Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933

<p>Abstract</p> <p>Background</p> <p>RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in <it>Escherichia coli </it>and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including <it>Salmonella </it>and <it>Vibrio </it>species. Though well characterized in non-pathogenic <it>E. coli </it>K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. <it>E</it>. <it>coli </it>O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of <it>E. coli </it>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 <it>rpoS </it>mutants of the <it>E. coli </it>O157:H7 EDL933 type strain.</p> <p>Results</p> <p>The <it>rpoS </it>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 <it>rpoS </it>resulted in impaired expression of genes responsible for stress response (e.g., <it>gadA</it>, <it>katE </it>and <it>osmY</it>), arginine degradation (<it>astCADBE</it>), putrescine degradation (<it>puuABCD</it>), fatty acid oxidation (<it>fadBA </it>and <it>fadE</it>), and virulence (<it>ler</it>, <it>espI </it>and <it>cesF</it>). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the <it>rpoS </it>mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the <it>rpoS </it>mutants under LEE induction conditions.</p> <p>Conclusion</p> <p>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.</p

Polymorphism and selection of rpoS in pathogenic Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Though RpoS is important for survival of pathogenic <it>Escherichia coli </it>in natural environments, polymorphism in the <it>rpoS </it>gene is common. However, the causes of this polymorphism and consequential physiological effects on gene expression in pathogenic strains are not fully understood.</p> <p>Results</p> <p>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 <it>E. coli </it>(VTEC) strains. Mutants (Suc⁺⁺) forming large colonies on succinate were isolated at a frequency of 10^-8mutants 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 <it>rpoS </it>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 <it>rpoS </it>was detected.</p> <p>Conclusion</p> <p>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 <it>rpoS </it>polymorphisms among <it>E. coli </it>isolates.</p

Stationary phase expression of the arginine biosynthetic operon argCBH in Escherichia coli

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

Role of RpoS in Virulence of Pathogens▿

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

Controlled Expression of an rpoS Antisense RNA Can Inhibit RpoS Function in Escherichia coli

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

Role of RpoS in the Virulence of Citrobacter rodentium▿

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 H2O2 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

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

Fecal pollution, commonly detected in untreated or less treated sewage, is associated with health risks (e.g., waterborne diseases and antibiotic resistance dissemination), ecological issues (e.g., release of harmful gases in fecal sludge composting, proliferative bacterial/algal growth due to high nutrient loads) and economy losses (e.g., reduced aqua farm harvesting). Therefore, the discharge of untreated domestic sewage to the environment and its agricultural reuse are growing concerns. The goals of fecal pollution detection include fecal waste source tracking and identifying the presence of pathogens, therefore assessing potential health risks. This review summarizes available biological fecal indicators focusing on host specificity, degree of association with fecal pollution, environmental persistence, and quantification methods in fecal pollution assessment. The development of practical tools is a crucial requirement for the implementation of mitigation strategies that may help confine the types of host-specific pathogens and determine the source control point, such as sourcing fecal wastes from point sources and nonpoint sources. Emerging multidisciplinary bacterial enumeration platforms are also discussed, including individual working mechanisms, applications, advantages, and limitations