ComFB, a widespread family of c-di-NMP receptor proteins

Abstract

Cyclic dimeric-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates a variety of cellular processes, including motility, biofilm formation, secretion, cell cycle progression, and development, and also contributes to the virulence of many bacterial pathogens. While the genes encoding c-di-GMP cyclases and hydrolases are readily identifiable in microbial genomes, known c-di-GMP receptor domains are quite few, with only PilZ and MshEN broadly distributed across bacterial phyla. Recently, a new c-di-GMP receptor, named CdgR or ComFB, has been identified in cyanobacteria and shown to regulate cell size and natural competence. We demonstrated that CdgR proteins exhibit sequence and structural similarity to the Bacillus subtilis late competence development protein ComFB, a conserved protein of unknown function associated with bacterial competence. This prompted us to hypothesize that ComFB and ComFB-like proteins could also serve as c-di-GMP receptors. Here, we comprehensively investigated the ComFB protein family and demonstrated that ComFB proteins are evolutionarily widespread among bacteria and function as a novel family of c-di-GMP receptors. We showed that ComFB proteins from Gram-positive bacteria (B. subtilis, Thermoanaerobacter brockii) and Gram-negative pathogens (Vibrio cholerae, Treponema denticola) bind c-di-GMP with high affinity. Several ComFB proteins also bind cyclic di-adenosine monophosphate (c-di-AMP), suggesting that ComFB represents a widely distributed bacterial protein family with dual specificity for c-di-GMP and c-di-AMP. Our physiological studies further showed that ComFB plays vital roles in controlling motility in a c-di-GMP-dependent manner in two phylogenetically distant bacteria, B. subtilis and the gram-negative Shewanella oneidensis, attesting to the biological relevance of ComFB as a c-di-GMP binding protein.

Keywords: ComFB superfamily; bacterial motility; c-di-AMP signaling; c-di-GMP signaling.

Fig. 1.. Sequence and structural conservation within the ComFB superfamily.

(A) Structural alignment of the dimeric forms of Bacillus subtilis ComFB (BsComFB, PDB: 4WAI, yellow and teal) and CdgR from Synechocystis (PDB: 8HJA, orange and red). The CdgR-bound c-di-GMP molecules are shown in stick mode with carbon atoms in blue. The c-di-GMP binding residues D53, N100, R101 and Y115 of CdgR are shown in stick mode with carbon atoms in green. (B) Sequence alignment of representative members of the ComFB superfamily (see full version in SI Appendix, Fig. S1). Proteins are shown under their UniProt identifiers, and secondary structure assignments (H, α-helix, E, β-strand) of ComFB and CdgR are shown with their PDB codes. The numbers indicate the positions of the start and end of the alignment and the lengths of the gaps between the aligned blocks. Conserved negatively (D, E) and positively (K, R) charged residues are shown in red and blue, respectively; nonpolar hydrophilic residues (N, Q, S, T) are in purple. Conserved hydrophobic residues are indicated with yellow shading, and conserved turn residues (G, P, S, A) are shaded green. Zinc-binding Cys residues of ComFB and the conserved Cys residues in other proteins are shown on a light blue background. The last sequence in the upper block represents the Pfam entry PF10719. The symbols in the “Function” line indicate [as specified in (51)]: d, residues responsible for protein dimerization; asterisks, residues involved in binding c-di-GMP; h and π, residues involved in hydrophobic interactions with the c-di-GMP ligand. The ComFB sequences are from the following organisms (top to bottom): Bacillus subtilis, Synechocystis, Thermoanaerobacter brockii, Vibrio cholerae, Treponema denticola, and Shewanella oneidensis. The last line shows the sequence of the N-terminal ComFB domain of a predicted diguanylate cyclase (Sll1170) from Synechocystis sp. (C) Cluster map of ComFB homologs. A set of 1,626 representative ComFB sequences (≤ 70% pairwise identity and ≥ 70% length coverage) was clustered using the CLANS tool (62) based on pairwise BLAST P-values. Dots represent individual sequences, colored according to their group. Line color intensity reflects sequence similarity, with darker lines indicating higher similarity. The analysis revealed four clusters: two within Cyanobacteriota, one comprising diverse phyla (e.g., Actinomycetota, Bacillota), and a distinct Pseudomonadota cluster, highlighting conserved c-di-GMP-binding residues across these diverse groups.

