent river mouths. Our results showed a 71.4 % (CPQ&#95;064) and 100 % (ORF18-mod) human sensitivity for CPQ&#95;064 and ORF18-mod, and a 100 % human specificity for both marker genes. CrAssphage was present in 57.7 % or 71.2 % of environmental water samples, with concentrations ranging from 1.45 to 5.14 log 10 gene copies per 100 mL water. Though concentrations of the two marker genes were strongly correlated, ORF18-mod features a higher human sensitivity and higher positive detection rates in environmental samples. Quantifiable crAssphage was mostly present in samples collected in June and July 2021 associated with higher rainfall. In addition, rivers had more frequent crAssphage presence and higher concentrations than their associated beaches, indicating more frequent and greater human fecal contamination in the rivers. However, crAssphage was more correlated with E. coli and Enterococcus at the beaches than in the rivers, suggesting human fecal sources may be more predominant in driving the increases in E. coli and Enterococcus at the beaches when impacted by river plumes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 non-exceedances for two urban beaches and their adjacent river water sources., Results: 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., Conclusion: 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., (© 2024. The Author(s).)

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%) missed health-risk days at Sunnyside and Marie Curtis Park East Beaches, respectively. The qPCR data also pointed to incorrect postings for 12 (31%) and 6 (16%) lost beach days at Sunnyside and Marie Curtis Park East Beaches, respectively., Conclusion: Application of a rapid Enterococcus qPCR method at two Toronto beaches revealed 5% of beach posting decisions were false negatives that missed health-risk days, while 23% of decisions were false positives resulting in lost beach days. Deployment of the rapid same-day qPCR method offers the potential to reduce both health risks and unnecessary beach postings., (© 2023. The Author(s).)

Infectious diseases continue to be a major cause of morbidity and mortality worldwide. Diseases cause perturbation of the host's immune system provoking a response that involves genes, proteins and metabolites. While genes are regulated by epigenetic or other host factors, proteins can undergo post-translational modification to enable/modify function. As a result, it is difficult to correlate the disease phenotype based solely on genetic and proteomic information only. Metabolites, however, can provide direct information on the biochemical activity during diseased state. Therefore, metabolites may, potentially, represent a phenotypic signature of a diseased state. Measuring and assessing metabolites in large scale falls under the omics technology known as "metabolomics". Comprehensive and/or specific metabolic profiling in biological fluids can be used as biomarkers of disease diagnosis. In addition, metabolomics together with genomics can be used to differentiate patients with differential treatment response and development of host targeted therapy instead of pathogen targeted therapy where pathogens are more prone to mutation and lead to antimicrobial resistance. Thus, metabolomics can be used for patient stratification, personalized drug formulation and disease control and management. Currently, several therapeutics and in vitro diagnostics kits have been approved by US Food and Drug Administration (FDA) for personalized treatment and diagnosis of infectious diseases. However, the actual number of therapeutics or diagnostics kits required for tailored treatment is limited as metabolomics and personalized medicine require the involvement of personnel from multidisciplinary fields ranging from technological development, bioscience, bioinformatics, biostatistics, clinicians, and biotechnology companies. Given the significance of metabolomics, in this review, we discussed different aspects of metabolomics particularly potentials of metabolomics as diagnostic biomarkers and use of small molecules for host targeted treatment for infectious diseases, and their scopes and challenges in personalized medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rahman and Schellhorn.)

Cyanobacteria (blue-green algae) can accumulate to form harmful algal blooms (HABs) on the surface of freshwater ecosystems under eutrophic conditions. Extensive HAB events can threaten local wildlife, public health, and the utilization of recreational waters. For the detection/quantification of cyanobacteria and cyanotoxins, both the United States Environmental Protection Agency (USEPA) and Health Canada increasingly indicate that molecular methods can be useful. However, each molecular detection method has specific advantages and limitations for monitoring HABs in recreational water ecosystems. Rapidly developing modern technologies, including satellite imaging, biosensors, and machine learning/artificial intelligence, can be integrated with standard/conventional methods to overcome the limitations associated with traditional cyanobacterial detection methodology. We examine advances in cyanobacterial cell lysis methodology and conventional/modern molecular detection methods, including imaging techniques, polymerase chain reaction (PCR)/DNA sequencing, enzyme-linked immunosorbent assays (ELISA), mass spectrometry, remote sensing, and machine learning/AI-based prediction models. This review focuses specifically on methodologies likely to be employed for recreational water ecosystems, especially in the Great Lakes region of North America.

