Riboswitches in eubacteria sense the second messenger cyclic di-GMP

Abstract

Cyclic di-guanosine monophosphate (di-GMP) is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms by which cyclic di-GMP regulates gene expression have remained a mystery. We found that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in messenger RNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pilus formation, and flagellum biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.

Fig. 1

A cyclic di-GMP aptamer from V. cholerae. (A) Sequence and structure of the Vc2 RNA from V. cholerae chromosome 2, and its proximity to the ORF of VC1722. Nucleotides shown correspond to the 110 Vc2 RNA construct. Bold numbers identify regions of ligand-mediated structure modulation as observed in B. Brackets identify the minimal 5′ or 3′ terminus (when the opposing terminus for 110 Vc2 RNA is retained) that exhibits structural modulation when tested with 10 nM cyclic di-GMP. Nucleotides in shaded boxes were mutated for studies depicted in Fig. 2A. (B) PAGE separation of RNA products generated by in-line probing of 5′ ³²P-labeled 110 Vc2 RNA. NR (no reaction); T1 (partial digest with RNase T1); ⁻OH (partial digest with alkali). RNA was incubated in the absence (−) or presence (+) of 100 μM cyclic di-GMP. (C) Plot of the normalized fraction of 110 Vc2 aptamer cleaved versus cyclic di-GMP concentration. Sites of structural modulation are as depicted in B. (D) Comparison of K_D values exhibited by 110 Vc2 aptamer for cyclic di-GMP (fig. S5) and various analogs. G (guanosine); pG, pGpG, pGpA (5′ phosphorylated mono- and dinucleotides); GpGpG (trinucleotide), AMP and GMP (adenosine- and guanosine monophosphate, respectively).

Fig. 2

Representative cyclic di-GMP aptamers are components of gene control elements. (A) Reporter fusion constructs carry wild-type (WT) or mutant (M1 through M3) riboswitches from V. cholerae (Vc2) in E. coli, or carry the equivalent WT and M3 riboswitches from B. cereus (Bc1 and Bc2) or C. difficile (Cd1) (fig. S6) in B. subtilis. (B) β-galactosidase reporter gene assays for reporter fusion constructs described in A. Maximum Miller units measured for the four aptamer representatives were 436, 47, 5 and 51, respectively. (C) B. subtilis cells carrying a β-galactosidase reporter construct fused to a wild-type (WT) Cd1 riboswitch and transformed with a plasmid lacking (−) or carrying a normal (+) or mutant (E170A) V. cholerae vieA gene encoding an EAL phosphodiesterase (PDE).