Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain

Abstract

Proteins containing GGDEF domains are encoded in the majority of sequenced bacterial genomes. In several species, these proteins have been implicated in biosynthesis of exopolysaccharides, formation of biofilms, establishment of a sessile lifestyle, surface motility, and regulation of gene expression. However, biochemical activities of only a few GGDEF domain proteins have been tested. These proteins were shown to be involved in either synthesis or hydrolysis of cyclic-bis(3'-->5') dimeric GMP (c-di-GMP) or in hydrolysis of cyclic AMP. To investigate specificity of the GGDEF domains in Bacteria, six GGDEF domain-encoding genes from randomly chosen representatives of diverse branches of the bacterial phylogenetic tree, i.e., Thermotoga, Deinococcus-Thermus, Cyanobacteria, spirochetes, and alpha and gamma divisions of the Proteobacteria, were cloned and overexpressed. All recombinant proteins were purified and found to possess diguanylate cyclase (DGC) activity involved in c-di-GMP synthesis. The individual GGDEF domains from two proteins were overexpressed, purified, and shown to possess a low level of DGC activity. The oligomeric states of full-length proteins and individual GGDEF domains were similar. This suggests that GGDEF domains are sufficient to encode DGC activity; however, enzymatic activity is highly regulated by the adjacent sensory protein domains. It is shown that DGC activity of the GGDEF domain protein Rrp1 from Borrelia burgdorferi is strictly dependent on phosphorylation status of its input receiver domain. This study establishes that majority of GGDEF domain proteins are c-di-GMP specific, that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria, and that it is highly regulated.

FIG. 1.

(A) Domain organization of the GGDEF domain proteins whose biochemical activities have been tested prior to this study. (B) Phylogenetic tree of Bacteria showing proteins investigated in this study. GAF, signal sensor domain often found in phytochromes and cyclic GMP-specific PDE; EAL, PDE domain probably specific to c-di-GMP; PAS, ubiquitous signal sensor domain; REC, receiver domain of response regulators from two-component systems (http://www.sanger.ac.uk/Software/Pfam; http://smart.embl-heidelberg.de).

FIG. 2.

R. sphaeroides DgcA protein (RSP3513). (A) Protein overexpression and purification (protein chip, Bioanalyzer; Agilent Technologies). Lane M, molecular mass markers in kilodaltons; lane 1, crude extract of E. coli cells prior to induction of expression of the MBP::DgcA fusion protein; lane 2, crude extract of E. coli cells after 2-h induction with IPTG; lane 3, pure protein after affinity chromatography and gel filtration. (B) Enzymatic activity of DgcA. Substrate (top; 0 min) and products (bottom; 60 min) of the reaction, separated by reversed-phase HPLC. Nicotine ADP was used as an internal control for quantification purposes. (C) Mass spectroscopy analysis of the product synthesized by MBP::DgcA. The top panel corresponds to chemically synthesized c-di-GMP. The lower panel corresponds to the product of the HPLC fraction, with retention time of 19 min (see Fig. 2A, bottom).

FIG. 3.

Synechocystis sp. Slr1143 protein. Top, full-length protein; middle, protein fragment corresponding to the CC-plus-GGDEF domains; bottom, protein fragment corresponding to the GGDEF domain. (A) Protein overexpression and purification. For details, see the legend to Fig. 2A. (B) Enzymatic activity. For details, see the legend to Fig. 2B. (C) Oligomeric state of MBP::Slr1143 and its derivatives assayed by FPLC. Marker, blue dextran.

FIG. 4.

B. burgdorferi Rrp1 protein. (A) Protein overexpression and purification. For details, see the legend to Fig. 2A. (B) Enzymatic activity. The substrate (left; 0 min) and products (right; 60 min) of the reaction mixture separated by HPLC are shown. MBP::Rrp1 (7.5 μM) was incubated with the indicated concentrations of acetyl phosphate (AcP) for 30 min at room temperature, followed by removal of AcP by dialysis. (C) Oligomeric state of the MBP::Rrp1 protein assayed by FPLC.