RpoS-dependent Genes Research Articles

Low temperature (23 °C) increases expression of biofilm-, cold-shock- and RpoS-dependent genes in Escherichia coli K-12

Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 degrees C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 degrees C. Another category of genes more highly expressed at 23 degrees C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 degrees C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 degrees C. Increased expression at 23 degrees C compared to 37 degrees C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and viability at 4 degrees C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 degrees C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

Altered acetate metabolism and biomass production in several Escherichia coli mutants lacking rpoS-dependent metabolic pathway genes

The stress responsive sigma factor RpoS regulates the expression of tktB and talAgenes of the non-oxidative pentose phosphate (PP) pathway, and fumCand acnA genes of the TCA cycle at the stationary phase of growth. In the present study, batch cultivations were performed using tktB, talA, fumC or acnA-knockout mutants of Escherichia coli to observe the metabolic changes at different phases of growth compared to the wild type strain. Although the specific growth rates of the mutants were similar to the wild type, acetate yield was nearly half in all mutants except the acnA mutant. Altered acetate yield in the mutants was also accompanied by variations in the biomass yield. While the biomass yield in both the tktB and talA mutants was increased by 13.8%, biomass was 5.5% and 13.8% lower in the fumC and acnA mutants, respectively. Upregulation of global regulators such as rpoS and soxRS, the acs, aceA, aceB genes, and several TCA cycle genes such as fumC, acnA and sucA, is consistent with higher acetate consumption and biomass yield in the tktB and talA mutants. On the other hand, the fumC and acnA mutants, with their impaired TCA cycles, were unable to utilize acetate for biomass production in spite of the higher expression of rpoS and soxRS.

RpoS regulation of gene expression during exponential growth of Escherichia coli K12

RpoS is a major regulator of genes required for adaptation to stationary phase in E. coli. However, the exponential phase expression of some genes is affected by rpoS mutation, suggesting RpoS may also have an important physiological role in growing cells. To test this hypothesis, we examined the regulatory role of RpoS in exponential phase using both genomic and biochemical approaches. Microarray expression data revealed that, in the rpoS mutant, the expression of 268 genes was attenuated while the expression of 24 genes was enhanced. Genes responsible for carbon source transport (the mal operon for maltose), protein folding (dnaK and mopAB), and iron acquisition (fepBD, entCBA, fecI, and exbBD) were positively controlled by RpoS. The importance of RpoS-mediated control of iron acquisition was confirmed by cellular metal analysis which revealed that the intracellular iron content of wild type cells was two-fold higher than in rpoS mutant cells. Surprisingly, many previously identified RpoS stationary-phase dependent genes were not controlled by RpoS in exponential phase and several genes were RpoS-regulated only in exponential phase, suggesting the involvement of other regulators. The expression of RpoS-dependent genes osmY, tnaA and malK was controlled by Crl, a transcriptional regulator that modulates RpoS activity. In summary, the identification of a group of exponential phase genes controlled by RpoS reveals a novel aspect of RpoS function.

DNA Microarray and Proteomic Analyses of the RpoS Regulon in Geobacter sulfurreducens

The regulon of the sigma factor RpoS was defined in Geobacter sulfurreducens by using a combination of DNA microarray expression profiles and proteomics. An rpoS mutant was examined under steady-state conditions with acetate as an electron donor and fumarate as an electron acceptor and with additional transcriptional profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS acts as both a positive and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism, including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and amino acid metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance and adaptation to nutrient limitation. Transcription of the cytochrome c oxidase operon, necessary for G. sulfurreducens growth using oxygen as an electron acceptor, and expression of at least 13 c-type cytochromes, including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis of a subset of the rpoS mutant proteome indicated that 15 major protein species showed reproducible differences in abundance relative to those of the wild-type strain. Protein identification using mass spectrometry indicated that the expression of seven of these proteins correlated with the microarray data. Collectively, these results indicate that RpoS exerts global effects on G. sulfurreducens physiology and that RpoS is vital to G. sulfurreducens survival under conditions typically encountered in its native subsurface environments.

