In Vitro Random Mutagenesis of the D1 Protein of the Photosystem II Reaction Center Confers Phototolerance on the Cyanobacterium Synechocystis sp. PCC 6803 (original) (raw)
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Proceedings of the National Academy of Sciences, 1993
We have studied photoinhibition of photosynthesis in the cyanobacterium Synechococcus sp. PCC 7942, which possesses two distinct forms of the photosystem II reaction-center protein D1 (D1:1 and D1:2). We report here that when cells adapted to a growth irradiance of 50 mumol.m-2.s-1 are exposed to an irradiance of 500 mumol.m-2.s-1, the normally predominant D1 form (D1:1) is rapidly replaced with the alternative D1:2. This interchange is not only complete within the first hour of photoinhibition but is also fully reversible once cells are returned to 50 mumol.m-2 x s-1. By using a mutant that synthesizes only D1:1, we show that the failure to replace D1:1 with D1:2 during photoinhibition results in severe loss of photosynthetic activity as well as a diminished capacity to recover after the stress period. We believe that this interchange between D1 forms may constitute an active component in a protection mechanism unique among photosynthetic organisms that enables cyanobacteria to eff...
1998
Current ambient UV-B levels can significantly depress productivity in aquatic habitats, largely because UV-B inhibits several steps of photosynthesis, including the photooxidation of water catalyzed by photosystem II. We show that upon UV-B exposure the cyanobacterium Synechococcus sp. PCC 7942 rapidly changes the expression of a family of three psbA genes encoding photosystem II D1 proteins. In wild-type cells the psbAI gene is expressed constitutively, but strong accumulations of psbAII and psbAIII transcripts are induced within 15 min of moderate UV-B exposure (0.4 W͞m 2). This transcriptional response causes an exchange of two distinct photosystem II D1 proteins. D1:1 is encoded by psbAI, but on UV-B exposure, it is largely replaced by the alternate D1:2 form, encoded by both psbAII and psbAIII. The total content of D1 and other photosystem II reaction center protein, D2, remained unchanged throughout the UV exposure, as did the content and composition of the phycobilisome. Wild-type cells suffered only slight transient inhibition of photosystem II function under UV-B exposure. In marked contrast, under the same UV-B treatment, a mutant strain expressing only psbAI suffered severe (40%) and sustained inhibition of photosystem II function. Another mutant strain with constitutive expression of psbAII and psbAIII was almost completely resistant to the UV-B treatment, showing no inhibition of photosystem II function and only a slight drop in electron transport. In Synechococcus the rapid exchange of alternate D1 forms, therefore, accounts for much of the cellular resistance to UV-B inhibition of photosystem II activity and photosynthetic electron transport. This molecular plasticity may be an important element in community-level responses to UV-B, where susceptibility to UV-B inhibition of photosynthesis changes diurnally.
Plant Physiology, 1987
psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertion of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic Dl polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.
Physiologia Plantarum, 1991
of photosynthesis and growth responses at different light levels in psbA gene mutants of the cyanobacterium Synechococcus.-Physioi. Plant. 82: 1-8. Photoinhibition of photosynthesis and growth responses at different light levels (iO, 120 and 250 [imol m-s"') were studied in psbA gene mutants R2S2C3 {psbAI gene present) and R2K1 (psbAUipsbAlIl genes present) of the cyanobacterium Synechococcus sp. PCC 7942 {Anacystis nidulans R2). Mutant R2K1 (possessing form II of the D1 protein of photosystem II) was much more resistant to photoinhibition than the mutant R2S2C3 (possessing form 1 of the Dl protein). At moderate inhibitory light levels (100 to 300 [xmol m-s"') this was largely ascribed to an increased resistance of the photosystem II reaction centres possessing form II of the Dl protein. However, at higher light levels the higher resistance of mutant R2K1 was assigned to a higher rate of photosystem II repair, i.e. turnover of the Di protein. Moreover, our results support the hypothesis that photoinhibition of photosystem I] and photoinhibitory induced quenching are due to separate processes. Results from growth experiments show that the R2K1 mutant has a slower growth rate than the R2S2C3 mntant but shows an increased survival under high light stress conditions. It is hypothesized that high resistance to photoinhibition, though allowing a better survival under high light, is not advantageous for optimal growth.
The Journal of biological chemistry, 1989
The cyanobacterium Synechocystis sp. PCC 6803 carries out oxygenic photosynthesis analogous to higher plants. Its photosystem I contains seven different polypeptide subunits. The cartridge mutagenesis technique was used to inactivate the psaD gene which encodes subunit II of photosystem I. A mutant strain lacking subunit II was generated by transforming wild type cells with cloned DNA in which psaD gene was interrupted by a gene conferring kanamycin resistance. The photoautotrophic growth of mutant strain is much slower than that of wild type cells. The membranes prepared from mutant cells lack subunit II of photosystem I. Studies on the purified photosystem I reaction center revealed that the complex lacking subunit II is assembled and is functional in P700 photooxidation but at much reduced rate. Therefore, subunit II of photosystem I is required for efficient function of photosystem I.
Journal of Bacteriology, 2000
We have identified genes in the unicellular cyanobacteriumSynechocystis sp. strain PCC 6803 that are involved with redox control of photosynthesis and pigment-related genes. The genes,rppA (sll0797) and rppB (sll0798), represent a two-component regulatory system that controls the synthesis of photosystem II (PSII) and PSI genes, in addition to photopigment-related genes. rppA (regulator of photosynthesis- and photopigment-related gene expression) andrppB exhibit strong sequence similarity to prokaryotic response regulators and histidine kinases, respectively. In the wild type, the steady-state mRNA levels of PSII reaction center genes increased when the plastoquinone (PQ) pool was oxidized and decreased when the PQ pool was reduced, whereas transcription of the PSI reaction center genes was affected in an opposite fashion. Such results suggested that the redox poise of the PQ pool is critical for regulation of the photosystem reaction center genes. In ΔrppA, an insertion mutation of...
Photosynth Res, 1999
Photosystem I (PSI) interacts with plastocyanin or cytochrome c 6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c 6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/ N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c 6 or an artificial electron donor was used. In contrast, cytochrome c 6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c 6 . Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wildtype PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.
Photosynthesis Research, 1996
Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. DI:I predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only DI:I have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing Dl:2. We show that when dark-adapted or under low to moderate light, cells with DI:I have higher non-photochemical quenching of PS II fluorescence (higher qN) than do cells with Dl:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697-698 nm in cells containing DI:I than in cells with Dl:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing DI:I than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D 1:1 confers capacity for extreme State II which may be of benefit under low and variable light. Abbreviations: D 1-t h e atrazine-binding 32 kDa protein of the PS II reaction centre core; D 1:1-the D 1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942; D 1:2-an alternate form of the D1 protein induced under excess excitation in Synechococcus sp.