Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Abstract

Two open reading frames denoted as cpcE and cpcF were cloned and sequenced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are located downstream of the cpcB2A2 gene cluster in the phycobilisome rod operon and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by insertion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which display 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 genes 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 isolated on sucrose density gradient from the R2EKM/R2FKM mutants. The faster sedimenting fraction consisted of intact phycobilisomes. The slower sedimenting biliprotein fraction was found to lack phycocyanin polypeptides, thus no free phycocyanin was detected in the mutants. Characterization of the phycocyanin from the mutants revealed that it was chromophorylated, had a lambda max similar to that from the wild type and could be assembled into the phycobilisome rods. Thus, although phycocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaining phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy transfer to the core.

The cyanobacterium Synechococcus resists UV-B by exchanging photosystem II reaction-center D1 proteins

“…The polyclonal antibodies against D1:1 and D1:2 are completely form-specific and show no detectable cross-reactivity (27). The antibodies against D1, D2, and total phycobilisome polypeptides are described elsewhere (29,42).…”

“…5). The antibody recognizes all the polypeptides from pure Synechococcus phycobilisomes, including the linker polypeptides, but does not crossreact with any other proteins from the cells (42). The new polypeptide, therefore, apparently shares antigenic determinants with normal constitutive phycobilisome polypeptides.…”

Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli : Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

“…Enzymes called bilin lyases are responsible for catalyzing the attachment of a bilin to the appropriate Cys residue for most PBPs (20,46,49,52,53,72,73,78). The ␣-phycocyanin (CpcA) PCB lyase is a CpcE/CpcF heterodimer (8,19,20,58,78). Orthologs of genes encoding these proteins occur in the genomes of all cyanobacteria that synthesize phycocyanin (PC).…”

Characterization of Cyanobacterial Biliverdin Reductase

“…If PCB is bound much more strongly to apo-allophycocyanin subunits and other PCB-bearing core polypeptides than it is to the apophycocyanin subunits, then this would account for the observed results. In cpcEF mutants (mutants in phycocyanin ␣ subunit PCB lyase), apophycocyanin subunits are rapidly degraded (37,38,40).…”