Genetic diversity of the submerged macrophyte Ceratophyllum demersum depends on habitat hydrology and habitat fragmentation.

The adaptability of plant populations to a changing environment depends on their genetic diversity, which in turn is influenced by the degree of sexual reproduction and gene flow from distant areas. Aquatic macrophytes can reproduce both sexually and asexually, and their reproductive fragments are spread in various ways (e.g. by water). Although these plants are obviously exposed to hydrological changes, the degree of vulnerability may depend on the types of their reproduction and distribution, as well as the hydrological differences of habitats. The aim of this study was to investigate the genetic diversity of the cosmopolitan macrophyte Ceratophyllum demersum in hydrologically different aquatic habitats, i.e. rivers and backwaters separated from the main river bed to a different extent. For this purpose, the first microsatellite primer set was developed for this species. Using 10 developed primer pairs, a high level of genetic variation was explored in C. demersum populations. Overall, more than 80% of the loci were found to be polymorphic, a total of 46 different multilocus genotypes and 18 private alleles were detected in the 63 individuals examined. The results demonstrated that microsatellite polymorphism in this species depends on habitat hydrology. The greatest genetic variability was revealed in populations of rivers, where flowing water provides constant longitudinal connections with distant habitats. The populations of the hydrologically isolated backwaters showed the lowest microsatellite polymorphism, while plants from an oxbow occasionally flooded by the main river had medium genetic diversity. The results highlight that in contrast to species that spread independently of water flow or among hydrologically isolated water bodies, macrophytes with exclusive or dominant hydrochory may be most severely affected by habitat fragmentation, for example due to climate change.

Planktonic and epilithic prokaryota community compositions in a large temperate river reflect climate change related seasonal shifts.

In freshwaters, microbial communities are of outstanding importance both from ecological and public health perspectives, however, they are threatened by the impact of global warming. To reveal how different prokaryotic communities in a large temperate river respond to environment conditions related to climate change, the present study provides the first detailed insight into the composition and spatial and year-round temporal variations of planktonic and epilithic prokaryotic community. Microbial diversity was studied using high-throughput next generation amplicon sequencing. Sampling was carried out monthly in the midstream and the littoral zone of the Danube, upstream and downstream from a large urban area. Result demonstrated that river habitats predominantly determine the taxonomic composition of the microbiota; diverse and well-differentiated microbial communities developed in water and epilithon, with higher variance in the latter. The composition of bacterioplankton clearly followed the prolongation of the summer resulting from climate change, while the epilithon community was less responsive. Rising water temperatures was associated with increased abundances of many taxa (such as phylum Actinobacteria, class Gammaproteobacteria and orders Synechococcales, Alteromonadales, Chitinophagales, Pseudomonadales, Rhizobiales and Xanthomonadales), and the composition of the microbiota also reflected changes of several further environmental factors (such as turbidity, TOC, electric conductivity, pH and the concentration of phosphate, sulphate, nitrate, total nitrogen and the dissolved oxygen). The results indicate that shift in microbial community responding to changing environment may be of crucial importance in the decomposition of organic compounds (including pollutants and xenobiotics), the transformation and accumulation of heavy metals and the occurrence of pathogens or antimicrobial resistant organisms.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants.

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium.

A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.

A single plasmid based CRISPR interference in Synechocystis 6803 - A proof of concept.

We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a "proof of concept". Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.

A novel enzyme of type VI sulfide:quinone oxidoreductases in purple sulfur photosynthetic bacteria.

Sulfide detoxification can be catalyzed by ancient membrane-bound flavoproteins, sulfide:quinone oxidoreductases (Sqr), which have important roles in sulfide homeostasis and sulfide-dependent energy conservation processes by transferring electrons from sulfide to respiratory or photosynthetic membrane electron flow. Sqr enzymes have been categorized into six groups. Several members of the groups I, II, III, and V are well-known, but type IV and VI Sqrs are, as yet, uncharacterized or hardly characterized at all. Here, we report detailed characterization of a type VI sulfide:quinone oxidoreductase (TrSqrF) from a purple sulfur bacterium, Thiocapsa roseopersicina. Phylogenetic analysis classified this enzyme in a special group composed of SqrFs of endosymbionts, while a weaker relationship could be observed with SqrF of Chlorobaculum tepidum which is the only type VI enzyme characterized so far. Directed mutagenesis experiments showed that TrSqrF contributed substantially to the sulfide:quinone oxidoreductase activity of the membranes. Expression of the sqrF gene could be induced by sulfide. Homologous recombinant TrSqrF protein was expressed and purified from the membranes of a SqrF-deleted T. roseopersicina strain. The purified protein contains redox-active covalently bound FAD cofactor. The recombinant TrSqrF enzyme catalyzes sulfur-dependent quinone reduction and prefers ubiquinone-type quinone compounds. Kinetic parameters of TrSqrF show that the affinity of the enzyme is similar to duroquinone and decylubiquinone, but the reaction has substantially lower activation energy with decylubiquinone, indicating that the quinone structure has an effect on the catalytic process. TrSqrF enzyme affinity for sulfide is low, therefore, in agreement with the gene expressional analyis, SqrF could play a role in energy-conserving sulfide oxidation at high sulfide concentrations. TrSqrF is a good model enzyme for the subgroup of type VI Sqrs of endosymbionts and its characterization might provide deeper insight into the molecular details of the ancient, anoxic, energy-gaining processes using sulfide as an electron donor.

