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Synechococcus sp. PCC 7002 was grown to steady state in optically thin turbidostat cultures under conditions for which light quantity and quality was systematically varied by modulating the output of narrow-band LEDs. Cells were provided photons absorbed primarily by chlorophyll (680 nm) or phycocyanin (630 nm) as the organism was subjected to four distinct mono- and dichromatic regimes. During cultivation with dichromatic light, growth rates were generally proportional to the total incident irradiance at values <275μmol photons m-2.s-1 and were not affected by the ratio of 630:680nm wavelengths. Notably, under monochromatic light conditions, cultures exhibited similar growth rates only when they were irradiated with 630nm light; cultures irradiated with only 680nm light grew at rates that were 60-70% of those under other light quality regimes at equivalent irradiances. The functionality of photosystem II and associated processes such as maximum rate of photosynthetic electron transport, rate of cyclic electron flow, and rate of dark respiration generally increased as a function of growth rate. Nonetheless, some of the photophysiological parameters measured here displayed distinct patterns with respect to growth rate of cultures adapted to a single wavelength including phycobiliprotein content, which increased under severely light-limited growth conditions. Additionally, the ratio of photosystem II to photosystem I increased ∼40% over the range of growth rates, although cells grown with 680nm light only had the highest ratios. These results suggest the presence of effective mechanisms which allow acclimation of Synechococcus sp. PCC 7002 acclimation to different irradiance conditions. [ABSTRACT FROM AUTHOR]

The biogenesis of thylakoid membranes in cyanobacteria is presently not well understood, but the vipp1 gene product has been suggested to play an important role in this process. Previous studies in Synechocystis sp. PCC 6803 reported that vipp1 (sll0617) was essential. By constructing a fully segregated null mutant in vipp1 (SynPCC7002_A0294) in Synechococcus sp. PCC 7002, we show that Vipp1 is not essential. Spectroscopic studies revealed that Photosystem I (PS I) was below detection limits in the vipp1 mutant, but Photosystem II (PS II) was still assembled and was active. Thylakoid membranes were still observed in vipp1 mutant cells and resembled those in a psaAB mutant that completely lacks PS I. When the vipp1 mutation was complemented with the orthologous vipp1 gene from Synechocystis sp. PCC 6803 that was expressed from the strong PcpcBA promoter, PS I content and activities were restored to normal levels, and cells again produced thylakoids that were indistinguishable from those of wild type. Transcription profiling showed that psaAB transcripts were lower in abundance in the vipp1 mutant. However, when the yfp gene was expressed from the PpsaAB promoter in the presence and the absence of Vipp1, no difference in YFP expression was observed, which shows that Vipp1 is not a transcription factor for the psaAB genes. This study shows that thylakoids are still produced in the absence of Vipp1 and that normal thylakoid biogenesis in Synechococcus sp. PCC 7002 requires expression and biogenesis of PS I, which in turn requires Vipp1. [ABSTRACT FROM AUTHOR]

Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved {gamma}-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of phycobiliproteins are discussed. [ABSTRACT FROM AUTHOR]

The sufR gene encodes a protein that functions as a transcriptional repressor of the suf regulon in cyanobacteria. It is predicted to contain an N-terminal helix loop helix DNA binding motif and a C-terminal Fe/S binding domain. Through immunoblotting assays of cell extracts, the sufR product in Synechocystis sp. PCC 6803 was shown to have a mass of ~25 kDa. This indicates that the second ATG in the open reading frame is the correct start codon and that sufR encodes a protein of 216 amino acids (SufR216) rather than the originally predicted 240 amino acids. Recombinant SufR216 harbored [4Fe-4S]2+,1+ clusters, which were present in a mixture of S = ½ and ⅜ ground spin states, and the holoprotein was a homodimer, containing 3.7 of non-heme irons and 3.5 labile sulfides per monomer. Thus, two [4Fe-4S]2+,1+ clusters are coordinated by each SufR216 homodimer. SufR216 bound to two DNA sequences in the regulatory region between the divergently transcribed sufR gene and the sufBCDS operon, and its binding affinity depended on the presence and redox state of the [4Fe-4S]2+,1+ clusters. A high affinity binding site, which controls sufRCDS expression, and a low affinity binding site, which controls sufR expression, were identified. The SufR binding sites, which are separated by 26 base pairs, each contain a perfect inverted repeat, CAAC-N6-GTTG, and are highly conserved in cyanobacteria. The Fe/S protein SufR thus functions both as a transcriptional repressor of the sufBCDS operon and as an autoregulator of sufR. [ABSTRACT FROM AUTHOR]

