New Functions and Subcellular Localization Patterns of c-di-GMP Components (GGDEF Domain Proteins) in B. subtilis

Abstract

The universal and pleiotropic cyclic dinucleotide second messenger c-di-GMP is most prominently known to inversely regulate planktonic and sessile lifestyles of Gram-negative species. In the Gram-positive model organism Bacillus subtilis, intracellular c-di-GMP levels are modulated by a concise set of three diguanylate cylases (DgcK, DgcP, DgcW) and one phosphodiesterase (PdeH). Two recent studies have reported the negative influence of the c-di-GMP receptor DgrA (PilZ domain protein) on swarming motility indicating a conserved role of this second messenger across the bacterial domain. However, it has been suggested that the degenerated GGDEF protein YdaK and the inactive EAL domain protein YkuI may also function as c-di-GMP receptors regulating potentially other processes than motility. Here we describe a novel c-di-GMP dependent signaling network in B. subtilis regulating the production of an unknown exopolysaccharide (EPS) that leads to strongly altered colony morphologies upon overproduction. The network consists of the c-di-GMP receptor YdaK and the c-di-GMP synthetase DgcK. Both proteins establish a spatially close signal-effector cluster at the membrane. The cytoplasmic DgcP synthetase can complement for DgcK only upon overproduction, while the third c-di-GMP synthetase, DgcW, of B. subtilis is not 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 regulatory circuits in a Gram-positive bacterium.

Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations

“…Instead, the recent development of cell-type-specific fluorescent reporters allows investigators to examine features of B. subtilis subpopulations and their relationship to c-di-GMP effector proteins, DGCs, and PDEs. (51)(52)(53).…”

“…It is possible that the primary purpose of c-di-GMP signaling in this organism is to influence the motility apparatus. Alternatively, it is possible that effector targets for c-di-GMP-binding proteins have yet to be identified, as might be suggested by the recently discovered link to the yda exopolysaccharide pathway (52,53). Overall, the abundance of c-di-GMP in a bulk ensemble of B. subtilis is likely averaged across the population, and we propose that single-cell analysis of c-di-GMP levels offers unique insight into how the abundance of this signaling molecule varies among cellular subpopulations.…”

“…YdaK is encoded within the ydaJKLMN operon. While the exact role of the yda operon has yet to be determined, the operon may be involved in the synthesis of an unknown exopolysaccharide, which may bolster the stress resistance of the biofilm under specific physiological conditions (52,53). Therefore, more remains to be understood regarding the role of c-di-GMP in regulating B. subtilis biofilm formation.…”

“…Elevated c-di-GMP levels have been shown to inhibit motility in this organism (49,50); however, it is currently unknown if sporulation and competence are also regulated by this signaling molecule. Developing evidence suggests that c-di-GMP may also affect the composition of extracellular polysaccharides during biofilm formation in this organism (52,53). Yet, the methods used to examine whether c-di-GMP affects biofilm formation have thus far incompletely considered that B. subtilis biofilms are heterogeneously composed of several subpopulations (4,5,56).…”

Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

“…Instead, spatially sequestering the signal (local pool) within multiprotein complexes at distinct cellular sites may result in highly specific signaling pathways. Evidence for this idea has come from the identification (i) of direct interactions of a DGC and its receptor protein in the Gram-negative organism Pseudomonas fluorescens (6); (ii) of a module of interacting DGCs, PDEs, and a DNA-binding protein acting as a signaling cascade in Escherichia coli (7); and (iii) of single, distinct localization points of DGCs and of effector proteins in B. subtilis (8,9). A third scenario has recently been suggested based on the identification of interaction hubs of DGCs and PDEs in E. coli, where a few DGCs and PDEs show multiple functional interactions in a tightly interconnected network (10,11).…”