Evolutionary relationships amongst Chlorobia and Ignavibacteria species/strains were examined using phylogenomic and comparative analyses of genome sequences. In a phylogenomic tree based on 282 conserved proteins, the named Chlorobia species formed a monophyletic clade containing two distinct subclades. One clade, encompassing the genera Chlorobaculum, Chlorobium, Pelodictyon, and Prosthecochloris, corresponds to the family Chlorobiaceae, whereas another clade, harboring Chloroherpeton thalassium, Candidatus Thermochlorobacter aerophilum, Candidatus Thermochlorobacteriaceae bacterium GBChlB, and Chlorobium sp. 445, is now proposed as a new family ( Chloroherpetonaceae fam. nov). In parallel, our comparative genomic analyses have identified 47 conserved signature indels (CSIs) in diverse proteins that are exclusively present in members of the class Chlorobia or its two families, providing reliable means for identification. Two known Ignavibacteria species in our phylogenomic tree are found to group within a larger clade containing several Candidatus species and uncultured Chlorobi strains. A CSI in the SecY protein is uniquely shared by the species/strains from this "larger Ignavibacteria clade". Two additional CSIs, which are commonly shared by Chlorobia species and the "larger Ignavibacteria clade", support a specific relationship between these two groups. The newly identified molecular markers provide novel tools for genetic and biochemical studies and identification of these organisms.

Cockayne syndrome group B protein (CSB), a member of the SWI/SNF superfamily, resides in an elongating RNA polymerase II (RNAPII) complex and regulates transcription elongation. CSB contains a C-terminal winged helix domain (WHD) that binds to ubiquitin and plays an important role in DNA repair. However, little is known about the role of the CSB-WHD in transcription regulation. Here, we report that CSB is dependent upon its WHD to regulate RNAPII abundance at promoter proximal pause (PPP) sites of several actively transcribed genes, a key step in the regulation of transcription elongation. We show that two ubiquitin binding-defective mutations in the CSB-WHD, which impair CSB's ability to promote cell survival in response to treatment with cisplatin, have little impact on its ability to stimulate RNAPII occupancy at PPP sites. In addition, we demonstrate that two cancer-associated CSB mutations, which are located on the opposite side of the CSB-WHD away from its ubiquitin-binding pocket, impair CSB's ability to promote RNAPII occupancy at PPP sites. Taken together, these results suggest that CSB promotes RNAPII association with PPP sites in a manner requiring the CSB-WHD but independent of its ubiquitin-binding activity. These results further imply that CSB-mediated RNAPII occupancy at PPP sites is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage.

For many bacteria, successful growth and survival depends on efficient adaptation to rapidly changing conditions. In Escherichia coli , the RpoS alternative sigma factor plays a central role in the adaptation to many suboptimal growth conditions by controlling the expression of many genes that protect the cell from stress and help the cell scavenge nutrients. Neither RpoS or the genes it controls are essential for growth and, as a result, the composition of the regulon and the nature of RpoS control in E. coli strains can be variable. RpoS controls many genetic systems, including those affecting pathogenesis, phenotypic traits including metabolic pathways and biofilm formation, and the expression of genes needed to survive nutrient deprivation. In this review, I review the origin of RpoS and assess recent transcriptomic and proteomic studies to identify features of the RpoS regulon in specific clades of E. coli to identify core functions of the regulon and to identify more specialized potential roles for the regulon in E. coli subgroups., (Copyright © 2020 Schellhorn.)

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.

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 identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet's groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources.

