Expression Patterns, Genomic Conservation and Input Into Developmental Regulation of the GGDEF/EAL/HD-GYP Domain Proteins in Streptomyces

Abstract

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.

c-di-AMP hydrolysis by a novel type of phosphodiesterase promotes differentiation of multicellular bacteria

“…RCK_C domains are established direct targets of c-di-AMP that have the I(L)I(L)X 2 DX 1 RX 5 NI(L)I(L) signature for ligand binding (Figure 6A) (31). We found the RCK_C-domain protein Vnz_28055 with a putative c-di-AMP binding motif (Figure 6A-B) in 93 Streptomyces species for which complete genome sequences are available (32). We purified N-terminally His-tagged Vnz_28055 and applied differential radial capillary action of ligand assay (DRaCALA) to probe interaction between Vnz_28055 and c-di-AMP.…”

“…Biochemical assays using radioactive-labeled substrates were conducted as described in (32). For diadenylate cyclase (DAC) assays, 5 µM 6xHis-tagged DisA Sven , DisA D86A or DisA Bsu were incubated with 83 nM [ 32 P]-ATP (Hartmann Analytic) in DisA cyclase buffer.…”

“…For detection of 3xFLAG-tagged DisA, Western blot analysis was performed as described in (32) using 5 µg total protein of S. venezuelae Δ disA expressing the FLAG-tagged disA allele from the Φ BT1 integration site under the control of the native promoter. Anti-FLAG primary antibody (Sigma) and the anti-mouse IgG-HRP (Thermo Fisher Scientific) were used for detection.…”

Specialized and shared functions of diguanylate cyclases and phosphodiesterases inStreptomycesdevelopment

“…In S. venezuelae , aerial mycelium formation is completely bypassed when c-di-GMP levels are too low, due to PDE overexpression (6). A phenotypically identical response can be caused through deleting the DGC-encoding gene cdgC – one of the 10 chromosomally-encoded GGDEF/EAL/HD-GYP genes in S. venezuelae (7, 8). Deletion of yet another DGC-encoding gene, cdgB , also leads to precocious sporulation; however, the cdgB mutant still undergoes the classical Streptomyces life cycle and forms spores on reproductive aerial hyphae like the wild type.…”

“…transition of aerial hyphae into chains of spores. Conversely, overexpressing the S. coelicolor DGC, CdgB, causes an opposing phenotype in S. venezuelae , in that it prolongs filamentous, vegetative growth (8); this process can be mimicked by deleting either rmdA or rmdB , which encode functional PDEs (9) (Fig. 1).…”