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.     

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.     

Sporulating bacteria prefers predation to cannibalism in mixed cultures

Predatory behavior, a property associated with ecosystems, is not commonly observed in microorganisms. However, cannibalistic tendencies have been observed in microorganisms under stress. For example, pure culture of Bacillus subtilis exhibits cannibalism under nutrient limitation. It has been proposed that a fraction of cells in the population produce Spo0A, a regulatory protein that is responsible for delaying sporulation. Cells containing spo0A would produce a killing factor by activating skf operon and an associated pump to export the factor. Cells that do not contain spo0A in the population are lysed. However in addition to the competition among the cells of B. subtilis, these cells also compete with other organisms for the limited nutrients. In this work, we report the cannibalistic behavior of B. subtilis in presence of Escherichia coli under severe nutritional limitation. We demonstrate that B. subtilis lyses cells of E. coli using an antibacterial factor under the regulation of Spo0A. Our experiments also suggest that B. subtilis prefers predation of E. coli to cannibalism in mixed cultures. B. subtilis also demonstrated predation in mixed cultures with other soil microorganisms, such as, Xanthomonas campestris, Pseudomonas aeruginosa and Acinetobactor lwoffi. This may offer B. subtilis a niche to survive in an environment with limited nutrients and under competition from other microorganisms.     

Bistability and biofilm formation in Bacillus subtilis

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.     

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.     

The effect of Spo0A and AbrB proteins on expression of the genes of guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis recombinant strains

Guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus are actively secreted under phosphate starvation by recombinant strains of Bacillus subtilis with native regulatory systems and by strains defective in some proteins of the Spo0A phosphorylation pathway. The level of expression of ribonuclease genes has been shown to increase approximately sixfold in recombinant strains with mutation in the spo0A gene and threefold in the spo0A/abrB mutants, as compared with native strains. These results demonstrate that the Spo0A protein regulates the production of ribonucleases and thus acts as a repressor, while the AbrB protein is an activator of expression of the genes encoding ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells.     

Analysis of non-polar deletion mutations in the genes of the spo0K (opp) operon of Bacillus subtilis

The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.     

Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies.

We have isolated a 1.0-kilobase fragment of the Bacillus subtilis chromosome which, when present in high-copy-number plasmids, caused a sporulation-proficient strain to become phenotypically sporulation deficient. This is referred to as the sporulation inhibition (Sin) phenotype. This DNA fragment, in multicopy, also inhibited the production of extracellular protease activity, which normally appears at the beginning of stationary growth. The origin of the fragment was mapped between the dnaE and spo0A genes on the B. subtilis chromosome, and its complete DNA sequence has been determined. By analysis of various deletions and a spontaneous mutant the Sin function was localized to an open reading frame (ORF) predicted from the DNA sequence. Inactivation of this ORF in the chromosome did not affect the ability of cells to sporulate. However, the late-growth-associated production of proteases and alpha-amylase was elevated in these cells. The predicted amino acid sequence of the protein encoded by this ORF had a DNA-binding domain, typically present in several regulatory proteins. We propose that the sin ORF encodes a regulatory protein that is involved in the transition from vegetative growth to sporulation.     

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.