Control of bacterial exoelectrogenesis by c-AMP-GMP

Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.Three major types of cyclic dinucleotide second messengers have been discovered in biological systems. The first to be found was c-di-GMP (Fig. 1A, Top), which serves as a regulator of diverse physiological changes in most bacterial lineages (1). The second, c-di-AMP (2), is an important signaling compound during bacterial sporulation and germination (3) and has also been found to regulate osmotic shock responses in Gram-positive bacteria (4, 5). More recently, a third type of cyclic dinucleotide called c-AMP-GMP (Fig. 1A, Bottom) was found to be required for Vibrio cholera virulence (6).Open in a separate windowFig. 1.Selective recognition of c-AMP-GMP by a natural aptamer. (A) Chemical structures of c-di-GMP and c-AMP-GMP. (B) Sequence and secondary structure of the 100 erfK RNA from G. metallireducens. P1, P2, and P3 identify base-paired substructures. M1 designates a mutant construct wherein position 20 is changed to G. Regions of constant, decreasing, and increasing RNA cleavage upon addition of ligand are indicated by yellow, red, and green circles, respectively. The arrowhead indicates the start of the RNA structure stability data derived from C. (C) PAGE analysis of in-line probing assays of 5′ ³²P-labeled 100 erfK in the presence (10 µM) of various cyclic dinucleotides. NR, T1, and ^‒OH designate lanes loaded with precursor RNA (Pre), RNA partially digested with RNase T1 (resulting in cleavage after G residues), and RNA partially digested with alkali (resulting in cleavage after every residue). Several RNase T1 cleavage product bands are labeled. Regions undergoing substantial change in spontaneous cleavage rates are labeled 1–4. The inosine-based analog of c-di-GMP is labeled c-di–IMP. (D) Plot of the fraction of riboswitch bound to ligand versus the log of the molar concentration (c) of the ligand. Data are derived from Fig. S2, and each point is the average of the normalized fraction of modulation at sites 1 and 2. Error bars indicate the SD of the average. Included are theoretical curves expected for one-to-one interaction between ligand and RNA for the K_D values given.Although bacterial c-AMP-GMP is formed via two 3′,5′-phosphodiester linkages, metazoans produce an analog with one 2′,5′-phosphodiester linkage and one 3′,5′-phosphodiester linkage (7–9). This natural structural variant has been identified as important for triggering innate immune responses in metazoans (7–11). Far greater diversity of cyclic dinucleotides is possible by varying either the nucleotide composition or the manner in which dinucleotides are joined. Therefore, many additional types of cyclic dinucleotides might await discovery in nature where they might regulate other critical cellular processes.Our approach to uncovering the chemical structures and biological functions of cyclic dinucleotide signaling compounds is to discover their RNA receptors. Three different riboswitch receptor classes have already been discovered that respond to c-di-GMP (12, 13) and c-di-AMP (5). Riboswitches are structured noncoding RNA (ncRNA) domains that selectively bind a small molecule or ion and thereby trigger a change in the expression of associated genes (14, 15). The majority of riboswitch ligands are composed of or derived from nucleotides, which supports the hypothesis that RNA-based metabolites and their riboswitch partners might descend from primordial RNA world organisms (16). If true, then cyclic dinucleotides and their receptors might be modern reflections of ancient signaling pathways. Regardless of their origins, the discovery of each new regulatory RNA motif that selectively recognizes a second messenger also reveals a superregulon for the signaling compound and the biology that it controls (5, 12, 13).Given that the diversity of natural cyclic dinucleotides might be greater than is currently known, and given that some riboswitch classes have undergone subtle changes to adapt to different ligands (17–20), we re-examined known cyclic dinucleotide riboswitch classes and their associated genes for evidence of unidentified ligands or riboswitch classes. Of special interest were those RNAs that both carry mutations in the core of the ligand-binding aptamer and have gene associations considered unusual for the parent riboswitch class. Through these efforts, we identified a subset of riboswitches within the c-di-GMP-I riboswitch class (12) that we hypothesized might bind a different ligand. In the current study, we demonstrate that these riboswitches recognize c-AMP-GMP and control a set of genes that are important for the utilization of iron(III) oxide in exoelectrogenesis.