Figure 2

Ectopic over-expression of HD-GYP genes in V. cholerae. Each gene encoding a putative HD-GYP domain protein, tagged with a 6-histidine sequence at the 3′ end, was expressed ectopically in V. cholerae using an IPTG-inducible promoter. (A) Production of each protein in V. cholerae after ~3 hours growth in the presence of 1 mM IPTG was assessed by western blot using anti-His6 antibodies. The predicted molecular weights of each protein, in kDa, are indicated at the bottom. Right: Expression of the large, predicted membrane protein VCA0895 was relatively poor; detection of the recombinant protein required loading more sample. (B) Motility of V. cholerae expressing each of the HD-GYP domain genes was assayed in soft agar medium as an indication of c-di-GMP hydrolysis, which augments flagellar motility of V. cholerae. Each strain was inoculated into motility medium with (grey bars) or without (white bars) 0.5 mM IPTG. The diameter of each motility swarm was measured after 20 hours incubation at room temperature. Shown are means and standard deviations for three biological replicates. (C) Biofilm formation by V. cholerae expressing each of the HD-GYP domain encoding genes was assessed as an indication of c-di-GMP hydrolysis, which decreases biofilm formation by V. cholerae. Each strain was grown in LB broth with (grey bars) or without (white bars) 0.5 mM IPTG to induce gene expression. Biofilm was assayed using standard crystal violet methods. Shown are means and standard deviations for three biological replicates. *P <0.05 by two-way ANOVA with Bonferroni’s post-test.