Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis.

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3'→5') GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.

Cyclic di-GMP cyclase SSFG_02181 from Streptomyces ghanaensis ATCC14672 regulates antibiotic biosynthesis and morphological differentiation in streptomycetes.

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.