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Bacillus subtilis is exposed to a variety of antimicrobial compounds in the soil. In this paper, we report on the response of B. subtilis to the fungal-related antimicrobials 6-brom-2-vinyl-chroman-4-on (chromanon) and 2-methylhydroquinone (2-MHQ) using proteome and transcriptome analyses. Chromanon, a derivative of aposphaerins from Aposphaeria species caused predominant protein damage in B. subtilis as indicated by the induction of the HrcA, CtsR, and Spx regulons. The expression profile of the ganomycin-related substance 2-MHQ was similar to that of catechol as reflected by the common induction of the thiol-specific oxidative stress response. Several putative ring-cleavage dioxygenases and oxidoreductases were differentially up-regulated by 2-MHQ, catechol, and chromanon including yfiDE, ydfNOP, yodED, ycnDE, yodC, and ykcA. The nitroreductase encoding yodC gene is induced in response to catechol, 2-MHQ, and chromanon, which depend on the MarR-type repressor YodB. The yfiDE (catDE) operon encodes a catechol-2,3-dioxygenase which is most strongly induced by catechol. The yodED (mhqED), ydfNOP (mhqNOP) operons, and ykcA (mhqA) respond most strongly to 2-MHQ and encode putative hydroquinone-specific extradiol dioxygenases. The ycnDE operon was most strongly induced by chromanon. Mutational analyses revealed that the putative hydroquinone-specific dioxygenases MhqO and MhqA confer resistance to 2-MHQ in B. subtilis.

Phenolic acids that are present in plant-soil ecosystems can be considered as toxins which induce specific stress responses in microorganisms. In this paper, we have analyzed the global response of the soil bacterium Bacillus subtilis to salicylic acid using proteomics and transcriptomics. The results demonstrate that salicylic acid caused predominantly the induction of the SigmaB-dependent general stress response in B. subtilis which is not related to the acidic conditions. Treatment of B. subtilis with growth-inhibitory concentrations of 4 mM salicylic acid caused protein damage in B. subtilis as reflected by the induction of the CtsR and Spx regulons. Both phenolic acid decarboxylases (pads) of B. subtilis padC and bsdBCD (yclBCD) were induced by 4 mM salicylic acid that were previously shown to be involved in decarboxylation and detoxification of different phenolic acids. Deletion of the putative LysR-type regulator encoded by the divergently transcribed bsdA (yclA) gene upstream of the bsdBCD operon revealed that BsdA is the transcriptional activator of bsdBCD expression in response to salicylic acid. Phenotype analysis of bsdA and padC single and double mutants demonstrated that both pads confer resistance to salicylic acid in B. subtilis.

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.

The bacterial signal recognition particle (SRP)-dependent pathway is believed to be a major targeting route for membrane proteins, as well as for subsets of secretory proteins. The present studies were aimed at an assessment of the role of two key components of SRP, namely Ffh and FtsY, in protein secretion by the Gram-positive bacterium Bacillus subtilis. Our results show that both components are important for the extracellular accumulation of proteins containing known signal peptides. Remarkably, extracellular accumulation of individual proteins was affected to different extents by depletion of Ffh or FtsY, at least under the conditions tested. Moreover, the observed Ffh or FtsY dependence of certain secretory proteins did not seem to correlate with signal peptide length or hydrophobicity. Although it is presently difficult to distinguish between direct and indirect effects, these findings suggest that other, yet unidentified, determinants in secretory proteins are also important for their SRP dependence. High-level production of homologous and heterologous secretory proteins was shown to result in elevated cellular Ffh and FtsY levels. This phenomenon is, most likely, due to post-transcriptional regulation. In conclusion, the present proteomic dissection of SRP-dependent extracellular protein accumulation provides exciting leads to identify novel determinants for interactions between secretory proteins and SRP.

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.