Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933 (original) (raw)
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Role of RpoS in Virulence of Pathogens
Infection and Immunity, 2010
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.
Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains
Applied and Environmental Microbiology, 2006
The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher -galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.
Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12
Molecular Genetics and Genomics, 2004
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. Keywords Stationary-phase regulation AE rpoS AE Starvation AE Stress AE Oligonucleotide arrays Communicated by A. M. Hirsch
RpoS in enterobacteria: genetic studies and physiological implications
2005
RpoS, the alternative sigma factor s , is important for bacterial survival under extreme conditions. Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence. To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species. A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri. Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E. coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers. The rpoS gene from E. cloacae and from K. cryocrescens was cloned and sequenced and a mutant allele was constructed in E. cloacae. Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential-and stationary-phase cells of both strains. E. cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.
Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli
Journal of …, 1998
During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than 100 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-lacZ operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an rpoS/rpoS ؉ merodiploid (rpoS background transformed with an rpoS-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an rpoS background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in Escherichia coli do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to otsA, katE, ecnB, and osmY-genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely, gabP, yhiUV, o371, o381, f186, and o215. Like many other free-living bacteria, Escherichia coli lives in environments that may change rapidly with respect to both nutrients and physical conditions. To survive stresses associated with starvation, E. coli expresses many stationary-phasespecific genes whose expression depends largely on an alternative sigma factor, s , encoded by rpoS (27, 30). Inactivation of this gene renders the cell sensitive to heat shock (25, 29), oxidative stress (25, 29), osmotic challenge (29), and near-UV light (40). Proteins that depend on RpoS include catalase HPII (33, 39, 42) and catalase HPI (32), exonuclease III (39), penicillin-binding proteins (15), and osmoprotective proteins (21, 22, 53). RpoS is required for virulence (17) and acid tolerance (6) in Salmonella typhimurium. Although the signal(s) giving rise to increased expression of RpoS itself is not completely understood, homoserine lactone (23), UDP-6-glucose (10), and weak acids, such as acetate (42), have been shown to be inducers of RpoS. Several approaches have been used to enumerate and identify RpoS-regulated functions. Many of these genes, however, are probably still unidentified. Two-dimensional gel electrophoresis studies of proteins expressed in wild-type and rpoS strains have revealed that the RpoS regulon is quite large (30). Mutagenesis with random lacZ (16, 51) or lux insertions (46), coupled with screening for RpoS-related characteristic phenotypes, has also been successfully employed to identify new RpoS-regulated genes (51). However, unlike other regulons, the RpoS regulon does not have a single unifying characteristic or differentiating phenotype that all members share. These factors, in addition to its suspected large size, have delayed complete characterization of the regulon. To circumvent the problems associated with the characteristics described above, we have employed a mutant identification scheme in which an rpoS null allele is introduced into strains containing random promoter-lacZ fusions to directly identify RpoS dependency. Since this procedure does not rely on a phenotype specific for the regulon (e.g., carbon starvation), this method should be of general use in the identification of members of any regulon for which a null allele of a positive-acting regulator is available. MATERIALS AND METHODS Bacterial strains, phage, and plasmid. The bacterial strains, phage, and plasmid used in this study are listed in Table 1. Chemicals and media. All chemicals were supplied by either Fisher Scientific, Ltd.
The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli
The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ(S)-dependent genes when compared to the wild-type rpoS(+) strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ(70)-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.
Polymorphism and selection of rpoS in pathogenic Escherichia coli
BMC Microbiology, 2009
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.
Applied and Environmental Microbiology, 2011
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.
RpoS-Regulated Genes of Escherichia coli Identified by Random lacZ Fusion Mutagenesis
Journal of Bacteriology, 2004
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.
Journal of Bacteriology, 2002
The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor ( S ), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose-and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher S levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on 70 and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than S for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.