CLONING OF THE CPCE AND CPCF GENES FROM SYNECHOCOCCUS SP PCC-6301 ANDTHEIR INACTIVATION IN SYNECHOCOCCUS SP PCC-7942

Two open reading frames denoted as cpcE and cpcF were cloned and seque nced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are loca ted downstream of the cpcB2A2 gene cluster in the phycobilisome rod op eron and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by inser tion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which displ ay a substantial reduction in spectroscopically detectable phycocyanin . The levels of beta- and alpha-phycocyanin polypeptides were reduced in the R2EKM and R2FKM mutants although the phycocyanin and linker gen es are transcribed at normal levels in the mutants as in the wild type indicating the requirement of the functional cpcE and cpcF genes for normal accumulation of phycocyanin. Two biliprotein fractions were iso lated on sucrose density gradient from the R2EKM/R2FKM mutants. The fa ster sedimenting fraction consisted of intact phycobilisomes. The slow er sedimenting biliprotein fraction was found to lack phycocyanin poly peptides, thus no free phycocyanin was detected in the mutants. Charac terization of the phycocyanin from the mutants revealed that it was ch romophorylated, had a lambda(max) similar to that from the wild type a nd could be assembled into the phycobilisome rods. Thus, although phyc ocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaini ng phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy trans fer to the core.