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A-C Construction of integrative vectors for insertional inactivation of the psaK, psaF, and psa¸genespsa¸genes in Syn. elongatus. For DNA sequences of the PSI genes see Mu¨hlenhoffMu¨hlenhoff et al. (1993). Antibiotic marker genes were obtaned from pRL161 (Km; Elhai and Wolk 1989), pBR325 (Cm), and pHP45 (Sm/Sp; Prentki and Krisch 1984). Restriction sites in parentheses were lost during construction. For cloning, the DNA fragments were inserted into the E. coli vectors pUC18 (psaF and psa¸) or pBSCM13> (psaK). Approximate fragment sizes are indicated by the bars
Source publication
DNA can be introduced into the thermophilic cyanobacteriumSynechococcus elongatus by electroporation or conjugation. Its genome can be readily manipulated through integrative transformation or by using promiscuous RSF1010-derived plasmids that can be transferred unaltered betweenEscherichia coli andSynechococcus elongatus. These vectors can therefo...
Context in source publication
Context 1
... were expected to be interrupted in the corresponding transformants FK1, KK2, LC1 and LS1 (Fig. 3). In case of the psaK locus, the 12-kb XbaI restriction fragment carrying the wild-type psaK gene is split into two fragments of 10 kb and 1.9 kb in DNA of strain KK2, where this gene has been interrupted by a Km cassette flanked by two XbaI sites (Figs. 1A, 2A). In wild-type DNA the psaF gene lies on a 1.2 kb NcoI restriction fragment, the second NcoI site being located 145 bp upstream of the ClaI site indicated in Fig. 1B. This fragment is replaced by two NcoI fragments of 1.7 kb and 0.9 kb in strain In strain LC1 the 1.3-kb EcoRI fragment harbouring the wild-type psaL gene is replaced by ...
Citations
... Standard methods were used for the isolation and cultivation of cyanobacteria. Samples were spread on plates (1.5% wt/vol Purified Agar) with different cyanobacterial media: BG11 (Rippka et al., 1979), Spirulina medium (hereafter UTEX, Starr and Zeikus, 1987), Castenholz-D medium (Mühlenhoff and Chauvat, 1996), and MDM (Watanabe, 1960;Supplementary Table S1). BG11 and MDM were also used without nitrogen combined sources (BG11 0 and MDM 0 , respectively). ...
Introduction
Microorganisms colonize a wide range of natural and artificial environments. Even though most of them are unculturable in laboratory conditions, some ecosystems are ideal niches for bioprospecting extremophiles with unique properties. Up today, there are few reports concerning microbial communities found on solar panels, a widespread, artificial, extreme habitat. Microorganisms found in this habitat belong to drought-, heat- and radiation-adapted genera, including fungi, bacteria, and cyanobacteria.
Methods
Here we isolated and identified several cyanobacteria from a solar panel. Then, some strains isolated were characterizated for their resistance to desiccation, UV-C exposition, and their growth on a range of temperature, pH, NaCl concentration or diverse carbon and nitrogen sources. Finally, gene transfer to these isolates was evaluated using several SEVA plasmids with different replicons to assess their potential in biotechnological applications.
Results and discussion
This study presents the first identification and characterization of cultivable extremophile cyanobacteria from a solar panel in Valencia, Spain. The isolates are members of the genera Chroococcidiopsis , Leptolyngbya , Myxacorys , and Oculatella all genera with species commonly isolated from deserts and arid regions. Four of the isolates were selected, all of them Chroococcidiopsis , and characterized. Our results showed that all Chroococcidiopsis isolates chosen were resistant up to a year of desiccation, viable after exposition to high doses of UV-C, and capable of being transformed. Our findings revealed that a solar panel is a useful ecological niche in searching for extremophilic cyanobacteria to further study the desiccation and UV-tolerance mechanisms. We conclude that these cyanobacteria can be modified and exploited as candidates for biotechnological purposes, including astrobiology applications.
... The thermophilic cyanobacterium T. vulcanus cells were grown at 45 °C under continuous white fluorescent illumination at a light intensity of 40 μmol photons m −2 s −1 in a DTN medium (Muhlenhoff and Chauvat 1996), either on 1.5% (w/v) agar plates or in liquid culture bubbled with air containing 3-5% (v/v) CO 2 . ...
