RpoS Levels Research Articles

An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation.

Expression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation. Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate. In contrast, ompC expression was higher under N limitation. The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes. The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures. A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor. RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC. Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression. Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate. Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways. pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation. Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC. In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins. Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.

Gene expression and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.

Gene expression and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms.

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.

Role of CspC and CspE in regulation of expression of RpoS and UspA, the stress response proteins in Escherichia coli.

Nine homologous proteins, CspA to CspI, constitute the CspA family of Escherichia coli. Recent studies are aimed at elucidating the individual cellular functions of these proteins. Two members of this family, CspC and CspE, are constitutively produced at 37 degrees C. In the present study, these two proteins were evaluated for their cellular role(s). The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE. RpoS is a regulatory element for osmY and dps. Further analysis showed a larger amount and greater stability of the rpoS mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE. This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself. Indeed, this upregulation is lost in the Delta rpoS strain. Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC. The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses. In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of E. coli.

Global adaptations resulting from high population densities in Escherichia coli cultures.

The scope of population density effects was investigated in steady-state continuous cultures of Escherichia coli in the absence of complications caused by transient environmental conditions and growth rates. Four distinct bacterial properties reflecting major regulatory and physiological circuits were analyzed. The metabolome profile of bacteria growing at high density contained major differences from low-density cultures. The 10-fold-elevated level of trehalose at higher densities pointed to the increased role of the RpoS sigma factor, which controls trehalose synthesis genes as well as the general stress response. There was an eightfold difference in RpoS levels between bacteria grown at 10(8) and at 10(9) cells/ml. In contrast, the cellular content of the DNA binding protein H-NS, controlling many genes in concert with RpoS, was decreased by high density. Since H-NS and RpoS also influence porin gene expression, the influence of population density on the intricate regulation of outer membrane composition was also investigated. High culture densities were found to strongly repress ompF porin transcription, with a sharp threshold at a density of 4.4 x 10(8) cells/ml, while increasing the proportion of OmpC in the outer membrane. The density-dependent regulation of ompF was maintained in rpoS or hns mutants and so was independent of these regulators. The consistently dramatic changes indicate that actively growing, high-density cultures are at least as differentiated from low-density cultures as are exponential- from stationary-phase bacteria.

Destruction by association

RpoS is a secondary sigma factor in Escherichia coli that controls the synthesis of many stationary phase and osmotically induced genes. Its expression is controlled at the levels of transcription, translation and protein stability. Although rapidly degraded in actively growing cells, RpoS is greatly stabilized at stationary phase and upon osmotic upshift. RssB, also referred to as SprE (or MviA in Salmonella typhimurium), negatively regulates RpoS stability in a ClpXP-dependent fashion. As RssB does not affect the levels or activity of ClpXP, it has been postulated that it alters the susceptibility of RpoS to ClpXP degradation. Despite its unique output domain, RssB clearly belongs to the family of response regulators.A turnover element directly or indirectly recognized by ClpXP was postulated to exist in RpoS between residues 172 and 188. Now, using site-directed mutagenesis, Becker et al.1xRegulation of RpoS proteolysis in Escherichia coli: the response regulator RssB is a recognition factor that interacts with the turnover element in RpoS. Becker, G. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6439–6444Crossref | PubMed | Scopus (129)See all References1 have localized this element to K173. K173, E174, V177 and Y178 were substituted with the corresponding residues present in the related, yet stable, RpoD. Of those tested, only the single K173E and the double K173E/V177K mutations eliminated RpoS turnover. Although both the K173E and V177K mutants accumulate RpoS in growing cells, RpoS levels can be further increased in the absence of RssB only for the V177K mutant. The in vitro binding of RssB to RpoS is stimulated in the presence of acetyl phosphate and can be disrupted by the K173E mutation, supporting the hypothesis that this residue is a site of interaction with RssB.Interestingly, the region including K173 is also important for RpoS activity. As the binding of RssB to RpoS is modulated by phosphorylation, RssB is not only able to tag RpoS for proteolysis but also to interfere with its function, even under conditions where proteolysis is inhibited2xRegulation of proteolysis of the stationary-phase sigma factor RpoS. Zhou, Y. et al. J. Bacteriol. 1998; 180: 1154–1158PubMedSee all References2. Co-immunoprecipitation of RpoS and RssB from cells should allow investigators to determine whether ClpX is also co-purified and, if so, if this is dependent on the function of RssB, its phosphorylation state or the growth phase. In the future, the isolation of functional, yet RssB-insensitive, RpoS mutants, locked gain-of-function RssB mutants, as well as second-site RssB suppressors of the existing RpoS mutants, will help define the novel output domain in RssB. Perhaps a unifying name for this unique regulator is long overdue.

The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE.

