CLONING AND CHARACTERIZING B SUBTILIS APASE GENES

我们对地衣芽孢杆菌的研究表明，有多种 植物营养碱性磷酸酶(APase)的结构基因 芽孢杆菌。在体外衍生的这些基因编码区的突变 两个串联基因不影响产孢酶。这些研究 旨在检验这样一种假设，即唯一的 生物化学上相同的APase蛋白在B.B. 地衣(1)膜伴生在小叶内侧 (外周)和外叶(整体)和(2)分泌的 多个结构基因在不同调控下的结果 控制力。我们建立了一套原生质体转化系统。 地衣并设计了制造染色体的实验 通过基因置换使克隆的APase基因发生突变。这些 实验导致了营养APase基因的突变 非法重组。对枯草芽孢杆菌的分析 拥有优越遗传系统的亲缘生物，证明了 相同的APase种类(可溶性种类：(1)分泌，(2) “周质”，和(3)胞浆(不活跃)，或膜物种： (1)盐和(2)可提取洗涤剂)作为回应 在相同的生长阶段中达到相同的生长条件 在地衣芽孢杆菌中观察到。鉴于这一技术的独特性 地衣芽孢杆菌APase系统与枯草芽孢杆菌共用 到枯草杆菌将允许构建突变体和分析 APase基因在同源细胞中的复杂调控 系统。作为复杂控制的例证，我们最近 表明枯草芽孢杆菌营养体APase基因的调控 不仅涉及磷酸调节蛋白的调控，而且还涉及早期 产孢量控制。我们已经从枯草杆菌中分离出11个克隆 它们在大肠杆菌中表达磷酸酶活性，目前 对其中的APase基因进行鉴定。实验概述如下 旨在增加我们对监管的理解 芽孢杆菌中时间基因的表达和蛋白质定位。(一) 定位整膜APase并验证以前的报道 外周膜APase的内切定位 完整和裂解原生质体的乳过氧化物酶125I标记。 定位产孢子形成APase。(Ii)鉴定APase基因 枯草杆菌磷酸酶克隆。(Iii)克隆调控基因 (SapA和SapB)产生产孢子型APase。(四) APase结构和调控基因的特征：序列， 转录起始图谱，需要RNA聚合酶全酶， 启动子LacZ在APase调控枯草杆菌突变体中的融合。(五) 构建仅保留1个APase基因的菌株以确定 每种基因产物的目的地和可能的细胞结合前体 与分泌的APase有关。

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.