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Subtle Changes Due to Mutations in the GGDEF Domain Result in Loss of Biofilm Forming Activity in the VC0395_0300 Protein from Vibrio cholerae, but No Major Change in the Overall Structure

[ Vol. 25 , Issue. 8 ]

Author(s):

Om Prakash Chouhan and Sumit Biswas*   Pages 740 - 747 ( 8 )

Abstract:


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.

Keywords:

Vibrio cholera, c-di-GMP, biofilm, site-directed mutagenesis, diguanylate cyclase, spectrofluorimetry.

Affiliation:

VISTA Lab, BITS, Pilani - K K Birla Goa Campus, Zuarinagar, Goa 403726, VISTA Lab, BITS, Pilani - K K Birla Goa Campus, Zuarinagar, Goa 403726

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