Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.     

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

Background  

The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.     

Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp.

Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.     

Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.     

Isolation and characterization of Burkholderia cenocepacia mutants deficient in pyochelin production: pyochelin biosynthesis is sensitive to sulfur availability

The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.     

Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two extracytoplasmic function sigma factors

Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C(55)-isoprenyl phosphate by de novo synthesis. We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant. A bcrC mutant strain had an eightfold-higher sensitivity to bacitracin. Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) sigma factors, sigma(X) or sigma(M). Bacitracin induced expression of bcrC, and this induction was dependent on sigma(M) but not on sigma(X). Under inducing conditions, expression was primarily dependent on sigma(M). As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive. A sigX sigM double mutant was similar to a bcrC mutant in sensitivity. These results support the suggestion that one function of B. subtilis ECF sigma factors is to coordinate antibiotic stress responses.     

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.