Aqueous-based coatings using combinations of polydopamine (PDA) (as bioadhesive) and grafted polyethylene glycol (PEG) (as antifouling agent) have been reported to reduce biofouling on multiple material surfaces. However, the achievable PEG grafting density and antifouling performance are limited, leaving exposed PDA to provide sites for attachment of proteins and cells. In the present work, we investigate the polymerization of dopamine on three substrate materials, polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass, to evaluate the utility of the PDA coatings for application to multiple materials. Additionally, we propose that the PDA-PEG method may be improved by "backfilling" with bovine serum albumin (BSA) as a blocker covering exposed PDA. AFM and ellipsometry studies revealed substantial differences in PDA thickness and roughness on each material despite their being modified under the same conditions. X-ray photoelectron spectroscopy (XPS) and water contact angle data revealed differences in PEG grafting on these materials as a consequence of varying PDA surface roughness, with the highest PEG coverage achieved on PC-PDA surfaces of intermediate roughness and lower PEG attachment on smoother PDMS-PDA surfaces. Fibrinogen adsorption experiments showed significantly less fouling on PDA-BSA surfaces compared to PDA-PEG for all three substrates, the larger BSA molecules presumably providing greater coverage of the PDA. On the PC and PDMS substrates, backfilling the PDA-PEG surfaces with BSA gave significant reductions in fibrinogen adsorption, with the lowest adsorption of 75 ng cm -2 achieved on PC-PDA-PEG/BSA.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Viruses are the most abundant microorganisms in the aquatic environment, yet the identification of viruses and assessing their diversity still remains a challenge. Here, we present a robust, routinely usable approach to identify viruses from two freshwater lakes of the lower Great Lakes region, Lake Ontario, and Lake Erie. We collected water samples from six different beaches of these two lakes during the summer period of 2012 and 2013, and separated into three distinct fractions, namely a bacterial fraction, a virus like particle (VLP) fraction, and a fraction of eDNA (environmental DNA). DNA extracted from all three fractions was sequenced and bioinformatic analyses of sequences revealed the presence of viruses from major viral families. The analyzed viral sequences were dominated by bacteriophage sequences, but also contained many plant and animal viruses. Within the context of this study, geographic location does not appear to have a major impact on viral abundance and diversity, since virome composition of both lakes were similar. Comparative analyses between eDNA and viral fractions showed that eDNA can be used in combination with VLP fractions to identify viruses from the environment.

The current taxonomy of the order Xanthomonadales is highly problematic and no comprehensive phylogenomic studies have been completed that include the most divergent members within the order. In this work, we have completed a phylogenomic analysis of a wide range of genomes, five of which were sequenced for the first time for this work, representing the vast majority of the diversity within the order Xanthomonadales. Using comparative genomic techniques, we have identified a large number of conserved signature inserts/deletions (CSIs) that are specifically found in different groups of related organisms, at different taxonomic levels, within the order. Our phylogenetic analyses do not support a monophyletic grouping of the members of the order Xanthomonadales and no CSIs were identified which are uniquely shared by all sequenced species within this order. However, our work has identified 10 CSIs which are specific to all members of the family Xanthomonadaceae and an additional 10 and 11 CSIs that are specific to one of two phylogenetically well-defined clades within the family Xanthomonadaceae. On the basis of the identified CSIs and the results of phylogenomic analyses, we propose a new taxonomic framework for the order Xanthomonadales. In this proposal, the families Algiphilaceae and Solimonadaceae (Nevskiaceae), which do not branch with the other members of the order Xanthomonadales, are transferred into the order Nevskiales ord. nov. The remaining members of the order Xanthomonadales are divided into two families: the family Xanthomonadaceae, containing the genus Xanthomonas and its closest relatives, and a new family, Rhodanobacteraceae fam. nov., containing the genus Rhodanobacter and its closest relatives. Additionally, we have also emended descriptions of the order Lysobacterales, the family Lysobacteraceae, and the family Nevskiaceae to indicate that they are earlier synonyms of the order Xanthomonadales, the family Xanthomonadaceae, and the family Solimonadaceae, respectively.

