Complete sequence and transcriptional analysis of the spo0F region of the Bacillus subtilis chromosome

Abstract

The total sequence of a 6,314-base-pair BgllM fragment of the BaciUus subtiUs chromosome containing the spoOF locus has been accomplished. Several genes of interest have been identified on this DNA fragment. The ctrA locus was recognized as coding for CTP synthetase by comparison of its deduced sequence with that of Escherichia coli CTP synthetase. A total of 53% of the residues are identical between the enzymes from these organisms. The spoOF locus was followed immediately by a locus, tsr, required for RNA synthesis in this organism. Stage 0 of the sporulation process is that time during which the bacterium decides to initiate those events required for the onset of sporulation. These initial steps in the sporulation process are under genetic control. Mutants have been obtained which are blocked at stage 0 of sporulation and are, presumably, deficient in regulation of the onset of the developmental sequence. These spoO mutations map in eight loci (14). Mutations in five of these loci, spoOA, spoOB, spoOE, spoOF, and spoOH, result not only in inhibition of sporulation, but also in the blockage of expression of a wide variety of genes (5). One approach to understanding the functions of the products of the spoO genes has been the identification of the gene products by cloning and sequencing. The spoOA (12, 18), spoOB (4, 10), spoOF (34, 39), and spoOH (8) genes have been characterized by this method.The spoOF gene was the first spoO gene cloned, although the early studies misidentified the spoOF open reading frame (31). The spoOF gene was found subsequently to code for a protein of Mr 14,286 with considerable amino acid sequence similarity to the amino-terminal portion of the spoOA product (34). This intriguing homology was discovered to be characteristic of a large variety of regulatory proteins that respond to signals from the environment (reviewed in reference 27). Overproduction of the spoOF gene product by placing the gene on a multicopy vector led to the paradoxical situation of sporulation inhibition by a protein required for sporulation (25,40 product, particularly whether it had any relationship with the genes closely linked to it on the chromosome.In this report, we extend the sequencing of the spoOF locus to include those genes surrounding it. We show that the spoOF locus is followed immediately by a locus, tsr, required for RNA synthesis in this organism. A pleiotropic suppressor mutation, rev4, of ribosomal mutations has been located in the cluster of genes surrounding the spoOF locus. The sequence of the linked ctrA locus allowed its identity as the gene for CTP synthetase. None of these loci appears to be directly related to spoOF expression. Studies of the mRNA produced by these genes has allowed the description of the transcription start sites and the promoters for these genes. Regulation of the genes was studied by lacZ fusions.MATERIALS AND METHODS Bacterial strains, media, and transformation. Escherichia coli DH5aL (F-endAl hsdR17 supE44 thi-J A-recAl gyrA96 relAl 4+80 dlacZAM15) was used for th...

Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

“…The amount of protein recovered in each sample was determined by amino acid analyses performed in duplicate (Packman & Berry, 1995). , 1989), Campylobacter jejuni (Burucoa et al, 1995), Schizosaccharomycespombe (Mutoh & Hayashi, 1994), Saccharomyces cerevisiue (Schwelberger et al, 1989), Neurospora crassa (Yamashita & Stuart, 1995), Corynebacterium glutamicum (von der Osten et al, 1989), Bacillussubtilis (Trach et al, 1988;Mitchell et al, 1992). and Rhodobactersphaeroides (Chen et al, 1991;Gibson et al, 1991) were aligned using the computer program PILEUP (Devereux et al, 1984) with a gap weight of 3.0and a gap length weight of 0.1.…”

An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter

“…Several of the pleC suppressors displayed conditional cell division defects (9) and were mapped to divJ (5), which, like pleC (10), encodes a histidine protein kinase, and to divK (6), which encodes the first essential response regulator identified in bacteria. DivK is a 125-residue polypeptide that belongs to the subfamily of single-domain response regulators that includes the chemotactic protein CheY of Salmonella typhimurium (11) and the sporulation protein SpoOF of Bacillus subtilis (12). Although DivJ and DivK appear to play central roles in regulating initiation of cell division in C. crescentus (6), the PleC kinase may be more directly involved in regulating motility, chemotaxis, and stalk formation (13).…”

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis

“…There is no known function for the orfU gene product, and its deletion causes no observable change in phenotype (21). A similar open reading frame with 33% identity to orfU was reported for the Clostridium acetobutylicum glucitol operon, and its function is unknown (lla).…”

“…When these were compared with sequences in the data base, we found that the amino acid sequence matched the predicted sequence of the orfY-tsr gene, which maps close to spoOF (21); the sequence of orfYtsr, which apparently codes for fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), has recently been revised (llb).…”