Starvation- and xenobiotic-related transcriptomic responses of the sulfanilic acid-degrading bacterium, Novosphingobium resinovorum SA1.

Novosphingobium resinovorum SA1 was the first single isolate capable of degrading sulfanilic acid, a widely used representative of sulfonated aromatic compounds. The genome of the strain was recently sequenced, and here, we present whole-cell transcriptome analyses of cells exposed to sulfanilic acid as compared to cells grown on glucose. The comparison of the transcript profiles suggested that the primary impact of sulfanilic acid on the cell transcriptome was a starvation-like effect. The genes of the peripheral, central, and common pathways of sulfanilic acid biodegradation had distinct transcript profiles. The peripheral genes located on a plasmid had very high basal expressions which were hardly upregulated by sulfanilic acid. The genomic context and the codon usage preference of these genes suggested that they were acquired by horizontal gene transfer. The genes of the central pathways were remarkably inducible by sulfanilic acid indicating the presence of a substrate-specific regulatory system in the cells. Surprisingly, the genes of the common part of the metabolic pathway had low and sulfanilic acid-independent transcript levels. The approach applied resulted in the identification of the genes of proteins involved in auxiliary processes such as electron transfer, substrate and iron transports, sulfite oxidases, and sulfite transporters. The whole transcriptome analysis revealed that the cells exposed to xenobiotics had multiple responses including general starvation-like, substrate-specific, and substrate-related effects. From the results, we propose that the genes of the peripheral, central, and common parts of the pathway have been evolved independently.

Characterization of the Rhodococcus sp. MK1 strain and its pilot application for bioremediation of diesel oil-contaminated soil.

Petroleum hydrocarbons and derivatives are widespread contaminants in both aquifers and soil, their elimination is in the primary focus of environmental studies. Microorganisms are key components in biological removal of pollutants. Strains capable to utilize hydrocarbons usually appear at the contaminated sites, but their metabolic activities are often restricted by the lack of nutrients and/or they can only utilize one or two components of a mixture. We isolated a novel Rhodococcus sp. MK1 strain capable to degrade the components of diesel oil simultaneously. The draft genome of the strain was determined and besides the chromosome, the presence of one plasmid could be revealed. Numerous routes for oxidation of aliphatic and aromatic compounds were identified. The strain was tested in ex situ applications aiming to compare alternative solutions for microbial degradation of hydrocarbons. The results of bioaugmentation and biostimulation experiments clearly demonstrated that - in certain cases - the indigenous microbial community could be exploited for bioremediation of oil-contaminated soils. Biostimulation seems to be efficient for removal of aged contaminations at lower concentration range, whereas bioaugmentation is necessary for the treatment of freshly and highly polluted sites.

Complete genome sequence of Novosphingobium resinovorum SA1, a versatile xenobiotic-degrading bacterium capable of utilizing sulfanilic acid.

Sulfanilic acid (4-aminobenzenesulfonic acid) is a sulfonated aromatic amine widely used in chemical industries for synthesis of various organic dyes and sulfa drugs. There are quite a few microbial co-cultures or single isolates capable of completely degrading this compound. Novosphingobium resinovorum SA1 was the first single bacterium which could utilize sulfanilic acid as its sole carbon, nitrogen and sulfur source. The strain has versatile catabolic routes for the bioconversion of numerous other aromatic compounds. Here, the complete genome sequence of the N. resinovorum SA1 strain is reported. The genome consists of a circular chromosome of 3.8 Mbp and four extrachromosomal elements between 67 and 1 759.8 kbp in size. Three alternative 3-ketoadipate pathways were identified on the plasmids. Sulfanilic acid is decomposed via a modified 3-ketoadipate pathway and the oxygenases involved form a phylogenetically separate branch on the tree. Sequence analysis of these elements might provide a genetic background for deeper insight into the versatile catabolic metabolism of various aromatic xenobiotics, including sulfanilic acid and its derivatives. Moreover, this is also a good model strain for understanding the role and evolution of multiple genetic elements within a single strain.

Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms.

Coregulated genes link sulfide:quinone oxidoreductase and arsenic metabolism in Synechocystis sp. strain PCC6803.

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer.

The cry-DASH cryptochrome encoded by the sll1629 gene in the cyanobacterium Synechocystis PCC 6803 is required for Photosystem II repair.

The role of the Syn-CRY cryptochrome from the cyanobacterium Synechocystis sp. PCC 6803 has been a subject of research for more than a decade. Recently we have shown that photolyase, showing strong homology with Syn-CRY is required for Photosystem II repair by preventing accumulation of DNA lesions under UV-B (Vass et al. 2013). Here we investigated if Syn-CRY is also involved in PSII repair, either via removal of DNA lesions or other mechanism? The Δsll1629 mutant lacking Syn-CRY lost faster the PSII activity and D1 protein during UV-B or PAR than the WT. However, no detectable damages in the genomic DNA were observed. The transcript levels of the UV-B and light stress indicator gene psbA3, encoding D1, are comparable in the two strains showing that Δsll1629 cells are not defective at the transcriptional level. Nevertheless 2D protein analysis in combination with mass spectrometry showed a decreased accumulation of several, mostly cytoplasmic, proteins including PilA1 and bicarbonate transporter SbtA. Δsll1629 cells exposed to high light also showed a limitation in de novo assembly of PSII. It is concluded that Syn-CRY is required for efficient restoration of Photosystem II activity following UV-B and PAR induced photodamage. This effect is not caused by retardation of DNA repair, instead the synthesis of new D1 (and D2) subunit(s) and/or the assembly of the Photosystem II reaction center complex is likely affected due to the lack of intracellular CO2, or via a so far unidentified pathway that possibly includes the PilA1 protein.

Assessing the Applicability of Singlet Oxygen Photosensitizers in Leaf Studies.

Singlet oxygen ((1) O2 ) is of special interest in plant stress physiology. Studies focused on internal, chlorophyll-mediated production are often complemented with the use of artificial (1) O2 photosensitizers. Here, we report a comparative study on the effects of Rose Bengal (RB), Methylene Violet (MVI), Neutral Red (NR) and Indigo Carmine (IC). These were infiltrated into tobacco leaves at concentrations generating the same fluxes of (1) O2 in solution. Following green light-induced (1) O2 production from these dyes, leaf photosynthesis was characterized by Photosystem (PS) II and PSI electron transport and oxidative damage was monitored as degradation of D1, a PSII core protein. Cellular localizations were identified on the basis of the dyes' fluorescence using confocal laser scanning microscopy. We found that RB and NR were both localized in chloroplasts but the latter had very little effect, probably due to its pH-dependent photosensitizing. Both RB and intracellular, nonplastid MVI decreased PSII electron transport, but the effect of RB was stronger than that of MVI and only RB was capable of damaging the D1 protein. Intercellularly localized IC had no significant effect. Our results also suggest caution when using RB as photosensitizer because it affects PSII electron transport.

The ability of cyanobacterial cells to restore UV-B radiation induced damage to Photosystem II is influenced by photolyase dependent DNA repair.

Damage of DNA and Photosystem-II are among the most significant effects of UV-B irradiation in photosynthetic organisms. Both damaged DNA and Photosystem-II can be repaired, which represent important defense mechanisms against detrimental UV-B effects. Correlation of Photosystem-II damage and repair with the concurrent DNA damage and repair was investigated in the cyanobacterium Synechocystis PCC6803 using its wild type and a photolyase deficient mutant, which is unable to repair UV-B induced DNA damages. A significant amount of damaged DNA accumulated during UV-B exposure in the photolyase mutant concomitant with decreased Photosystem-II activity and D1 protein amount. The transcript level of psbA3, which is a UV-responsive copy of the psbA gene family encoding the D1 subunit of the Photosystem-II reaction center, is also decreased in the photolyase mutant. The wild-type cells, however, did not accumulate damaged DNA during UV-B exposure, suffered smaller losses of Photosystem-II activity and D1 protein, and maintained higher level of psbA3 transcripts than the photolyase mutant. It is concluded that the repair capacity of Photosystem-II depends on the ability of cells to repair UV-B-damaged DNA through maintaining the transcription of genes, which are essential for protein synthesis-dependent repair of the Photosystem-II reaction center.

Characterization of stress responses of heavy metal and metalloid inducible promoters in synechocystis PCC6803.

