Our work with B. licheniformis has shown there are multiple structural genes for vegetative alkaline phosphatase (APase) in Bacillus. In vitro-derived mutations in the coding region of these two tandem genes do not affect sporulation APase. These studies were designed to examine the hypothesis that the unique distribution of biochemically identical APase proteins in B. licheniformis (1) membrane associated on the inner leaflet (peripherally) and outer leaflet (integrally) and (2) secreted is the result of multiple structural genes under different regulatory control. We developed a protoplast transformation system for B. licheniformis and designed experiments to make chromosomal mutations in the cloned APase genes by gene replacement. These experiments resulted in mutations of the vegetative APase genes via illegitimate recombination. Analysis of B. subtilis, a closely related organism with a superior genetic system, demonstrates that the same APase species (soluble species: (1) secreted, (2) """"""""periplasmic,"""""""" and (3) cytosol (inactive), or membrane species: (1) salt and (2) detergent extractable) are produced in response to the same growth conditions during the same phases of growth as observed in B. licheniformis. Given that the unique aspects of the B. licheniformis APase system are shared by B. subtilis a switch to B. subtilis will permit construction of mutants and the analysis of the complex regulation of the APase genes in a homologous system. Illustrative of the complex control, we have recently shown that the regulation of B. subtilis vegetative APase genes involve not only the phosphate regulon control, but also early sporulation control. We have isolated 11 clones from B. subtilis which express phosphatase activity in E. coli and are currently identifying the APase genes among them. Experiments outlined below are designed to increase our understanding of the regulation of temporal gene expression and protein localization in Bacillus. (I) Localize the integral membrane APase and verify previously reported endo-location of the peripheral membrane APase using lactoperoxidase 125I-labeling of intact and lysed protoplasts. Localize sporulation APase. (II) Identify the APase genes among the B. subtilis phosphatase clones. (III) Clone regulatory genes (sapA and sapB) for sporulation APase production. (IV) Characterize APase structural and regulatory genes: sequence, transcriptional start mapping, RNA polymerase holoenzyme required, promoter lacZ fusions in APase regulatory B. subtilis mutants. (V) Construct strains retaining only 1 APase gene to determine destination of each gene product and possible cell-bound precursor to the secreted APase.
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