Chromosomally and plasmid-encoded gene clusters for CO2 fixation (cfx genes) in Alcaligenes eutrophus

Abstract

Two sets of structural genes (cfxL, cfxS) for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were localized in the genome of Alcaligenes eutrophus H16 by means of hybridization probes. One set is encoded on a chromosomal 12 kb EcoRI fragment, the other on an 11 kb EcoRI fragment of the megaplasmid pHG1. These fragments have previously been shown to carry the chromosomal and plasmid copies of the also reiterated phosphoribulokinase (PRK) gene (cfxP). Restriction mapping of the cloned fragments showed that, on each of the two genetic entities, the three cfx genes are located in a very similarly organized cluster. The closely linked Rubisco genes are separated from the downstream PRK gene by about 3.8 kb. The two cfx regions are highly homologous as deduced from cross-hybridization experiments. The Rubisco genes were found to be cotranscribed into a 2.1 kb cfxL-cfxS mRNA. Their expression is regulated at the transcriptional level. Rubisco and PRK genes were subcloned and expressed in Escherichia coli under the control of the lac promoter of pUC plasmids. Since active recombinant enzymes with the structural properties of the authentic enzymes were formed, the ribosome binding sites of the genes are recognized in E. coli. A low level of expression of the Rubisco genes occurred even without lac promoter control, indicating some activity of the cfxL-cfxS promoters in the foreign host.