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 I 1 reaction center complex and is found in a11 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 posttranslationally modified at the carboxyl terminus, probably through processing. A detailed biophysical analysis of the chimeric photosystem I 1 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 S p ) 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 I 1 reaction center.

Cyanobacterial flv4-2 Operon-Encoded Proteins Optimize Light Harvesting and Charge Separation in Photosystem II

“…Previously, it was shown that a specific D1 copy (D1:2 form) encoded by the psbA3 gene in Synechococcus elongatus and Thermosynechococcus elongatus is responsible for a temperature downshift of the B and Q bands in TL curves (Sane et al, 2002;Sander et al, 2010;Sugiura et al, 2014). This functional difference is related to the presence of a glutamate residue instead of a glutamine at position 130 in the D1:2 protein sequence, which interacts with a key pheophytin cofactor (Nixon et al, 1991;Rappaport et al, 2002). Contrary to Synechococcus, the psbA3 gene of Synechocystis does not encode this specific D1:2 form, but both the psbA2 and psbA3 genes encode identical D1 proteins (for a review on D1 forms see Mulo et al, 2009;, which do not possess a Q130E mutation.…”

Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII‐H subunit

“…The choice of this substitution was suggested by the relative ease of targeting genes into a cyanobacterial genome compared with the nuclear or chloroplast genomes of plants, as required for the reverse combination. Hybrid PSIIs constructed in this way have already been used to study the function of essential components of the core, such as D1 and CP47 polypeptides [15,16].…”

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

“…This may suggest that the extension is not important in the binding of the substrate to the processing enzyme. The fact that the psbA gene product of a higher plant is processed by the enzyme in cyano- 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.…”