c-di-GMP inhibits the RNA unwinding activity of PlzA in E. coli
RNA chaperone activity has been assayed in vivo utilizing a transcription antitermination assay (74,84-86). E. coli strain RL211 harbors the chloramphenicol (Cm) acyltransferase gene (cat ) preceded by a Rho-independent trpL transcriptional terminator. The terminator is a stem-loop followed by a poly(U) stretch that efficiently terminates transcription of the cat gene and results in susceptibility of the RL211 strain to Cm. However, proteins with RNA chaperone unwinding activity melt or unfold the trpL terminator leading to antitermination, transcription of the cat gene and resistance to Cm (Fig. 3A). We sought to determine if PlzA had antitermination activity in vivo in E. coli using the RL211 strain. Plasmids carrying stpA and plzA under the control of the lac promoter were transformed separately into the RL211 strain. Cultures were grown overnight in the absence of Cm and then spotted onto plates without (LB) and with Cm (Fig. 3B). As expected, all strains grew similarly on the LB plates (Fig. 3B). RL211 was unable to grow in the presence of Cm and growth was restored in the presence of Cm in the strain producing StpA, which is known to have RNA unwinding activity (71,87,88). The strain producing PlzA had minimal growth in the presence of Cm (Fig. 3B). We postulated that c-di-GMP may be inhibiting RNA unwinding in E. coli, an effect similar to what we observed in the strand displacement assay in vitro (Fig. 2).E. coli harbors 12 genes encoding diguanylate cyclases that generate c-di-GMP (89). Therefore, we tested the ability of a PlzA mutant that is unable to bind c-di-GMP (PlzA R145D) and a mutant with increased affinity for c-di-GMP (PlzA R145K) to rescue growth in Cm (53). Expression of the PlzA R145D mutant, which cannot bind c-di-GMP, rescued growth in Cm, while expression of PlzA R145K, which binds c-di-GMP with a high affinity, did not rescue growth in Cm (Fig. 3B). Taken together, these data suggest that PlzA has antitermination activity and this activity is inhibited by c-di-GMP in vivo , at least in a heterologous system. Therefore, both the RNA displacement and unwinding activities of PlzA, but not its RNA annealing activity, are modulated by c-di-GMP.