We have recently identified the molecular nature of diguanylate cyclases (DGC) in bacterial cells. DGC proteins are widely distributed in the bacterial kingdom with many bacterial genomes containing a large number of dgc paralogs. This suggests that c-diGMP plays a prominent role in bacterial signaling. Increasing evidence links c-diGMP to the regulation of bacterial community behavior and surface associated traits required for multicellular activities like biofilm formation. However, the molecular mechanisms involved in regulated synthesis, breakdown and downstream control of this signaling compound are still completely elusive.

We propose to use Caulobacter crescentus and its inherent developmental characteristics as a model to study c-di-GMP signaling mechanisms in bacteria. C. crescentus contains 11 DGC proteins, one of which, PleD, is involved in the controlled remodeling of the cell poles during the transition from a planktonic swarmer cell to a surface attached stalked cell. We have shown that the PleD diguanylate cyclase is activated by phosphorylation through asymmetrically positioned sensor kinases and concomitantly segregates to the site of action at the cell pole. How activation of PleD is coupled to its subcellular localization and how the synthesis of c-di-GMP controls polar development is unclear. Based on our own work and based on data published by other labs, we propose that, as for cAMP and cGMP, cellular levels of c-di-GMP are controlled in bacteria by specific DGCs and phosphodiesterases (PDE). We hypothesize that one mechanism that allows cells to maintain the specificity of different c-di-GMP signaling pathways operating in parallel (bacteria encode up to 50 DGCs) is through spatial constriction of the DGC and the generation of a local c-diGMP readout. Alternatively, different c-di-GMP pathways might converge to a central c-diGMP pool and DGC circuits might be separated by their differential expression patterns.

PleD is the best-understood DGC protein so far and we propose to use it as a model protein to understand synthesis and control of c-di-GMP production in bacteria. We will analyze how PleD activation is coupled to the production of c-di-GMP and to its subcellular localization. We will test the hypothesis of DGCs acting as local pace makers, by investigating the relationship between PleD localization and stalked pole development. Furthermore, we plan to analyze other members of the DGC family in C. crescentus in order to define general regulatory principles of c-di-GMP signaling like feedback inhibition and specific spatial organization. We will also focus on the identification of PDEs and on their role in c-di-GMP signaling. Finally, we propose to elucidate how the novel messenger c-di-GMP functions in vivo by identifying downstream targets of c-di-GMP and analyzing their role in cell function. Together, this will lead to a basic understanding of how levels of c-di-GMP are controlled in the cell and how these alterations are transmitted into a coordinated response.

We will use a combination of biochemical, cell biology, genetics, and molecular biology techniques to establish a model for c-di-GMP metabolism and activity in time and space. We have established collaborations with organic chemists, structural biologists, and experts in molecular modeling in order to have access to c-di-GMP and some of its chemically modified versions, to solve the three-dimensional structure of novel key components of c-di-GMP signaling, and to make predictions about binding properties of physiological and chemotherapeutical ligands. Our work will contribute to the understanding of bacterial biofilm formation and the basic cellular control mechanisms involved in this process. Biofilms are medically important, accounting for many of the microbial infections of humans, animals and plants. Microbes in biofilms show a dramatically increased resistance against antibiotics and are difficult for the host’s immune system to develop an appropriate response. Interference with biofilm formation is thus an attractive strategy to counteract microbial infections. One of the targets exploited in an attempt to counteract biofilm formation is density dependent signaling based on homoserine lactones, as it has been shown that biofilm formation is dependent on the production of these compounds. c-di-GMP appears to be a central regulatory molecule orchestrating surface attachment and colonization of bacteria. Interference with c-di-GMP signaling thus offers attractive chemotherapeutic strategies to counteract biofilm formation in medically relevant environments.