MHYT, a new integral membrane sensor domain.

MHYT, a new conserved protein domain with a likely signaling function, is described. This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098. In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins. In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein. A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.

New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis.

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis.

The sigmaB transcription factor of the bacterium Bacillus subtilis controls the synthesis of over 100 general stress proteins that are induced by growth-limiting conditions. Genetic evidence suggests that RsbR modulates the phosphorylation state of the RsbS antagonist in the signaling pathway that regulates sigmaB activity in response to environmental stresses that limit growth. According to the current model, the phosphorylated RsbS antagonist is unable to complex RsbT, which is then released to initiate a signaling cascade that ultimately activates sigmaB. Here, we show that the RsbR protein itself has no kinase activity but instead stimulates RsbS phosphorylation by the RsbT serine kinase in vitro. We further show that in addition to its previously known serine kinase activity directed toward the RsbS antagonist, purified RsbT also possesses a threonine kinase activity directed toward residues 171 and 205 of the RsbR modulator. Threonine residues 171 and 205 were each found to be important for RsbR function in vivo, and phosphorylation of these residues abolished the ability of RsbR to stimulate RsbT kinase activity in vitro. These results are consistent with a model in which RsbR modulates the kinase activity of RsbT directed toward its RsbS antagonist in vivo, either specifically in response to environmental signals or as part of a feedback mechanism to prevent continued signaling.

General stress transcription factor sigmaB and sporulation transcription factor sigmaH each contribute to survival of Bacillus subtilis under extreme growth conditions.

The general stress response of the bacterium Bacillus subtilis is controlled by the sigmaB transcription factor. Here we show that loss of sigmaB reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol. We further show that loss of the developmental transcription factor sigmaH also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.

Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis.

Bacillus subtilis responds to signals of environmental and metabolic stress by inducing over 40 general stress genes under the control of the sigma B transcription factor. sigma B activity is regulated post-translationally by a multi-component network composed of two coupled partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, each containing a serine phosphatase (X or U), an antagonist protein (S or V), and a switch protein/serine kinase (T or W). The upstream module (X-S-T) is required to transmit signals of environmental stress. In contrast, the downstream module (U-V-W) is required to transmit signals of energy stress as well as the environmental signals conveyed to it from the upstream module. Until now the function of the rsbR gene product was unknown. RsbR shares significant sequence similarity with the RsbS and RsbV antagonist proteins whose phosphorylation states control key protein-protein interactions within their respective modules. Here we present evidence that RsbR is associated with RsbS in the upstream, environmental-sensing module. To investigate RsbR function, we constructed deletion and point mutations within rsbR and tested their effects on expression of sigma B-dependent reporter fusions, both singly and in combination with other rsb mutations. To determine the possible interaction of RsbR with other Rsb proteins, we tested the ability of wild-type or mutant RsbR to activate transcription in the yeast two-hybrid system in conjunction with other Rsb regulators. On the basis of this genetic analysis, we conclude that RsbR is a positive regulator which modulates sigma B activity in response to salt and heat stress. Our data further suggest that: (i) RsbR influences the antagonist function of RsbS by direct protein-protein interaction; and (ii) this interaction with RsbS is likely controlled by the phosphorylation state of RsbR.

[Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis]. / Klonirovanie genov, kontroliruiushchikh biosintez batsitratsina u Bacillus licheniformis.

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.

[Isolation and characteristics of a plasmid from the thermotolerant strain of Bacillus licheniformis]. / Vydelenie i kharakteristika plazmidy iz termotolerantnogo shtamma Bacillus licheniformis.

A 8.3 kb cryptic plasmid was isolated from the thermotolerant strain of Bacillus licheniformis 28KA and designated pLT83. The replicative (rep) region was localized on the plasmid map. The pLT83 plasmid labelled in vitro with an antibiotic resistance determinant is able to replicate in B. subtilis cells. The pLT83 plasmid replicates stably in B. licheniformis strain at higher temperatures (37-60 degrees C) than in B. subtilis cells (37-50 degrees C). The plasmid and its derivatives may be used as vectors for gene cloning in B. subtilis and B. licheniformis cells.

[Fusion of Bacillus subtilis and Bacillus licheniformis protoplasts. Interspecies recombination resulting from protoplast fusion]. / Sliianie protoplastov Bacillus subtilis i Bacillus licheniformis. Mezhvidovaia rekombinatsiia v rezul'tate sliianiia protoplastov.

Recombinants between B. subtilis and B. licheniformis were prepared by fusion of the bacterial protoplasts. Genetically marked strains SB25 trp C hisH and 168 ade-met-leu- of B. subtilis and 1001 ura-thr- and 1001 met- of B. licheniformis were used as the parent strains. The recombinants were selected with the indirect method followed by analysis of their nutrient requirements and cultural and morphological features. All the hybrids acquired the specific properties of B. subtilis. Apparently, their formation was based on the whole chromosome of B. subtilis and recombination of separate fragments of B. licheniformis with it. Hybrids with prototrophic properties with respect to one, two or three markers of the initial strains were detected independent of the genotype of the B. subtilis parent strains. Moreover, the protoplast fusion resulted in formation of hybrids which were prototrophic with respect to the amino acid markers of B. subtilis and deficient with respect to homoserine and thiamine or only thiamine, whereas the initial strains were not auxotrophic with respect to homoserine and thiamine. Thi-Hom- and a number of the prototrophic recombinants were characterized by the capacity for increased synthesis of riboflavin lacking in the initial cultures. Homologous and heterologous transformation appeared to be possible in the recombinants of the Thi-Hom- phenotype, while transformation of the initial strain SB25 by the intergenocytic markers was possible in reciprocal crossings. It is concluded that contrary to transformation of isolated DNA, protoplast fusion may result in formation of interspecies recombinants of B. subtilis and B. licheniformis with respect to different operones of amino acid synthesis.