CLONING AND CHARACTERIZING B. LICHENIFORMIS APASE GENES

我们的初步结果已经确定至少有2个结构性的

Our preliminary results have established there are at least 2 structural APase genes in B. licheniformis, APase I and APase II, and that the 2 genes are transcribed by different RNA polymerase holoenzymes. We will determine if these are the only 2 genes responsible for the APase species previously localized and isolated from this organism, by using the cloned genes to construct a APase I-APase II double mutant of B. licheniformis. If the double mutant is not APase negative, we will clone the other APase gene(s) and include this cloned gene(s) in all the proposed experiments below. These experiments are designed to examine the hypothesis that the unique distribution of alkaline phosphatase in B. licheniformis ((1) membrane-associated (integral--ecto-protein; peripheral--ecto- and endo-protein) and (2) secreted - primarily in stationary growth) is the result of similar multiple structural genes under different regulatory control. We propose: 1. To determine if there are more than 2 APase structural genes in the B. licheniformis genome and construct B. licheniformis MC14 mutants which retain only 1 functional APase gene. 2. To analyze the transcriptional regulation of each clone APase gene. 3. To determine the DNA sequence of each APase gene and compare the functional domains of the APase proteins as determined from the derived amino acid sequence. 4. To determine the final destination of the APase in APase mutants which retain only 1 APase gene.