The second messenger bis‐3,5‐cyclic di‐guanosine monophosphate (c‐di‐GMP) determines when
Streptomycesinitiate 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. venezuelaeis 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 ∆ rmdBstrains revealed that the c‐di‐GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelaegenes being differentially expressed in the cdgCmutant. 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 Streptomycesaerial hyphae and spores.