The characterization of a cyclic-di-GMP (c-Di-GMP) pathway leads to a new tool for studying c-Di-GMP metabolic genes.

Second messengers play an integral role in regulating a wide variety of pathways in response to triggers from the extracellular milieu. The intracellular second messenger cyclic diguanylate (c-di-GMP) has been shown to regulate microbial adaptation to extracellular environments in myriad ways. Primarily studied in Gram-negative bacteria thus far, c-di-GMP regulates motility, biofilm formation, virulence, and more (reviewed in references 1 to 3). Specifically, elevated c-di-GMP favors a sessile lifestyle, often stimulating biofilm formation. Conversely, reduced c-diGMP promotes biofilm dispersal and motility, and low c-di-GMP can activate virulence pathways in numerous bacterial pathogens. In comparison, little is known about the functions of c-di-GMP in Gram-positive bacteria. Determining how and under what circumstances bacteria regulate c-di-GMP levels, and the outcomes of c-di-GMP modulation, is important for understanding the role of c-di-GMP in bacterial cell signaling and adaptation to changing extracellular cues. Yet a full appreciation of c-di-GMP signaling pathways is often hindered by the fact that many bacterial genomes encode multiple, sometimes several dozen, c-di-GMP synthase (called diguanylate cyclase, or DGC) and hydrolase (called phosphodiesterase, or PDE) enzymes. There are many examples of single-gene mutations that result in detectable changes in some aspect of bacterial physiology (4–10), but often such mutations do not result in observable phenotypic alterations. Lack of gene expression, lack of enzyme activity, redundancy among c-di-GMP metabolic enzymes, and compensatory effects of other DGC and/or PDE enzymes under the conditions tested are some of the reasons that a mutation in a single DGC or PDE gene may not cause detectable changes in phenotype. In the current issue of the Journal of Bacteriology, Gao et al. present a systematic analysis of the principal c-di-GMP signaling components of the model Gram-positive bacterium Bacillus subtilis (11). Taking advantage of the relatively simple c-di-GMP signaling network of B. subtilis, the authors describe a mutant B. subtilis strain that may be used to analyze putative DGC enzymes for activity using an easily assayed motility phenotype as a readout. In the highlighted study (11), Gao et al. aimed to fully examine the in vitro and in vivo functions of putative c-di-GMP signaling components of B. subtilis, including four putative DGCs (identified based on the presence of a GGDEF diguanylate cyclase domain), two predicted PDEs (identified based on the presence of an EAL c-di-GMP phosphodiesterase domain), one protein with tandem GGDEF and EAL domains, and a putative c-di-GMP receptor containing a PilZ domain. Recently, Chen et al. used genetic approaches to implicate one PDE, YuxH (renamed PdeH by Gao et al.) and the PilZ domain c-di-GMP receptor YpfA (renamed DgrA by Gao et al.) in regulation of B. subtilis motility and biofilm formation, indicating that these processes are controlled by c-diGMP (12). In that work, deletion of a single PDE gene, pdeH, led to a significant reduction of B. subtilis motility. The motility defect was complemented by the expression in trans of wild-type pdeH, but not an allele of pdeH expected to encode an inactive PDE, suggesting that the decreased motility was specifically due to increased c-di-GMP as a result of the loss of PdeH enzymatic activity (12). In the current study (11), Gao et al. built on that work by providing evidence that the reduced motility of a B. subtilis pdeH mutant is due to increased intracellular c-di-GMP. Whereas c-diGMP was undetectable in the wild-type parent by liquid chromatography followed by tandem mass spectrometry (LC–MS-MS) (at least under the growth conditions used), c-di-GMP was measurable in the pdeH mutant, indicating that loss of PdeH activity leads to accumulation of c-di-GMP. In addition, the authors show that three of the five GGDEF domain proteins and one of the three EAL domain proteins of B. subtilis are enzymatically active in vitro and in vivo. Overexpressing any of the three DGCs that were active in vitro in B. subtilis drove c-di-GMP to detectable levels. Interestingly, mutation of the GGDEF domain-encoding genes, individually or in combination, had no effect on motility of B. subtilis, despite encoding functional DGCs. These results corroborate the findings of Chen et al. (12). The lack of phenotype associated with these mutations could be because the encoded DGCs regulate pathways other than motility. Alternatively, c-di-GMP already may be adequately low under the growth conditions used to allow maximal motility. Thus, further reduction of c-di-GMP by inactivation of DGCs would thus have no discernible effect. Having established the set of enzymes capable of modulating intracellular c-di-GMP levels, the authors then investigated the mechanisms by which c-di-GMP could be sensed in the cell. They focused on a putative c-di-GMP receptor protein containing a PilZ domain, DgrA. Previously, it was shown that deletion of dgrA restores wild-type levels of motility to a pdeH mutant, suggesting that DgrA mediates the effect of increased c-di-GMP on motility (12). Further analysis revealed that DgrA interacts with the MotA motor protein (12), consistent with previous reports in other bacteria that c-di-GMP can act through PilZ domain receptors to block motility via interaction with MotA or FliG (13–15). Gao et al. used size exclusion chromatography and isothermal titration calorimetry to show that DgrA directly binds c-di-GMP in vitro. DgrA is the only PilZ domain protein predicted in B. subtilis, but it is likely that