The role of the second messenger cyclic di-GMP in Bacillus subtilis

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The bacterial second messenger c-di-GMP represents an integral key regulator in the control of bacterial motility and biofilm formation. In this context, an increase in intracellular c-di-GMP production correlates with a sessile lifestyle, whereas low c-di-GMP levels favor planktonic cell behavior. Intracellular c-di-GMP levels are controlled by the antagonistic activity of c-di-GMP specific synthetases (diguanylate cyclases, DGCs) and hydrolases (phosphodiesterases, PDEs). Bacteria contain diverse c-di-GMP binding receptors/effectors, which exert the regulatory functions of this signaling molecule. A given bacterial genome typically encodes several paralogous copies of DGCs and PDEs. This lead to the question of how cells cope with such a multiplicity of signaling components and guarantee that specificity within certain signaling modules is mediated. Two general models for signal specificity through functional sequestration are currently discussed: the so-called local and global pool signaling hypotheses. Spatially sequestering the signal (pool) in multi-protein complexes at distinct cellular site may result in highly specific signaling pathways. Temporal and/ or conditional separation through differential expression and activation of DGCs/ PDEs/ output systems respectively, could have a distinct impact on the global c-di-GMP pool. This work investigates the role of c-di-GMP and its players in the Gram-positive model organism B. subtilis, which possesses a relatively small c-di-GMP signaling equipment. In particular, the obtained findings define a novel c-di-GMP signaling pathway regulating the production of an unknown exopolysaccharide (EPS) and furthermore imply that local and global signaling pools potentially operate in B. subtilis to regulate motility and exopolysaccharide production. The proposed c-di-GMP receptor YdaK resides in the putative EPS synthesis operon ydaJKLMN. Artificial YdaJ-N induction results in strongly altered colony biofilms, increased Congo Red staining and provokes furthermore cell clumping, which provides indirect evidence of EPS production. The putative EPS synthase components YdaM/ YdaN and YdaK co-localize to clusters predominantly at the cell poles and are statically positioned at this subcellular site, suggesting that exopolysaccharide production takes place at distinct sites of the membrane. The potential glycosyl hydrolase YdaJ is not essential for the generation of the above-described phenotypes, whereas the presence of YdaK is required, implying an involvement of the second messenger c-di-GMP. To approach the potential regulation of exopolysaccharide production through c-di-GMP via YdaK, different combinations of overexpression and deletion mutants of the operon and of dgc genes, respectively, were generated. Importantly, the presence of dgcK was shown to be indispensable for the production of the unknown EPS, thereby revealing a new function for one of the three known DGC enzymes. DgcK and YdaK partially co-localize to the same subcellular positions at the cell membrane implying close proximity of these players, which strongly suggests that YdaK receives its activation signal directly from the spatially close DgcK in agreement with the local pool hypothesis. The cytoplasmic DgcP synthetase can complement for DgcK only upon overproduction, while the third c-di-GMP synthetase, DgcW, seems not to be part of the signaling pathway. Removal of the regulatory EAL domain from DgcW reveals a distinct function in biofilm formation. Therefore, our study is compatible with the local pool signaling hypothesis, but shows that in case of the yda operon, this can easily be overcome by overproduction of non-cognate DGCs, indicating that global pools can also confer signals to this regulatory circuit. Furthermore, indications are provided within this study that all three DGCs might cooperate in inhibition of motility via the c-di-GMP receptor DgrA indicating that DgrA depends on globally elevated c-di-GMP levels.