Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis.

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.     

Mutational analysis of the helix-turn-helix region of Bacillus subtilis response regulator DegU, and identification of cis-acting sequences for DegU in the aprE and comK promoters

The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development. Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively. Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity. This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter. We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions. Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation. Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE. Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.     

Isolation and characterization of mutations in the gene encoding an endogenous Bacillus subtilis beta-galactosidase and its regulator.

We have isolated mutations that appear to inactivate the gene (lacA) encoding an endogenous beta-galactosidase activity in Bacillus subtilis and in a closely linked negative regulatory element (lacR). Both genes map to the hisA-thrA region. The lacA mutations may help to avoid some of the problems arising from the use of the Escherichia coli lacZ gene as a reporter gene in B. subtilis.     

Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions.

We constructed in-frame translational fusions of the Escherichia coli lacZ gene with four genes (sspA, sspB, sspD, and sspE) which code for small, acid-soluble spore proteins of Bacillus subtilis, and integrated these fusions into the chromosomes of various B. subtilis strains. With single copies of the fusions in wild-type B. subtilis, beta-galactosidase was synthesized only during sporulation, with the amounts accumulated being sspB much greater than sspE greater than or equal to sspA greater than or equal to sspD. Greater than 97% of the beta-galactosidase was found in the developing forespore, and the great majority was incorporated into mature spores. Less than 2% of the maximum amount of beta-galactosidase was made when these fusions were introduced into B. subtilis strains blocked in stages 0 and II of sporulation, as well as in some stage III mutants. Other stage III mutants, as well as stage IV and V mutants, had no effect on beta-galactosidase synthesis. Increasing the copy number of the sspA-, sspD-, or sspE-lacZ fusions (up to 17-fold for sspE-lacZ) in wild-type B. subtilis resulted in a parallel increase in the amount of beta-galactosidase accumulated (again only in sporulation and with greater than 95% in the developing forespore), with no significant effect on wild-type small, acid-soluble spore protein production. Similarly, the absence of one or more wild-type ssp genes or the presence of multiple copies of wild-type ssp genes had no effect on the expression of the lacZ fusions tested. These data indicate that these ssp-lacZ fusions escape the autoregulation seen for the intact sspA and sspB genes. Strikingly, the kinetics of beta-galactosidase synthesis were identical for all four ssp-lacZ fusions and paralleled those of glucose dehydrogenase synthesis. Similarly, all asporogenous mutants tested had identical effects on both glucose dehydrogenase and ssp-lacZ fusion expression.     

A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis

A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.     

Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis 168

An additional amylase, besides the typical alpha-amylase, was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the beta-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing beta-cyclodextrin (beta-CD), maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the beta-CD hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the beta-CD hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing beta-CD in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and beta-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.