Genetic Analysis of Global Streptomyces Antibiotic Regulation

Champness 9604055链霉菌是丝状土壤细菌，具有两个使它们成为具有研究吸引力的原核生物的特征：它们发育成复杂的、多细胞的、分化的菌落，并产生大量的次生代谢产物，其中许多用作抗生素。抗生素的产生通常与形态分化有关，始于营养生长和前孢子分化菌丝的产生之间的过渡。这项研究的长期目标是发现调控抗生素合成的基因，了解它们的功能，并确定它们在控制抗生素基因表达的调控网络中的功能关系。这项提案的近期目标集中于在之前的NSF资助期中发现的三种遗传因素：Absa、AbbB和Mia。研究将沿着以下方向进行：1.确定Absa编码的信号转导系统对抗生素基因表达实施全球负调控的机制。遗传学、分子遗传学和生物化学实验将：a)表征AbsA1/A2“传感器-激酶”和反应调节蛋白的重要功能区域和相互作用；b)确定蛋白质磷酸化在AbsA介导的调节中的作用；2.AbB基因及其可能的基因产物RNaseIII将通过确定RNaseIII(AbbB)无效表型、对天蓝色链霉菌RNaseIII的功能研究以及候选RNaseIII靶标的鉴定来表征。3.对190个核苷酸的Mia序列进行鉴定，以确定该序列的功能区，该序列对抗生素基因的表达具有多拷贝抑制作用。这项工作的结果有望阐明链霉菌基因表达的调控程序，并扩大我们对微生物多细胞分化的理解。

Champness 9604055 Streptomycetes, filamentous soil bacteria, possess two characteristics that make them attractive prokaryotes for study: they develop into complex, multicellular, differentiated colonies and they produce a vast array of secondary metabolites, many of which are useful as antibiotics. Antibiotic production is usually coupled to morphological differentiation, beginning at the transition between vegetative growth and production of pre-spore differentiated hyphae. The long term goal of this research is to discover the genes that regulate antibiotic synthesis, to understand how they function, and to determine their functional relationships in the regulatory network controlling antibiotic gene expression. The immediate objectives of this proposal focus on three genetic elements discovered in previous NSF funding periods: absA, absB and mia. Research will proceed along the following lines: 1. The mechanism by which the absA-encoded signal transduction system exerts global negative control over antibiotic gene expression will be determined. Genetic, molecular genetic and biochemical experiments will: a) characterize functionally important regions and interactions of the AbsA1/A2 "sensor-kinase" and response regulator proteins; b) establish the role of protein phosphorylation in AbsA-mediated regulation; and c) explore the genetic pathway through which absA regulates expression of the antibiotic "pathway-specific regulators." 2. The absB gene and its probable gene product, an RNaseIII, will be characterized through determination of the RNaseIII (absB) null phenotype, functional studies on the S. coelicolor RNaseIII, and identification of candidate RNaseIII targets. 3. The 190 nt mia sequence, which exerts multi-copy inhibition of antibiotic gene expression, will be characterized to determine the functional regions of the sequence. The results of the proposed work promise to elucidate aspec ts of the regulatory programs controlling streptomycete gene expression, and to expand our understanding of microbial multicellular differentiation.