Background: Cyclic-di-GMP (c-di-GMP) is a ubiquitous secondary messenger molecule
in bacteria synthesized by diguanylate cyclases. This universal messenger regulates diverse cellular
functions in bacteria at the transcriptional, translational and posttranslational levels. The cellular
functions regulated by c-di-GMP include cell motility, cell cycle progression, virulence, biofilm
formation, antibiotic production and other unknown functions. The VC0395_0300 protein from the
chromosome I of the Vibrio cholerae classical strain O395, serotype O1 has been established to be
a diguanylate cyclase with a necessary role in biofilm formation.
Objective: Mutations in the central position of the GGEEF active site of VC0395_0300 protein
have been created by site-directed mutagenesis. The conditions for maximum production of mutated
protein have been optimized. While there is a significant loss-of-biofilm-forming activity in
the mutants, the basis for the same needed an investigation at the structural level.
Methods: Subsequently, the mutant proteins have been characterized using spectrofluorimetry and
circular dichroism spectroscopy.
Results: While the unfolding pattern of the mutant proteins shows some changes with respect to the
wild type, the overall structure of the protein does not show significant changes due to the mutagenesis,
despite the absence of biofilm formation in the mutants.
Conclusion: This led us to conclude that whatever changes that occur in the mutated proteins, do
not disturb the GGEEF domain architecture, but are restricted to the local architecture, and are
hence, subtle in nature.