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.

Keywords: EAL; GGDEF; HD-GYP; Streptomyces; c-di-GMP; development.

FIGURE 1

Expression profiles of c-di-GMP metabolism genes and proteins during submerged sporulation of S. venezuelae. (A) Expression data were extracted from microarray analyses in Bibb et al. (2012) and normalized with quantile normalization and median polish using the RMA method. Expression values are shown as Log2. (B) Western blot analysis of FLAG-tagged proteins. GGDEF/EAL/HD-GYP-domain proteins were FLAG-tagged and expressed in the corresponding mutants. Protein samples were harvested after indicated time of growth and following amounts of total protein were used for Western blotting: 5 μg for CdgB-FLAG; 10 μg for CdgE-FLAG, RmdB-FLAG and HdgB-FLAG; 15 μg for CdgA-FLAG, CdgC-FLAG, and RmdA-FLAG; 20 μg for CdgD-FLAG, CdgF-FLAG, HdgA-FLAG. co, wildtype sample as control.

FIGURE 2

Morphological differentiation is accelerated by the deletion of cdgB and cdgC and delayed by the deletion of rmdA and rmdB. (A) Time-course development of S. venezuelae wildtype and mutant macrocolonies grown on MYM agar for 5 days at 30°C. (B) Phase contrast microscopy images showing coverslip imprints from the upper layer of the macrocolonies grown for 30 h and 50 h in (A).

FIGURE 3

CdgC is an active DGC. (A) Synthesis of c-di-GMP from GTP was assayed in vitro using purified cytosolic fraction of CdgC (ΔTM-CdgC; TM: transmembrane) and PleD^∗ and visualized by thin layer chromatography. (B) The cdgC mutant and a strain carrying a chromosomal cdgC version with the ALLEF motif in the active site show green wildtype morphology when complemented with cdgC from the pMS82 vector but remain gray and do not form aerial mycelium with the empty vector. Overexpression of CdgB can partially suppress the cdgC deletion. Strains were grown for 3 days on MYM agar.

FIGURE 4

Distribution of c-di-GMP turnover genes in Streptomyces spp. Species are sorted in alphabetical order and the model streptomycete, S. venezuelae, is highlighted in red. The conservation of each gene across Streptomyces spp. is shown next to each gene name. Black boxes in each column represent the presence of gene orthologs calculated using the BPGA pipeline or blastp. White boxes represent absence of orthologous genes. TM, transmembrane.