scholarly journals Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803 by Combinatorial Mutagenesis

2000 ◽  
Vol 182 (9) ◽  
pp. 2453-2460 ◽  
Author(s):  
Anna T. Keilty ◽  
Svetlana Y. Ermakova-Gerdes ◽  
Wim F. J. Vermaas
Keyword(s):  
Photosystem Ii ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Functional Requirements ◽  
Coding Region ◽  
Functional Roles ◽  
Loop Region ◽  
Combinatorial Mutagenesis ◽  
D2 Protein ◽  
Pcc 6803

ABSTRACT The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 inSynechocystis sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.

scholarly journals Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803

2001 ◽  
Vol 183 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Svetlana Ermakova-Gerdes ◽  
Zhenbao Yu ◽  
Wim Vermaas
Keyword(s):  
Photosystem Ii ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Terminal Part ◽  
Photoautotrophic Growth ◽  
Intragenic Complementation ◽  
Novel Method ◽  
Α Helix ◽  
D2 Protein ◽  
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 Synechocystissp. 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 QA-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-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 Synechocystissp. strain PCC 6803 was developed to facilitate genetic analysis ofpsbDI 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.

scholarly journals A reversibly induced CRISPRi system targeting Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

2020 ◽  
Author(s):  
Deng Liu ◽  
Virginia M. Johnson ◽  
Himadri B. Pakrasi
Keyword(s):  
Photosystem Ii ◽  
Sole Carbon Source ◽  
Model Organism ◽  
Inducible Promoter ◽  
New Strategy ◽  
Photosynthetic Complexes ◽  
Promoter System ◽  
Synechocystis Sp ◽  
D2 Protein ◽  
Pcc 6803

ABSTRACTThe cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited Photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased Photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.

scholarly journals Photosystem II antenna modules CP43 and CP47 do not form a stable ‘no reaction centre complex’ in the cyanobacterium Synechocystis sp. PCC 6803

2022 ◽  
Author(s):  
Martina Bečková ◽  
Roman Sobotka ◽  
Josef Komenda
Keyword(s):  
Photosystem Ii ◽  
High Light ◽  
Oxygenic Photosynthesis ◽  
Reaction Centre ◽  
Wild Type ◽  
Migration Patterns ◽  
Separate Protein ◽  
Antenna Module ◽  
D2 Protein ◽  
Pcc 6803

AbstractThe repair of photosystem II is a key mechanism that keeps the light reactions of oxygenic photosynthesis functional. During this process, the PSII central subunit D1 is replaced with a newly synthesized copy while the neighbouring CP43 antenna with adjacent small subunits (CP43 module) is transiently detached. When the D2 protein is also damaged, it is degraded together with D1 leaving both the CP43 module and the second PSII antenna module CP47 unassembled. In the cyanobacterium Synechocystis sp. PCC 6803, the released CP43 and CP47 modules have been recently suggested to form a so-called no reaction centre complex (NRC). However, the data supporting the presence of NRC can also be interpreted as a co-migration of CP43 and CP47 modules during electrophoresis and ultracentrifugation without forming a mutual complex. To address the existence of NRC, we analysed Synechocystis PSII mutants accumulating one or both unassembled antenna modules as well as Synechocystis wild-type cells stressed with high light. The obtained results were not compatible with the existence of a stable NRC since each unassembled module was present as a separate protein complex with a mutually similar electrophoretic mobility regardless of the presence of the second module. The non-existence of NRC was further supported by isolation of the His-tagged CP43 and CP47 modules from strains lacking either D1 or D2 and their migration patterns on native gels.

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