Targeted random mutagenesis to identify functionally important residues in the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803

Abstract

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q(A)-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane alpha-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.

FIG. 1

Representation of the wild-type sequence of the region of D2 that had been exposed to targeted random mutagenesis (top) and of the sequences in the various mutants (below). An asterisk above the wild-type sequence indicates a residue that has been mutated in one of the mutants indicated here. Minus signs above residues of the wild-type sequence indicate that these residues could not be changed by the method employed, and arrows delineate the region corresponding to the part of psbDI that was single stranded during the targeted random-mutagenesis experiment. Two residues that closely interact with Q_A or nonheme iron are underlined. These are Trp253 (presumed to be located between pheophytin and Q_A) and His268 (a putative ligand to nonheme iron). Residues the mutation of which appears to give rise to impaired PSII activity are in boldface. An asterisk in one of the mutant sequences indicates a stop codon. The four deletion (del.) plasmids that were used for functional complementation (see text) are indicated at the bottom.

FIG. 2

Schematic representation of recombination events that are likely to have taken place to generate the G285S mutant as a result of transformation of the D2R4 strain with DNA from D2R12. The sequence at residues 288 and 310 is the wild-type sequence in strain D2R4, but the mutations present at residues 295, 302, and 308 in strain D2R4 were not retained. The solid horizontal line indicates the strain from which a particular region seems to originate in the G285S mutant, and crosses indicate the approximate locations where crossovers appear to have taken place. Note that a fourth crossover occurred at a position before codon 260.

FIG. 3

Variable fluorescence yield recorded as a function of time after illumination in the absence (A) and presence (B) of 10 μM DCMU in intact cells of the S254F mutant (crosses) and the control (squares). The difference in signal-to-noise ratio between the two graphs is due to the fact that the measurements in the absence of DCMU were done with PSI-less strains and the measurements in the presence of DCMU were done with PSI-containing strains. The choice of background strain did not impact the decay kinetics of variable fluorescence.

FIG. 4

Inhibition of oxygen evolution in intact cells upon addition of artificial quinones. (A) Effects of DCBQ (closed symbols) and DMBQ (open symbols) on oxygen evolution in intact wild-type (triangles), S254F (squares), and G258D (circles) cells. (B) Inhibition of oxygen evolution in wild-type (triangles), S254F (squares), and G258D (circles) cells by different concentrations of DQ. No exogenous electron acceptors were added, except for 0.5 mM K₃Fe(CN)₆, which does not penetrate the cells and which was present to keep the quinones oxidized.