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.     

AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.     

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.     

Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.