Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion.

Bacillus subtilis strains carrying div-341 or sacU mutations, or both, have been characterized to reveal the roles of both genes in the initiation of sporulation, as well as in cell division and exoenzyme secretion. Both mutations were closely linked by transformation and caused the pleiotropic effects on sporulation and sporulation-associated events. Some sacU mutations (sacUh) resulted in hyperproduction of exoenzymes, reduced autolysis, and an ability to sporulate in the presence of excess nutrients. The div-341 mutation, on the other hand, resulted in filamentous growth at a higher temperature (45 degrees C) and showed spo0 properties at an intermediate permissive temperature (37 degrees C) in the usual sporulation medium. However, the div-341 strain sporulated better than wild-type strain at 37 degrees C in the presence of excess nutrients. Exoenzyme production and autolysis were reduced at 37 degrees C in the div-341 strain. A double mutant with sacUh32 and div-341 showed the complex phenotypes. It showed the sacUh32 property of autolysis and exoenyzme secretion. It showed the sacUh32 property of sporulation at 30 degrees C and the div-341 property at 37 degrees C. Slow growth and defective spore outgrowth of the div-341 strain at 37 degrees C were not observed in the double-mutant strain. Based on pleiotropic phenotypes and close linkages of both mutations, we discuss the relationship between the sacU and div-341 genes and their roles in sporulation, exoenzyme secretion, and cell division.     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.     

Impaired cell division and sporulation of a Bacillus subtilis strain with the ftsA gene deleted.

The ftsZ and ftsA genes of Bacillus subtilis are organized in a simple operon expressed from promoter sequences immediately upstream of ftsA. The promoter-distal ftsZ gene is an essential septation gene. In this report, it is shown that the promoter-proximal ftsA gene can be deleted in a previously constructed strain in which the essential gene, ftsZ, is under the control of the inducible spac promoter. Absence of the ftsA gene product resulted in a very filamentous morphology indicating an important role for ftsA in cell division. Also, growth was severely impaired, and viability and sporulation were reduced. The defective sporulation phenotype correlated with a deficiency in the processing of pro-sigma E to its active form.     

Molecular cloning of a Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion

The wild type div-341+ gene of Bacillus subtilis was cloned in a temperate phage rho 11, and was recloned in a smaller temperate phage phi 105. The resulting Div+ transducing phage carried a 3 kilobase Cfr13I digested chromosomal fragment which showed Div+ transforming activity and contained the whole div-341+ gene which is involved in cell division, sporulation, exoenzyme secretion, competent cell formation, and autolysis. A partial restriction map of the fragment was established. The merodiploid system of the div-341+ gene, wild type gene on the phage genome and mutant gene on the chromosome, resulted in the suppression of mutant phenotypes and indicated that the wild type div-341+ gene is dominant over mutant gene.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.     

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.     

Nucleotide sequence of the Bacillus subtilis developmental gene spoVE

We have determined the nucleotide sequence of a 1159 bp DNA fragment containing the spoVE locus of Bacillus subtilis. The locus contained a single open reading frame of 293 codons. On the basis of the predicted amino acid sequence, the product of the spoVE gene is believed to be a protein with an Mr of 31,539. The amino-terminal portion of the spoVE gene was used to construct a translational fusion with the lacZ' gene. The hybrid spoVE-lacZ' gene was shown to be expressed in Escherichia coli and, therefore, it seems reasonable to conclude that the proposed open reading frame for the spoVE gene does indeed function in vivo.