The highly pleiotropic stage 0 sporulation locus of Bacilus subtilis, spoOA, has been cloned in bacteriophage X, subcloned in plasmids, and sequenced. The locus was found to code for a protein of 29,691 Da. Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus. The transcription start site was preceded by a promoter resembling &37-dependent promoters. Two mutations originally assigned to a second locus, spoOC, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced. To study the regulatory mechanisms that control the initiation of sporulation in bacteria, the approach taken in this laboratory has been to isolate mutants defective in the initiation process. These mutants are the stage 0 mutants and the mutations are designated by the genotypic symbol spoO. Nine spoO genes have been found among several hundred mutations mapped (1, 2). The majority of the stage 0 mutations map in five loci that are unlinked on the Bacillus subtilis chromosome. Stage 0 mutants may be blocked in any one of the processes leading to the initiation of development: the formation of an intracellular metabolic signal, the mechanisms by which this signal is associated with the switch mechanism, or the transmission of a metabolic signal to the transcription machinery. In any case it is clear that all of the stage 0 mutants have gross pleiotropic effects on the synthesis of a wide variety of gene products associated with the sporulation process (3). This result may be a consequence of the inability of SpoO mutants to permit transcription from a wide variety of promoters (4, 5). The most pleiotropic of the stage 0 mutations occur in the spoOA locus. In this report, we describe the cloning and sequencing of the spoOA locus. Nuclease S1 Protection Assay. Nuclease S1 mapping of the spoOA locus and the JH703 deletion was performed by the method of Berk and Sharp (10), with some modification. Several single end-labeled DNA probes complementary to the spoOA mRNA were prepared as follows. The EcoRI or Bgl II site of pJF1642 (this plasmid carries the equivalent fragment to pJF1361, plus several hundred bases upstream, in pUC9) were 32P-labeled at their 5' termini by using [y-32P]ATP (ICN, crude, >7000 Ci/mmol; 1 Ci = 37 GBq) and T4 polynucleotide kinase in a reaction previously described (11). The EcoRI site of pJF1599 was 3' end-labeled by using [a-32P]dATP (Amersham; >800 Ci/mmol) and E. coli DNA polymerase I large fragment. End-labeled fragments were isolated from a polyacrylamide gel after digestion at the HindIII site in the polylinker region of the two plasmids. [The sizes of the probes were 1.6 kilobases (kb) from the 5' endlabeled EcoRI fragment, 1.06 kb from the 5' end-labeled Bgl II fragment, and 0.67 kb from the 3' end-labeled EcoRI fragment from pJF1599.] B. subtilis mRNA (100 ,ug) from W168 or JH703 and approximately 40,000 cpm of end-labeled DNA probe were coprecipitated with ethanol, dried briefly under v...

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...

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