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.     

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.     

S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus

Using a two-plasmid system, we recently identified sigma(E)-dependent promoters directing expression of the sigma(E) regulon genes in Salmonella enterica serovar Typhimurium (S. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the sigma(E)-dependent rpoEp3 promoter directing expression of rpoE. This two-plasmid system was previously optimized to identify nucleotides critical for the rpoEp3 promoter activity. However, two highly conserved nucleotides in the sigma(E) consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the rpoEp3 promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAACtt) and -10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the sigma(E) consensus sequence. The expression of the wild-type and mutated rpoEp3 promoters was confirmed in S. Typhimurium and was found to exhibit a different pattern of sigma(E) activation compared with Escherichia coli, with a peak rpoEp3 promoter activity in early stationary phase followed by a decrease in late stationary phase.     

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.     

Listeria monocytogenes sigma B regulates stress response and virulence functions

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.     

Detection of sialoglycomolecules in five plant trypanosomatids and in an insect phytophagous isolate

A mutation in the cell division gene ftsK causes super-induction of sigma(70)-dependent stress defense genes, such as uspA, during entry of cells into stationary phase. In contrast, we report here that stationary phase induction of sigma(S)-dependent genes, uspB and cfa, is attenuated and that sigma(S) accumulates at a lower rate in ftsK1 cells. Ectopic overexpression of rpoS restored induction of the rpoS regulon in the ftsK mutant, as did a deletion in the recA gene. Thus, a mutation in the cell division gene, ftsK, uncouples the otherwise coordinated induction of sigma(S)-dependent genes and the universal stress response gene, uspA, during entry into stationary phase.     

Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.     

RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high levels of sigma(s)

RpoS-dependent promoters require ppGpp for induction in the stationary phase. This has been thought to be a simple consequence of final sigma(S) itself requiring ppGpp for its production. By using four model promoters requiring final sigma(S) for normal induction in the stationary phase, we demonstrate that final sigma(S)-dependent promoters require ppGpp even in the presence of high levels of final sigma(S) produced ectopically. Similar to final sigma(70)-dependent promoters under positive control by ppGpp, the requirement of final sigma(S)-dependent promoters for this alarmone is bypassed by specific "stringent" mutations in the beta-subunit of RNA polymerase. The results suggest that stationary phase induction of both final sigma(S)- and final sigma(70)-dependent genes requires the stringent control modulon and that stringency confers dual control on the RpoS regulon by affecting promoter activity and the levels of the required final sigma-factor.