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In Bacillus subtilis, activity of the general stress transcription factor B is controlled posttranslationally by a regulatory network that transmits signals of environmental and metabolic stress. These signals include heat, ethanol, or osmotic challenge, or a sharp decrease in cellular energy levels, and all ultimately control B activity by influencing the binding decision of the RsbW anti-factor. In the absence of stress, RsbW binds to B and prevents its association with RNA polymerase core enzyme. However, following stress, RsbW binds instead to the RsbV anti-anti-factor, thereby releasing B to direct transcription of its target genes. These two principal regulators of B activity are encoded in the eight-gene sigB operon, which has the gene order rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sigB-rsbX (where rsb stands for regulator of sigma B). Notably, the predicted rsbS product has significant amino acid identity to the RsbV anti-anti-factor and the predicted rsbT product resembles the RsbW anti-factor. To determine the roles of rsbS and rsbT, null or missense mutations were constructed in the chromosomal copies of each and tested for their effects on expression of a B -dependent reporter fusion. On the basis of this genetic analysis, our principal conclusions are that (i) the rsbS product is a negative regulator of B activity, (ii) the rsbT product is a positive regulator, (iii) RsbS requires RsbT for function, and (iv) the RsbS-RsbT and RsbV-RsbW pairs act hierarchically by a common mechanism in which key protein-protein interactions are controlled by phosphorylation events.In response to stress and starvation signals, the gram-positive bacterium Bacillus subtilis expresses a large set of genes termed the general stress regulon, which is primarily under control of the alternative transcription factor B (3,8,16,19,20,24,36). The activity of B is itself controlled posttranslationally by a multicomponent signal transduction pathway which conveys signals of environmental stress, such as heat shock, osmotic stress, or ethanol challenge, and signals of metabolic stress, such as the drop in cellular energy levels that occurs upon challenge with uncouplers of oxidative phosphorylation or upon entry into the stationary growth phase (2,4,6,7,9,34,35).All of the known regulators in the B signal transduction pathway are encoded by genes in the sigB operon, which also contains the B structural gene (20, 37). As shown in Fig. 1, these genes are termed rsb, for regulator of sigma B. Previous genetic and biochemical data indicate that the RsbW antifactor is the primary regulator, binding directly to B and maintaining it in a transcriptionally inactive complex (4, 5, 9).B is released from this complex by the action of the RsbV anti-anti-factor, which apparently sequesters RsbW by direct protein-protein interaction (4, 9, 13). Additional in vitro and in vivo experiments have demonstrated that the RsbW antifactor can associate with either B or RsbV and that the binding decision of RsbW is controlled by the phosphorylation state of RsbV (1,...

No abstract

hilA encodes an activator of Salmonella enterica serovar Typhimurium virulence genes and is transcriptionally modulated by environmental conditions. We show that H-NS represses hilA under low-osmolarity conditions. H-NS, HU, and Fis also appear to affect the derepression of hilA by HilD. Modulation of hilA by counteracting repressing and derepressing mechanisms may allow Salmonella serovar Typhimurium to regulate its virulence genes in response to different situations in vivo.

SummarySalmonella typhimurium is a Gram-negative enteric pathogen that can infect intestinal epithelial cells and induce inflammation of the intestinal mucosa. These processes are mediated by a type III secretion system (TTSS), which is encoded on Salmonella pathogenicity island 1 (SPI1). Previous studies showed that four SPI1-encoded transcriptional regulators, HilD, HilC, HilA and InvF, act in an ordered fashion to coordinately activate expression of the SPI1 TTSS. HilD and HilC derepress hilA transcription. HilA activates invF as well as SPI1 genes that encode components of the TTS apparatus. InvF then activates genes that encode proteins secreted by the SPI1 TTS apparatus. In this scheme, HilD and HilC indirectly activate expression of the SPI1 TTS apparatus and its secreted substrates by affecting hilA expression. Here, we report that HilD and HilC can also activate expression of a subset of SPI1 genes independently of HilA. Our studies show that HilD and HilC activate transcription of invF from a promoter that is far upstream of its HilA-dependent promoter. This activation is most probably through direct binding of HilD and HilC to sequences upstream and downstream of this alternative HilA-independent promoter. We conclude that HilD and HilC have a second role in SPI1 gene regulation that is separate from their role in co-ordinating expression of the SPI1 TTSS through hilA .

The general stress response of Bacillus subtilis is triggered by a variety of environmental and metabolic stresses which activate the aB transcription factor. Among the more than 100 genes controlled b y aB.(the csb genes), the functions identified thus far include resistance to oxidative stress, resistance to protein denaturation and resistance to osmotic stress. T o understand the breadth of functions in which csb genes participate, the transcriptional organization and predicted products of two such genes previously identified in a screen for oB-dependent lacZ fusions were analysed. The csb-22::Tn917lacZ and csb-34::Tn917lacZ fusions are unusual among csb genes in that their expression appears to be completely dependent upon aB. By plasmidintegration experiments, fusion analyses and site-directed mutagenesis, stressinducible, aB-dependent promoters for both these fusions were identified. The csb-34 fusion marked an ORF (yxcC or csbC) which by sequence analysis lay in a monocistronic transcriptional unit. This ORF encoded a predicted 461-residue product which had high identity with Class I sugar transporters of the major facilitator superfamily. It was speculated that the csbC product could serve either a nutritional or an osmotic protection function. In contrast, the csb-22 fusion identified an ORF (ywmG or csbD) which appeared to be the second gene of a two-gene operon. This ORF encoded a predicted 62-residue product which resembled a small Escherichia coli protein of unknown function. The aBdependent promoter lay immediately upstream from csbD and appeared to be an internal promoter for the operon.

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Natural antimicrobials can be added to help extend the shelf life of milk and reduce contamination potential, especially in developing countries. The levels of lactoferrin found in raw milk have not been thoroughly studied in regard to specific antibacterial activity against key pathogens. This study aimed to investigate various concentrations of lactoferrin as a biopreservative measure against Salmonella enterica and Escherichia coli O157:H7. Our results indicate that lactoferrin has potential as a biopreservative. These results will assist in the reduction of milk-borne illnesses in developing countries that may lack means for proper refrigeration. Highlights• In the absence of proper refrigeration of raw milk, bacterial pathogens such as Salmonella and E. coli O157:H7 may cause illness. • Growth of each pathogen in raw milk was measured using a novel tetrazolium salt reduction assay.• Growth of Salmonella and E. coli O157:H7 was inhibited by lactoferrin in raw bovine milk.• Lactoferrin represents a safe means of milk preservation in developing countries lacking proper refrigeration.