SUMMARY Secretory proteins perform a variety of important“ remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various“ secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only∼ 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities.

SummaryDisulphide bond formation catalysed by thioldisulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition.

The development of genetic competence in the Grampositive eubacterium Bacillus subtilis is a complex postexponential process. Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B. subtilis Systematic Gene Function Analysis program. Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor. BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation. Consistent with this, BdbC and BdbD are needed for the secretion of the Escherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B. subtilis. Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants. In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations. Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery.