CHARACTERIZATION OF THE DUPLICATE RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE GENES AND CBB PROMOTERS OF ALCALIGENES-EUTROPHUS

Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alca ligenes eutrophus H16 is genetically determined by two highly homologo us cbb operons, one of which is located on the chromosome and the othe r on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies i n divergent orientation only 167 bp upstream of the chromosomal operon acid controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,S-bisphosphate car boxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2 .1-kb cbbLS transcripts by primer extension and by nuclease S1 treatme nt revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derive d cbb operon promoter showed similarity to sigma(70)-dependent promote rs of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transc riptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma(70)-dependent promo ters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon pr omoters were strongly regulated in response to autotrophic versus hete rotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal an d plasmid-borne cbb promoters of A. eutrophus H16 are functionally equ ivalent despite minor structural differences.