Gaza Salih

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition.

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and FV/FM was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of FV/FM after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.

[11] Use of Synechocystis 6803 to study expression of a psbA gene family

Publisher Summary This chapter discusses the use of Synechocystis 6803 to study expression of a psb A Gene family. The unicellular cyanobacterium Synechocystis 6803 offers an excellent model system for mutagenesis studies of the reaction center proteins in Photosystem II (PSII) and their genes for at least three reasons: (1) The reaction center in Synechocystis 6803 is very similar, both at the structural and the functional level, to that of higher plants; (2) the Synechocystis 6803 cells can be easily transformed and the added DNA is incorporated into the chromosome by homologous recombination; and (3) the cells can grow heterotrophically as well as photoautotrophically. Therefore, mutants that partially or completely lack PSII activity can be cultured and analyzed. Studies on regulation of psb A gene expression in Synechocystis 6803 display several interesting features. The psb A genes are differentially expressed in a light-dependent manner with approximately 95% of the transcripts being produced by psb A2 and the rest by psb A3. Inactivation of the highly expressed psb A2 leads to an eightfold up-regulation of psb A3.

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PPi) in three phototrophic bacteria. Formation of PPi was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PPi synthesis in Rps. viridis was 0.15 μmol PPi formed/(min*μmol Bacteriochlorophyll) at 23°C. The synthesis of PPi was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PPi synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PPi after short light flashes was also studied. The yield of PPi after a single 1 ms flash was equivalent to approximately 1 μmol PPi/500 μmol Bacteriochlorophyll. In Rps. viridis chromatophores, PPi was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.

Engineering of N-terminal threonines in the D1 protein impairs photosystem II energy transfer in Synechocystis 6803

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 with N‐terminal changes in the photosystem (PSII) II D1 protein were analysed by flash‐induced oxygen evolution, chlorophyll a fluorescence decay kinetics and 77 K fluorescence emission spectra. The data presented here show that mutations of the Thr‐2, Thr‐3 and Thr‐4 in D1 do not influence the oxygen evolution. A perturbation on the acceptor side was observed and the importance of the N‐terminal threonines for an efficient energy transfer between the phycobilisome and PSII and for stability of the PSII complex was demonstrated.

Constructed deletions in lumen-exposed regions of the D1 protein in the cyanobacterium Synechocystis 6803: Effects on D1 insertion and accumulation in the thylakoid membrane, and on Photosystem II assembly

Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in Δ(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Δ(V58-D61) or Δ(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Δ(P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the β subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.

Site-specific mutations of the N-terminal threonines in the D1 protein affects photoautotrophic growth but not D1 protein stability in Synechocystis 6803

The cyanobacterium Synechocystis 6803 was engineered to produce a D1 protein where one or more of the N-terminal threonines at positions 2, 3 and 4 were replaced by other amino acid residues. No phenotypic effects were found for the T2S or T2L mutations, whereas the T2V, T2L;T4V and T2V;T3V;T4V mutations resulted in reduced photoautotrophic growth rate and oxygen evolving activity. The mutant strain T2V;T3V;T4V exhibited an oxygen evolution activity that was only half of that for the wild-type strain. Despite of that, both accumulation and stability of the D1 protein in the thylakoid membrane appeared unaffected in the mutant.

Determination of the Intracellular Concentration of Inorganic Pyrophosphate in Rhodospirillum rubrum

Rhodospirillum rubrum is a purple, non-sulphur phototrophic bacterium. This organism is capable of catalyzing the synthesis of inorganic pyrophosphate (PPi) upon illumination. The phosphorylation of Pi to PPi is catalyzed by the membrane bound proton translocating inorganic pyrophosphatase (the H+-PPase) (1). PPi is also formed during the biosynthesis of cellular constituents such as proteins and nucleic acids. Beside the H+-PPase there is also a soluble PPase in R. rubrum (soluble PPases are found in all types of cells). The soluble enzyme only catalyze the hydrolysis of PPi not the synthesis, When PPi is hydrolyzed by the soluble PPase, the energy liberated upon hydrolysis of the anhydride bond is lost.

Light-Driven Inorganic Pyrophosphate Synthesis in Phototrophic Bacteria

It is well known that inorganic pyrophosphate (PPi) is formed during biosynthesis of cellular constituents such as proteins, polysaccharides, nucleic acids and lipids. However, PPi can also be formed by oxidative phosphorylation in mitochondria from different sources and by photophosphorylation in some phototrophs, such as Rhodospirillum rubrum.