... Mutations of the D1 protein was made in the psbA 3 gene in a strain in which, two neighboring genes psbA 1 and psbA 2 are deleted by replacing a fragment from the 64 nucleotide upstream psbA 1 to the 1029 nucleotide of psbA 2 with a chloramphenicol (Cm)-resistance cassette (1036 bp) at the position between Alf II and NheI sites (Fig. 1a). Transformation was performed by electroporation (Muhlenhoff and Chauvat 1996;Katoh et al. 2001;Kirilovsky et al. 2004), and single colonies of the psbA 1 and psbA 2 deletion mutant (named WT' here) were selected on a Cm-containing (4 μg Cm mL −1 ) DTN agar plate and maintained in the presence of 6 μg mL −1 Cm). The deletion mutant was confirmed by sequencing the PCR product amplified from the genomic DNA of the transformants using primers F1 (5′-ACG GAG GTT TGC GTG TTG TCG GTG AG-3′) and R1(5′-CTG TTG GAA TCG GGA CGG GTT GAG GG-3′) (Fig. 1b). ...
Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63–91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ.
... All the PCR fragments obtained were purified by a DNA gel extraction kit (Axygen) and cloned into the pUC18 plasmid with the chloramphenicol-resistant gene cassette located between the His tag and the 3′-flanking fragment. This plasmid was named His-psb28-pUC18 and introduced into the WT and psbV-deleted mutant (ΔPsbV) 42 cells of T. vulcanus by natural transformation as described previously 56,57 . After the appearance of the transformants in two to five weeks, the clones were segregated by growing on agar plates containing 5 μg ml −1 chloramphenicol at least three times. ...
Photosystem II (PSII) is a multisubunit pigment–protein complex and catalyses light-induced water oxidation, leading to the conversion of light energy into chemical energy and the release of dioxygen. We analysed the structures of two Psb28-bound PSII intermediates, Psb28–RC47 and Psb28–PSII, purified from a psbV-deletion strain of the thermophilic cyanobacterium Thermosynechococcus vulcanus, using cryo-electron microscopy. Both Psb28–RC47 and Psb28–PSII bind one Psb28, one Tsl0063 and an unknown subunit. Psb28 is located at the cytoplasmic surface of PSII and interacts with D1, D2 and CP47, whereas Tsl0063 is a transmembrane subunit and binds at the side of CP47/PsbH. Substantial structural perturbations are observed at the acceptor side, which result in conformational changes of the quinone (QB) and non-haem iron binding sites and thus may protect PSII from photodamage during assembly. These results provide a solid structural basis for understanding the assembly process of native PSII.
... RSF1010 and its derivatives were shown to be efficiently transferred by conjugation from E. coli to cyanobacteria where they replicate autonomously, even though they contain no cyanobacterial replicon. These cyanobacteria were, namely, Synechocystis PCC 6803 [426], Synechococcus NKBG15041C, Pseudanabaena NKBG040605C [427], Synechocystis PCC 6714, Synechococcus strains PCC7942 and PCC6301 [428], Thermosynechococcus elongatus PB1 [429], marine Synechococcus strains sp. WH7803, WH8102 and WH8103 [430], Gloeobacter violaceus PCC 7421 [431,432], Prochlorococcus MIT9313 [433], Nostoc (Anabaena) PCC 7120, Nostoc punctiforme ATCC29133 (also registered as PCC 73102) [255,434,435], Leptolyngbya BL0902 [436] and Cyanothece PCC 7425 [410]. ...
Cyanobacteria are widely-diverse, environmentally crucial photosynthetic prokaryotes of great interests for basic and applied science. Work to date has focused mostly on the three non-nitrogen fixing unicellular species Synechocystis PCC 6803, Synechococcus PCC 7942, and Synechococcus PCC 7002, which have been selected for their genetic and physiological interests summarized in this review. Extensive “omics” data sets have been generated, and genome-scale models (GSM) have been developed for the rational engineering of these cyanobacteria for biotechnological purposes. We presently discuss what should be done to improve our understanding of the genotype-phenotype relationships of these models and generate robust and predictive models of their metabolism. Furthermore, we also emphasize that because Synechocystis PCC 6803, Synechococcus PCC 7942, and Synechococcus PCC 7002 represent only a limited part of the wide biodiversity of cyanobacteria, other species distantly related to these three models, should be studied. Finally, we highlight the need to strengthen the communication between academic researchers, who know well cyanobacteria and can engineer them for biotechnological purposes, but have a limited access to large photobioreactors, and industrial partners who attempt to use natural or engineered cyanobacteria to produce interesting chemicals at reasonable costs, but may lack knowledge on cyanobacterial physiology and metabolism.
