Mutational Analysis of the cbb Operon (CO 2 Assimilation) Promoter of Ralstonia eutropha

Abstract

ABSTRACT P L promoters direct the transcription of the duplicated cbb operons from the facultative chemoautotroph Ralstonia eutropha H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO 2 assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. P L was altered in its presumed hexameric −35 and/or −10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the ς 70 consensus promoter of Escherichia coli . P L :: lacZ transcriptional fusions of various promoter variants were assayed in transconjugant strains of R. eutropha as well as in corresponding cbbR deletion mutants. Mutations increasing the similarity of the −35 and/or −10 box to the consensus sequence stimulated P L activity to various extents, whereas mutations deviating from the consensus decreased the activity. The length of the spacer region also proved to be critical. The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active P L . All improved P L mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive. They were also activated in the cbbR mutants. The activity of the overlapping, divergently oriented cbbR promoter was less affected by the mutations. The half- and full-consensus P L mutants were comparably active in E. coli . Two major conclusions were drawn from the results: (i) the location and function of P L were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of the R. eutropha cbb operons.