Detailed phylogenetic and comparative genomic analyses are reported on 140 genome sequenced cyanobacteria with the main focus on the heterocyst-differentiating cyanobacteria. In a phylogenetic tree for cyanobacteria based upon concatenated sequences for 32 conserved proteins, the available cyanobacteria formed 8-9 strongly supported clades at the highest level, which may correspond to the higher taxonomic clades of this phylum. One of these clades contained all heterocystous cyanobacteria; within this clade, the members exhibiting either true (Nostocales) or false (Stigonematales) branching of filaments were intermixed indicating that the division of the heterocysts-forming cyanobacteria into these two groups is not supported by phylogenetic considerations. However, in both the protein tree as well as in the 16S rRNA gene tree, the akinete-forming heterocystous cyanobacteria formed a distinct clade. Within this clade, the members which differentiate into hormogonia or those which lack this ability were also separated into distinct groups. A novel molecular signature identified in this work that is uniquely shared by the akinete-forming heterocystous cyanobacteria provides further evidence that the members of this group are specifically related and they shared a common ancestor exclusive of the other cyanobacteria. Detailed comparative analyses on protein sequences from the genomes of heterocystous cyanobacteria reported here have also identified eight conserved signature indels (CSIs) in proteins involved in a broad range of functions, and three conserved signature proteins, that are either uniquely or mainly found in all heterocysts-forming cyanobacteria, but generally not found in other cyanobacteria. These molecular markers provide novel means for the identification of heterocystous cyanobacteria, and they provide evidence of their monophyletic origin. Additionally, this work has also identified seven CSIs in other proteins which in addition to the heterocystous cyanobacteria are uniquely shared by two smaller clades of cyanobacteria, which form the successive outgroups of the clade comprising of the heterocystous cyanobacteria in the protein trees. Based upon their close relationship to the heterocystous cyanobacteria, the members of these clades are indicated to be the closest relatives of the heterocysts-forming cyanobacteria.

Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.

Bacterial adaptation to suboptimal nutrient environments, including host and/or extreme environments, is subject to complex, coordinated control involving many proteins and RNAs. Among the γ-proteobacteria, which includes many pathogens, the RpoS regulon has been a key focus for many years. Although the RpoS regulator was first identified as a growth phase-dependent regulator, our current understanding of RpoS is now more nuanced as this central regulator also has roles in exponential phase, biofilm development, bacterial virulence and bacterial persistence, as well as in stress adaptation. Induction of RpoS can also exert substantial metabolic effects by negatively regulating key systems including flagella biosynthesis, cryptic phage gene expression and the tricarboxylic acid cycle. Although core RpoS-controlled metabolic functions are conserved, there are substantial differences in RpoS regulation even among closely related bacteria, indicating that regulatory plasticity may be an important aspect of RpoS regulation, which is important in evolutionary adaptation to specialized environments.

Dietary antioxidants are essential nutrients that inhibit the oxidation of biologically important molecules and suppress the toxicity of reactive oxygen or nitrogen species. When the total antioxidant capacity is insufficient to quench these reactive species, oxidative damage occurs and contributes to the onset and progression of chronic diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer. However, epidemiological studies that examine the relationship between antioxidants and disease outcome can only identify correlative associations. Additionally, many antioxidants also have prooxidant effects. Thus, clinically relevant animal models of antioxidant function are essential for improving our understanding of the role of antioxidants in the pathogenesis of complex diseases as well as evaluating the therapeutic potential and risks of their supplementation. Recent progress in gene knockout mice and virus-based gene expression has potentiated these areas of study. Here, we review the current genetically modified animal models of dietary antioxidant function and their clinical relevance in chronic diseases. This review focuses on the 3 major antioxidants in the human body: vitamin C, vitamin E, and uric acid. We examine genetic models of vitamin C synthesis (guinea pig, Osteogenic Disorder Shionogi rat, Gulo(-/-) and SMP30(-/-) mouse mutants) and transport (Slc23a1(-/-) and Slc23a2(-/-) mouse mutants), vitamin E transport (Ttpa(-/-) mouse mutant), and uric acid synthesis (Uox(-/-) mouse mutant). The application of these models to current research goals is also discussed.

