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

Abstract

PL promoters direct the transcription of the duplicatedcbb operons from the facultative chemoautotrophRalstonia eutropha H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO2 assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. PL 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 ς70 consensus promoter of Escherichia coli. PL::lacZ transcriptional fusions of various promoter variants were assayed in transconjugant strains ofR. eutropha as well as in corresponding cbbRdeletion mutants. Mutations increasing the similarity of the −35 and/or −10 box to the consensus sequence stimulated PLactivity 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 PL. All improved PL 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 cbbRpromoter was less affected by the mutations. The half- and full-consensus PL mutants were comparably active inE. coli. Two major conclusions were drawn from the results: (i) the location and function of PL were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of theR. eutropha cbb operons.

New Insight into the Role of the Calvin Cycle: Reutilization of CO2 Emitted through Sugar Degradation

“…Another mutant strain, H16G∆ cbbR , was constructed by deletion of cbbR on chromosome 2, encoding a common transcriptional activator for the two cbb operons. It has been reported that a cbbR -deleted strain of R. eutropha was incapable of growing autotrophically owing to insufficient induction of the cbb genes 26 . Indeed, qRT-PCR analysis demonstrated that expression levels of cbbL (encoding Rubisco large subunit), cbbP (encoding phosphoribulokinase) and cbbF (encoding fructose-1,6-bisphosphatase I/sedoheptulose-1,7-bisphosphatase) in H16G∆ cbbR incubated with glucose were approximately one-hundredth compared to those in the parent strain H16G ( Fig.…”

Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16

“…Finally, it encodes the Strep epitope downstream of the artificial cbb c -P L -M9 promoter (Fig. 2b), which directs high constitutive expression levels in R. eutropha (Jeffke et al, 1999). Restriction sites downstream of the strep-tag sequence facilitate the construction of fusion genes (Fig.…”

“…The derivatives pGP1655 and pGP1656 of plasmid pBBR1MCS-3 were assembled in several steps. First, the cbb c -P L -M9 promoter was amplified by PCR using primers DK15/DK16 and plasmid pBKM9 (Jeffke et al, 1999) as the template. This DNA fragment was used to replace the XbaI-KpnI fragment of plasmid pGP788 K, resulting in plasmid pGP1651.…”

Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases

“…Regions of interest were amplified from PAK genomic DNA and ligated into a cloning vector. A section of the coding sequence was then deleted (and replaced by an antibiotic resistance cassette in some cases) and this inactivated operon was then cloned into the suicide plasmid pNHG1 (Jeffke et al ., 1999). To inactivate ccoNOQP ‐1, a 1554 bp BstB I to EcoR I fragment was replaced by a ΩGm r cassette to create strain Cco1.1 (Fig.…”