Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

The long-chain fatty acid sensor, PsrA, modulates the expression of rpoS and the type III secretion exsCEBA operon in Pseudomonas aeruginosa

The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5 β-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC . DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N -(butyryl)- l -homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa .     

Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli

The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background.     

Significance of rpoS during maturation of Escherichia coli biofilms

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.     

Cloning,sequencing, and functional studies of the rpoS gene from Vibrio harveyi

The Vibrio harveyi rpoS gene which encodes an alternative sigma factor (sigma(s) or sigma(38)), has been cloned and characterized. The predicted protein sequence is closely related to RpoS proteins in other bacteria with up to 86% sequence identity. A rpoS null mutant of V. harveyi was constructed and the phenotype studied. Comparison of the properties of the V. harveyi wild type and rpoS deletion mutant showed that rpoS affected the ability of the cells to survive only under specific types of environmental stresses. The rpoS null mutant had a lower survival rate compared to the wild type parental strain at high concentrations of ethanol and in the stationary phase. In contrast to other bacteria, deletion of rpoS in V. harveyi did not affect the resistance of the cells to high osmolarity or hydrogen peroxide, suggesting the existence of alternative systems in V. harveyi responsible for resistance to these stresses. RpoS appears not to be involved in the control of luminescence in V. harveyi even though it is implicated in regulation of other acyl-homoserine dependent quorum sensing systems.     

The LysR-like regulator LeuO in Escherichia coli is involved in the translational regulation of rpoS by affecting the expression of the small regulatory DsrA-RNA

The translation of rpoS , which encodes the general stress sigma factor, σ^S, in Escherichia coli , is stimulated by various stress conditions. Regulatory factors involved in this control are the RNA-binding Hfq (HF-I) protein, the histone-like protein H-NS and the small regulatory DsrA-RNA (with the last being specifically required for increased rpoS translation at low temperature). Here, we report the characterization of a transposon insertion mutant (Tn 10 -8) with reduced σ^S levels that led to the identification of an additional factor involved in the regulation of rpoS translation, the LysR-like regulator LeuO. Tn 10 -8 decreases rpoS translation predominantly at low growth temperature. The mutation results in similarly strongly reduced DsrA-RNA expression and does not affect rpoS expression in a dsrA null mutant background, indicating that it affects rpoS translation via DsrA-RNA. Tn 10 -8 is inserted 26 bp upstream of the leuO open reading frame, which encodes a putative LysR-like regulator of unknown function. Instead of being a leuO null mutation, Tn 10 -8 activates leuO expression as a result of the p_out promoter on IS 10 _L reading into leuO , indicating that LeuO represses dsrA and thereby reduces rpoS translation at low temperature. LeuO does not contribute to temperature regulation of dsrA since its own expression is rather low and not temperature dependent. In a mutant deficient for H-NS, however, leuO is strongly derepressed. We conclude that rpoS translation is controlled by a regulatory network that includes Hfq, H-NS, LeuO and DsrA-RNA. In this network, H-NS plays a dual role by interfering with rpoS translation in general and, via LeuO, influencing the synthesis of its own low-temperature antagonist, DsrA-RNA.     

MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.     

rpoS Mutations and Loss of General Stress Resistance in Escherichia coli Populations as a Consequence of Conflict between Competing Stress Responses

The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor (phi(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 phi(S) levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on phi(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 phi(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.     

Suggested interrelationships of RNA-polymerase sigma S subunit and nitrogen control system in Pseudomonas chlororaphis

The effect of mutation in rpoS gene encoding sigma S subunit of RNA-polymerase on the capacity of Pseudomonas chlororaphis 449 to assimilate nitrogen was investigated. It has been shown that mutant cells with knocked-out rpoS gene had significantly lower capacity to utilize the nitrogen sources such as alanine, proline, histidine, arginine, urea, and ammonium and glutamine synthetase was downregulated in their cell free extracts. Both defects were abolished by glutamine supplementation to the medium. It is suggested that in Pseudomonas chlororaphis the association of the nitrogen control system and the system of gene expression is regulated by RNA-polymerase sigma S subunit, which can be responsible for cell adaptation at nitrogen supply limitation.     

Identification and characterization of a high-affinity zinc uptake system in Neisseria gonorrhoeae

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 rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P 相似文献   

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.     

Antitoxin MqsA helps mediate the bacterial general stress response

Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).