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Enteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmental Escherichia coli isolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. The rpoS sequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after plating rpoS+ isolates on succinate, RpoS mutants could be readily selected from environmental E. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than their rpoS+ isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects for rpoS mutations in environmental E. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival of E. coli in the environment.

The RpoS sigma factor in proteobacteria regulates genes in stationary phase and in response to stress. Although of conserved function, the RpoS regulon may have different gene composition across species due to high genomic diversity and to known environmental conditions that select for RpoS mutants. In this study, the distribution of RpoS homologs in prokaryotes and the differential dependence of regulon members on RpoS for expression in two gamma-proteobacteria (Escherichia coli and Pseudomonas aeruginosa) were examined. Using a maximum-likelihood phylogeny and reciprocal best hits analysis, we show that the RpoS sigma factor is conserved within gamma-, beta-, and delta-proteobacteria. Annotated RpoS of Borrelia and the enteric RpoS are postulated to have separate evolutionary origins. To determine the conservation of RpoS-dependent gene expression across species, reciprocal best hits analysis was used to identify orthologs of the E. coli RpoS regulon in the RpoS regulon of P. aeruginosa. Of the 186 RpoS-dependent genes of E. coli, 50 proteins have an ortholog within the P. aeruginosa genome. Twelve genes of the 50 orthologs are RpoS-dependent in both species, and at least four genes are regulated by RpoS in other gamma-proteobacteria. Despite RpoS conservation in gamma-, beta-, and delta-proteobacteria, RpoS regulon composition is subject to modification between species. Environmental selection for RpoS mutants likely contributes to the evolutionary divergence and specialization of the RpoS regulon within different bacterial genomes.

Understanding mechanisms of bacterial pathogenesis is critical for infectious disease control and treatment. Infection is a sophisticated process that requires the participation of global regulators to coordinate expression of not only genes coding for virulence factors but also those involved in other physiological processes, such as stress response and metabolic flux, to adapt to host environments. RpoS is a key response regulator to stress conditions in Escherichia coli and many other proteobacteria. In contrast to its conserved well-understood role in stress response, effects of RpoS on pathogenesis are highly variable and dependent on species. RpoS contributes to virulence through either enhancing survival against host defense systems or directly regulating expression of virulence factors in some pathogens, while RpoS is dispensable, or even inhibitory, to virulence in others. In this review, we focus on the distinct and niche-dependent role of RpoS in virulence by surveying recent findings in many pathogens.

Background: RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other gamma-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain., Results: The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (twofold, P<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions., Conclusion: Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains.

RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA, and rpoE) and genes in metabolic pathways (e.g., lysA, lysC, and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in Luria-Bertani media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media.

Citrobacter rodentium is a mouse enteropathogen that is closely related to Escherichia coli and causes severe colonic hyperplasia and bloody diarrhea. C. rodentium infection requires expression of genes of the locus of enterocyte effacement (LEE) pathogenicity island, which simulates infection by enteropathogenic E. coli and enterohemorrhagic E. coli in the human intestine, providing an effective model for studying enteropathogenesis. In this study we investigated the role of RpoS, the stationary phase sigma factor, in virulence in C. rodentium. Sequence analysis showed that the rpoS gene is highly conserved in C. rodentium and E. coli, exhibiting 92% identity. RpoS was critical for survival under heat shock conditions and during exposure to H(2)O(2) and positively regulated the expression of catalase KatE (HPII). The development of the RDAR (red dry and rough) morphotype, an important virulence trait in E. coli, was also mediated by RpoS in C. rodentium. Unlike E. coli, C. rodentium grew well in the mouse colon, and the wild-type strain colonized significantly better than rpoS mutants. However, a mutation in rpoS conferred a competitive growth advantage over the wild type both in vitro in Luria-Bertani medium and in vivo in the mouse colon. Survival analysis showed that the virulence of an rpoS mutant was attenuated. The expression of genes on the LEE pathogenicity island, which are essential for colonization and virulence, was reduced in the rpoS mutant. In conclusion, RpoS is important for the stress response and is required for full virulence in C. rodentium.

