Regulation of ribulose bisphosphate carboxylase expression in Rhodospirillum rubrum: characteristics of mRNA synthesized in vivo and in vitro

Abstract

The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium. The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%). Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein. RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E. coli or R. rubrum RNA polymerase. With both enzymes there was excellent synthesis of RuBPCase mRNA, but no signfficant synthesis of RuBPCase was detected. The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped. A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R. rubrum. However, transcripts synthesized from pRR117 in vitro or from E. coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site. Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry.

Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus

“…Reactions with restriction and modifying enzymes were carried out under the conditions recommended by the suppliers. Double-stranded DNA fragments were radioactively labeled with [␣- 32 P]dCTP with a random primer labeling kit (Life Technologies, Eggenstein, Germany), and oligonucleotides were labeled with [␥ 32 P]ATP be means of the polynucleotide kinase reaction. DNA sequencing was conducted on both strands by the dideoxy chain termination method (40) with a T7 DNA polymerase sequencing kit (Pharmacia, Freiburg, Germany).…”

Complementation analysis and regulation of CO2 fixation gene expression in a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum

“…The high levels of regulated expression by the upstream cbbM sequence in R. sphaeroides, as well as mRNA initiation mapping data (32), substantiate that this region contains a genuine promoter. In addition, the lack of promoter activity exhibited by plasmid pRR523 (Fig.…”

“…3B). R. rubrum cbbM promoter activity was thus evident for all plasmids introduced in R sphaeroides, except pRPS523, which includes 327 bp in front of the cbbM translational start codon and within which a cbbM transcriptional initiation site has been mapped (32). The identity of the upstream gene proximal to cbbM as cbbR thus implicates CbbR as a requirement for cbbM expression in the R sphaeroides host strain (Fig.…”

“…(The recently described designation cbb has been proposed for structural genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway in bacteria [46].) Previous physiological studies had quantified the repression and derepression of RubisCO synthesis in R rubrum (39 vivo analysis of specific transcripts and verified that derepression occurred at the transcriptional level (32).…”

“…Under photoheterotrophic growth conditions the reduction level of the organic substrate is of paramount importance (32,39). Similar growth conditions are important in controlling RubisCO expression in another purple, nonsulfur bacterium, Rhodobacter sphaeroides, which encodes two distinct RubisCO enzymes.…”