Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP)

Abstract

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

Keywords: bacterial signaling; cyclic dinucleotides; fluorescent biosensor; second messengers; surface sensing.

Fig. 1.

In vivo fluorescent biosensor screen of 29 Geobacter GGDEF genes reveals a cAG synthase. (A) Average fluorescence measured by flow cytometry (n = 3; 10,000 cells per run) of E. coli BL21 (DE3) Star cells coexpressing the cdiG-selective biosensor Dp-Spinach2 (blue) or cAG-selective biosensor Gm0970-p1-4delA-Spinach (red) along with GGDEF domain proteins from G. sulfurreducens strain PCA, diguanylate cyclase WspR, cAG synthase DncV, or empty vector. Blue and red stars denote significant (P < 0.01) fluorescence turn-on by Student’s t test above control signal (i.e., significant signal above pCOLA background for the cdiG sensor; above WspR for the cAG sensor). (B) LC/MS analysis of E. coli cell extracts overexpressing constructs shown or empty vector; see SI Appendix, Fig. S1 for protein domain color scheme. Shown are the MS spectra from integrating the retention time region containing all three cyclic dinucleotides. Expected masses are for cdiG (m/z = 691), cAG (m/z = 675), and cdiA (m/z = 659).

Fig. 2.

GSU1658 produces different cyclic dinucleotides depending on ATP-to-GTP ratios. (A) Cellulose TLC analysis of radiolabeled products from enzymatic reactions with 1:1 ATP-to-GTP substrates in excess and doped with trace amounts of α-³²P-labeled ATP or α-³²P-labeled GTP. Before loading, reactions were quenched with alkaline phosphatase to digest unreacted nucleotides, resulting in production of inorganic phosphate (P_i). Residue R393 is located in the putative I-site. (B) Representative LC/MS analysis of an enzymatic assay performed with MBP-GSU1658 R393A at 3:1 ratio of ATP to GTP in comparison with a standard containing all three product CDNs at equal concentrations. Shown is the MS spectrum from integrating the retention time region containing all three cyclic dinucleotides. Expected masses are for cdiG (m/z = 691), cAG (m/z = 675), and cdiA (m/z = 659). (C) Analysis of product ratios for MBP-GSU1658 R393A at different ATP-to-GTP ratios based on LC/MS analysis and comparison with CDN standard to account for different ionization efficiencies. Average values of two replicate runs are shown.

Fig. 3.

Identification of specificity position in Hypr GGDEF active site. (A) Cellulose TLC of radiolabeled products from enzymatic reactions with 1:1 ATP-to-GTP substrates in excess and doped with trace amounts of α-³²P-labeled GTP (full TLC and results for α-³²P-labeled ATP are shown in SI Appendix, Fig. S10). Residue R393 is located in the putative I-site, S347 is located in the nucleotide binding site, and D52 is the putative phosphorylation site in the Rec domain. (B) Nucleotide binding region of PleD in complex with nonhydrolyzable GTP analog (Protein Data Bank 2V0N, ref. 26). Hydrogen bonding contacts between the guanine base and key protein residues are shown as dotted lines.

Fig. 4.

Validation of Hypr activity in select Deltaproteobacteria and Acidobacteria. (A) LC/MS analysis of E. coli cell extracts overexpressing candidate Hypr enzymes; see SI Appendix, Fig. S1 for protein domain color scheme and SI Appendix, Fig. S12 for corresponding protein gel. Bd, Bdellovibrio bacteriovorus; Ddes, Desulfovibrio desulfuricans; Mxan, Myxococcus xanthus; Cabther, Candidatus Chloracidobacterium thermophilum. Shown are the MS spectra from integrating the retention time region containing all three cyclic dinucleotides. Expected masses are for cdiG (m/z = 691), cAG (m/z = 675), and cdiA (m/z = 659). (B) LC/MS analysis of M. xanthus cell extracts from surface- or liquid-grown samples. Shown is the extracted ion trace for cAG (m/z = 675.1072; ppm < 10 cutoff) normalized to the weight of extracted cells. A second biological replicate is shown in SI Appendix, Fig. S15.

Fig. 5.

Expanded understanding of the function and evolution of cAG signaling. Levels of the second messenger cAG are regulated by the activity of cAG synthases and phosphodiesterases (PDEs) in response to primary environmental signals. Effectors that bind cAG then propagate downstream effects on bacterial physiology. In V. cholerae, DncV serves as the synthase and variant HD-GYP domains as the phosphodiesterases, but no effectors are known. We have shown that diverse Deltaproteobacteria and Acidobacteria contain Hypr GGDEF enzymes that can act as cAG synthases and control various processes through cAG riboswitches (Geobacter) and other unidentified effectors. An asterisk indicates organisms confirmed to have endogenous cAG. Text in red indicates new information put forth in this paper.