Expression of a Higher Plant psbA Gene in Synechocystis 6803 Yields a Functional Hybrid Photosystem II Reaction Center Complex

Abstract

The psbA gene codes for the D1 polypeptide of the photosystem II reaction center complex and is found in all photosynthetic organisms that carry out oxygenic photosynthesis. Here we describe the construction and characterization of a strain of the cyanobacterium Synechocystis sp PCC 6803 in which the three endogenous psbA genes are replaced by a single psbA gene from the chloroplast genome of the higher plant Poa annua. The resulting chimeric strain, KWPAS, grows photoautotrophically with a doubling time of 26 hours compared with 20 hours for wild-type Synechocystis 6803. The mutant oxidizes water to oxygen at light-saturated rates comparable with wild type, despite differences in 15% of the primary structure of D1 between these species. RNA gel blot analysis indicates the presence in KWPAS of a psbA transcript of approximately 1.25 kilobases, consistent with the chloroplast promoter also acting as a promoter in Synechocystis. By using antibodies specific for the carboxyl-terminal extension of the D1 polypeptide of higher plants, we showed that the D1 polypeptide synthesized by KWPAS is post-translationally modified at the carboxyl terminus, probably through processing. A detailed biophysical analysis of the chimeric photosystem II complex indicated that the rates of forward electron transfer are similar to wild type. The rates of charge recombination between the donor and acceptor sides of the reaction center are, however, accelerated by as much as a factor of nine (QA- to S2) and are the most likely explanation for the lower rate of photoautotrophic growth in the mutant. We conclude that the psbA gene from a higher plant can be expressed in cyanobacteria and its product processed and assembled into a functional chimeric photosystem II reaction center.

Recognition signal for the C‐terminal processing protease of D1 precursor protein in the photosystem II reaction center An analysis using synthetic oligopeptides

“…bacterial cells [16], the D1 precursor protein of which contains largely different C-terminal extension compared to that of higher plants, further supports this interpretation. In order to test this possibility, the inhibitory effect of a polypeptide, corresponding to the Cterminal 9 amino acids (P-9) to the processing activity, using an in vitro translated D1 precursor protein, was examined in the experiment shown in Fig.…”

Engineering of an alternative electron transfer path in photosystem II

“…psbA1 potentially encodes a D1 protein that is different in three amino acids (D1′) and has been considered as a pseudogene (30). In the TD34 Syn strain, each of the three psbA genes has been replaced by antibiotic resistance cassettes (31). This enables the replacement of a single cassette with a wt or mutated copy of psbA, using heterologous recombination (Fig.…”

Inactivation of the Open Reading Frame slr0399 in Synechocystis sp. PCC 6803 Functionally Complements Mutations near the QA Niche of Photosystem II

“…In the case of mutants that are impaired in photosynthesis, pseudorevertants with improved photosynthetic function and with secondary mutations in genes coding for proteins with known function have been isolated and analyzed (for example, see Refs. [5][6][7][8][9][10][11]. These genes may be identical to the ones carrying the original mutations or may be at a different locus.…”