REGULATION OF CO2 ASSIMILATION IN RALSTONIA-EUTROPHA - PREMATURE TRANSCRIPTION TERMINATION WITHIN THE CBB OPERON

In the facultatively chemoautotrophic bacterium Ralstonia eutropha (fo rmerly Alcaligenes eutrophus), most genes required for CO2 assimilatio n via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectivel y, of strain H16, These operons are subject to tight control exerted b y a promoter upstream of the 5'-terminal cbbL gene that is regulated b y the activator CbbR. The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK, Nevertheless, mar ked differential expression of the promoter proximal ribulose-1,5-bisp hosphate carboxylase-oxygenase genes cbbLS and the remaining distal ge nes occurs within the operons, Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression. Nuclease S1 mappin g identified a major 3' end and a minor 3' end of the relatively stabl e cbbLS partial transcript just downstream of this structure. Moreover , operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional terminatio n downstream of cbbS rather than increased the segmental stability of the cbbLS transcript, Premature transcription termination thus represe nts an important mechanism leading to differential gene expression wit hin the cbb operons.