Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I is one of the two multisubunit pigment-protein complexes in the thylakoid membranes of cyanobacteria. Subunit III of photosystem I complex was isolated from a mutant of the cyanonbacterium Synechocystis sp PCC 6803, which lacks subunit II. The sequence of its NH2-terminal residues was determined and corresponding oligonucleotide probes were used to isolate the gene encoding this subunit. The gene, designated as psaF, codes for a mature protein of 15705 Da that is synthesized with a 23-amino acid extension. The deduced amino acid sequence is homologous to subunit III from spinach and Chlamydomonas reinhardtii. The presequence of subunit III shows characteristics typical of bacterial presequences and exhibits remarkable amino acid identity around the proteolytic processing site when compared to corresponding regions from the precursors of eukaryotic subunit III. There are two conserved hydrophobic regions in the mature subunit III which may cross or interact with thylakoid membrane. The gene psaF exists as a single copy in the genome and is expressed as a monocistronic RNA. A stable mutant strain in which the gene psaF was replaced by a gene conferring resistance to kanamycin was generated by targeted mutagenesis. Photoautotrophic growth of the mutant strain was comparable with that of the wild type suggesting that function of subunit III is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Addition of more MgSO4 to BG11 medium enhanced growth of the mutant strain but not of the wild type cells.     

Targeted deletion of psaJ from the cyanobacterium Synechocystis sp. PCC 6803 indicates structural interactions between the PsaJ and PsaF subunits of photosystem I

Photosystem I catalyzes the light-driven oxidation of plastocyanin or cytochrome c ₆ and the reduction of ferredoxin or flavodoxin. PsaJ is a 4.4 kDa hydrophobic subunit of photosystem I from cyanobacteria and chloroplasts. To investigate the function of PsaJ, we generated a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 in which the psaJ gene is replaced by a gene for chloramphenicol resistance. Deletion of psaJ led to a reduction in the steady state RNA level from psaF which is located upstream from psaJ. Immunoquantification using an anti-PsaF antibody revealed a significant decrease in the amount of PsaF in membranes of the mutant strain. Trimeric photosystem I complexes isolated from the mutant strain using n-dodecyl -D-maltoside lacked PsaJ, contained ca. 80% less PsaF, but maintained wild-type levels of other photosystem I subunits. In contrast, the photosystem I purified using Triton X-100 contained less than 2% PsaF when compared to the wild type, showing the more extractable nature of PsaF in PsaJ-less photosystem I in the presence of Triton X-100. PsaE was more accessible to removal by NaI in a mutant strain lacking PsaF and PsaJ than in the wild type. The presence of PsaF in photosystem I from the PsaJ-less strain did not alter the increased susceptibility of PsaE to removal by NaI. These results indicate an interaction between PsaJ and PsaF in the organization of the complex.     

PsaK2 subunit in photosystem I is involved in state transition under high light condition in the cyanobacterium Synechocystis sp. PCC 6803

To avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II or to photosystem I (PSI) upon high light acclimation by the process so-called state transition. We found that an alternative PSI subunit, PsaK2 (sll0629 gene product), is involved in this process in the cyanobacterium Synechocystis sp. PCC 6803. An examination of the subunit composition of the purified PSI reaction center complexes revealed that PsaK2 subunit was absent in the PSI complexes under low light condition, but was incorporated into the complexes during acclimation to high light. The growth of the psaK2 mutant on solid medium was inhibited under high light condition. We determined the photosynthetic characteristics of the wild type strain and the two mutants, the psaK1 (ssr0390) mutant and the psaK2 mutant, using pulse amplitude modulation fluorometer. Non-photochemical quenching, which reflects the energy transfer from phycobilisome to PSI in cyanobacteria, was higher in high light grown cells than in low light grown cells, both in the wild type and the psaK1 mutant. However, this change of non-photochemical quenching during acclimation to high light was not observed in the psaK2 mutant. Thus, PsaK2 subunit is involved in the energy transfer from phycobilisome to PSI under high light condition. The role of PsaK2 in state transition under high light condition was also confirmed by chlorophyll fluorescence emission spectra determined at 77 K. The results suggest that PsaK2-dependent state transition is essential for the growth of this cyanobacterium under high light condition.     

PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions

A PsbQ homologue has been found associated with photosystem II complexes in Synechocystis sp. PCC 6803 where it is involved in optimal photoautotrophic growth and water splitting under CaCl(2)-depleted conditions [Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175]. By inactivating psbQ in strains carrying photosystem II-specific mutations, we have identified stringent requirements for PsbQ in vivo. Whereas under nutrient-replete conditions the DeltaPsbQ mutant was similar to wild type, a strain lacking PsbQ and PsbV was not photoautotrophic, exhibiting decreased oxygen evolution and decreased photosystem II assembly compared to the DeltaPsbV mutant. Combining the removal of PsbU and PsbQ introduced an altered requirement for Ca(2+) and Cl(-), and photoautotrophic growth of the DeltaPsbQ strain was prevented in nutrient-limiting media depleted in Ca(2+), Cl(-), and iron. Unlike other photosystem II extrinsic proteins PsbQ did not participate in the acquisition of thermotolerance; however, photoautotrophic growth at elevated temperatures was impaired in this mutant. Growth of the DeltaPsbV:DeltaPsbQ mutant was restored at pH 10.0: in contrast, an additional deletion between Arg-384 and Val-392 in the CP47 protein of photosystem II prevented recovery at alkaline pH. When conditions prevented photoautotrophy in strains lacking PsbQ, photoheterotrophic growth was indistinguishable to wild type, indicating that photosystem II had been inactivated. These data substantiate a role for PsbQ in optimizing photosystem II activity in Synechocystis sp. PCC 6803 and establish an absolute requirement for the subunit under specific biochemical and physiological conditions.     

