The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

The PhoB regulatory system modulates biofilm formation and stress response in El Tor biotype Vibrio cholerae

The PhoBR regulatory system is required for the induction of multiple genes under conditions of phosphate limitation. Here, we examine the role of PhoB in biofilm formation and environmental stress response in Vibrio cholerae of the El Tor biotype. Deletion of phoB or hapR enhanced biofilm formation in a phosphate-limited medium. Planktonic and redispersed biofilm cells of the Δ phoB mutant did not differ from wild type for the expression of HapR, suggesting that PhoB negatively affects biofilm formation through an HapR-independent pathway. The Δ phoB mutant exhibited elevated expression of exopolysaccharide genes vpsA and vpsL compared with the wild type. Deletion of hapR enhanced the expression of the positive regulator vpsT , but had no effect on the expression of vpsR . In contrast, deletion of phoB enhanced the expression of the positive regulator vpsR , but had no effect on the expression of hapR and vpsT . The Δ phoB mutant was more sensitive to hydrogen peroxide compared with the wild type and with an isogenic Δ rpoS mutant. Conversely, the Δ phoB mutant was more resistant to acidic conditions and high osmolarity compared with the wild type and with an isogenic Δ rpoS mutant. Taken together, our data suggest that phosphate limitation induces V. cholerae to adopt a free-swimming life style in which PhoB modulates environmental stress response in a manner that differs from the general stress response regulator RpoS.     

Polyamines and glutamate decarboxylase-based acid resistance in Escherichia coli

The expression of gadA and gadB, which encode two glutamate decarboxylases (GADs) of Escherichia coli, is induced by an acidic environment and participate in acid resistance. In this study, we constructed a polyamine-deficient mutant and investigated the role of polyamines in acid resistance. The expression of gadA and gadB was shown to be dependent on polyamines. For that reason, the polyamine-deficient mutant was completely devoid of GAD activity and was very susceptible to low pH if large amounts of polyamines were not provided. We also showed that the polyamine-deficient mutant contained higher cAMP levels than the isogenic polyamine-proficient wild type, and cAMP negatively regulated the expression of gadA and gadB. Therefore, introduction of the cya (encoding adenylate cyclase) mutation allele into the polyamine-deficient mutant resulted in the increment of GAD activity and thus restored the reduced acid resistance of the mutant. The positive regulators, H-NS (histone-like protein, encoded by the hns gene) and RpoS (alternative RNA polymerase sigma subunit, encoded by rpoS gene), also significantly governed the expression of gadA and gadB, respectively. However, polyamines did not regulate either the intracellular H-NS level or rpoS expression under these culture conditions. These results strongly suggest that there are at least two different regulatory systems in acid resistance, one is positive regulation via a H-NS/RpoS system and the other is negative regulation via a polyamine/cAMP system.     

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.     

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.     

Regulation of the homologous two-component systems KvgAS and KvhAS in Klebsiella pneumoniae CG43

In Klebsiella pneumoniae CG43, deletion of the sensor gene kvgS reduced the kvgAS expression in M9 medium with 0.2 mM paraquat, 0.2 mM 2,2-dihydropyridyl, or 300 mM NaCl. This result shows an autoregulatory role of KvgS and a stress-responsive expression of the two-component system (2CS). The kvgS deletion also appeared to decrease the expression of kvhAS, paralogous genes of kvgAS. Additionally, measurements of the promoter activity in kvgA(-) mutant revealed that KvgA is probably an activator for the expression of kvgAS and kvhAS. The subsequent electrophoretic mobility shift assay, indicating a specific binding of the recombinant KvgA to the putative promoters P(kvgAS) and P(kvhAS), also supported an interacting regulation between the 2CSs. In P(kvgAS) and P(kvhAS), the presence of RpoS binding elements suggested an RpoS-dependent regulation. Nevertheless, the rpoS deletion reduced the expression of kvgAS but increased that of kvhAS. Moreover, the kvgA deletion reduced the expression of katG and sodC. The overexpression of KvhA altered the susceptibility to fosfomycin and an increasing activity of UDP-N-acetylglucosamine enolpyruvyl transferase, the target protein of fosfomycin, which suggesting a regulation by KvhA. Taken together, these indicated that the two 2CSs probably belong to different regulatory circuits of the RpoS regulon.