Control of bacterial exoelectrogenesis by c-AMP-GMP

Abstract

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.

Keywords: GEMM; Geobacter; c-di-GMP; riboswitch; second messenger.

Fig. 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.

Fig. 2.

Numerous aptamers with a G20A variation bind c-AMP-GMP and discriminate against c-di-GMP. (A) K_D values for various natural riboswitch aptamer variants. (B) Consensus sequences and secondary structures of RNAs predicted to selectively bind c-AMP-GMP (Left) and the c-di-GMP-I riboswitch aptamer. Nucleotides depicted in red, black, and gray, respectively, are present in at least 97, 90, and 75% of representatives. The c-AMP-GMP consensus comprises 107 sequences, whereas the c-di-GMP-I consensus comprises 99 sequences from Deltaproteobacteria. Circles designate the presence of a nucleotide at the indicated position with frequencies of 97, 90, 75, and 50% as depicted by red, black, gray, and open circles, respectively. Base pairs shaded in green exhibit phylogenetic evidence of covariation. The nucleotides corresponding to positions 20 and 92 are indicated by arrowheads. Additional annotations are as described for Fig. S1. Validated and predicted c-AMP-GMP riboswitches typically carry an internal loop in P1.

Fig. 3.

Regulation of transcription by a c-AMP-GMP riboswitch. (A) The genes downstream of the pilM riboswitch in G. metallireducens. Genes in black boxes are putatively involved in exoelectrogenesis. Gene sizes and gaps are not drawn to scale. (B) Predicted sequence and secondary structure of 127 pilM from G. metallireducens in the presence of c-AMP-GMP. The predicted expression platform consists of an intrinsic terminator stem highlighted in gray followed by a run of U residues. (C) Single-round in vitro transcription assays using a DNA template corresponding to the riboswitch depicted in B in the absence (‒) or presence of various cyclic dinucleotides at the concentrations indicated. (Top) PAGE separation of transcripts where FL and T designate full-length and terminated products, respectively. (Bottom) Plot of the fraction of full-length RNA transcripts produced as determined from the PAGE image.

Fig. 4.

The superregulons for c-di-GMP and c-AMP-GMP in Deltaproteobacteria. Genes controlled by riboswitches with a guanosine at position 20 are assigned to c-di-GMP control (46 genes), whereas those with an adenosine are assigned to the c-AMP-GMP control (134 genes). Hypothetical genes are not listed. See Table S2 for the assignment of genes to various categories.