Putative protein VC0395_0300 from Vibrio cholerae is a diguanylate cyclase with a role in biofilm formation

Abstract

The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.

Diguanylate Cyclases in Vibrio cholerae: Essential Regulators of Lifestyle Switching

“…A DGC with a GGEEF domain was reported by our group in 2017 (Bandekar et al, 2017;Chouhan and Biswas, 2018). While VC0395_0300 was shown to synthesize c-di-GMP actively and had an essential role to play in the biofilm formation of V. cholerae, mutations at the central positions of the GGEEF sequence were detrimental to the functional activity of the protein (Chouhan et al, 2016).…”

“…Cellular c-di-GMP levels are regulated by the synthesis of activities of c-di-GMP by diguanylate cyclases (DGCs), and degraded by phosphodiesterases (Römling et al, 2013;Bandekar et al, 2017). Apart from Mycobacterium smegmatis (only two DGCs) (Kumar and Chatterjee, 2008), there is an abundance of DGCs in different bacterial systems (Römling et al, 2013;Chouhan et al, 2016).…”