RecE-dependent lysogenic induction in the absence of repressor in Bacillus subtilis non-complementing diploids

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of phi 105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent phi 105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.     

The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A.

SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase. Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE.     

Temporal regulation of the Bacillus subtilis early sporulation gene spo0F

The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class. One of these, spo0F, codes for a protein of 14,000 daltons. We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays. spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels. The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect. In most respects, the spo0F gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P. Zuber and R. Losick, J. Bacteriol. 169:2223-2230, 1987).     

Critical roles of spo0A and spo0H in vegetative alkaline phosphatase production in Bacillus subtilis.

Growth conditions established to optimize vegetative alkaline phosphatase production and stability in Bacillus subtilis were used to compare alkaline phosphatase synthesis and secretion in isogenic strains JH646 (spo0A12) and JH646MS (spo0A12 abrB15). A mutation in spo0A blocked vegetative alkaline phosphatase production, and a second mutation at the abrB locus resulted in hyperinduction of vegetative alkaline phosphatase. Phosphate regulation of vegetative alkaline phosphatase synthesis was unaffected in the double mutant. spo0H, on a multicopy plasmid, partially overcame the spo0A effect.     

New suppressor mutation sur0B of spo0B and spo0F mutations in Bacillus subtilis

Two extragenic suppressor mutations, sur0B20 and sur0F1, which restore the sporulation of spo0B or spo0F mutants of Bacillus subtilis to the wild-type level, were obtained. These suppressor mutations were located in the spo0A gene. Their location is close to that of the sof-1 mutation, which suppresses spo0B, spo0E and spo0F mutations. However, spo0 strains bearing the sur0B20 mutation differed in several phenotypic characteristics from spo0 mutants bearing the sof-1 suppressor. Nucleotide sequence analysis revealed that the sur0B20 and sur0F1 mutations resulted in Glu14 to Val and Asn12 to Lys conversion, respectively, in the spo0A gene. This result indicates that sur0B20 is a new suppressor of spo0b and spo0F mutations, whereas sur0F1 is identical to sof-1.     

Identification of a Bacillus subtilis spo0H allele that is necessary for suppression of the sporulation-defective phenotype of a spo0A mutation.

A mutation in Bacillus subtilis spo0A codon 97 suppressed the sporulation defect caused by the spo0A9V mutation. The suppressor activity of the codon 97 mutation was evident only in the presence of a novel spo0H allele. Our results suggest that the spo0A gene product interacts with the sigma factor subunit of RNA polymerase.     

The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.     

Identification of the Product of Sporulation Gene spo0B in Bacillus subtilis

A 1.4-megadalton EcoRI restriction fragment carrying Bacillus subtilis sporulation gene spo0B was cloned from the specialized transducing phage, φ 105spo0B, into a unique EcoRI site of plasmid vector pUB110, and four plasmids having a deletion in the 1.4-megadalton EcoRI fragment were constructed. Analysis of the polypeptides synthesized in B. subtilis minicells harboring these plasmids and the sporulation ability of strain UOT0436 (spo0B136 recE4) harboring these plasmids showed that the spo0B gene product is a polypeptide of 24,000 daltons. Two-dimensional polyacrylamide gel analysis showed that the isoelectric point of this protein is almost neutral.     

Cloning of sporulation genes spo0A and spo0C of Bacillus subtilis onto rho 11 temperate bacteriophage.

A HindIII fragment harboring the intact spo0C gene of Bacillus subtilis was cloned with rho 11 temperate bacteriophage as a vector. Transformation experiments with the DNA from rho 11 dspo0C+ specialized transducing phage showed that the spo0C gene resides on a 5.3-megadalton fragment generated by HindIII digestion. The 5.3-megadalton fragment also contains the intact spo0A gene, but not spoIIIA, spoIIIB, or spoIVB.     

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.     

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.