The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium

Abstract

Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.

Figure 1. Identification and disruption of diguanylate cyclases

A. The Ddis genome contained a single gene with GGDEF domain. BLAST search identified orthologs in Dlac, Ppal and Asub, and 13 monophyletic genes in Dfas. A phylogenetic tree was constructed from aligned Dictyostelid and prokaryote GGDEF sequences and annotated with the domain architecture of the proteins. B. Ddis DgcA was ablated by homologous recombination. Dgca- and wild-type cells were incubated on non-nutrient agar to follow developmental progression. C. Ddis dgca- cells were transformed with fusion constructs of the A15 promoter with either DdisDgcA or PpalDgcA and developed for 22 h. Bar: 1 mm.

Figure 2. DgcA expression pattern

A. Total RNA was isolated during development of Ddis wild-type (WT) and dgca- cells on non-nutrient agar. DgcA RNA levels were measured by qRT-PCR with DgcA specific primers DgcAf and DgcAr (Supplementary Table S1). B-D. Ddis cells were transformed with a fusion of 1 kb DgcA 5′ intergenic sequence and the LacZ reporter gene. β-galactosidase activity was visualized with Xgal in fixed aggregates (B), slugs (C) and fruiting bodies (D). Bar: 100 μm.

Figure 3. Biological role of c-di-GMP

A. DgcA- cells were mixed with 0 or 10% wild-type cells and developed for 24 h, or dgca- slugs were exposed to 1 mM c-di-GMP and observed after 10 h. B. The wild-type and dgca- RNA time series (Fig. 2A) was used to amplify ecmA, ecmB, pspA and SpiA by qRT-PCR using gene-specific primers (Table S1). C. Dissociated aggregates of dgca- (open circles, diamonds, filled circles) or dmta- (triangles), transformed with ST-gal, were incubated at 10⁶ cells/ml for 8 h with increasing concentrations of c-di-AMP (diamonds), c-di-GMP (open circles, triangles) or c-di-GMP and 1 mM cAMP (filled circles), followed by β-galactosidase assay. DgcA-/ST-gal was additionally incubated for variable time periods with (filled squares) or without (open squares) 1 μM c-di-GMP before assay. Means and s.e.m. (n=9) D/E. Ddis V12M2 cells were incubated in monolayers with 10 μM c-di-GMP, 100 nM DIF and/or 1 mM cAMP. After 30 h cells were photographed (D) and the proportion of stalk cells to total cells was determined (E). Means and SD (n=2).

Figure 4. Bioassay of DGC activity

A. Preboiled or active immunoprecipitates of the ΔTL-DgcA-YFP fusion protein were incubated with 0.1 mM GTP and 10 mM MgCl₂ at 22°C. After boiling, reaction mixtures were tested for ST-gal induction activity. c-di-GMP concentrations in the mixtures were estimated by comparison with a dose-response curve of ST-gal induction by c-di-GMP, and standardized on the amount of cells from which the immunoprecipitate was derived. B. DGC activity was measured for 60 min with combinations of 0.1 mM GTP and 10 mM MgCl₂ as indicated. Data represent means and s.e.m. of two experiments assayed in triplicate.