Fig. S1. BdcA does not catalyse c-di-GMP degradation. 31P NMR spectrum for GMP and for c-di-GMP with and without BdcA E50Q. The 85% phosphoric acid was used as an external standard for the chemical shift at 0 p.p.m.

Fig. S2. Swiss model for evolved BdcA. The E50 residue is indicated in yellow, the E50V variant is indicated in red, and the E50Q variant is indicated in blue. The typical phosphodiesterase for c-di-GMP contains EALXR for coordinating Mg2+, Q/R/D/D for c-di-GMP binding and T/E for catalysis. The degenerate phosphodiesterase domains of BdcA are indicated: EAL in pink as part of the EALXR motif, Q49/D136/D180 in turquoise as part of the Q/R/D/D motif, and E220 in orange as part of the T/E motif. The amino and carboxy termini are marked as N and C.

Fig. S3. Phylogenetic tree of BdcA.

Table S1. Normalized biofilm formation (OD540/OD620) in LB medium at 37°C for mutations in uncharacterized genes that are related to AI-2 transporter TqsA (Herzberg et al., 2006).

Table S2. DNA oligonucleotides used in this study. N represents A, T, G or C, while S represents G and C.

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