Cyclic di-GMP in the control of multicellular differentiation in antibiotic-producing bacteria Streptomyces.

链霉菌是临床上重要的抗生素和人类医学中使用的其他天然产物最丰富的来源。这些次生代谢物的产生在时间上和遗传上与一个复杂的发育生命周期相协调，包括作为分枝菌丝的营养菌丝生长和通过在称为气生菌丝的特殊生殖结构上形成的孢子扩散。我们最近发现，由ggdef结构域蛋白产生并被EAL-或hd - gypp结构域蛋白降解的核苷酸第二信使c-di-GMP在控制链霉菌发育中起着至关重要的作用。我们在链霉菌属中鉴定出发育主调控因子BldD是第一个c-di-GMP效应物，并证明BldD与四聚体形式的c-di-GMP结合，导致转录因子二聚，并在营养生长过程中有效抑制产孢基因。然而，我们对链霉菌中c-二gmp信号传导成分的了解仍然非常有限。提出的工作目的是系统地分析和表征委内瑞拉链霉菌中所有GGDEF， EAL， HD-GYP结构域蛋白的分子细节功能和调控方面。我的初步数据显示，四种c-二gmp代谢酶对分化有影响，并受到多种发育调节因子的控制，但其潜在机制尚不清楚。此外，在我的下拉实验中，我确定了BldD是c-di-GMP效应蛋白，并富集了多种其他假定的c-di-GMP结合蛋白。本提案计划验证这些候选c-di-GMP效应器并揭示其功能。总之，本研究旨在发现这些c-di-GMP信号成分的作用机制，阐明它们在细胞调节通路中的位置，揭示它们的生理作用。这项工作将极大地提高我们对链霉菌生理学和c-di-GMP信号在涉及多细胞分化和次生代谢物产生的新生理背景下的理解，并有助于更好地利用链霉菌的基因工程来生产抗生素。

Streptomyces are the most abundant source of clinically important antibiotics and other natural products used in human medicine. The production of these secondary metabolites is temporally and genetically coordinated with a complex developmental life cycle involving growth as vegetative mycelium of branching hyphae and dispersion via spores formed on specialized reproductive structures called aerial hyphae. We have recently discovered that the nucleotide second messenger c-di-GMP, which is produced by GGDEF-domain proteins and degraded by either EAL- or HD-GYP-domain proteins, plays a crucial role in controlling Streptomyces development. We identified the developmental master regulator BldD as a first c-di-GMP effector in the genus Streptomyces and demonstrated that BldD binds a tetrameric form of c-di-GMP, which results in transcription factor dimerization and effective repression of sporulation genes during vegetative growth. However, our knowledge about c-di-GMP signalling components in Streptomyces is still very limited. The aim of the proposed work is to systematically analyse and characterize in molecular detail the functions and regulatory aspects of all GGDEF, EAL, HD-GYP domain proteins in Streptomyces venezuelae. My initial data show that four of the c-di-GMP metabolizing enzymes have an impact on differentiation and are under the control of multiple developmental regulators but the underlying mechanisms are not understood. Moreover, in my pull-down experiments, which led to the identification of BldD as a c-di-GMP effector protein, multiple other putative c-di-GMP binding proteins were enriched. To validate these candidates as c-di-GMP effectors and to uncover their function(s) is planned in this proposal. Altogether, the proposed research aims at finding the mechanism(s) of action of these c-di-GMP signalling components, elucidation of their position(s) in cellular regulatory pathways and uncovering of their physiological role(s). This work will greatly improve our understanding of Streptomyces physiology and c-di-GMP signalling in a new physiological context involving multicellular differentiation and secondary metabolite production and can contribute to a better exploitation of genetic engineering in Streptomyces for the production of antibiotics.