THE CBB OPERONS OF THE FACULTATIVE CHEMOAUTOTROPH ALCALIGENES-EUTROPHUS ENCODE PHOSPHOGLYCOLATE PHOSPHATASE

The two highly homologous cbb operons of Alcaligenes eutrophus H16 tha t are located on the chromosome and on megaplasmid pHG1 contain genes encoding several enzymes of the Calvin carbon reduction cycle. Sequenc e analysis of a region from the promoter-distal part revealed two open reading frames, designated cbbT and cbbZ, at equivalent positions wit hin the operons. Comparisons with known sequences suggested cbbT to en code transketolase (TK; EC 2.2.1.1) as an additional enzyme of the cyc le. No significant overall sequence similarities were observed for cbb Z. Although both regions exhibited very high nucleotide identities, 93 % (cbbZ) and 96% (cbbT), only the chromosomally encoded genes were het erologously expressed to high levels in Escherichia coli. The molecula r masses of the observed gene products, CbbT (74 kDa) and CbbZ (24 kDa ), correlated well with the values calculated on the basis of the sequ ence information. TK activities were strongly elevated in E. coli clon es expressing cbbT, confirming the identity of the gene. Strains of E. coli harboring the chromosomal cbbZ gene showed high levels of activi ty of 2-phosphoglycolate phosphatase (PGP; EC 3.1.3.18), a key enzyme of glycolate metabolism in autotrophic organisms that is not present i n wild-type E. coli. Derepression of the cbb operons during autotrophi c growth resulted in considerably increased levels of TK activity and the appearance of PGP activity in A. eutrophus, although the pHG1-enco ded cbbZ gene was apparently not expressed. To our knowledge, this stu dy represents the first cloning and sequencing of a PGP gene from any organism.