The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway

The link between cell division defects and the induction of the universal stress response is demonstrated to operate via the RecA regulator of the SOS response. An insertion in the cell division gene ftsK upregulates uspA in a recA-dependent manner. Unlike true SOS response genes, this upregulation only occurs in growth-arrested cells and is LexA independent. Thus, besides ppGpp-dependent starvation signals, DNA aberrations transduce RecA-dependent signals to the uspA promoter, which only affect the promoter during stasis. Further, we show that ftsK itself, like uspA, is induced in stationary phase and that this induction requires the stringent control modulon rather than activated RecA. Thus, ftsK, like uspA, is regulated by at least two global regulators: ppGpp of the stringent control network and RecA of the SOS modulon. We suggest that UspA is a new bona fide member of the RecA-dependent DNA protection and repair system, as mutants lacking functional UspA were found to be sensitive to UV irradiation and mitomycin C exposure. Moreover, the UV sensitivity of uspA mutants is enhanced in an additive manner by the ftsK1 mutation.     

A mutation in the ftsK gene of Escherichia coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA.

An insertional mutation in ftsK, encoding an Escherichia coli product similar to the sporulation protein SpoIIIE of Bacillus subtilis, results in uspA overexpression in stationary phase and impairs cell division. The ftsK1::cat insertion mutant forms chains which are the result of inhibited cell-cell separation, while chromosome synthesis and partitioning appear to be normal as judged by flow cytometry and electron and light microscopy in combination with DNA staining. The cells of the chains are attached to each other by a small envelope structure, and unlike in a spoIIIE mutant of B. subtilis, there is no DNA trapped in the division plane. In addition, plasmids harboring a truncated ftsK allele lacking the last 195 bp of the gene cause chain formation in wild-type cells. While the mutant cells grow at essentially the same rate as the parent in complex and defined minimal media, they are sensitive to stresses. Specifically, the mutant failed to grow at elevated salt concentrations and survived stationary phase poorly. The phenotypes of the ftsK1::cat mutant are complemented by the 3' end (spoIIIE-like half) of the ftsK locus. In contrast, the 5' end of the ftsK locus reported to complement ftsK44(Ts) phenotypes does not complement the phenotypes of the ftsK1::cat mutant.     

Paraquat regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12 is SoxRS independent but modulated by sigma S.

We report the first example of a gene, hmp, encoding a soluble flavohemoglobin in Escherichia coli K-12, which is up-regulated by paraquat in a SoxRS-independent manner. Unlike what is found for other paraquat-inducible genes, high concentrations of paraquat (200 microM) were required to increase the level of hmp expression, and maximal induction was observed only after 20 min of exposure to paraquat. Neither a mutation in soxS nor one in soxR prevented the paraquat-dependent increase in phi(hmp-lacZ) expression, but either mutant allele delayed full expression of phi(hmp-lacZ) activity after paraquat addition. Induction of hmp by paraquat was demonstrated in aerobically grown cultures during exponential growth and the stationary phase, thus revealing two Sox-independent regulatory mechanisms. Induction of hmp by paraquat in the stationary phase was dependent on the global regulator of stationary-phase gene expression, RpoS (sigma S). However, a mutation in rpoS did not prevent an increase in hmp expression by paraquat in exponentially growing cells. Induction of sigma S in the exponential phase by heat shock also induced phi(hmp-lacZ) expression in the presence of paraquat, supporting the role of sigma S in one of the regulatory mechanisms. Mutations in oxyR or rob, known regulators of several stress promoters in E. coli, had no effect on the induction of hmp by paraquat. Other known superoxide-generating agents (plumbagin, menadione, and phenazine methosulfate) were not effective in inducing hmp expression.     

The alternative sigma factor, sigmaS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, sigma(S), influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS-negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that sigma(S) might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.     

Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.     

Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli.

The rpoS (katF) gene of Escherichia coli encodes a putative sigma factor (sigma S) required for the expression of a variety of stationary phase-induced genes, for the development of stationary-phase stress resistance, and for long-term starvation survival (R. Lange and R. Hengge-Aronis, Mol. Microbiol. 5:49-59, 1991). Here we show that the genes otsA, otsB, treA, and osmB, previously known to be osmotically regulated, are also induced during transition into stationary phase in a sigma S-dependent manner. otsA and otsB, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, are involved in sigma S-dependent stationary-phase thermotolerance. Neither sigma S nor trehalose, however, is required for the development of adaptive thermotolerance in growing cells, which might be controlled by sigma E.     

Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S.

The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells. Stationary-phase induction of bolA is dependent on sigma S. The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter.     

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.