Fig. 2.. Genomic neighborhoods of selected ComFB/CdgR family proteins.

Genomic fragments are listed with the organism names, GenBank accession numbers, and genomic coordinates. Gene sizes are drawn approximately to scale, and gene names are from GenBank, RefSeq, and/or the COG database. ComFB genes are in red, other competence-related genes are in pink (GC indicates comGC and GD stands for comGD [also known as FimT]), flagella-related genes are in orange, pili-related genes are in green, signal transduction genes are in yellow, metabolic genes are in various shades of blue, poorly characterized genes are in grey or white. The graph displays fragments of the following genomes: (A) Bacillus subtilis 168; (B) Synehocystis sp.; (C) Thermoanaerobacter tengcongensis MB4; (D) Allochromatium vinosum DSM 180; (E) Desulfohalobium retbaense DSM 5692; (F) Treponema denticola ATCC 35405; (G) Vibrio cholerae O1 biovar El Tor str. N16961. The genomic fragments for B. subtilis and T. denticola are shown in reverse complement.

Fig. 3.. Structural gallery of representative ComFB domain-containing proteins from various species.

α-helices in the ComFB domain are colored red, β-strands are in yellow, and the remainder of the protein in grey. For proteins with two ComFB domains, one domain is shown in lighter shades. The structures are AlphaFold2 predictions from UniProt/AlphaFold DB, except for Bacillus subtilis ComFB (PDB: 4WAI). The species represented include Synechocystis (Slr1970, Sll1170, Slr1505, and Sll1739; UniProt accessions P74113, P74197, P73943, and P73385, respectively), Bacillus subtilis (P39146), Vibrio cholerae (Q9KM28), Treponema denticola (Q73MV1), Thermoanaerobacter brockii (E8USF0), Fischerella muscicola (A0A2N6JYB2), Trichormus variabilis (Q3M730), Synechococcus sp. PCC 7502 (K9SSQ8), and Pseudanabaena sp. PCC 7367 (K9SGA4). Several of these proteins also contain additional features, such as N- or C-terminal extensions (e.g., Slr1505), coiled-coil segments (e.g., PspA), or other domains: Sll1739 and Slr1970 have uncharacterized α-helical bundle domains, T. denticola contains an Ig-like domain, and Sll1170 possesses DUF1816, PAS, and GGDEF domains. Other domain architectures of ComFB-containing proteins are shown in SI Appendix (Fig. S5).

Fig. 4.. Isothermal titration calorimetry (ITC) analysis of c-di-GMP binding to phylogenetically different ComFB proteins.

Upper panels show the raw ITC data in the form of heat produced during the titration of c-di-GMP on different ComFB proteins; lower panels show the binding isotherms and the best-fit curves according to the one binding site model. (A-D) ITC analysis of c-di-GMP binding to B. subtilis or T. brockii ComFB proteins in the absence (A,C) or presence of 150 μM c-di-AMP (B,D). (E,F) ITC analysis of c-di-GMP binding to V. cholerae (E) or T. denticola (F) ComFB proteins.

Fig. 5.. ComFB inhibits swimming motility.

(A-C) Effect of ComFB on B. subtilis motility. Low concentration (0.3%) agar plates were toothpick inoculated in their centers and incubated at 33° C. Panels (A) and (B) were derived from independent experiments. For panel (A), the plates were incubated until shortly after the cyclase null strains (dgcP, dgcW and dgcK deletion) reached the edge of the plate. For panel B, the plates were incubated until the empty vector strain reached the edge of the plate. (C) Immunoblot analysis of ComFB showing that the comFB (K41A) and cyclase mutations did not interfere with the expression of ComFB. Upper panel, a loading control using antiserum raised against elongation factor G (α-EFG). The strengths of the ComFB signals were normalized to those of EFG (lower panel). (D-E) Effect of ComFB on S. oneidensis motility. Independent swimming assays were conducted as described in (SI Appendix, Materials and Methods). (D) phenotypic analysis of comFB overexpression and marker-less deletion (ΔcomFB) strains. (D,E) Plasmids carrying comFB or empty vector without comFB (pBAD33-EVC) were expressed in wild-type and ΔcomFB backgrounds. Complementation of ΔcomFB was achieved be re-introducing comFB back into S. oneidensis genome. Significance ****: P value <0.0001 in Student’s t test, in comparison to wild-type. Whiskers: standard deviation. ns.: not significant.