Morphological Differentiation in a Filamentous Bacterium

原核生物形态分化的最引人入胜和最引人注目的例子之一是由色链霉菌和其他链霉菌形成的气生菌丝。这些丝状细菌通过形成一个类似霉菌的多核菌丝分支网络生长，这些多核菌丝被称为基质菌丝。气生菌丝由菌丝组成，菌丝生长在远离菌落表面的空气中，形成茂密的毛发状细丝草坪，最终蜕变成色素孢子链。空中菌丝体的形成取决于所谓的秃顶（即“秃顶”）基因，这种基因的突变产生光滑、无毛的菌落。秃顶突变体的一个显著特性是，当它们生长在邻近但不与其他秃顶突变体接触时，它们能够重新获得形成空中菌丝体的能力。这种被称为细胞外互补的表型拯救，其特点是单向的，一个突变体作为供体，另一个作为受体。气生菌丝转化为色素孢子链取决于所谓的whi（“白色”）基因，这种基因的突变产生的气生菌丝缺乏成熟孢子所特有的灰色色素。一些bld和whi基因已被克隆和鉴定，但尚不可能将发育基因组装成途径或构建一个控制该生物形态分化的完整图景。本项目的主要目标是通过转座子标记对参与气生菌丝和孢子形成的链霉菌基因进行全基因组筛选，从而方便地克隆和表征发育基因。设计了一种转座子介导的诱变程序，并应用于发现7个发育基因和基因簇。一个这样的集群，其中插入导致whi突变表型由三个功能未知的基因组成。每个基因都是位于染色体广泛分散位置的旁系基因大家族的创始成员。另一个新发现的基因的插入失活导致聚酮色素生产的深刻转变，从基质菌丝体特征的蓝色色素（放线素）的合成到孢子特征的灰色色素的错误合成。然而，在先前未表征的基因中另一个插入突变导致空中菌丝形成的严重阻断。这些和其他新发现的基因的调控和功能将被研究。此外，将通过大规模的插入诱变来寻找参与形态分化的其他基因。此外，作为一种补充策略，将创建一个转座子衍生物，产生与绿色荧光蛋白基因的融合。这样一个产生融合的转座子应该可以根据发育基因表达的时间和定位来发现发育基因。

One of the most fascinating and dramatic examples of morphological differentiation among prokaryotes is the formation of an aerial mycelium by Streptomyces coelicolor and other streptomycetes. These filamentous bacteria grow by the formation of a mold-like, branching network of multinucleoid hyphae called the substrate mycelium. The aerial mycelium consists of hyphae that grow into the air away from the colony surface to form a luxuriant lawn of hair-like filaments that eventually metamorphose into chains of pigmented spores. The formation of the aerial mycelium depends on so-called bld (i.e., "bald") genes, mutants of which produce smooth, hairless colonies. A striking property of bld mutants is their capacity to regain the ability to form an aerial mycelium when grown near, but not in contact with, certain other bld mutants. This phenotypic rescue, which is called extracellular complementation, is characteristically unidirectional, with one mutant acting as a donor and the other as a recipient. The conversion of aerial hyphae into chains of pigmented spores depends on so-called whi ("white") genes, mutants of which produce an aerial mycelium that lacks the gray pigment characteristic of mature spores. Some bld and whi genes have been cloned and characterized, but it is not yet possible to assemble developmental genes into pathways or to construct an integrated picture for the control of morphological differentiation in this organism. The principal goal of this project is to carry out a genome wide screen for additional Streptomyces genes involved in aerial mycelium and spore formation by transposon tagging so that developmental genes can be conveniently cloned and characterized. A procedure for carrying out transposon-mediated mutagenesis in S. coelicolor was devised and applied to the discovery of seven developmental genes and gene clusters. One such cluster in which an insertion causes a whi mutant phenotype consists of three genes of unknown function. Each gene is a founding member of a large family of paralogous genes located at widely scattered sites in the chromosome. Insertional inactivation of another newly discovered gene causes a profound switch in polyketide pigment production, from the synthesis of the blue pigment (actinhorodin) characteristic of the substrate mycelium to the mislocalized synthesis of the gray pigment characteristic of spores. Yet another insertional mutation in a previously uncharacterized gene causes a severe block in aerial mycelium formation. The regulation and function of these and other newly discovered genes will be investigated. Further, additional genes involved in morphological differentiation will be sought by carrying out insertional mutagenesis on a large scale. Also, as a complementary strategy, a transposon derivative will be created that generates fusions to the gene for the Green Fluorescent Protein. Such a fusion-generating transposon should make it possible to discover developmental genes on the basis of the timing and localization of their expression.