Strain of Synechocystis PCC 6803 with Aberrant Assembly of Photosystem II Contains Tandem Duplication of a Large Chromosomal Region

Abstract

Cyanobacterium Synechocystis PCC 6803 represents a favored model organism for photosynthetic studies. Its easy transformability allowed construction of a vast number of Synechocystis mutants including many photosynthetically incompetent ones. However, it became clear that there is already a spectrum of Synechocystis “wild-type” substrains with apparently different phenotypes. Here, we analyzed organization of photosynthetic membrane complexes in a standard motile Pasteur collection strain termed PCC and two non-motile glucose-tolerant substrains (named here GT-P and GT-W) previously used as genetic backgrounds for construction of many photosynthetic site directed mutants. Although, both the GT-P and GT-W strains were derived from the same strain constructed and described by Williams in 1988, only GT-P was similar in pigmentation and in the compositions of Photosystem II (PSII) and Photosystem I (PSI) complexes to PCC. In contrast, GT-W contained much more carotenoids but significantly less chlorophyll (Chl), which was reflected by lower level of dimeric PSII and especially trimeric PSI. We found that GT-W was deficient in Chl biosynthesis and contained unusually high level of unassembled D1-D2 reaction center, CP47 and especially CP43. Another specific feature of GT-W was a several fold increase in the level of the Ycf39-Hlip complex previously postulated to participate in the recycling of Chl molecules. Genome re-sequencing revealed that the phenotype of GT-W is related to the tandem duplication of a large region of the chromosome that contains 100 genes including ones encoding D1, Psb28, and other PSII-related proteins as well as Mg-protoporphyrin methylester cyclase (Cycl). Interestingly, the duplication was completely eliminated after keeping GT-W cells on agar plates under photoautotrophic conditions for several months. The GT-W strain without a duplication showed no obvious defects in PSII assembly and resembled the GT-P substrain. Although, we do not exactly know how the duplication affected the GT-W phenotype, we hypothesize that changed stoichiometry of protein components of PSII and Chl biosynthetic machinery encoded by the duplicated region impaired proper assembly and functioning of these multi-subunit complexes. The study also emphasizes the crucial importance of a proper control strain for evaluating Synechocystis mutants.

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly

“…The Synechocystis sp PCC 6803 glucose-tolerant substrain GT-P was used as the wild type (Tichý et al, 2016). The DrubA strain was constructed by replacing the slr2033 gene (36-339 bp) by a zeocin antibiotic resistance cassette.…”

Association of Psb28 and Psb27 Proteins with PSII-PSI Supercomplexes upon Exposure of Synechocystis sp. PCC 6803 to High Light

“…Since this disagreement could be caused by differences in the genetic backgrounds of the two WT strains used to make the mutants, we constructed two new Psb28-1-and Psb28-2-less mutants using a different WT strain. In our original study (Dobá ková et al, 2009), we used a WT strain very similar to the glucose-tolerant GT-O2 strain (Morris et al, 2014), which was subsequently shown to exhibit partial Chl depletion upon autotrophic growth conditions (GT-W; Tichý et al, 2016). The new set of mutants was instead constructed in the GT-P strain similar to the GT-Kazusa strain (Supplemental Figure 6), which contains a similar content of Chl as the standard motile PCC 6803 strain (Tichý et al, 2016).…”

“…In our original study (Dobá ková et al, 2009), we used a WT strain very similar to the glucose-tolerant GT-O2 strain (Morris et al, 2014), which was subsequently shown to exhibit partial Chl depletion upon autotrophic growth conditions (GT-W; Tichý et al, 2016). The new set of mutants was instead constructed in the GT-P strain similar to the GT-Kazusa strain (Supplemental Figure 6), which contains a similar content of Chl as the standard motile PCC 6803 strain (Tichý et al, 2016). The resulting psb28 mutants were found to have a similar cellular Chl content as the control and contained very similar levels of PSI and both monomeric and dimeric PSII core complexes as revealed by CN gel electrophoresis ( Figure 6) in agreement with Sakata et al (2013).…”

“…The non-motile, glucose-tolerant strain of Synechocystis sp. PCC 6803 GT-P (Tichý et al, 2016) was used in this study as a WT strain and as a background for the generated deletion mutant strains (DPsb28-1, DPsb28-2, DPsb28-1/DPsb28-2, and DPsb27/DPsb28-1) and FLAGtagged mutants (Psb28-1-FLAG, FLAG-Psb28-1, Psb28-2-FLAG, and FLAG-Psb28-2).…”

An Improved Natural Transformation Protocol for the Cyanobacterium Synechocystis sp. PCC 6803

“…Synechocystis sp. PCC 6803 (hereafter Synechocystis) is widely used as a model cyanobacterium for fundamental and applied research because it is naturally transformable (Grigorieva and Shestakov, 1982;Kufryk et al, 2002), can grow heterotrophically on glucose (Anderson and McIntosh, 1991), and has a publicly available genome sequence (Ikeuchi, 1996;Morris et al, 2014;Tichý et al, 2016). Exogenous DNA molecules introduced into Synechocystis via natural transformation may replicate independently on a suitable plasmid (Mermet-Bouvier et al, 1993) or be incorporated into the genome via double homologous recombination (Grigorieva and Shestakov, 1982;Shestakov et al, 1985).…”