Figure 3

Heterologous expression of HD-GYP genes in E. coli . Each gene encoding a putative HD-GYP domain protein, tagged with a 6-histidine sequence at the 3′ end, was expressed ectopically in E. coli BL21. (A) Production of each protein 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) Lysates of E. coli BL21 expressing each of the HD-GYP domain-encoding genes were tested for the ability to hydrolyze c-di-GMP as described in the Methods. Buffer only (“B”) and lysates from E. coli BL21 with vector only were included as negative controls. The radiolabeled c-di-GMP substrate has a relative mobility (R_f) of ~0.3 and appears for all samples at t =0, when the reaction was initiated by addition of [³²P]-c-di-GMP. The GMP reaction product appears at R_f ~0.65.