The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo-). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo- mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram-negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide-transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP-binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide-binding protein, which is periplasmically located in Gram-negative species, has a signal sequence characteristic of lipoproteins with an amino-terminal lipo-amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram-negative bacteria is the recycling of cell-wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signalling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.     

The primary role of comA in establishment of the competent state in Bacillus subtilis is to activate expression of srfA.

The establishment of genetic competence in Bacillus subtilis requires the genes of the competence regulon which function in the binding, processing, and transport of DNA. Their expression is governed by multiple regulatory pathways that are composed of the comA, comP, sin, abrB, spo0H, spo0K, spo0A, degU, and srfA gene products. Among these, srfA is thought to occupy an intermediate position in one of the pathways that controls late competence gene expression. The full expression of srfA requires the gene products of comP, comA, and spo0K. To determine the role of these genes in the regulation of competence development, the expression of the srfA operon was placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter Pspac and the expression of the Pspac-srfA construct was examined in mutants blocked in early competence. By monitoring the IPTG-induced expression of Pspac-srfA with a srfA-lacZ operon fusion, it was observed that srfA expression was no longer dependent on the products of comP, comA, and spo0K. Production of the lipopeptide antibiotic surfactin in Pspac-srfA-bearing cells was induced in the presence of IPTG and was independent of ComP and ComA. Competence development was induced by IPTG and was independent of comP, comA, and spo0K in cells carrying Pspac-srfA. These results suggest that the ComP-ComA signal transduction pathway as well as Spo0K is required for the expression of srfA in the regulatory cascade of competence development. Studies of Pspac-srfA also examined the involvement of srfA in the growth stage-specific and nutritional regulation of a late competence gene.     

Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.     

Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation

Summary
Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second-site revertants can be found that restore sporulation and pep-tide transport. These second-site mutations were found in a second oligopeptide transport system, app , in which the peptide-binding protein, AppA, is mutant owing to a frame-shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD-appF-appA-appB-appC , with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra- and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app ⁺opp⁻ are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The pheno-types raised some speculation about the subunit configuration of the Opp system.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

Genetic analysis of the non-sporulating phenotype of brewer's yeasts

The ploidies and sporulation abilities of six brewer's yeasts were examined. One (YB11-1) out of the six was triploid and sporulating, another (IFO2031) was haploid, and the others (IFO1167, IFO2003, S341 and YB3-7) were diploid and non-sporulating. The five non-sporulating strains did not have the premeiotic DNA synthesis. Their non-sporulating phenotypes were genetically analyzed by examining the sporulation abilities of hybrids between brewer's yeasts and standard genetic strains of Saccharomyces cerevisiae. All non-sporulating brewer's yeasts complemented 32 sporulation-deficient mutations (spoT–spoT23, spo1–spo5, spo7, spo8, spo10, and spo11). Hybrids between brewer's yeasts and haploid or diploid strains homozygous for the mating-type locus had poor or no sporualtion. On the contrary, hybrids between brewer's yeasts and diploid strains heterozygous for the mating-type locus sporulated at a high frequency. These results indicated that the non-sporulating phenotype of brewer's yeasts was caused by a deficiency of the mating-type genes rather than by mutations of sporulation genes. The Southern hybridization probed with the MATa gene showed polymorphisms in mating-type genes of brewer's yeasts.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.