ANALYSIS OF THE GENES FORMING THE DISTAL PARTS OF THE 2 CBB CO2 FIXATION OPERONS FROM ALCALIGENES-EUTROPHUS

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of t he genes (cbb genes) encoding enzymes of the Calvin carbon reduction c ycle are organized within two highly homologous cbb operons, one locat ed on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searc hes in databases and heterologous expressions of the subcloned genes i n Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate k inase, and a class II fructose-1,6-bisphosphate aldolase, respectively . The aldolase could be grouped together with the enzymes from Rhodoba cter sphaeroides and Bacillus subtilis as a new subtype of class II al dolases. A phenotypic complementation analysis with a cbb operon mutan t of A. eutrophus showed that the cbbG product is essential for autotr ophic growth of the organism, whereas the products of cbbK and cbbA ca n apparently be substituted by isoenzymes encoded elsewhere on the chr omosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operon s discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.