Transcriptional responses of Escherichia coli rpoS- BW25113 vs. wild-type BW25113 under 15% ethanol shock in log phase
ABSTRACT: RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli.RpoS also acts as a global regulator for stress control of gene expression, and actually dose so in log stage and stationary stage. To further understand the effect of environmental stresses on in ethanologenic strains, DNA microarrys was used to analyze the expression profiles of E. coli and its rpoS mutant strain. Overall design: BW25113（rpoS-）and BW25113 were selected at log stages and stationary stage of early development for RNA extraction and hybridization on Affymetrix microarrays. To that end, we hand-selected BW25113 and BW25113（rpoS-） according at different treatments: BW25113（rpoS-）at log stage (BW25113(rpoS-) log), BW25113 at log stage (BW25113 log),BW25113（rpoS-）atstationary stage ( BW25113(rpoS-) stationary), BW25113 at stationary stage ( BW25113 stationary)
INSTRUMENT(S): [E_coli_2] Affymetrix E. coli Genome 2.0 Array
Project description:RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli.RpoS also acts as a global regulator for stress control of gene expression, and actually dose so in log stage and stationary stage. To further understand the effect of environmental stresses on in ethanologenic strains, DNA microarrys was used to analyze the expression profiles of E. coli and its rpoS mutant strain. BW25113（rpoS-）and BW25113 were selected at log stages and stationary stage of early development for RNA extraction and hybridization on Affymetrix microarrays. To that end, we hand-selected BW25113 and BW25113（rpoS-） according at different treatments: BW25113（rpoS-）at log stage (BW25113(rpoS-) log), BW25113 at log stage (BW25113 log),BW25113（rpoS-）atstationary stage ( BW25113(rpoS-) stationary), BW25113 at stationary stage ( BW25113 stationary)
Project description:Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.
Project description: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.
Project description:Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.
Project description:The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS(+) wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH(2)O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log(10) in sterile ddH(2)O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log(10) in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS(+) and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.
Project description:Transcriptional profiling of L. pneumophila JR32 (wild-type) and rpoS- (LM1376) strains grown to stat phase compared to cultures grown to log phase Keywords: growth phase Overall design: For each of the two strains, six test samples and six reference samples were analyzed and averaged. The test sample being JR32 or rpoS- in stat phase and the reference sample being JR32 or rpoS- in log phase.
Project description:To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.
Project description:Balancing of reducing equivalents is a fundamental issue in bacterial metabolism and metabolic engineering. Mutations in the key metabolic genes ldhA and pflB of Escherichia coli are known to stall anaerobic growth and fermentation due to a buildup of intracellular NADH. We observed that the rate of spontaneous mutation in E. coli BW25113 (DeltaldhA DeltapflB) was an order of magnitude higher than that in wild-type (WT) E. coli BW25113. We hypothesized that the increased mutation frequency was due to an increased NADH/NAD(+) ratio in this strain. Using several redox-impaired strains of E. coli and different redox conditions, we confirmed a significant correlation (P < 0.01) between intracellular-NADH/NAD(+) ratio and mutation frequency. To identify the genetic basis for this relationship, whole-genome transcriptional profiles were compared between BW25113 WT and BW25113 (DeltaldhA DeltapflB). This analysis revealed that the genes involved in DNA repair were expressed at significantly lower levels in BW25113 (DeltaldhA DeltapflB). Direct measurements of the extent of DNA repair in BW25113 (DeltaldhA DeltapflB) subjected to UV exposure confirmed that DNA repair was inhibited. To identify a direct link between DNA repair and intracellular-redox ratio, the stringent-response-regulatory gene relA and the global-stress-response-regulatory gene rpoS were deleted. In both cases, the mutation frequencies were restored to BW25113 WT levels.