... To generate the ΔPsbV strain of T. vulcanus, a plasmid vector, which includes a disrupted psbV gene from the 53rd nucleotide in the promoter region to the stop codon, a streptomycinresistant gene cassette, as well as the upstream and downstream of psbV for homologous recombination, was constructed (49). The plasmid was introduced into the wild type cells of T. vulcanus by either electroporation or natural transformation as previously described (62,63). After the transformants appeared in 2 to 5 wk, they were segregated by culturing on agar plates containing 10 μg/mL streptomycin. ...
Significance
Photosystem II (PSII) is a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthetic organisms. Several inactive PSII intermediates are involved in the biogenesis of the multisubunit PSII complexes as well as in their repair after unavoidable oxidative damage targeted to PSII under light. Psb27 is one of the assembly factors associated with inactive PSII intermediate and plays important roles in the biogenesis/repair of PSII. Here, we report the structure of a dimeric Psb27-PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus by cryo-electron microscopy. Our results reveal the location and binding properties of Psb27 in the intermediate PSII and show the structural differences between the intermediate and native PSII. These results provide important clues for the roles of Psb27 in the biogenesis/repair of PSII.
... Cells of the thermophilic cyanobacterium T. vulcanus were grown in a DTN medium (Muhlenhoff and Chauvat 1996), either on 1.5% (w/v) agar plates or in liquid culture, under continuous illumination with white fluorescent lamp at a light intensity of 40 μmol photons m −2 s −1 at 45 °C. The liquid culture was continuously bubbled with air containing 3-5% (v/v) CO 2 . ...
... The plasmids containing the site-directed mutations were transformed into T. vulcanus cells by electroporation (Kirilovsky et al. 2004;Muhlenhoff and Chauvat 1996). Single colonies were selected on Cm-containing (4 μg mL −1 ) DTN agar plates. ...
PsbO-D158 is a highly conserved residue of the PsbO protein in photosystem II (PSII), and participates in one of the hydrogen-bonding networks connecting the manganese cluster with the lumenal surface. In order to examine the role of PsbO-D158, we mutated it to E, N or K in Thermosynechococcus vulcanus and characterized photosynthetic properties of the mutants obtained. The growth rates of these three mutants were similar to that of the wild type, whereas the oxygen-evolving activity of the three mutant cells decreased to 60–64% of the wild type. Fluorescence kinetics showed that the mutations did not affect the electron transfer from QA to QB, but slightly affected the donor side of PSII. Moreover, all of the three mutant cells were more sensitive to high light and became slower to recover from photoinhibition. In the isolated thylakoid membranes from the three mutants, the PsbU subunit was lost and the oxygen-evolving activity was reduced to a lower level compared to that in the respective cells. PSII complexes isolated from these mutants showed no oxygen-evolving activity, which was found to be due to large or complete loss of PsbO, PsbV and PsbU during the process of purification. Moreover, PSII cores purified from the three mutants contained Psb27, an assembly co-factor of PSII. These results suggest that PsbO-D158 is required for the proper binding of the three extrinsic proteins to PSII and plays an important role in maintaining the optimal oxygen-evolving activity, and its mutation caused incomplete assembly of the PSII complex.
... The plasmids constructed were introduced into the wildtype cells (WT) of T. vulcanus by either electroporation or natural transformation as previously described (Iwai et al. 2004;Muhlenhoff and Chauvat 1996;Onai et al. 2004). After appearance of the transformants in 2-5 weeks, they were segregated by growing on agar plates containing 10 µg/ ml streptomycin for at least twice. ...
