Cyclic diguanylate (c-di-GMP) is a bacterial second messenger of growing recognition involved in the regulation of a number of complex physiological processes. This review describes the biosynthesis and hydrolysis of c-di-GMP and several mechanisms of regulation of c-di-GMP metabolism. The contribution of c-di-GMP to regulating biofilm formation and motility, processes that affect pathogenesis of many bacteria, is described, as is c-di-GMP regulation of virulence gene expression. Finally, ways in which c-di-GMP may mediate these regulatory effects are proposed.

Cyclic diguanylate (c-di-GMP) is an allosteric activator and second messenger implicated in the regulation of a variety of biological processes in diverse bacteria. In Vibrio cholerae, c-di-GMP has been shown to inversely regulate biofilm-specific and virulence gene expression, suggesting that c-di-GMP signaling is important for the transition of V. cholerae from the environment to the host. However, the mechanism behind this regulation remains unknown. Recently, it was proposed that the PilZ protein domain represents a c-di-GMP-binding domain. Here we show that V. cholerae PilZ proteins bind c-di-GMP specifically and are involved in the regulation of biofilm formation, motility, and virulence. These findings confirm a role for PilZ proteins as c-di-GMP-sensing proteins within the c-di-GMP signaling network.

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNAbinding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.In the past few years, it has become increasingly clear that small untranslated RNAs (sRNAs) regulate many diverse cellular processes. In Escherichia coli, sRNAs have already been shown to modulate sigma factor production, iron utilization, acid resistance, porin expression, and the response to oxidative stress, and functions still need to be defined for a majority of its more than 50 sRNAs (reviewed in reference 2). The modes of action of sRNAs have also been shown to be diverse. In general, sRNA-based regulation is thought to depend upon base pairing between short regions of complementary sequence in the sRNA and its target mRNA(s), although sRNAs that interact with proteins (e.g., CsrB and 6S RNA) have also been described (32,40). Pairing between an sRNA and an mRNA can promote or inhibit mRNA translation or increase or decrease mRNA stability, depending upon the RNAs involved (7,21,22,27). The location of the target sequence within an mRNA probably influences the events that follow binding to an sRNA; still, it is not fully understood how sRNA binding has such a range of consequences.Many of the sRNAs that interact with mRNAs also interact with the RNA-binding protein Hfq. Hfq has homology to eukaryotic Sm proteins and appears to function as an RNA chaperone (12,25,43). Hfq interacts with both sRNAs and mRNAs and can foster formation of sRNA:mRNA complexes. mRNAbinding sRNAs generally have reduced stability in strains lacking Hfq, and their regulatory roles are typically impaired in an hfq strain background (12,25,36). We recently reported that Vibrio cholerae lacking hfq is severely attenuated in its ability to colonize the small intestines of suckling mice and thus is largely avirulent in this commonly used model host for study of cholera pathogenesis (8). This finding suggested that one or more sRNAs might be critical for V. cholerae virulence. Lenz et al. subsequently identified fo...