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

Abstract

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q A function, has an apparently mobile Q A , and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q A function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q A in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.The cyanobacterium Synechocystis sp. PCC 6803 is a useful molecular genetic system to study oxygenic photosynthesis and cell physiology of photosynthetic microorganisms. The organism is unique in that it combines several desirable properties: (i) Synechocystis sp. PCC 6803 is spontaneously transformable and incorporates exogenous DNA into its genome via doublehomologous recombination (1, 2); (ii) the strain can grow (photo)heterotropically, thus enabling the creation of Synechocystis sp. PCC 6803 strains impaired in photosystem I (PS I) 1 and/or photosystem II (PS II) function (for a recent review, see Ref.3); and (iii) the entire genome sequence of Synechocystis sp. PCC 6803 is known (4). Because of these properties, a variety of molecular genetic approaches have been applied to study the role of particular proteins in this organism.One of these approaches is the mapping and characterization of pseudorevertants, which carry second-site mutations restoring viability under conditions that are lethal for the original mutants. 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...

Discovery of a Chlorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

“…Ycf39 has previously been suggested to act as a chaperone helping to insert quinone into mutated Q A binding pockets of PSII (Ermakova-Gerdes and Vermaas, 1999). However, Ycf39 is not actually needed for Q A insertion into wild-type PSII (ErmakovaGerdes and Vermaas, 1999), so it seems more likely that binding of Ycf39 protein on the cytoplasmic surface of RCII might have indirect effects on the access/binding of quinone to the Q A binding pocket.…”

A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase

“…Ycf39 belongs to family of atypical short-chain alcohol dehydrogenase/reductases, which have an NAD(P)H binding motif near the N terminus but lack the canonical Tyr residue critical for activity of typical shortchain alcohol dehydrogenase/reductases (Kallberg et al, 2002). The function of Ycf39 in Synechocystis has not been established; deletion of the cognate slr0399 gene had no effect on cell viability, but it did complement mutations near to the Q A quinone acceptor of PSII (Ermakova-Gerdes and Vermaas, 1999). This observation led to speculation that Ycf39 is a chaperone-like protein involved in quinone insertion into the PSII complex (Ermakova-Gerdes and Vermaas, 1999).…”

“…The function of Ycf39 in Synechocystis has not been established; deletion of the cognate slr0399 gene had no effect on cell viability, but it did complement mutations near to the Q A quinone acceptor of PSII (Ermakova-Gerdes and Vermaas, 1999). This observation led to speculation that Ycf39 is a chaperone-like protein involved in quinone insertion into the PSII complex (Ermakova-Gerdes and Vermaas, 1999). Inactivation of the Arabidopsis Ycf39 homolog Hcf244 greatly decreased accumulation of PSII core proteins, and although the mechanism of Hcf244 action was not explained, it appears to be connected to the synthesis of the D1 protein (Link et al, 2012).…”

HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

“…The repair cycle of PSII requires the participation of hundreds of auxiliary proteins (Mulo et al, 2008;Nickelsen and Rengstl, 2013), and those in higher plants are different from those of more primitive photosynthetic organisms (Nixon et al, 2010). However, some PSII assembly and repair factors are conserved in cyanobacteria and higher plants, including HIGH CHLOROPHYLL FLUORESCENCE136 (Komenda et al, 2008), ALBINO3 (Luirink et al, 2001;Göhre et al, 2006), Psb27 (Nowaczyk et al, 2006;Komenda et al, 2012), Psb28 (Dobáková et al, 2009;Sakata et al, 2013), Psb29 (Keren et al, 2005), and YCF39 (Ermakova-Gerdes and Vermaas, 1999), as well as immunophilins, such as CYCLOPHILIN38 (CYP38)/ THYLAKOID LUMEN PEPTIDYLPROLYL ISOMERASE OF 40 kD (TLP40) . Presumably, therefore, the roles of these factors in PSII biogenesis or maintenance have been retained during evolution.…”