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

威利9628767链霉菌属。是革兰氏阳性土壤细菌，它们经历了与丝状真菌相似的形态和生理分化的时间和空间调节周期。在链霉菌的生命周期中，产生三种类型的细胞。穿透底物的营养菌丝体形成向上生长的毛状气生菌丝。抗生素的生物合成伴随着气生菌丝体的形成。气生菌丝体随后分化为单核孢子。在天蓝色链霉菌中已鉴定出两类发育控制基因：WHI和BLD基因。Whi基因控制气生菌丝体中孢子的发育；因此，Whi基因只从气生菌丝体中突变。BLD基因参与了营养菌丝体向气生菌丝体的分化。BLD基因的突变会导致只形成底物菌丝体的菌落生长；这些突变中的许多也会多向性地阻止抗生素的形成。BLD突变体还阻止了一种小的形态发生肽SapB的合成，SapB似乎直接参与了气生菌丝的形成。BLD突变体在外源应用纯化的SapB后恢复了气生菌丝的形成，证明了这一点。更多的证据支持这样一种观点，即SapB是由非核糖体合成的，推测是由类似于多肽抗生素的多酶复合体合成的。SapB在天蓝色链霉菌形态发生中的作用是什么？营养感应在营养菌丝向气生菌丝体分化中的作用是什么？这项研究计划拨款旨在通过确定特定参与SapB生物合成和/或气生菌丝形成的营养调节的基因来解决这些问题。这种方法是基于观察到纯化的SapB的加入缓解了形态野生型天蓝色链霉菌J1501在LB琼脂中生长时对气生菌丝形成的抑制。这种气生菌丝形成的恢复允许在天蓝色链霉菌J1501细胞中筛选高拷贝表达的基因组文库，试图过度生产SapB生物合成所必需的基因，从而模拟这一现象。来自互补菌落的质粒将被提纯并插入亚克隆，以确定所选表型所需的最小尺寸。然后，将对亚克隆的插入片段进行测序和分析，以便可能将推测的功能分配给特定的基因产物。互补序列可能编码1)SapB结构基因或2)调控或结构基因，这些基因是推测的SapB多肽合成酶操纵子的一部分和/或3)不直接参与SapB生物合成或调节但参与形态分化的基因。对这些基因的识别将提供初步数据，作为完整研究计划的基础。***

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. ***