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpcT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin β-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin β-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin β-subunit. [ABSTRACT FROM AUTHOR]

Cyanobacteria have versatile electron transfer pathways and many of the proteins involved are functional in both respiratory and photosynthetic electron transport. Examples of such proteins include the cytochrome b6 f complex, NADH dehydrogenase and cytochrome oxidase complexes. In this study we have cloned and sequenced two gene clusters from the marine cyanobacterium Synechococcus sp. PCC 7002 that potentially encode heme-copper cytochrome oxidases. The ctaCIDIEI and ctaCIIDIIEII gene clusters are most similar to two related gene clusters found in the freshwater cyanobacterial strain Synechocystis sp. PCC 6803. Unlike Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002 does not have a cydAB-like gene cluster which encodes a quinol oxidase. The ctaCIDIEI and ctaCIIDIIEII gene clusters were transcribed polycistronically, although the levels of transcripts for the ctaCIIDIIEII gene cluster were lower than those of the ctaCIDIEI gene cluster. The ctaDI and ctaDII coding sequences were interrupted by interposon mutagenesis and full segregants were isolated and characterized for both single and double mutants. Growth rates, chlorophyll and carotenoid contents, oxygen consumption and oxygen evolution were examined in the wild type and mutant strains. Differences between the wild type and mutant strains observed in 77 K fluorescence spectra and in pulse-amplified modulated (PAM) fluorescence studies suggest that the cyanobacterial oxidases play a role in photoinhibition and high light tolerance in Synechococcus sp. PCC 7002. [ABSTRACT FROM AUTHOR]

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center. [ABSTRACT FROM AUTHOR]

In previous work, some members of our group isolated mutant strains of Synechocystis sp. strain PCC 6803 in which point mutations had been inserted into the psaC gene to alter the cysteine residues to the F[sub A] and F[sub B] iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem. 272:8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll a and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 µmol m[sup -2] s[sup -1]). Two separate suppressor strains of C14S[sub PsaC], termed C14S[sub PsaC]-R62 and C14S[sub Psac]-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp. strain PCC 6803 genome named sll0088. C14S[sub PsaC]-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482→C change in sll0088, and C14S[sub PsaC]-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in sil0088. These suppressor strains showed near-wild-type levels of chlorophyll a and photosystem I, yet the serine oxygen ligand to F[sub B] was retained as shown by the retention of the S ≥ 3/2 spin state of the [4Fe-4S] cluster. The inactivation of sll0088 by insertion of a kanamycin resistance cartridge in the primary C14S[sub PsaC] mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in psaC gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an sll0088 deletion mutant. The sll0088 gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms,... [ABSTRACT FROM AUTHOR]

Examines the solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009. Comparison between PsaE from the Nostoc sp. strain and Synechococcus sp. strain PCC 7002; Thermodynamic stability of Nostoc sp. strain; Determination of second representative structure to anticipate protein folding and explore interaction.

A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of Fx, FB, and FA was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and tubA genes were insertionally inactivated. In isolated PSI trimers, the decay of P700+ measured in the near-IR and the decay of A1- measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 ps. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9-. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the sP700 product of P700+A0- charge recombination via the TO spin level in those PSI complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q- polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the As binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of Fx and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the AS site makes the menB tubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I. [ABSTRACT FROM AUTHOR]

Cyanobacteria are ubiquitous in nature and have developed numerous strategies that allow them to live in a diverse range of environments. Certain cyanobacteria synthesize chlorophylls d and f to acclimate to niches enriched in far-red light (FRL) and incorporate paralogous photosynthetic proteins into their photosynthetic apparatus in a process called FRL-induced photoacclimation (FaRLiP). We characterized the macromolecular changes involved in FRL-driven photosynthesis and used atomic force microscopy to examine the supramolecular organization of photosystem I associated with FaRLiP in three cyanobacterial species. Mass spectrometry showed the changes in the proteome of Chroococcidiopsis thermalis PCC 7203 that accompany FaRLiP. Fluorescence lifetime imaging microscopy and electron microscopy reveal an altered cellular distribution of photosystem complexes and illustrate the cell-to-cell variability of the FaRLiP response. [ABSTRACT FROM AUTHOR]

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, Mössbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-FX core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [FA/FB]- to P700+ and lightinduced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro. [ABSTRACT FROM AUTHOR]

All phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their β-subunits. Methylation of this Asn to produce γ-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-³H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the β-subunits of phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding a-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo. [ABSTRACT FROM AUTHOR]