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.

De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly

ABSTRACTBacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to “kill pathogens without detectable resistance” (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455–459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these “resistance-proof” compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

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.