A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis.

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.

Induction of sigma(B)-dependent general stress genes by amino acid starvation in a spo0H mutant of Bacillus subtilis.

Solely sigma(B)-dependent genes like gsiB and gspA are not significantly induced in amino acid-starved wild-type cells, since amino acid starvation does not trigger activation of sigma(B). The general stress gene yvyD is subject to the control of both sigma(B) and sigma(H) therefore displaying induction in response to amino acid starvation at the sigma(H)-dependent promoter. Surprisingly, the proteins YvyD, GsiB and GspA were significantly induced in amino acid-starved cells of a strain lacking sigma(H) activity. Transcriptional studies provided evidence that sigma(B)-dependent transcription is indeed induced in a spo0H mutant during amino acid starvation and depends on RsbP but not on RsbU indicating that the stress signal transduction is not required for this induction. A similar phenomenon of sigma(B) activation was observed in amino acid-starved cells of a spo0A deletion mutant. The sigma(B)-dependent transcription in a spo0H mutant further needs an active RelA protein which is responsible for strong repression of house-keeping genes after amino acid starvation (stringent response). Our data indicate that in the absence of sigma(H) and under conditions which provoke the stringent response, RsbP-dependent levels of active sigma(B) can more effectively compete for increased levels of free RNA polymerase core enzyme leading to the induction of the probably strongest sigma(B)-dependent genes.

The stringent response, sigmaH-dependent gene expression and sporulation in Bacillus subtilis.

Recently, we found that transcription of the yvyD gene of Bacillus sibtilis from a sigmaH-dependent promoter is induced in response to amino acid starvation. This induction occurred in the wild type, but not in the relA mutant, suggesting that ppGpp might be involved in the activa tion of sigmaH. In order to substantiate this finding we looked for other sigmaH-dependent genes which also required an active relA gene product for activation in amino acid-starved cells. Three additional transcription units, namely spo0A, spoVG and the ytxGHI operon (also known as the csb40 operon), were found to be under the dual control of RelA and sigmaH in amino acid-starved cells, but many other sigmaH-dependent genes were not activated under these conditions. Of particular interest is the observation that spo0A is one of the genes that need a functional relA gene product for sigmaH-dependent gene activation. Furthermore, in sporulation medium, both Spo0A accumulation and phosphorylation are delayed in the relA deletion strain, leading to a delay in sporulation. A comparison between a relA point mutant and a relA deletion mutant revealed that the relA deletion mutant exhibits a more pronounced delay in the appearance of spores. These results suggest that the stringent response is not required for sporulation per se, but rather enhances the efficiency of sporulation.

The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH.

During a search by computer-aided inspection of two-dimensional (2D) protein gels for sigmaB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis. In addition to the typical sigmaB-dependent, stress- and starvation-inducible pattern, yvyD is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/or spo0H gene, two promoters were mapped upstream of the yvyD gene. The sigmaB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a sigmaH-dependent promoter is responsible for yvyD transcription following amino acid limitation. Analysis of Northern blots revealed that yvyD is transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and relA mutant cells showed that the YvyD protein is not synthesized in the relA mutant background. It was concluded that the stringent response plays a role in the activation of sigmaH. The yvyD gene product is homologous to a protein which might modify the activity of sigma54 in gram-negative bacteria. The expression of a sigmaL-dependent (sigmaL is the equivalent of sigma54 in B. subtilis) levD-lacZ fusion is upregulated twofold in a yvyD mutant. This indicates that the yvyD gene product, being a member of both the sigmaB and sigmaH regulons, might negatively regulate the activity of the sigmaL regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the sigmaB and sigmaH regulons on one side and the sigmaL regulon on the other.

Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study.

A two-dimensional (2-D) gel electrophoresis study of Bacillus subtilis strain 168 identified 20 proteins that are strongly induced in response to phosphate starvation. The induction of nine of these phosphate-starvation-induced (Psi) proteins was dependent on a functional PhoR protein. PhoR is the histidine sensor-kinase component of a phosphate-concentration-sensing two-component regulatory system which, together with its partner response regulator PhoP, controls the expression of genes in the Pho regulon. Genes encoding PhoR-dependent Psi proteins are therefore likely to be members of the Pho regulon. Spo0A approximately P, the response regulator of the signal transduction pathway required for the induction of sporulation, has previously been shown to negatively affect the induction of the Pho regulon by repressing the phoP-phoR operon. The induction pattern of some PhoR-dependent Psi proteins was altered in a spo0A mutant such that their synthesis continued for longer than was found with the wild-type. The most abundant Psi protein, Psi1-3, was characterized by N-terminal sequencing of internal peptide fragments and shown to have a high similarity to an Escherichia coli protein which is involved in phosphate uptake during phosphate starvation.