Phosphoenolpyruvate is a signal metabolite in transcriptional control of the cbb CO2 fixation operons in Ralstonia...

The two highly homologous cbb operons of the facultative chemoautotroph Ralstonia eutropha H16 encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle. Their transcriptional regulation was investigated both in vitro and in vivo to identify a metabolic signal involved in this process. For this purpose an in vitro transcription system employing the DNA-dependent RNA polymerase purified from R. eutropha was established. The enzyme from Escherichia coli was also used in verifying comparative studies. Plasmid DNA carrying the control region of the chromosomal cbb operon served as template. In the homologous as well as the heterologous system specific transcripts synthesized under the control of the operon promoter PcbbL were observed, depending on the structure of the tested promoter variant as well as the presence or absence of the activator protein CbbR. Unlike mutationally improved PcbbL variants, the wild-type promoter remained inactive, even in the presence of CbbR together with various potential signal metabolites. CbbR stimulated PcbbL mutants with intermediate basal activity. Phosphoenolpyruvate (PEP) was identified as a negative effector of CbbR that inhibited PcbbL-directed transcription and increased the operator-binding affinity of the protein. This CbbR-mediated inhibition was confirmed by assaying wild-type PcbbL operon fusions in glucose- or succinate-grown cells of E. coli, which contain greatly different concentrations of PEP. It is concluded that at least one additional protein must participate in the overall control of the cbb operons in R. eutropha.