Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis

A spoIIA::lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the 'later' loci tested. The beta-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spo0J, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.     

Glucose-resistant sporulation in Bacillus subtilis crsA47 mutants does not depend on promoter switching at the spo0A gene

We have found that sporulation in Bacillus subtilis crsA47 mutants does not require the sigma(H)-dependent promoter of the spo0A gene. This implies that the glucose-resistant sporulation phenotype of this strain is not related to the switch from the vegetative-stage sigma(A)-dependent promoter to the sigma(H)-dependent promoter at the spo0A gene.     

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.     

Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis

A spoVAA::lacZ gene fusion has been used to study expression of the spoVA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known spo0 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.     

Tyrocidine-induced modulation of the DNA conformation in Bacillus brevis

Using the [3H]trimethylpsoralen photobinding method [Sinden, R.R., Carlson, J.O. & Pettijohn, D.E. (1980) Cell 21, 773-783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension-relaxing and tension-inducing activities. Addition of tyrocidine to vegetative cells reduced by one-half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross-links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA-tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.     

Cloning of an early sporulation gene in Bacillus subtilis.

A 0.8-megadalton BglII restriction fragment of Bacillus licheniformis cloned into the BglII site of plasmid pBD64 can complement spo0H mutations of Bacillus subtilis. The clone was isolated by selecting for the Spo+ phenotype and antibiotic resistance, using the helper system described by Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980). The insert is functional in both orientations and thus presumably has its own promoter. A deletion generated within the 0.8-megadalton insert by HindIII restriction and subsequent religation eliminates the ability of the cloned fragment to complement spo0H mutations. The cloned B. licheniformis deoxyribonucleic acid segment specifies the synthesis, in minicells, of a polypeptide of approximately 27,000 daltons. This protein is observed with both orientations, but not when the HindIII deletion is present in the cloned B. licheniformis chromosomal fragment. We have also demonstrated that ribonucleic acid complementary to the cloned B. licheniformis sporulation gene is transcribed in B. licheniformis both during vegetative growth and sporulation.     

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.     

In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.     

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.