To proliferate, antibiotic-producing Streptomyces undergo a complex developmental transition from vegetative growth to the production of aerial hyphae and spores. This morphological switch is controlled by the signaling molecule cyclic bis-(3′,5′) di-guanosine-mono-phosphate (c-di-GMP) that binds to the master developmental regulator, BldD, leading to repression of key sporulation genes during vegetative growth. However, a systematical analysis of all the GGDEF/EAL/HD-GYP proteins that control c-di-GMP levels in Streptomyces is still lacking. Here, we have FLAG-tagged all 10 c-di-GMP turnover proteins in Streptomyces venezuelae and characterized their expression patterns throughout the life cycle, revealing that the diguanylate cyclase (DGC) CdgB and the phosphodiesterase (PDE) RmdB are the most abundant GGDEF/EAL proteins. Moreover, we have deleted all the genes coding for c-di-GMP turnover enzymes individually and analyzed morphogenesis of the mutants in macrocolonies. We show that the composite GGDEF-EAL protein CdgC is an active DGC and that deletion of the DGCs cdgB and cdgC enhance sporulation whereas deletion of the PDEs rmdA and rmdB delay development in S. venezuelae. By comparing the pan genome of 93 fully sequenced Streptomyces species we show that the DGCs CdgA, CdgB, and CdgC, and the PDE RmdB represent the most conserved c-di-GMP-signaling proteins in the genus Streptomyces.

The second messenger bis‐3,5‐cyclic di‐guanosine monophosphate (c‐di‐GMP) determines when Streptomyces initiate sporulation. c‐di‐GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor σWhiG. However, functions of the development‐specific diguanylate cyclases (DGCs) CdgB and CdgC, and the c‐di‐GMP phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF‐EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c‐di‐GMP to 5′pGpG. Despite having an equivalent GGDEF‐EAL domain arrangement, RmdA cleaves c‐di‐GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA, and ∆rmdB strains revealed that the c‐di‐GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c‐di‐GMP‐controlled targets determining the timing and mode of sporulation are genes involved in cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores.

25Levels of the second messenger bis-3´-5´-cyclic di-guanosinemonophosphate (c-di-GMP) 26 determine when Streptomyces initiate sporulation to survive under adverse conditions. c-di-27 GMP signals are integrated into the genetic differentiation network by the regulator BldD and 28 the sigma factor σ WhiG . However, functions of the development-specific c-di-GMP 29 diguanylate cyclases (DGCs) CdgB and CdgC, and the phosphodiesterases (PDEs) RmdA and 30 RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL 31 domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-32 GMP to 5´pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA 33 cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL 34 motif is crucial for the in vivo function of both enzymes since strains expressing protein 35 variants with an AAA motif instead of EAL are delayed in development, similar to null 36 mutants. Global transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA and ∆rmdB strains revealed 37 that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20 % 38 of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest 39 that the major c-di-GMP-controlled targets determining the timing and mode of sporulation 40 are genes involved cell division and the production of the hydrophobic sheath that covers 41Streptomyces aerial hyphae and spores. Altogether, this study provides a global view of the c-42 di-GMP-dependent genes that contribute to the hyphae-to-spores transition and sheds light on 43 the shared and specific functions of the key enzymes involved in c-di-GMP metabolism in S. 44 venezuelae. 45 46 245 words 47 48 Importance 49 50Streptomyces are important producers of clinical antibiotics. The ability to synthesize these 51 natural products is connected to their developmental biology, which includes a transition from 52 filamentous cells to spores. The widespread bacterial second messenger c-di-GMP controls 53 this complex switch and is a promising tool to improve antibiotic production. Here, we 54 analyzed the enzymes that make and break c-di-GMP in S. venezuelae by studying the 55 genome-wide transcriptional effects of the DGCs CdgB and CdgC and the PDEs RmdA and 56RmdB. We found that the c-di-GMP specified by these enzymes has a global regulatory role. 57 However, despite shared enzymatic activities, the four c-di-GMP enzymes have specialized 58 inputs into differentiation. Altogether, we demonstrate that altering c-di-GMP levels through 59 the action of selected enzymes yields characteristically distinct transcriptional profiles; this 60 can be an important consideration when modulating c-di-GMP for the purposes of natural 61 product synthesis in Streptomyces. 62 63 143 words 64 65 66 Streptomyces development is controlled by a complex network of Bld and Whi 100 regulators. Strains mutated in bld genes fail to develop aerial hyphae, while deletion of whi 101 genes blocks t...