Inability to synthesize vitamin C, because of a deficiency in gulonolactone oxidase (GULO) expression, is a genetic deficiency shared by a small number of animals including humans. Although the most overt symptom of vitamin C deficiency, scurvy, can be readily corrected by modest consumption of vitamin C, there is increasing interest in the effect of high-level administration in treating human disease. Using a previously derived Gulo-expressing vector, which produces murine GULO under the control of the murine cytomegalovirus (mCMV) promoter, we constructed and validated a recombinant helper-dependent adenovirus (HDAd-mCMV-Gulo) that can be used to correct this genetic defect. A human liver cell line (Hep G2) infected with the HDAd-mCMV-Gulo vector expressed GULO in a time- and gene dose-dependent manner. These cells also produced ascorbic acid when exogenous gulonolactone was supplemented in the medium. Likewise, Gulo(-/-) mice treated with HDAd-mCMV-Gulo at 2 x 10(11) VP expressed GULO in the liver and produced ascorbic acid. Serum ascorbic acid concentrations in Gulo(-/-) mice injected with GULO-expressing HDAd were elevated to levels comparable to those of wild-type mice (62 +/- 15 microM) after 4 days of infection and were maintained at significantly higher levels compared with those in untreated Gulo(-/-) mice for at least 23 days. A similar elevation was observed in urine and tissue ascorbic acid concentrations in vector-treated animals. In conclusion, we demonstrate here that gene therapeutic HDAd-mCMV-Gulo vectors can mediate the expression of GULO and endogenous production of ascorbic acid in human cells and in Gulo(-/-) transgenic mice. Taken together, these data show that a gene therapy approach can be successfully employed in the treatment and further study of vitamin C deficiency in scurvy-prone mammals.

RpoS is a major regulator of genes required for adaptation to stationary phase in E. coli. However, the exponential phase expression of some genes is affected by rpoS mutation, suggesting RpoS may also have an important physiological role in growing cells. To test this hypothesis, we examined the regulatory role of RpoS in exponential phase using both genomic and biochemical approaches. Microarray expression data revealed that, in the rpoS mutant, the expression of 268 genes was attenuated while the expression of 24 genes was enhanced. Genes responsible for carbon source transport (the mal operon for maltose), protein folding (dnaK and mopAB), and iron acquisition (fepBD, entCBA, fecI, and exbBD) were positively controlled by RpoS. The importance of RpoS-mediated control of iron acquisition was confirmed by cellular metal analysis which revealed that the intracellular iron content of wild type cells was two-fold higher than in rpoS mutant cells. Surprisingly, many previously identified RpoS stationary-phase dependent genes were not controlled by RpoS in exponential phase and several genes were RpoS-regulated only in exponential phase, suggesting the involvement of other regulators. The expression of RpoS-dependent genes osmY, tnaA and malK was controlled by Crl, a transcriptional regulator that modulates RpoS activity. In summary, the identification of a group of exponential phase genes controlled by RpoS reveals a novel aspect of RpoS function.

Vitamin C is required for collagen synthesis and biosynthesis of certain hormones and recommended dietary intake levels are largely based these requirements. However, to function effectively as an antioxidant (or a pro-oxidant), relatively high levels of this vitamin must be maintained in the body. The instability of vitamin C combined with its relatively poor intestinal absorption and ready excretion from the body reduce physiological availability of this vitamin. This inability to maintain high serum levels of vitamin C may have serious health implications and is particularly relevant in the onset and progression of degenerative disease, such as cancer and cardiovascular disease (CVD), which have a strong contributing oxidative damage factor. In this review, we examine recent studies on the regulation of transport mechanisms for vitamin C, related clinical ramifications, and potential implications in high-dose vitamin C therapy. We also evaluate recent clinical and scientific evidence on the effects of this vitamin on cancer and CVD, with focus on the key mechanisms of action that may contribute to the therapeutic potential of this vitamin in these diseases. Several animal models that could be utilized to address unresolved questions regarding the feasibility of vitamin C therapy are also discussed.

