The stringent response inBacillus subtiliswas characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type andrelAmutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response todl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in therelAmutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknowny-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

The phosphate starvation response in Bacillus subtilis was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ς ^B , the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhF confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a ς ^A promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed with phoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in both phoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ς ^B -dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ς ^B -dependent manner and in the sigB mutant probably via ς ^H . YxiE was induced by phosphate starvation independently of ς ^B and PhoPR.

The Gram-positive soil bacteriumBacillus subtilisresponds to oxidative stress by the activation of different cellular defence mechanisms. These are composed of scavenging enzymes as well as protection and repair systems organized in highly sophisticated networks. In this study, the peroxide and the superoxide stress stimulons ofB. subtiliswere characterized by means of transcriptomics and proteomics. The results demonstrate that oxidative-stress-responsive genes can be classified into two groups. One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species. Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress. Similarly, both kinds of stress stimulated the activation of the stringent response. The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide. Several genes encoding proteins of unknown function could be assigned to one of these groups.

The ς ^B -dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the ς ^S regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the ς ^B regulon of Staphylococcus aureus , especially in virulence control, ς ^B -dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of ς ^B . N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their ς ^B dependency, and moreover, about 7 additional ς ^B -dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 ς ^B -dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC -homologous gene was localized on the S. aureus sequence solely transcribed by ς ^B . In contrast, a second clpC -homologous gene in S. aureus forming an operon with ctsR , yacH , and yacI was ς ^B independently expressed.