Synthesis of the OmpF porin of Escherichia coli is regulated in response to environmental and growth phase signals. In order to identify constituents of the various regulatory pathways involved in modulating ompF transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased ompF'-lacZ activity were identified as previously described. Mutations mapping to a previously identified gene of unknown function, lrhA, were obtained. We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of ompF. In addition to altered growth phase regulation of ompF, these lrhA mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels. We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease. LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE.

High levels of transcription factor RpoS (σ S) in mviA mutants negatively affect 1,2-propanediol-dependent transcription of the cob/pdu regulon of Salmonella typhimurium LT2

Expression of the cobalamin biosynthetic ( cob) and 1,2-propanediol utilization ( cob/pdu) regulon of Salmonella typhimurium LT2 is controlled at the transcriptional level by global and specific regulatory proteins. In this paper we show that mutations in the mviA gene negatively affect cob/pdu transcription in response to 1,2-propanediol in the environment. The effects of mviA mutations were consistent with its role in the regulation of RpoS levels in the cell. Null mutations in rpoS eliminated the negative effect of mviA mutations on cob/pdu transcription, and restored growth on succinate, propionate and 1,2-propanediol. In addition, mviA mutants were deficient in the utilization of succinate, propionate and 1,2-propanediol as carbon and energy sources.

High levels of transcription factor RpoS (sigma S) in mviA mutants negatively affect 1,2-propanediol-dependent transcription of the cob/pdu regulon of Salmonella typhimurium LT2.

Expression of the cobalamin biosynthetic (cob) and 1,2-propanediol utilization (cob/pdu) regulon of Salmonella typhimurium LT2 is controlled at the transcriptional level by global and specific regulatory proteins. In this paper we show that mutations in the mviA gene negatively affect cob/pdu transcription in response to 1,2-propanediol in the environment. The effects of mviA mutations were consistent with its role in the regulation of RpoS levels in the cell. Null mutations in rpoS eliminated the negative effect of mviA mutations on cob/pdu transcription, and restored growth on succinate, propionate and 1,2-propanediol. In addition, mviA mutants were deficient in the utilization of succinate, propionate and 1,2-propanediol as carbon and energy sources.

Importance of RpoS and Dps in survival of exposure of both exponential- and stationary-phase Escherichia coli cells to the electrophile N-ethylmaleimide.

The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.

Roles of DnaK and RpoS in starvation-induced thermotolerance of Escherichia coli.

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these dnaK mutant phenotypes closely resemble those of rpoS (sigma38) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30 degrees C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (sigma32) suppressors of the dnaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved E. coli.

Central regulatory role for the RpoS sigma factor in expression of Salmonella dublin plasmid virulence genes.

The plasmid virulence genes spvABCD of Salmonella spp. are regulated by SpvR and the stationary-phase sigma factor RpoS. The transcription of spv genes is induced during the post-exponential phase of bacterial growth in vitro. We sought to investigate the relationship between growth phase and RpoS in spv regulation. rpoS insertion mutations were constructed in S. dublin Lane and plasmid-cured LD842 strains, and the mutants were found to be attenuated for virulence and deficient in spv gene expression. We utilized the plasmid pBAD::rpoS to express rpoS independent of the growth phase under the control of the arabinose-inducible araBAD promoter. SpvA expression was induced within 2 h after the addition of 0.1% arabinose, even when bacteria were actively growing. This suggested that the level of RpoS, instead of the growth phase itself, controls induction of the spv genes. However, RpoS did not activate transcription of spvA in the absence of SpvR protein. Using a constitutive tet promoter to express spvR, we found that the spvA gene can be partially expressed in the rpoS mutant, suggesting that RpoS is required for SpvR synthesis. We confirmed that spvR is poorly expressed in the absence of RpoS. With an intact rpoS gene, spvR expression is not dependent on an intact spvR gene but is enhanced by spvR supplied in trans. We propose a model for Salmonella spv gene regulation in which both RpoS and SpvR are required for maximal expression at the spvR and spvA promoters.

The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium.

The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor sigma S encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon sigma S for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, sigma S proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by sigma S and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When sigma S concentrations are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.

Cloning, analysis and expression of an rpoS homologue gene from Pseudomonas aeruginosa PAO1.

A homologue of the rpoS gene of Escherichia coli was cloned from Pseudomonas aeruginosa PAO1 by hybridization with an oligodeoxyribonucleotide probe designed from an amino-acid stretch conserved among the principal sigma factors of eubacteria. Two open reading frames, the pcm gene and the orf-297 of unknown function, were found in the upstream region of rpoS, and in the same order as in E. coli. The rpoS gene of P. aeruginosa was expressed in E. coli and complemented the catalase deficiency of the rpoS mutant of E. coli. The RpoS protein of P. aeruginosa was identified by Western blot analysis in both P. aeruginosa (Pa) and the transformed E. coli. Levels of RpoS of Pa increased drastically at the onset of the stationary growth phase.