Cyclic di-AMP metabolism and functions in Streptomyces

革兰氏阳性土壤细菌链霉菌属于自然界最有能力的天然产品工厂，是用于医疗、兽医和农业用途的各种抗生素和其他生物活性分子的重要来源。这些次生代谢物的产生在时间上和遗传上与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体生长，以及通过在被称为气生菌丝的特殊生殖结构上形成的孢子传播。我们最近发现，在淡色链霉菌中，含有二腺苷酸环化酶(DAC)结构域的蛋白DISA产生核苷酸第二信使环状双AMP，它通过与rpfA基因5-未翻译前导区(UTR)上的核糖开关结合来控制细胞壁裂解酶RpfA的水平。然而，我们对c-diamp的生物合成、周转及其在链霉菌生理中的作用的了解仍然非常有限。这项在SPP1879中提出的工作的目的是对DISA和c-diAMP在金黄色葡萄球菌中的作用进行详细的分子分析和表征。我们将确定DISA表达和活性的条件，并确定这种蛋白和c-diAMP如何对链霉菌所采用的分化和逆境生存策略做出贡献。由于链霉菌不包含任何已经表征的c-di-AMP结合蛋白，我们将对c-di-AMP效应蛋白进行全球筛选，这将使我们能够鉴定出一种潜在的新型c-di-AMP降解磷酸二酯酶和一类新的效应物。此外，在我们之前未发表的研究中，我们发现，到目前为止，斑腿沙门氏菌产生一种至今尚未确定的环二核苷酸。对这种化合物的详细分析和表征将导致在细菌中发现一种新型的基于核苷酸的第二信使。综上所述，这项工作将极大地提高我们对链霉菌生理和胁迫生存机制的理解。并有助于在涉及多细胞分化和次生代谢物产生的新生理背景下更好地了解c-di-AMP信号。

The Gram-positive soil bacteria streptomycetes belong to natures most competent natural product factories and are an important source of diverse antibiotics and other bioactive molecules for medical, veterinary and agricultural use. 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 shown that in Streptomyces venezuelae the diadenylate cyclase (DAC) domain-containing protein DisA produces the nucleotide second messenger cyclic di-AMP which controls the levels of the cell wall lytic enzyme RpfA by binding to a riboswitch in the 5-untranslated leader region (UTR) of the rpfA gene. However, our knowledge about the biosynthesis, turnover and functions of c-di-AMP in the physiology of streptomycetes is still very limited. The aim of the proposed work within the SPP 1879 is to analyse and characterise in molecular detail the roles of DisA and c-di-AMP in S. venezuelae. We will determine the conditions under which DisA is expressed and active and define how this protein and c-di-AMP contribute to differentiation and stress survival strategies employed by Streptomyces spp. Since streptomycetes do not contain any of the already characterised c-di-AMP-binding proteins, we will perform a global screen for c-di-AMP effector proteins, which will allow us to identify a potentially novel type of c-di-AMP-degrading phosphodiesterases and a new class of effectors. Further, in our previous, unpublished studies we found, that S. venezuelae produces an up to now uncharacterised cyclic di-nucleotide. Detailed analysis and characterisation of this compound will lead to the discovery of a novel type of nucleotide-based second messengers in bacteria. Altogether, this work will greatly improve our understanding of the physiology and stress survival mechanisms in Streptomyces spp. and contribute to a better knowledge of c-di-AMP signalling in a new physiological context involving multicellular differentiation and secondary metabolite production.