Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168.

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.