Abstract
The cyanobacterium Synechocystis sp. PCC 6803 has three copies of the psbA gene encoding the D1 reaction centre protein of Photosystem II (PS II). We have designed a mutagenesis system that allows introduction of mutations into the constitutively expressed psbA2 copy without affecting the expression of any flanking genes. A triple deletion strain was constructed in which psbA1 and psbA3 were removed by markerless deletions and psbA2 was replaced by a chloramphenicol-resistance cassette. A vector was then designed to enable the reintroduction of psbA2 in the chromosome using a kanamycin-resistance cassette as a selectable marker. This system was used to generate a control strain, which had an unmodified copy of psbA2, and two mutant strains which contained a copy of psbA2 where the codon for D1-Glu244 had been mutated to code for a His or Asp. The D1-Glu244 residue was targeted as it has been hypothesised to participate in a hydrogen-bond network that is required for protonation of the PS II secondary plastoquinone electron acceptor Q
B
. While the phenotype of the control strain was similar to wild type, the E244H mutant exhibited impaired oxygen evolution and altered electron transfer between the primary quinone acceptor Q
A
and Q
B
. However, substitution of Glu-244 by Asp resulted in a mutant more closely resembling the control strain.