GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.     

Identification of a non-haem catalase in Salmonella and its regulation by RpoS (sigmaS)

We report the identification and functional analysis of katN, a gene encoding a non-haem catalase of Salmonella enterica serotype Typhimurium. katN, which is not present in Escherichia coli, is located between the yciGFE and yciD E. coli homologues in the Salmonella genome. Its predicted protein product has a molecular weight of 31 826 Da and is similar to the Mn-catalases of Lactobacillus plantarum and Thermus spp. Its product, KatN, was visualized as a 37 kDa protein in E. coli maxicells. A KatN recombinant protein, containing six histidine residues at its C-terminus, was purified, and its catalase activity was observed on a non-denaturing polyacrylamide gel. KatN was also visualized by catalase activity gel staining of bacterial cell extracts. Its expression was shown to be regulated by growth phase and rpoS. Northern blotting indicated that kat forms an operon with the upstream yciGFE genes. A putative rpoS-regulated promoter was identified upstream of yciG. Southern blotting revealed that katN is conserved within Salmonella serovars. katN homologues were found in Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens. A katN mutation did not appear to affect the hydrogen peroxide (H2O2) response of Salmonella. However, the expression of katN increased the H2O2 resistance of unadapted cells in the exponential phase and of rpoS mutants in stationary phase. Thus, KatN may contribute to hydrogen peroxide resistance in Salmonella in certain environmental conditions.     

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.     

Effect of sigmaS on sigmaE-directed cell lysis in Escherichia coli early stationary phase

The sigmaE regulon has been shown to perform a novel function that causes dead-cell lysis specific to the early stationary phase in addition to its well-known role in the extracytoplasmic stress response in Escherichia coli. Here, the effect of sigmaS as a general stress-responsive sigma factor on sigmaE-directed cell lysis was investigated. The lysis phenomena were observed in both rpoS mutant and parental strains constitutively expressing active sigmaE, but the former lysis occurred at a relatively early stage compared to the latter. Based on these results and experiments with hydrogen peroxide, we propose that some stresses generate living but non-culturable cells, which are subject to sigmaE-directed cell lysis.     

The molecular basis of selective promoter activation by the sigmaS subunit of RNA polymerase

Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, sigmaS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, sigma70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing sigmaS or sigma70, or by both holoenzymes. How these promoter features impose sigmaS selectivity is further discussed. Moreover, additional pathways allow sigmaS to compete more efficiently than sigma70 for limiting amounts of core RNAP (E) and thereby enhance EsigmaS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes.     

Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors

σ ^S, the stationary phase sigma factor of Escherichia coli and Salmonella , is regulated at multiple levels. The σ^S protein is unstable during exponential growth and is stabilized during stationary phase and after various stress treatments. Degradation requires both the ClpXP protease and the adaptor RssB. The small antiadaptor protein IraP is made in response to phosphate starvation and interacts with RssB, causing σ^S stabilization under this stress condition. IraP is essential for σ^S stabilization in some but not all starvation conditions, suggesting the existence of other anti-adaptor proteins. We report here the identification of new regulators of σ^S stability, important under other stress conditions. IraM (inhibitor of RssB activity during Magnesium starvation) and IraD (inhibitor of RssB activity after DNA damage) inhibit σ^S proteolysis both in vivo and in vitro . Our results reveal that multiple anti-adaptor proteins allow the regulation of σ^S stability through the regulation of RssB activity under a variety of stress conditions.     

The response regulator RssB, a recognition factor for sigmaS proteolysis in Escherichia coli, can act like an anti-sigmaS factor

sigmaS (RpoS) is the master regulator of the general stress response in Escherichia coli. Several stresses increase cellular sigmaS levels by inhibiting proteolysis of sigmaS, which under non-stress conditions is a highly unstable protein. For this ClpXP-dependent degradation, the response regulator RssB acts as a recognition factor, with RssB affinity for sigmaS being modulated by phosphorylation. Here, we demonstrate that RssB can also act like an anti-sigma factor for sigmaS in vivo, i.e. RssB can inhibit the expression of sigmaS-dependent genes in the presence of high sigmaS levels. This becomes apparent when (i) the cellular RssB/sigmaS ratio is at least somewhat elevated and (ii) proteolysis is reduced (for example in stationary phase) or eliminated (for example in a clpP mutant). Two modes of inhibition of sigmaS by RssB can be distinguished. The 'catalytic' mode is observed in stationary phase cells with a substoichiometric RssB/sigmaS ratio, requires ClpP and therefore probably corresponds to sequestering of sigmaS to Clp protease (even though sigmaS is not degraded). The 'stoichiometric' mode occurs in clpP mutant cells upon overproduction of RssB to levels that are equal to those of sigmaS, and therefore probably involves binary complex formation between RssB and sigmaS. We also show that, under standard laboratory conditions, the cellular level of RssB is more than 20-fold lower than that of sigmaS and is not significantly controlled by stresses that upregulate sigmaS. We therefore propose that antisigma factor activity of RssB may play a role under not yet identified growth conditions (which may result in RssB induction), or that RssB is a former antisigma factor that during evolution was recruited to serve as a recognition factor for proteolysis.     

Evidence that RpoS (sigmaS) in Borrelia burgdorferi is controlled directly by RpoN (sigma54/sigmaN)

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.     

Interactions between the 2.4 and 4.2 regions of sigmaS, the stress-specific sigma factor of Escherichia coli, and the -10 and -35 promoter elements

The σ^s subunit of Escherichia coli RNA polymerase holoenzyme (Eσ^S) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by Eσ^S and the housekeeping Eσ⁷⁰ is as yet not clearly understood. We used a genetic approach to investigate the interaction of σ^S with its target promoters. Starting with down-promoter variants of a σ^S promoter target, osmEp, altered in the –10 or –35 elements, we isolated mutant forms of σ^S suppressing the promoter defects. The activity of these suppressors on variants of osmEp and ficp, another target of σ^S, indicated that σ^S is able to interact with the same key features within a promoter sequence as σ⁷⁰. Indeed, (i) σ^S can recognize the –35 element of some but not all its target promoters, through interactions with its 4.2 region; and (ii) amino acids within the 2.4 region participate in the recognition of the –10 element. More specifically, residues Q152 and E155 contribute to the strong preference of σ^S for a C in position –13 and residue R299 can interact with the –31 nucleotide in the –35 element of the target promoters.     

Abrogation of ospAB constitutively activates the Rrp2‐RpoN‐RpoS pathway (sigmaN‐sigmaS cascade) in Borrelia burgdorferi

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2‐RpoN‐RpoS pathway. Complementing the ospAB mutant with a wild‐type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2‐RpoN‐RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC‐negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2‐RpoN‐RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2‐RpoN‐RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.