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. 2012 Nov 30;32(3):354–368. doi: 10.1038/emboj.2012.315

Figure 5.

Figure 5

Mutations in PgaD render the PgaCD complex constitutively active and independent of c-di-GMP. (A) Predicted topology of PgaD. Positions of c-di-GMP-independent (orange) and loss-of-function mutations (red) within the most conserved region of PgaD (grey) are indicated. Sites of C-terminal PgaD truncations are marked by triangles. IM, inner membrane; PP, periplasm. Transmembrane helices were predicted using the TMHMM server (Sonnhammer et al, 1998). (B) Biofilm formation of strains expressing C-terminally truncated pgaD alleles as a function of cellular c-di-GMP concentrations. The last residue of each mutant is indicated (A). Δ7 strains harbouring individual pgaD alleles contained plasmids with an IPTG-inducible copy of the heterologous DGC wspR (pwspR) or control plasmids (vector). Expression of plasmid-borne pgaD alleles was induced with 0.2% (left graph) and 0.02% L-arabinose (right graph). Error bars are standard deviations of the mean of 3–6 replicas. (C) Contribution of pgaD mutants to biofilm formation is shown in the control strain (black bars) and the Δ7 mutant (grey bars). Isolated constitutive alleles are underlined. Error bars are standard deviations of the mean of 3–6 replicas. (D) Immunoblot analysis of steady-state levels of wild-type and mutant forms of PgaD–3 × Flag in the control strain and the Δ7 mutant. (E) C-di-GMP-dependent PgaCD GT activity. Membranes of a Δ7 mutant containing either PgaD wild-type or mutant forms were incubated with (black bars) or without c-di-GMP (grey bars) in the presence of 300 μM UDP-GlcNAc. PgaD mutant variants were expressed as PgaCD fusion proteins. A representative data set of two independent experiments is shown with standard errors. a.u., arbitrary unit. (F) The PgaD mutant N75D,K76E is strongly impaired in c-di-GMP binding. Relative amounts of PgaD wild-type or mutant forms captured in the presence (black bars) or absence of excess c-di-GMP (grey bars) are shown as an average of two independent experiments with standard deviations. PgaD variants were expressed as PgaCD fusion proteins. Figure source data can be found in Supplementary data.