title: Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.
abstract: Vibrio cholerae, the cause of seven noted pandemics, leads a dual lifecycle-one in the human host in its virulent form, and the other as a sessile, non-virulent bacterium in aquatic bodies in surface biofilms. Surface biofilms have been attributed to be associated with a ubiquitous protein domain present in all branches of bacteria, known as the GGD(/E)EF domain. While the diguanlyate cyclase activities of these proteins are universally established, the role of these proteins as diguanlyate-specific phosphodiesterases in conjunction with a EAL domain has also been reported. The VC0395_0300 protein from V. cholerae which shows biofilm forming abilities also acts as a phosphodiesterase. Interestingly, this GGD(/E)EF protein contains a EAL site in the reverse orientation. We attempted to mutate the GGEEF signature along the sequence by site-directed mutagenesis. The resultant mutants (Sebox5-7) did not show much difference in phosphodiesterase activity in comparison with the wild type protein (Sebox3), indicating the independence of the phosphodiesterase activity of the protein from the GGD(/E)EF domain. However, the ability of the mutants to form surface biofilm was significantly lesser in the case of mutations in the three central positions of the signature domain.
authors.affiliation: VISTA Lab, BITS, Pilani, KK Birla Goa Campus, Zuarinagar, Goa, 403726, India.
authors.forename: Om Prakash
authors.initials: OP
authors.lastname: Chouhan
authors.affiliation: VISTA Lab, BITS, Pilani, KK Birla Goa Campus, Zuarinagar, Goa, 403726, India.
authors.forename: Divya
authors.initials: D
authors.lastname: Bandekar
authors.affiliation: Department of Microbiology, University of Kalyani, Kalyani, West Bengal, 741245, India.
authors.forename: Mousumi
authors.initials: M
authors.lastname: Hazra
authors.affiliation: VISTA Lab, BITS, Pilani, KK Birla Goa Campus, Zuarinagar, Goa, 403726, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Baghudana
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India.
authors.forename: Saugata
authors.initials: S
authors.lastname: Hazra
authors.affiliation: VISTA Lab, BITS, Pilani, KK Birla Goa Campus, Zuarinagar, Goa, 403726, India. sumit@goa.bits-pilani.ac.in.
authors.forename: Sumit
authors.initials: S
authors.lastname: Biswas
keywords: Biofilm; Diguanylate cyclase; Motility; Phosphodiesterase; Site directed mutagenesis