Expression of the ςB-Dependent General Stress Regulon Confers Multiple Stress Resistance in Bacillus subtilis

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Analysis of the role of RsbV, RsbW, and RsbY in regulating {sigma}B activity in Bacillus cereus

The alternative sigma factor sigma(B) is an important regulator of the stress response of Bacillus cereus. Here, the role of the regulatory proteins RsbV, RsbW, and RsbY in regulating sigma(B) activity in B. cereus is analyzed. Functional characterization of RsbV and RsbW showed that they act as an anti-sigma factor antagonist and an anti-sigma factor, respectively. RsbW can also act as a kinase on RsbV. These data are in line with earlier functional characterizations of RsbV and RsbW homologs in B. subtilis. The rsbY gene is unique to B. cereus and its closest relatives and is predicted to encode a protein with an N-terminal CheY domain and a C-terminal PP2C domain. In an rsbY deletion mutant, the sigma(B) response upon stress exposure was almost completely abolished, but the response could be restored by complementation with full-length rsbY. Expression analysis showed that rsbY is transcribed from both a sigma(A)-dependent promoter and a sigma(B)-dependent promoter. The central role of RsbY in regulating the activity of sigma(B) indicates that in B. cereus, the sigma(B) activation pathway is markedly different from that in other gram-positive bacteria.     

Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.     

Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine

Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to sigmaB-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy.