Identification of cbbBc as an additional distal gene of the chromosomal cbb CO2 fixation operon from Ralstonia eutropha

Abstract

Ralstonia eutropha (formerly Alcaligenes eutrophus) strain H16 possesses two highly homologous cbb operons encoding most of the Calvin cycle enzymes. One copy of the operon is located on the chromosome, the other on the megaplasmid pHG1 of the organism. Sequence analysis of the region downstream of the presumptive 3′-terminal gene (cbbA _c) of the chromosomal operon revealed the presence of an open reading frame comprising 2,274 bp. Evidence is presented that this open reading frame is an additional distal gene (designated cbbB _c) of the operon. In contrast to the other genes of the operon, cbbB _c is not duplicated in the plasmid-borne operon. The deduced amino acid sequence of the cbbB _c product (757 residues, molecular mass 83.17 kDa) showed the highest similarity to the large catalytic subunits of various bacterial formate dehydrogenases (FDH), suggesting that cbbB _c might represent a structural FDH gene of R. eutropha. However, the properties of a cbbB _c mutant strain indicated that the potential gene product is not related to known FDH of the organism. Transcriptional analysis in the homologous host and heterologous expression in Escherichia coli demonstrated that cbbB _c is an active gene, which apparently has no essential function in the autotrophic metabolism of R. eutropha. The gene is a novel member of cbb operons in autotrophic bacteria.