In several biotechnological applications of living bacterial cells with inducible gene expression systems, the extent of overexpression and the specificity to the inducer are key elements. In the present study, we established the concentration ranges of Zn(2+), Ni(2+), Co(2+), AsO(2)(-), and Cd(2+) ions that caused significant activation of the respective promoters of Synechocystis sp. without concomitant unspecific stress responses. The low expression levels can be increased up to 10-100-fold upon treatments with Cd(2+), AsO(2)(-), Zn(2+), and Co(2+) ions and up to 800-fold upon Ni(2+) treatment. These results facilitate the development of conditional gene expression systems in cyanobacteria.

A unique regulation of the expression of the psbA, psbD, and psbE genes, encoding the 01, 02 and cytochrome b559 subunits of the Photosystem II complex in the chlorophyll d containing cyanobacterium Acaryochloris marina.

Photosynthetic electron transport, chromatic photoacclirnation and expression of the genes encoding the 01, 02, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a 01' isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the 01 and the abundant isoform of the 02 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a 02' isoform whose primary structure is different from the abundant 02 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of 01 and 02 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha-alpha homodimer organization of cytochrome b559 in Photosystem II complexes.

The Translational Medicine Ontology and Knowledge Base: driving personalized medicine by bridging the gap between bench and bedside.

BACKGROUND: Translational medicine requires the integration of knowledge using heterogeneous data from health care to the life sciences. Here, we describe a collaborative effort to produce a prototype Translational Medicine Knowledge Base (TMKB) capable of answering questions relating to clinical practice and pharmaceutical drug discovery. RESULTS: We developed the Translational Medicine Ontology (TMO) as a unifying ontology to integrate chemical, genomic and proteomic data with disease, treatment, and electronic health records. We demonstrate the use of Semantic Web technologies in the integration of patient and biomedical data, and reveal how such a knowledge base can aid physicians in providing tailored patient care and facilitate the recruitment of patients into active clinical trials. Thus, patients, physicians and researchers may explore the knowledge base to better understand therapeutic options, efficacy, and mechanisms of action. CONCLUSIONS: This work takes an important step in using Semantic Web technologies to facilitate integration of relevant, distributed, external sources and progress towards a computational platform to support personalized medicine. AVAILABILITY: TMO can be downloaded from http://code.google.com/p/translationalmedicineontology and TMKB can be accessed at http://tm.semanticscience.org/sparql.

Superoxide anion radicals generated by methylviologen in photosystem I damage photosystem II.

The effect of superoxide anion radicals on the photosynthetic electron transport chain was studied in leaves and isolated thylakoids from tobacco. Superoxide was generated by methylviologen (MV) in the light at the acceptor side of photosystem I (PSI). In isolated thylakoids, the largest damage was observed at the level of the water-splitting activity in photosystem II (PSII), whereas PSI was hardly affected at the light intensities used. Addition of reactive oxygen scavengers protected PSII against damage. In leaves in the presence of MV, the quantum yield of PSII decreased during illumination whereas the size of the P(700) signal remained constant. There was no D1 protein loss in leaves illuminated in the presence of MV and lincomycin, but a modification to a slightly higher molecular mass was observed. These data show that PSII is more sensitive to superoxide or superoxide-derived reactive oxygen species (ROS) than PSI. In our experiments, this susceptibility was not because of any action of the ROS on the translation of the D1 protein or on the repair cycle of photosystem.

Transcriptional regulation of the bidirectional hydrogenase in the cyanobacterium Synechocystis 6803.

To identify optimal conditions for renewable hydrogen production from sunlight and water we have studied transcriptional changes of the hoxEFUYH genes encoding the bidirectional hydrogenase in the cyanobacterium Synechocystis PCC 6803. Transcript abundance detection by real time polymerase chain reaction was supplemented with variable chlorophyll fluorescence measurements to monitor redox changes of the photosynthetic electron transport chain. Our main observations are: (i) abundance of hox transcripts decreases in the dark and recovers in the light. (ii) Inhibition of the Calvin cycle by glycolaldehyde suppresses hox gene transcription, which can be restored by the addition of electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea and dibromothymoquinone. (iii) The transcript levels of all hox genes are increased in anoxia, with additional induction of hoxEF in darkness or in the presence of dibromothymoquinone. (iv) Plastoquinone pool redox changes are not correlated with hox transcript level changes. (v) Changes in the transcript levels of lexA and sll0359 genes, encoding putative regulators of hox genes, are only partly correlated with transcript changes of hox genes under different conditions. Our data demonstrate a previously unrecognized light- and oxygen-dependent regulation of hox gene transcription in Synechocystis PCC 6803, which is related to photosynthetic electron transport and to unidentified oxygen and redox sensors. We also conclude that neither LexA nor Sll0359 are likely to be exclusive regulators of hox gene transcription.