Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms.

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN_1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

Biofilms are complex microbial architectures that encase microbial cells in a matrix comprising self-produced extracellular polymeric substances. Microorganisms living in biofilms are much more resistant to hostile environments than their planktonic counterparts and exhibit enhanced resistance against the microbicides. From the human perspective, biofilms can be classified into beneficial, neutral, and harmful. Harmful biofilms impact food safety, cause plant and animal diseases, and threaten medical fields, making it urgent to develop effective and robust strategies to control harmful biofilms. In this review, we discuss various strategies to control biofilm formation on infected tissues, implants, and medical devices. We classify the current strategies into three main categories: (i) changing the properties of susceptible surfaces to prevent biofilm formation; (ii) regulating signalling pathways to inhibit biofilm formation; (iii) applying external forces to eradicate the biofilm. We hope this review would motivate the development of innovative and effective strategies for controlling harmful biofilms.

Cyclic diadenylate (c-di-AMP) is a widespread second messenger in bacteria and archaea that is involved in the maintenance of osmotic pressure, response to DNA damage, and control of central metabolism, biofilm formation, acid stress resistance, and other functions. The primary importance of c-di AMP stems from its essentiality for many bacteria under standard growth conditions and the ability of several eukaryotic proteins to sense its presence in the cell cytoplasm and trigger an immune response by the host cells. We review here the tertiary structures of the domains that regulate c-di-AMP synthesis and signaling, and the mechanisms of c-di-AMP binding, including the principal conformations of c-di-AMP, observed in various crystal structures. We discuss how these c-di-AMP molecules are bound to the protein and riboswitch receptors and what kinds of interactions account for the specific high-affinity binding of the c-di-AMP ligand. We describe seven kinds of non-covalent–π interactions between c-di-AMP and its receptor proteins, including π–π, C–H–π, cation–π, polar–π, hydrophobic–π, anion–π and the lone pair–π interactions. We also compare the mechanisms of c-di-AMP and c-di-GMP binding by the respective receptors that allow these two cyclic dinucleotides to control very different biological functions.

C-di-GMP riboswitches are structured RNAs located in the 5′-untranslated regions (5′-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results showed that this natural triple-tandem riboswitch controlled the expression of the reporter gene more stringently and digitally than the double-tandem or single riboswitch. A sandwich-like dual-fluorescence reporter was further constructed by fusing the Bc3-5 RNA gene between the two fluorescence protein genes amcyan and turborfp. This reporter strain was found to exhibit detectable fluorescence color changes under bright field in response to intracellular c-di-GMP level altered by induced expression of diguanylate cyclase (DGC) PleD. Using this system, two putative membrane-bound DGCs from B. thuringiensis and Xanthomonas oryzae were verified to be functional by replacing pleD with the corresponding DGC genes. This report represented the first native triple-tandem riboswitch that was applied to serve as a riboswitch-based dual-fluorescence reporter for the efficient and convenient verification of putative DGC activity in vivo.