Genes required for cytochrome c synthesis in Bacillus subtilis

Cytochromes of c-type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram-negative bacteria. Corresponding genes are not found in the genome sequences of Gram-positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram-positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram-positive bacteria, cyanobacteria, epsilon-proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram-negative bacteria (System I) and mitochondria (System III).     

Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis.

The gram-positive, endospore-forming bacterium Bacillus subtilis contains several membrane-bound c-type cytochromes. We have isolated a mutant pleiotropically deficient in cytochromes c. The responsible mutation resides in a gene which we have named ccdA (cytochrome c defective). This gene is located at 173 degrees on the B. subtilis chromosome. The ccdA gene was found to be specifically required for synthesis of cytochromes of the c type. CcdA is a predicted 26-kDa integral membrane protein with no clear similarity to any known cytochrome c biogenesis protein but seems to be related to a part of Escherichia coli DipZ/DsbD. The ccdA gene is cotranscribed with two other genes. These genes encode a putative 13.5-kDa single-domain response regulator, similar to B. subtilis CheY and Spo0F, and a predicted 18-kDa hydrophobic protein with no similarity to any protein in databases, respectively. Inactivation of the three genes showed that only ccdA is required for cytochrome c synthesis. The results also demonstrated that cytochromes of the c type are not needed for growth of B. subtilis.     

Bacillus subtilis StoA Is a thiol-disulfide oxidoreductase important for spore cortex synthesis

Bacillus subtilis is an endospore-forming bacterium. There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood. Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins. CcdA is the only B. subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis. In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform. Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency. StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore. Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases. The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation. Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA. Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.     

Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes

Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.     

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 dithiol:disulfide oxidoreductases DsbA and DsbB of Rhodobacter capsulatus are not directly involved in cytochrome c biogenesis,but their inactivation restores the cytochrome c biogenesis defect of CcdA-null mutants

The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli, are thought to supply the reducing equivalents required for the biogenesis of c-type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA-null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps(-)) and do not produce any active cytochrome c oxidase (Nadi(-)) due to a pleiotropic cytochrome c deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps(+) Nadi(+) colonies that produce c-type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps(+) growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a dsbA ccdA double mutant was shown to be Ps(+) Nadi(+), establishing that in R. capsulatus the inactivation of dsbA suppresses the c-type cytochrome deficiency due to the absence of ccdA. Next, the ability of the wild-type dsbA allele to suppress the Ps(+) growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA-independent ccdA revertants. Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps(+) phenotypes could be suppressed by the wild-type allele of dsbB. As with dsbA, a dsbB ccdA double mutant was also Ps(+) Nadi(+) and produced c-type cytochromes. Therefore, the absence of either DsbA or DsbB restores c-type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of R. capsulatus are Ps(+) and produce c-type cytochromes, unlike their E. coli counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in R. capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions.     

Novel processing and localization of catA, ccdA associated thiol-disulfide oxidoreductase, in protein hyper-producing bacterium Brevibacillus choshinensis

Previously, we have cloned ccdA and its associated thiol-disulfide oxidoreductase gene, catA, in Brevibacillus choshinensis. CcdA is known to be an integral membrane protein and its associated oxidoreductase homologues are believed to be membrane anchoring proteins, both providing reducing equivalents across the membrane to control correct disulfide bond formation. Here, we found that CatA is first localized as a membrane bound form and then slowly released into the cellular periphery and culture medium with cleavage at a novel processing site.     

The Two CcdA Proteins of Bacillus anthracis Differentially Affect Virulence Gene Expression and Sporulation

The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.     

Identification of ccdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete deficiency in c-type cytochromes

A transposon Tn5-mob insertional mutant of Paracoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently. ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn5-mob was inserted into ccdA. The deduced ORFA product showed no similarity to any protein in databases. However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of P. pantotrophus is 42% identical to a protein predicted by ORF2, and its location within the sox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.     

Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K

The bacterial endospore cortex peptidoglycan is synthesized between the double membranes of the developing forespore and is required for attainment of spore dehydration and dormancy. The Bacillus subtilis spoVB, spoVD and spoVE gene products are expressed in the mother cell compartment early during sporulation and play roles in cortex synthesis. Here we show that mutations in these genes block synthesis of cortex peptidoglycan and cause accumulation of peptidoglycan precursors, indicating a defect at the earliest steps of peptidoglycan polymerization. Loss of spoIV gene products involved in activation of later, sigma(K)-dependent mother cell gene expression results in decreased synthesis of cortex peptidoglycan, even in the presence of the SpoV proteins that were synthesized earlier, apparently due to decreased precursor production. Data show that activation of sigma(K) is required for increased synthesis of the soluble peptidoglycan precursors, and Western blot analyses show that increases in the precursor synthesis enzymes MurAA, MurB, MurC and MurF are dependent on sigma(K) activation. Overall, our results indicate that a decrease in peptidoglycan precursor synthesis during early sporulation, followed by renewed precursor synthesis upon sigma(K) activation, serves as a regulatory mechanism for the timing of spore cortex synthesis.     

A three-protein signaling pathway governing immunity to a bacterial cannibalism toxin

We describe a three-protein signal-transduction pathway that governs immunity to a protein toxin involved in cannibalism by the spore-forming bacterium Bacillus subtilis. Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells. Killing is mediated by the exported toxic protein SdpC. We report that extracellular SdpC induces the synthesis of an immunity protein, SdpI, that protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein.     

Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis.

Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore. The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized. The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores. This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure. Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane. Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B. subtilis chromosome. This is a region where there are no other known spo, ger, or PBP genes. In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis. In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5*. The two PBPs could be distinguished by immunoassays.     

Characterization of a sporulation gene, spoIVA, involved in spore coat morphogenesis in Bacillus subtilis.

Mutations in the spoIVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a sigma E-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spoIVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spoIVA mutations, would be in accord with the hypothesis that sigma E is only active after septation and in the mother cell compartment.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Regulatory studies on the promoter for a gene governing synthesis and assembly of the spore coat in Bacillus subtilis

gerE is a regulatory gene of Bacillus subtilis that governs the synthesis and assembly of the spore coat and is required for the production of spores that are lysozyme-resistant and germination-proficient. We report the identification of the promoter for gerE and studies on the regulation of its expression. We show that gerE is switched on at the fourth hour of sporulation (stage-V) and that this expression is restricted to the mother-cell chamber of the sporangium. Dependency studies in which the level of gerE expression was measured in 36 different developmental mutants indicate that efficient expression of gerE requires the products of almost all spo0-IV genes tested as well as certain spoV genes. On the basis of its time of induction, compartmentalization of expression and pattern of dependence on other spo genes, gerE is inferred to be regulated co-ordinately with the previously studied spore coat protein gene cotA. gerE and cotA may be members of a developmental regulon of genes whose products are involved in the assembly of the spore coat.