Starvation for Different Nutrients in Escherichia coli Results in Differential Modulation of RpoS Levels and Stability

Levels of RpoS increase upon glucose starvation in Escherichia coli, which leads to the transcription of genes whose products combat a variety of stresses. RpoS stability is a key level of control in this process, as SprE (RssB)-mediated degradation is inhibited under glucose starvation. Starvation for ammonia or phosphate also results in increased stress resistance and induction of RpoS-dependent genes. However, we demonstrate that RpoS levels following ammonia starvation are only slightly increased compared to growing cells and are 10-fold below the levels observed under glucose or phosphate limitation. This difference is largely due to regulated proteolysis of RpoS, as its stability in ammonia-starved cells is intermediate between that in logarithmic-phase cells and glucose-starved cells. Use of an rpoS construct that is devoid of the gene's native transcriptional and translational control regions reveals that stability differences are sufficient to explain the different levels of RpoS observed in logarithmic phase, ammonia starvation, and glucose starvation. Under phosphate starvation, however, rpoS translation is increased. The cellular response to nutrient limitation is much more complex than previously appreciated, as there is not simply one response that is activated by starvation for any essential nutrient. Our data support the hypothesis that SprE activity is the key level at which ammonia and glucose starvation signals are transmitted to RpoS, and they suggest that carbon source and/or energy limitation are necessary for full inactivation of the SprE pathway.

Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12

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.

Controlled induction of the RpoS regulon in Escherichia coli, using an RpoS-expressing plasmid.

RpoS, an alternative sigma factor produced by many gram-negative bacteria, primarily controls genes that are expressed in stationary phase in response to nutrient deprivation. To test the idea that induction of RpoS in the exponential phase, when RpoS is not normally expressed, increases RpoS-dependent gene expression, we constructed a plasmid carrying the rpoS gene under the control of an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible T7lac promoter. Northern and Western analyses revealed that levels of RpoS mRNA and protein, respectively, increased in response to the inducer IPTG. Assays of changes in RpoS-dependent functions (catalase activity and glycogen accumulation), confirmed that induced RpoS was functional in exponential phase and was sufficient for the expression of RpoS-dependent functions. Controlled expression of RpoS and RpoS-dependent genes by plasmid-encoded rpoS may thus offer a useful tool for the study of RpoS-dependent gene expression.

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.

Silencing of the Escherichia coli bgl operon by RpoS requires Crl.

Silencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS. Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter. However, the expression level of activated bgl operon derivatives varies with the strain background. Here it is shown that the repression of the bgl operon by RpoS requires Crl. Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes. In a Crl-negative strain RpoS had no effect on the bgl operon. The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E. coli strains. Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.

High magnetic field enhances stationary phase-specific transcription activity of Escherichia coli

When Escherichia coli B was aerobically grown at 37°C under inhomogeneous 5.2–6.1 Tesla (T) magnetic fields in the superconducting magnet biosystem (SBS), the cell number in the stationary phase after the growth had leveled off, was about 3 times higher than that under a geomagnetic field. When the E. coli defective in the rpoS gene which encodes a sigma factor, σ s of RNA polymerase and is specifically expressed in the stationary phase was cultivated at 37°C in SBS, such enhancement of cell survival was significantly reduced. The E. coli cells carrying rpoS–lacZ fusion gene or other rpoS dependent genes fused with lacZ were grown, significant increase in the activity of β-galactosidase was observed in the stationary phase under high magnetic field. These data suggest that enhancement of the transcription activity in stationary phase is involved in the higher survival of the cells under magnetic field.

Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli.

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.

Role of RPOS Regulon in Resistance to Oxidative Stress and Near- UV Radiation In ΔOXYR Suppressor Mutants of ESCHERICHIA COLI