Catalase is a commonly assayed enzyme found in many bacteria and eukaryotes. In this report, we examined the applicability of a kinetic microassay to quantify catalase from two different sources. The assay was found to be linear over a wide range (0.1-1.0 units), but was limited at high values (>1 unit) by oxygen evolution. Nonetheless, the microassay allows simultaneous evaluation of many samples (up to 96) in a short time (<5 min) and is thus well-suited to applications, such as high-throughput screening, where many parallel assays are required.

Man, with other primates, lost the ability to synthesize vitamin C through an inactivating mutation of the gene encoding gulonolactone oxidase (GULO) millions of years ago. Though the consequences of this prehistoric loss must have been favorable (and thus selected for) at the population level, the inability to produce vitamin C may have serious health implications for modern humans, especially for those conditions in which antioxidants (like vitamin C) have been implicated as potential therapeutic agents. Two general types of recent findings regarding vitamin C have made re-evaluation of this important nutrient imperative. First, vitamin C is now known to be involved in several novel physiological phenomena including stem cell differentiation and respiratory development, which likely require pharmacological levels of vitamin C. Secondly, the growing recognition that many ageing-related diseases, including heart disease, neural degeneration and cancer, may have a contributing oxidative damage factor that might be reduced by dietary antioxidants such as vitamin C. In this paper, we hypothesize that high serum-level vitamin C provides important, broad-ranging therapeutic benefits in treating ageing-related degenerative diseases. This hypothesis can be readily tested using traditional and newly-developed genetically-engineered animal models.

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.

A series of Escherichia coli mutants, constructed originally by Cupples and Miller [C.G. Cupples, J.H. Miller, A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345-5349], provides a unique system for quantifying base-change mutations, and the repair processes that limit their establishment, in bacteria under selective and non-selective conditions. We focussed on one strain in which a T-->G replacement inactivates the lacZ gene. Reversions of this strain can occur through oxidation of G, leading to G-->T transversions. We show that spontaneous reversions occurred both in lactose (selective) and glucose (non-selective) medium. The number of revertants per viable cell was much greater in medium containing lactose or both sugars than glucose alone. In glucose medium, the rate of reversion was highest below 0.6% glucose and strongly inhibited at and above that level. Evidence that reversions occurred through G-->T transversions in both lactose and glucose media came from two observations: by sequence analysis of a series of revertants and by comparing the reversion rates in strains possessing and lacking the mutM gene (encoding formamidopyrimidine DNA glycosylase, FPG). However, the rate of reversion was stimulated by reducing O2 to 1% and inhibited or delayed by increasing O2 to 90%. In mutM- cells grown on glucose medium, the proportion of revertants increased over a 5-day period. In contrast, in mutM+ cells, revertants appeared primarily during the first 2-3 days after plating; few new revertants appeared in the following days. These data imply that base excision repair initiated by FPG was less effective in the first 2 days and more effective later in stationary phase.

Background: Arginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression., Results: In this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 microM L-arginine), a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited), levels of expression were only one third of those observed in deltaargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in deltaargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS., Conclusion: The results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

The alternative sigma factor RpoS controls the expression of many stationary-phase genes in Escherichia coli and other bacteria. Though the RpoS regulon is a large, conserved system that is critical for adaptation to nutrient deprivation and other stresses, it remains incompletely characterized. In this study, we have used oligonucleotide arrays to delineate the transcriptome that is controlled by RpoS during entry into stationary phase of cultures growing in rich medium. The expression of known RpoS-dependent genes was confirmed to be regulated by RpoS, thus validating the use of microarrays for expression analysis. The total number of positively regulated stationary-phase genes was found to be greater than 100. More than 45 new genes were identified as positively controlled by RpoS. Surprisingly, a similar number of genes were found to be negatively regulated by RpoS, and these included almost all genes required for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a physically contiguous group of genes located in the Rac prophage region. Negative regulation by RpoS is thus much more extensive than has previously been recognized, and is likely to be an important contributing factor to the competitive growth advantage of rpoS mutants reported in previous studies.

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.

Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene. The reasons for this apparent selective loss and the nature of the selective agent are not well understood. In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations. Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function. All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function. Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected. To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template. Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene. Types of mutations detected included single or multiple base deletions, insertions, and transversions. No transition mutations were identified. All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+. Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level. These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene.

l-Gulono-gamma-lactone oxidase (GULO) is a critical enzyme present in most mammalian species that is required for the terminal step in vitamin C biosynthesis. Primates are absolutely dependent on exogenously supplied dietary vitamin C due to inactivation of the Gulo gene by mutation over 40 million years ago. In this study, we report the cloning and expression of the murine l-gulono-gamma-lactone oxidase cDNA and gene. The cDNA (2.3 kb) encodes an open reading frame of 440 amino acids that shows high homology to the rat l-gulono-gamma-lactone oxidase (>94%). The Gulo gene is 22 kb long and contains 12 exons. The 11 introns range in size from 479 to 5641 bp. Northern blot analysis revealed high expression of Gulo transcript in the liver. To investigate whether metabolic loss of vitamin C biosynthesis in human cells can be corrected by heterologous expression of GULO, we constructed a first-generation adenoviral vector expressing the murine GULO cDNA under the transcriptional control of the murine cytomegalovirus (MCMV) early promoter. Low rescue efficiency of Gulo-expressing adenoviral constructs and reduced viral growth in HEK293 cells were observed, suggesting that overexpression of Gulo may be inhibitory to cell growth. Placement of a removable stuffer fragment flanked by lox sites between the MCMV promoter and the Gulo gene resulted in efficient vector rescue and normal viral replication in parental HEK293 cells and high-level expression of Gulo in HEK293 cells expressing Cre recombinase. Cells infected with Gulo-expressing vectors overexpressed an FAD-containing protein that corresponded in size to that predicted for recombinant GULO protein and expressed a functional enzyme as measured by the conversion of l-gulono-gamma-lactone to ascorbic acid in cell-free extracts. The cloning of the murine Gulo cDNA and the construction of Gulo-expressing adenoviral vectors are vital steps toward determining the role of vitamin C in basic metabolism and in disease.

RpoS, an alternative sigma factor produced by many gram-negative bacteria, primarily controls genes that are expressed in stationary phase in response to nutrient deprivation. To test the idea that induction of RpoS in the exponential phase, when RpoS is not normally expressed, increases RpoS-dependent gene expression, we constructed a plasmid carrying the rpoS gene under the control of an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible T7lac promoter. Northern and Western analyses revealed that levels of RpoS mRNA and protein, respectively, increased in response to the inducer IPTG. Assays of changes in RpoS-dependent functions (catalase activity and glycogen accumulation), confirmed that induced RpoS was functional in exponential phase and was sufficient for the expression of RpoS-dependent functions. Controlled expression of RpoS and RpoS-dependent genes by plasmid-encoded rpoS may thus offer a useful tool for the study of RpoS-dependent gene expression.

We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents.

The stationary phase expression of many conserved, adaptive bacterial proteins is dependent on RpoS, a second vegetative sigma factor. The regulation of RpoS itself, however, is complex and not fully understood, particularly at the level of transcription. In this report, we show that the observed hydrogen peroxide sensitivity of a mutant defective in expression of barA, a bacterial virulence factor, can be explained by a reduction in catalase activity, an RpoS-controlled function. Levels of katE mRNA, encoding the major catalase of Escherichia coli, were much lower in the barA mutant, suggesting that BarA is required for the expression of this RpoS-regulated gene. Expression of another RpoS-regulated gene, osmY, was also found to be severely reduced in the barA mutant. Employing Western analyses with anti-RpoS antisera and Northern analyses using probes specific for rpoS, we found that BarA is required for the exponential phase induction of RpoS itself. Operon lacZ fusion expression studies and Northern analyses indicate that BarA itself is maximally expressed in early exponential phase cultures immediately preceding the transcriptional induction of RpoS. Results of primer extension studies indicate that exponential phase expression from the rpoSp1 promoter is reduced by more than 85% in a barA mutant but could be efficiently complemented by a plasmid-borne copy of barA in trans. These results suggest that regulatory signals that are operant in exponentially growing cultures play an important role in effecting stationary phase gene expression.

In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane-bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (sigmas), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10-fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild-type and rpoS- strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.