Untersuchungen zur genetischen Regulation der CO<sub>2</sub>-Assimilation in <i>Ralstonia</i> spp.

Abstract

Autotrophic CO2 assimilation in Ralstonia eutropha H16 and Ralstonia metallidurans CH34 involves the enzymes of the Calvin-Benson-Bassham (CBB) cycle. The expression of the two highly homologous cbb operons, cbbc and cbbp, in R. eutropha H16 is mainly controlled by the LysR type transcriptional regulator CbbR. The sigma70-dependent promoters of the cbb operon (pcbbL) and the cbbRgene (pcbbR) had been already verified within the cbb control region in earlier studies. Binding experiments with the cbb operator, site-directed mutageneses and activity determinations of pcbbL in wild type strain H16 and a cbbR deletion mutant suggested that the cbb expression is controlled by at least one additional regulator.To investigate the cbbgene regulation in R. eutropha H16 homologous regulator genes of CO2 fixation in other bacteria, like the two-component system RegBA, were deleted. Additional potential cbb regulators were isolated by DNA affinity chromatography using the cbb control region and by binding interaction with CbbR employing a two-hybrid system. Deletion mutants of the corresponding genes showed no alteration of the autotrophic phenotype. The gene frcR, encoding one of the proteins isolated by DNA affinity chromatography and belonging to the ROK family of transcription regulators, forms an operon together with the genes of a potential fructose transporter system, a sugar kinase, a glucose-6-phosphate dehydrogenase (G6PD) and a phosphoglucose isomerase (PGI). Activities of G6PD and PGI were elevated in a frcR deletion mutant, but still depended on the specific growth conditions. Thus, FrcR seems to be a repressor of the frcoperon which probably is controlled by at least one additional regulator. In contrast there was no significant influence of FrcR on the regulation of the cbbgenes.Activator CbbR is essential for cbbgene expression in R. eutropha H16. However its activating function is inhibited by binding the negative effector phosphoenolpyruvate, a signal metabolite of the central carbon metabolism. Site-directed mutageneses of CbbR suggested a structural function of the amino acid residues A175 and W274 and a role of residues G203, G205 and R135 in the effector binding, confirming the differential function of CbbR. A cbbR gene is encoded immediatly upstream of each of the two heterologous cbb operons (cbbI and cbbII) of R. metallidurans CH34. It was shown that CbbRI as well as CbbRII bind to the two cbb control regions. A cbbRII deletion mutant exhibited strongly reduced autotrophic growth, suggesting preferential regulation of the cbb operons by their associated CbbR proteins. These findings indicate significant differences in cbb gene regulation in these two related organisms.