Escherichia coliΔoxyR mutants are hyper-sensitive to oxidative agents but this sensitivity is reversed to hyper-resistance in ΔoxyR suppressor strains (ΔoxyRsup; Greenberg, J.T. and Demple, B. 1988. EMBO J. 7:2611- 2618). Also, ΔoxyR mutants have increased mutation rates that are also reversed in ΔoxyRsup. We now report that the rpoS regulon may have a role in determining hyper-resistance and loss of hyper-mutability of ΔoxyRsup. ΔoxyRsup cells were also resistant to near-ultraviolet radiation (near-UV) and survived longer in stationary phase than ΔoxyR cells. In ΔoxyRsup cells elevated β-galactosidase expression from a rpoS::lacZ promoter fusion and significant overproduction of RpoS protein was observed. These increases were accompanied by substantial elevation in transcription of rpoS-dependent genes as determined by β-galactosidase expression from katE::lacZ, dps::lacZ, and xthA::lacZ promoters. Catalase HPI and HPII activities were also increased. When rpoS::Tn10 was transduced into ΔoxyRsup, phenotypes switched back to hyper-sensitive, hyper-mutable and reduced catalases I and II. Individual ΔoxyR colonies exhibited significant clonal variability in β-galactosidase expression from rpoS::lacZ promoter. These results provide further evidence of the functional and regulatory overlap between two major anti-oxidant defense systems of bacteria.

Expression of Salmonella typhimurium rpoS and rpoS-dependent genes in the intracellular environment of eukaryotic cells.

Adaptation to the intracellular environment of host cells is crucial for the pathogenesis of Salmonella infections. The alternative sigma factor RpoS is a global regulator of gene expression during starvation and stress conditions and is required for virulence in Salmonella spp. We have used lacZ reporter fusions to rpoS and rpoS-dependent genes to study rpoS regulation after entry of Salmonella typhimurium into macrophages and epithelial cells. The results demonstrate that expression of an rpoS::lacZ translational fusion increases rapidly in S. typhimurium after phagocytosis. Activity of RpoS also increases after bacterial entry into both macrophages and epithelial cells, as demonstrated by the induction of the rpoS-regulated genes katE and spvB. A control rpoS-independent promoter for neomycin resistance does not show significant induction after cell entry. These results demonstrate that the regulatory system mediated by RpoS in S. typhimurium is activated by the intracellular environment of eukaryotic cells.

AldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp

Escherichia coli aldB was identified as a gene that is negatively regulated by Fis but positively regulated by RpoS. The complete DNA sequence determined in this study indicates that aldB encodes a 56.3-kDa protein which shares a high degree of homology with an acetaldehyde dehydrogenase encoded by acoD of Alcaligenes eutrophus and an aldehyde dehydrogenase encoded by aldA of Vibrio cholerae and significant homology with a group of other aldehyde dehydrogenases from prokaryotes and eukaryotes. Expression of aldB is maximally induced during the transition from exponential phase to stationary phase. Its message levels are elevated three- to fourfold by a fis mutation and abolished by an rpoS mutation. In addition, the expression of an aldB-lacZ fusion was decreased about 20-fold in the absence of crp. DNase I footprinting analysis showed that five Fis binding sites and one Crp binding site are located within the aldB promoter region, suggesting that Fis and Crp are acting directly to control aldB transcription. AldB expression is induced by ethanol, but in contrast to that of most of the RpoS-dependent genes, the expression of aldB is not altered by an increase in medium osmolarity.

Osmotic regulation of rpoS-dependent genes in Escherichia coli

The rpoS gene, which encodes a putative alternative sigma factor (sigma S), is essential for the expression of a variety of stationary-phase-induced genes as well as for stationary-phase-specific multiple-stress resistance. As previously shown for the otsA and otsB genes (R. Hengge-Aronis, W. Klein, R. Lange, M. Rimmele, and W. Boos, J. Bacteriol. 173:7918-7924, 1991), we demonstrate here that additional rpoS-controlled genes (bolA, csi-5) as well as at least 18 proteins on two-dimensional O'Farrell gels could be induced in growing cells by osmotic upshift via an rpoS-dependent mechanism. Also, rpoS-dependent thermotolerance and resistance against hydrogen peroxide could be osmotically stimulated. In contrast, the expression of glgS, while exhibiting strong stationary-phase induction, was only weakly increased by elevated osmolarity, and several rpoS-dependent proteins previously identified on two-dimensional gels were not osmotically induced. During osmotic induction of rpoS-dependent genes, rpoS transcription and the level of sigma S remained unchanged. We conclude that osmotically regulated genes represent a subfamily within the rpoS regulon that requires differential regulation in addition to that provided by sigma S.