Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis.

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.     

Bacillus subtilis YqjG is required for genetic competence development

Members of the evolutionary conserved Oxa1/Alb3/YidC family have been shown to play an important role in membrane protein insertion, folding and/or assembly. Bacillus subtilis contains two YidC-like proteins, denoted as SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable, but SpoIIIJ is essential for spore formation and YqjG cannot complement this activity. To elucidate the role of YqjG, we determined the membrane proteome and functional aspects of B. subtilis cells devoid of SpoIIIJ, YqjG or both. The data show that SpoIIIJ and YqjG have complementary functions in membrane protein insertion and assembly. The reduced levels of F(1)F(O) ATP synthase in cells devoid of both SpoIIIJ and YqjG are due to a defective assembly of the F(1)-domain onto the F(0)-domain. Importantly, for the first time, a specific function is demonstrated for YqjG in genetic competence development.     

Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.     

The Bacillus subtilis ureABC operon.

The Bacillus subtilis ureABC operon encodes homologs of the three subunits of urease enzymes of the family Enterobacteriaceae. Disruption of ureC prevented utilization of urea as a nitrogen source and resulted in a partial growth defect in minimal medium containing limiting amounts of arginine or allantoin as the sole nitrogen source.     

CodY is required for nutritional repression of Bacillus subtilis genetic competence.

The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.     

The Bacillus subtilis spoIIG operon encodes both sigma E and a gene necessary for sigma E activation.

A sporulation-specific sigma factor of Bacillus subtilis (sigma E) is formed by a proteolytic activation of a precursor protein (P31). Synthesis of the precursor protein is shown to be abolished in B. subtilis mutants with plasmid insertions as far as 940 base pairs upstream of the P31 structural gene (sigE), and processing of P31 to sigma E is blocked by a deletion in this upstream region. These results substantiate the view that sigE is the distal member of a 2-gene operon and demonstrate that the upstream gene (spoIIGA) is necessary for sigma E formation.     

X-ray study of competence development in Bacillus subtilis

Summary Pre-competent and competent cultures of Bacillus subtilis were x-irradiated before and after centrifugal separation of cells in a Renografin density gradient. Pre-competent cultures have no cells at the radiosensitivity of the cells in the bulk competent culture, but there is a substantial fraction of cells in a multi-target state, with heterogeneous target numbers. On reaching maximal competence, the survival becomes entirely linear in radiosensitivity. Irradiation of the separated competent cells shows that competence development correlates with disappearance of multi-target cells from the non-competent band of cells and the appearance of single-target cells in the competent band at a radiosensitivity equal to that of the bulk competent culture. Thus the multi-target state may be a required stage in the development of competence in this system.     

The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system

The Bacillus subtilis LiaRS two-component system (TCS) responds to perturbations of the cell envelope induced by lipid II-interacting antibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce the liaRS TCS. In addition to insertions in liaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of the yydFGHIJ operon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that the yydFGHIJ operon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent P_liaI promoter only in the presence of the yydFGH genes. Cells harboring the complete yydFGHIJ operon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.     

Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.     

The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis.

Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase.     

A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon

An insertion mutation was isolated that resulted in derepressed expression of the Bacillus subtilis dipeptide transport operon (dpp) during the exponential growth phase in rich medium. DNA flanking the site of insertion was found to encode an operon (codVWXY) of four potential open reading frames (ORFs). The deduced product of the codV ORF is similar to members of the λ Int family; CodW and CodX are homologous to HsIV and HsIU, two putative heat-shock proteins from Escherichia coli, and to LapC and LapA, two gene products of unknown function from Pasteurella haemolytica. CodX also shares homology with a family of ATPases, including CIpX, a regulatory subunit of the E. coli ClpP protease. CodY does not have any homologues in the databases. The insertion mutation and all previously isolated spontaneous cod mutations were found to map In codY. In-frame deletion mutations in each of the other cod genes revealed that only codY is required for repression of dpp in nutrient-rich medium. The cody mutations partially relieved amino acid repression of the histidine utilization (hut) operon but had no effect on regulation of certain other early stationary phase-induced genes, such as spoVG and gsiA.