Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.

Functional characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway.

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.