RPG: A Novel Genetic Screen for the Identification of Genes Involved in Streptomyces Morphogenesis

RPG：用于鉴定参与链霉菌形态发生的基因的新型遗传筛选

• 批准号: 9628767
• 负责人: Joanne Willey
• 金额: $ 1.53万
• 依托单位: Hofstra University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9628767&HistoricalAwards=false
• 关键词: RPG; Novel; Genetic; Screen; Identification

Willey 9628767 The Streptomycete spp. are gram positive soil bacteria that undergo a temporally and spatially regulated cycle of morphological and physiological differentiation similar to that of the filamentous fungi. During the streptomycete life cycle three cell types are made. The substrate-penetrating vegetative mycelia give rise to the upwardly growing hair-like aerial mycelia. Antibiotic biosynthesis is concomitant with the onset of the aerial mycelium formation. The aerial mycelium subsequently differentiate into uninucleate spores. Two classes of developmentally controlled genes have been identified in Streptomyces coelicolor: the whi and bld genes. The whi genes control the development of spores form the aerial mycelium; whi mutants thus from only the aerial mycelium. The bld genes are involved in the differentiation of the vegetative mycelium into the aerial mycelium. Mutations in bld genes result in the growth of colonies that form only the substrate mycelium; many of these mutants are pleiotropically blocked in antibiotic formation as well. bld mutants are also blocked in the synthesis of a small morphogenetic peptide, SapB, which appears to be directly involved in aerial mycelium formation. This is evidenced by the restoration of aerial mycelium formation by bld mutants following the exogenous application of purified SapB. Additional evidence supports the notion that SapB is synthesized nonribosomally, presumably by a multienzyme complex in analogy with the peptide antibiotics. What is the function of SapB in S. coelicolor morphogenesis and what is the role of nutrient sensing in the differentiation of the vegetative to the aerial mycelium? This research planning grant seeks to address these questions by the identification of genes involved specifically in SapB biosynthesis and/or the nutritional regulation of aerial mycelium formation. This approach is based on the observation that the addition of purified SapB relieved the suppression of aerial mycelium formation in the morphologically wild type strain S. coelicolor J1501 when grown in LB agar media. This restoration of aerial mycelium formation permits the screening of a genomic library expressed in high copy in S. coelicolor J1501 cells in attempt to overproduce genes essential for SapB biosynthesis and thereby mimic this phenomenon. Plasmid from complemented colonies will be purified and inserts subcloned to establish the minimum size required for the selected phenotype. Subcloned inserts will then be sequenced and analyzed so that a putative function might be assigned to specific gene products. Complementing sequences might encode either i) the SapB structural gene or ii) regulatory or structural genes that are part of a presumed SapB peptide synthetase operon and/or iii) genes not directly involved in SapB biosynthesis or regulation but nonetheless involved in morphological differentiation. The identification of such genes would then provide preliminary data on which a full research proposal will be based. ***

Willey 9628767链霉菌属是革兰氏阳性土壤细菌，其经历类似于丝状真菌的形态和生理分化的时间和空间调节循环。 在链霉菌的生命周期中，有三种细胞类型。 穿透基质的营养菌丝产生向上生长的毛状气生菌丝。 抗生素的生物合成是伴随着气生菌丝体形成的开始。 气生菌丝体随后分化成单核孢子。 天蓝色链霉菌中有两类发育控制基因：whi和bld基因。 whi基因控制孢子从气生菌丝体的发育;因此，whi仅从气生菌丝体突变。 bld基因参与营养菌丝体向气生菌丝体的分化。 bld基因的突变导致只形成基质菌丝体的菌落生长;这些突变体中的许多在抗生素形成方面也被多效性阻断。 bld突变体还在小的形态发生肽SapB的合成中被阻断，SapB似乎直接参与气生菌丝体的形成。 这是证明了恢复气生菌丝体形成的bld突变体后，外源性应用纯化的SapB。 另外的证据支持这样的观点，即SapB是非核糖体合成的，推测是由类似于肽抗生素的多酶复合物合成的。 SapB在S. coelicolor形态发生和营养感应在营养菌丝体向气生菌丝体分化中的作用是什么？ 这项研究计划资助旨在通过鉴定特别参与SapB生物合成和/或气生菌丝体形成的营养调控的基因来解决这些问题。 这种方法是基于观察到的纯化的SapB的加入解除了抑制气生菌丝体形成的形态野生型菌株S。coelicolor J1501在LB琼脂培养基中生长时， 这种气生菌丝体形成的恢复允许筛选在S. coelicolor J1501细胞试图过量产生SapB生物合成所必需的基因，从而模拟这种现象。 将纯化来自互补菌落的质粒，并亚克隆插入物，以确定所选表型所需的最小大小。 亚克隆的插入片段将被测序和分析，以便将推定的功能分配给特定的基因产物。 互补序列可以编码i）SapB结构基因或ii）作为推测的SapB肽合成酶操纵子的一部分的调节或结构基因和/或iii）不直接参与SapB生物合成或调节但仍然参与形态分化的基因。 这些基因的鉴定将提供初步数据，作为全面研究建议的基础。 ***