RpoS in enterobacteria: genetic studies and physiological implications (original) (raw)
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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.
rpoS Mutants in Archival Cultures of Salmonella enterica Serovar Typhimurium
Journal of Bacteriology, 2000
Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.
Journal of bacteriology, 2014
The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, wit...
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.
Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933
BMC Genomics, 2009
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.
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.
Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes
Journal of Applied Microbiology, 1999
A . F ER R EI RA , L. RE N DA NO , M. WI E DM AN N AN D K . J. BO O R. 1999. The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical s S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of s S sequence conservation to include other Gram-negative bacteria, the E. coli s S sequences were compared with those from diverse Gram-negative organisms; s S sequence identities ranged from 50·2 to 99·7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates ×10% after 2 h exposure to pH 2·5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.
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.
Current Research in Microbiology and Infection, 2020
This study aimed to detect presence of RpoS gene in Escherichia coli O157:H7 and non-O157 and investigate their survival pattern in different water treatment methods. A total of fifteen serologically and molecularly identified E. coli was selected from a previous work, out of which eight were Escherichia coli O157 and seven were E. coli non-O157. From among these, S30 and S89 identified isolates served as presentative E. coli O157:H7 and non-O157 respectively for survival studies. The water treatment methods used employed included: use of silver, lime, storage, acidification (low pH), high temperature and Moringa oleifera. Survival pattern of the test organisms under the influence of these methods were carried out using standard techniques. Molecular detection of stress response gene, RpoS, in the fifteen (15) test organisms was performed following manufacturer’s instruction. Results showed that for both test organisms, silver was bactericidal at high concentration while storage all...
Induction of RpoS Degradation by the Two-Component System Regulator RstA in Salmonella enterica
Journal of Bacteriology, 2007
Bacterial survival in diverse and changing environments relies on the accurate interplay between different regulatory pathways, which determine the design of an adequate adaptive response. The proper outcome depends on a precise gene expression profile generated from the finely tuned and concerted action of transcriptional factors of distinct regulatory hierarchies. Salmonella enterica serovar Typhimurium harbors multiple regulatory systems that are crucial for the bacterium to cope with harsh extra- and intracellular environments. In this work, we found that the expression of Salmonella RstA, a response regulator from the two-component system family, was able to downregulate the expression of three RpoS-controlled genes ( narZ , spvA , and bapA ). Furthermore, this downregulation was achieved by a reduction in RpoS cellular levels. The alternative sigma factor RpoS is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host ...