PsbV (cytochrome c550) is one of the three extrinsic proteins of photosystem II (PSII) and functions to maintain the stability and activity of the Mn4CaO5 cluster, the catalytic center for water oxidation. PsbV-Y137 is the C-terminal residue of PsbV and is located at the exit of a hydrogen-bond network mediated by the D1-Y161-H190 residue pair. In order to examine the function of PsbV-Y137, four mutants, PsbV-Y137A, PsbV-Y137F, PsbV-Y137G, and PsbV-Y137W, were generated with Thermosynechococcus vulcanus (T. vulcanus). These mutants showed growth rates similar to that of the wild-type strain (WT); however, their oxygen-evolving activities were different. At pH 6.5, the oxygen evolution rates of Y137F and Y137W were almost identical to that of WT, whereas the oxygen evolution rates of the Y137A, Y137G mutants were 64% and 61% of WT, respectively. However, the oxygen evolution in the latter two mutants decreased less at higher pHs, suggesting that higher pHs facilitated oxygen evolution probably by facilitating proton egress in these two mutants. Furthermore, thylakoid membranes isolated from the PsbV-Y137A, PsbV-Y137G mutants exhibited much lower levels of oxygen-evolving activity than that of WT, which was found to be caused by the release of PsbV. In addition, PSII complexes purified from the PsbV-Y137A and PsbV-Y137G mutants lost all of the three extrinsic proteins but instead bind Psb27, an assembly cofactor of PSII. These results demonstrate that the PsbV-Tyr137 residue is required for the stable binding of PsbV to PSII, and the hydrogen-bond network mediated by D1-Y161-H190 is likely to function in proton egress during water oxidation.
... Man erhält ein niedrig aufgelöstes Modell, das das gemessene Protein abbildet.Um den Detergensgürtel um PSI beschreiben zu können, wurden PSI-Monomere und -Trimere in 5 mM MES-NaOH (pH 6,0) mit 60 mM MgSO 4 und 0,02 % DDM umgepuffert. Dieser Puffer wurde genutzt, um die SAXS-Messungen mit vergangenen Messungen vergleichen zu können(Müh und Zouni 2008). Die Konzentration der Proteine betrug 15 µM P 700 (Monomer, 5 g Protein · l -1 ) und 30 µM P 700 (Trimer, 10 g Protein · l -1 ). ...
Photosystem I (PSI) aus dem thermophilen Cyanobakterium Thermosynechococcus elongatus ist ein transmembraner Protein-Pigment-Superkomplex der photosynthetischen Elektronentransportkette. Er wandelt die Energie des Lichts in elektrische Energie mit einer Quanteneffizienz von nahezu 100 % um. Dazu uberträgt PSI Elektronen von Plastocyanin bzw. Cytochrom c6 (Cyt c6) auf Ferredoxin. Die Struktur des PSI wurde bereits 2001 mit einer Auflösung von 2,5 Å beschrieben (Jordan et al. 2001). Es lässt sich zur Generierung von Photoströmen auf Elektrodenoberflächen assemblieren und zur Produktion von Biokraftstoffen mit Enzymen koppeln. Die elektrische Kontaktierung des PSI mit Elektrodenoberflächen kann durch Komplexierung mit dem mitochondrialem Cytochrom c aus Pferdeherz (Cyt cHH) erhöht werden. Aufgrund der Nutzbarkeit dieses Proteinkomplexes sollte geklärt werden, wie PSI und Cyt cHH wechselwirken und wie sich die Interaktion von der des nativen PSI-Cyt c6-Komplexes unterscheidet. Deshalb lag der Fokus meiner Arbeit darauf, die Bindung des Cyt c6 und seines Analogons Cyt cHH an PSI mit kinetischen, kalorimetrischen, theoretischen und strukturellen Methoden zu untersuchen. Das Cyt c6 bindet im reduzierten Zustand an PSI und verringert nach erfolgtem Elektronentransfer seine Affinität. Das Cyt cHH bindet dagegen sowohl im reduzierten als auch im oxidierten Zustand an PSI. Mit Hilfe der kinetischen Messungen habe ich Bedingungen identifiziert, unter denen PSI mit dem jeweiligen Cytochrom c einen stabilen Komplex eingeht. Mit Hilfe eines rigid-body dockings wurden potenzielle Bindungsstellen der beiden Cytochrome berechnet. Fur Cyt c6 ergab sich eine spezifische Bindungsstelle, die eine gute Übereinstimmung mit den von mir gemessenen Kinetiken sowie mit weiteren Literaturdaten zeigt. Diese Bindungsstelle korreliert mit der veröffentlichten Kostruktur des bakteriellen Reaktionszentrums mit Cyt c2 aus Rhodobacter sphaeroides. Demgegenüber sind mehrere Cyt cHH-Bindungsstellen ...
... 80 However, the lack of PsaF in Cyanobacteria has been shown in numerous studies to have no effect on reduction of the complex via native or non-native cytochromes, indicating that PsaF is not involved in cytochrome docking or electron transfer in Cyanobacteria. [81][82][83][84][85] As shown in Fig. 2, SMA copolymers possess a welldocumented drawback, most formulations exhibit a sensitivity to divalent cations and become insoluble as pH drops, with more hydrophobic (less charged) polymers aggregating closer to neutral pH. [51][52][53]74,86,87 When using a buffer consisting of 150 mM KCl, devoid of divalent ions and pH ¼ 9.5 (room temperature) for laser ash photolysis measurements, the reduction rate decreased for both PSI-SMALP and PSI-DDM ( Fig. 7A and B). ...
Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus (Te) is the largest membrane protein complex to have had its structure solved by X-ray diffraction. This trimeric complex has 36 protein subunits, over 380 non-covalently bound cofactors and a molecular weight of ∼1.2 MDa. Previously, it has been isolated and characterized in a detergent micelle using the non-ionic detergent n-dodecyl-β-D-maltoside (DDM). We have now succeeded in isolating this complex without the use of detergents, using styrene–maleic acid (SMA) alternating copolymer. Intriguingly, a partially esterified copolymer formulation (SMA 1440, Cray Valley) was found to be most efficient in cyanobacterial thylakoid membranes. A host of biochemical, biophysical and functional assays have been applied to characterize this non-detergent form of PSI, referred to as a SMA Lipid Particle (SMALP). The PSI-SMALP has a lower sedimentation coefficient compared to PSI-DDM, suggesting decreased density or a more extended particle shape. We show the 77 K fluorescence maximum for PSI is red shifted in PSI-SMALP compared to PSI-DDM, suggesting a more native orientation of PsaA/B associated chlorophyll. We report that PSI-SMALPs are functional despite the selective loss of one transmembrane subunit, PsaF. This loss may reflect a more labile interaction of the PSI core and PsaF, or a selective displacement during copolymer insertion and/or assembly. PSI-SMALP exhibited decreased reduction kinetics with native recombinant cytochromes c6, while non-native horse heart cytochrome c shows faster reduction of PSI-SMALP compared to PSI-DDM. This is the largest membrane protein isolated using SMA copolymers, and this study expands the potential use of this approach for the isolation and characterization of large supramolecular complexes.
... We focused our attention on several phylogenetically-distant cyanobacteria (Supplementary Table S16) because they are presently well studied thanks to their powerful genetics (for example Synechocystis PCC 6803, Synechococcus PCC 7002, and Synechococcus PCC 7942). Furthermore, they should also be increasingly investigated in the near future because of their interesting natural properties (Supplementary Table S16) and the likely possibility that they could be manipulated with a broad-host-range of RSF1010 plasmids that have been shown to replicate in various cyanobacteria since first being reported (Marraccini et al., 1993;Mühlenhoff and Chauvat, 1996;Tolonen et al., 2006a,b;Araki et al., 2013;Taton et al., 2014). ...
Cyanobacteria are widely-diverse prokaryotes that colonize our planet. They use solar energy to assimilate huge amounts of atmospheric CO2 and produce a large part of the biomass and oxygen that sustain most life forms. Cyanobacteria are therefore increasingly studied for basic research objectives, as well as for the photosynthetic production of chemicals with industrial interests. One potential approach to reduce the cost of future bioproduction processes is to couple them with wastewater treatment, often polluted with urea, which in any case is cheaper than nitrate. As of yet, however, research has mostly focused on a very small number of model cyanobacteria growing on nitrate. Thus, the genetic inventory of the cyanobacterial phylum is still insufficiently employed to meaningfully select the right host for the right purpose. This review reports what is known about urea transport and catabolism in cyanobacteria, and what can be inferred from the comparative analysis of the publicly available genome sequence of the 308 cyanobacteria. We found that most cyanobacteria mostly harbor the genes encoding the urea catabolytic enzymes urease (ureABCDEFG), but not systematically, together with the urea transport (urtABCDE). These findings are consistent with the capacity of the few tested cyanobacteria that grow on urea as the sole nitrogen source. They also indicate that urease is important for the detoxification of internally generated urea (re-cycling its carbon and nitrogen). In contrast, several cyanobacteria have urtABCDE but not ureABCDEFG, suggesting that urtABCDE could operate in the transport of not only urea but also of other nutrients. Only four cyanobacteria appeared to have the genes encoding the urea carboxylase (uc) and allophanate hydrolase (ah) enzymes that sequentially catabolize urea. Three of these cyanobacteria belongs to the genera Gloeobacter and Gloeomargarita that have likely diverged early from other cyanobacteria, suggesting that the urea carboxylase and allophanate hydrolase enzymes appeared in cyanobacteria before urease.
