Morphological Differentiation in a Filamentous Bacterium

原核生物形态分化最令人着迷和戏剧性的例子之一是天蓝色链霉菌和其他链霉菌气生菌丝的形成。这些丝状细菌通过形成霉菌样的多核菌丝分支网络而生长，称为基质菌丝体。气生菌丝体由菌丝组成，这些菌丝远离殖民地表面生长到空气中，形成毛发状细丝的繁茂草坪，最终变形为有色孢子链。气生菌丝体的形成取决于所谓的bld（即，“秃”）基因，其突变体产生光滑、无毛的菌落。bld突变体的一个显著特性是，当它们生长在某些其它bld突变体附近但不与它们接触时，它们能够重新获得形成气生菌丝体的能力。这种表型拯救被称为细胞外互补，其特征是单向的，一个突变体作为供体，另一个作为受体。气生菌丝转化为带色素孢子链取决于所谓的whi（“白色”）基因，其突变体产生缺乏成熟孢子所特有的灰色色素的气生菌丝体。一些bld和whi基因已被克隆和鉴定，但它还不可能组装成通路的发育基因或构建一个完整的图片，在这种生物体的形态分化的控制。该项目的主要目标是通过转座子标记进行基因组范围的筛选，以获得参与气生菌丝体和孢子形成的其他链霉菌基因，从而可以方便地克隆和表征发育基因。一种在S. coelicolor被设计并用于发现七个发育基因和基因簇。一个这样的簇，其中插入导致whi突变表型由三个未知功能的基因组成。每个基因都是一个旁系同源基因大家族的创始成员，这些基因位于染色体上广泛分散的位点。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，从基底菌丝体特征的蓝色色素（放线菌素）的合成到孢子特征的灰色色素的错误定位合成。另一个先前未表征的基因中的插入突变导致气生菌丝体形成的严重阻断。这些和其他新发现的基因的调节和功能将被研究。此外，将通过大规模进行插入诱变来寻找参与形态分化的其他基因。此外，作为补充策略，将产生转座子衍生物，其产生与绿色荧光蛋白基因的融合物。这样一个融合转座子应该有可能发现发育基因的基础上的时间和本地化的表达。

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.