BaciUlus subtilis has an alkaline phosphatase (APase) gene

The first alkaline phosphatase (APase) structural gene mutant of Bacilus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide. The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of Bacillus species produce alkaline phosphatase (APase) when phosphate becomes growth limiting (termed vegetative) as well as during sporulation, when phosphate supplies are abundant. We have shown that there are at least two different APase proteins responsible for vegetative APase activity (9) and present evidence here that at least two are produced during sporulation. When the vegetative APases are derepressed in culture medium which does not support sporulation, the two secreted proteins that are responsible for the major APase activity are APase III and APase IV. APase III has a native molecular weight of 80,000 and is active as a dimer. APase IV is active as a monomer and has a native molecular weight of 45,000 (9).During the process that leads to formation of a mature spore, APase synthesis is derepressed as a developmental stage-specific event unrelated to the supply of phosphate in the medium (4-6, 11, 33). The function of the sporulation APase proteins in producing a mature exospore or later in the germination process is unknown because previously the only mutations that eliminated sporulation APase were in regulatory genes that block all developmental events past stage II.In order to understand the function and regulation of the multiple alkaline phosphatase proteins that are synthesized in Bacillus subtilis, we have isolated, purified, and sequenced the amino termini of two of these proteins (9). These sequences allowed us to synthesize oligomers that were used to probe genomic libraries. We report here the cloning, partial sequence, and insertional inactivation of one of those APases, APase III. We also present evidence that the APase III protein, unlike APase IV, is synthesized during both vegetative and sporulation-derepressing conditions. Analysis of the phoAIII strain, the first structural APase mutant in Bacillus species, indicates that this gene * Corresponding author.responds to phosphate starvation as well as to the developmental signals of sporulation. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis JH646MS (trpC2 pheAl spoOA12 abrB15) (from J. Hoch) was used to isolate the APase proteins. B. subtilis 168 (trpC2) was the wild-type strain from which the gene was cloned and in which the structural mutations were made. The plasmid pJM103 (J. Hoch), derived from a ligation between pUC19 and the gram-positive chloramphenicol resistance gene, was used in cloning and construction of the structural mutant. The recipient Escherichia coli strain used for cloning, subcloning, and culturing the plasmid was DH5a

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