A regulatory role of the PetM subunit in a cyanobacterial cytochrome b6f complex

To investigate the function of the PetM subunit of the cytochrome b6f complex, the petM gene encoding this subunit was inactivated by insertional mutagenesis in the cyanobacterium Synechocystis PCC 6803. Complete segregation of the mutant reveals a nonessential function of PetM for the structure and function of the cytochrome b6f complex in this organism. Photosystem I, photosystem II, and the cytochrome b6f complex still function normally in the petM- mutant as judged by cytochrome f re-reduction and oxygen evolution rates. In contrast to the wild type, however, the content of phycobilisomes and photosystem I as determined from 77 K fluorescence spectra is reduced in the petM- strain. Furthermore, whereas under anaerobic conditions the kinetics of cytochrome f re-reduction are identical, under aerobic conditions these kinetics are slower in the petM- strain. Fluorescence induction measurements indicate that this is due to an increased plastoquinol oxidase activity in the mutant, causing the plastoquinone pool to be in a more oxidized state under aerobic dark conditions. The finding that the activity of the cytochrome b6f complex itself is unchanged, whereas the stoichiometry of other protein complexes has altered, suggests an involvement of the PetM subunit in regulatory processes mediated by the cytochrome b6f complex.     

Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I reaction center of the cyanobacterium Synechocystis sp. PCC 6803 contains seven different polypeptide subunits. The subunit with a molecular mass of about 8 kDa was isolated, and the sequence of its amino-terminal residues was determined. Oligonucleotide probes corresponding to this sequence were used to isolate the gene encoding this subunit. The gene, termed as psaE, codes for a polypeptide with a mass of 8075 Da. It is present as a single copy in the genome and is transcribed as a monocistronic messenger. The amino acid sequence of the 8-kDa subunit deduced from the gene sequence shows high homology with the deduced amino acid sequence of subunit IV of photosystem I from spinach. The DNA fragment sequenced in these studies also contains two other unidentified major open reading frames. A stable deletion mutation for the psaE gene was generated by transforming Synechocystis sp. PCC 6803 with a cloned DNA in which the psaE gene for 8-kDa subunit was replaced by a gene conferring resistance to kanamycin. The mutant strain shows minor differences in growth under photoautotrophic conditions and in the photosystem I activity in comparison to the wild type.     

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.     

Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.     

Inactivation of ycf33 results in an altered cyclic electron transport pathway around photosystem I in Synechocystis sp. PCC6803

ycf33 encodes a small protein with a molecular mass of 7.5 kDa and is found from cyanobacteria to higher plants. A ycf33 deletion mutant was constructed in Synechocystis sp. PCC6803 and characterized. The mutant showed a higher phycobilisome/chlorophyll ratio than the wild type and a higher photosystem II/photosystem I fluorescence ratio measured at 77 K. Under photoautotrophic conditions, the growth rates were not much different from those of the wild type. Cyclic electron transport activities around photosystem I were not much different between the wild type and the mutant. However, the effects of diphenyleneiodonium, an inhibitor of flavoprotein, on cyclic electron transport in the mutant were different from those in the wild type; it was severely inhibited in the wild type but not much in the mutant. Together with the effects of nitrite, which accepts electrons from ferredoxin via nitrite reductase and those of HgCl2, it was suggested that the pathway of cyclic electron transport is altered in the mutant.     

Molecular cloning and sequencing of the psaD gene encoding subunit II of photosystem I from the cyanobacterium, Synechocystis sp. PCC 6803

Photosystem I reaction center was isolated from the cyanobacterium, Synechocystis sp. PCC 6803, in a form which contains seven different polypeptide subunits. One of the subunits, with a molecular mass of about 16 kDa, was isolated, and protein sequence information was obtained for the amino terminus and several tryptic peptides. Oligonucleotide probes, corresponding to these sequences, were used to probe a genomic library, and the gene, designated psaD, encoding subunit II was cloned and sequenced. The gene encodes a polypeptide with a mass 15,644 Da, which exhibits a high degree of similarity to subunit II from tomato, as well as amino acid sequences reported from barley photosystem I. In addition to this gene, three large open reading frames were identified. Two remain unidentified, and the third is highly homologous to anthranilate synthase, component 1 from Escherichia coli and Saccharomyces cerevisiae.     

Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.     

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.     

Kinetic Behavior of Electron Paramagnetic Resonance Signal I. II Comparison of Wild Type and Mutant (ac-206) Chlamydomonas reinhardtii

The electron paramagnetic resonance (EPR) characteristics of wild type Chlamydomonas reinhardtii are compared with those of a mutant strain (ac-206) which lacks cytochrome 553. The steady-state signals I and II are similar but differ in their responses to light of long and short wavelengths, reflecting the fact that the electron transport chain linking photosystems I and II is interrupted. The kinetic behavior of signal I is simpler in the mutant, which lacks induction effects prominent in the wild type. The decay of the signal when light ceases is not dependent on the length or intensity of illumination in the mutant, whereas it is in the wild type. These data can be interpreted in terms of signal I being a reflection of cyclic flow in a pathway which does not involve cytochrome 553 in the mutant, whereas in the wild type there is also a contribution of electrons from photosystem II.     

Arabidopsis mutants deleted in the light-harvesting protein Lhcb4 have a disrupted photosystem II macrostructure and are defective in photoprotection

The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C₂S₂ particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。