Thermophilic Cyanobacterium Synechococcus Research Articles

Physiological and spectroscopical changes of the thermophilic cyanobacterium Synechococcus elongatus under iron stress and recovery culture

The iron in cyanobacterial cells contributes to energy conversion via electron transport chains, so its availability directly influences all metabolic pathways. Besides exploring cell adaptations and changes occurring with iron insufficient stress, the present work examines profoundly for the first time the metabolic changes accompanied with iron recovery for the thermophilic cyanobacterium Synechococcus elongatus (Nag., var. thermalis Geitl. Strain Kovrov 1972/8). S. elongatus cells were cultivated on two iron deprivation levels, iron-limited (45 nM Fe) and iron-deficient (4.5 nM Fe). Growth, carotenoids/Chlorophyll-a (Car/Chl-a) ratio, transmitting electron images, pigments fractionation analysis, and cell activity were checked. The obtained results demonstrated an astounding decrease in Chl-a content, an increment in β-carotene content, leading to rising the Car/Chl-a ratio, a reduction of phycobilins content, a reduction in cell diameters, a decrease of energy transfer, a depletion of the electron transport chain, and a reduction of photosynthesis and respiration processes under inadequate iron condition. Likewise, the photochemical activity of photosystem II (PSII), determined by Fv/Fm ratio and thermoluminescence estimations, demonstrated that PSII was disabled under iron pressure. With iron recovery by 200 nM Fe, cells regained full metabolic activity within 24 h. Despite the fact that photosynthesis and respiration activity exhibited nearly a similar behavior, S. elongatus under iron-deficiency began to recuperate the activity of the photosynthetic apparatus quicker than the respiratory machinery by regaining their pigment content and activity.

Effect of light colour and photoperiod on biomass growth and phycocyanin production by Synechococcus PCC 6715

The effect of light colour and light regime on growth and production of the thermostable C-phycocyanin (PC) by the thermophilic cyanobacterium Synechococcus 6715 in the tubular photobioreactor has been analysed. The highest specific growth rate (1.918 d−1) and biomass concentration (5.11 gVS ⋅L−1) were observed under constant illumination of the red light. However, the PC concentration in volatile solids (e.g blue light 30.68 ± 0.8 mgPC⋅gVS−1 PP and 21.7 ± 1 mgPC⋅gVS−1 CI) as well as per photobioreactor unit volume (e.g red light 122.66 ± 2.28 mgPC⋅L−1 PP and 74.71 ± 8.43 mgPC⋅L−1 PP) was higher in the 16L:8D photoperiod. The obtained PC purity was higher in the case of photoperiod (≈1.5). PCC6715 lacks genes encoding phycoerythrins what suggests T1 type of pigmentation. Although changes in biomass pigmentation were not significant, the strain was able to adapt its photosystem what can be used in the optimization of PC production by application of different light colours.

Ecological Divergence with Gene Flow in a Thermophilic Cyanobacterium.

How ecological diversity is maintained and distributed within populations is a longstanding question in microbial ecology. In the thermophilic cyanobacterium Synechococcus B', high observed levels of recombination are predicted to maintain ecological variation despite the simultaneous action of diverse selective pressures on different regions of the genome. To investigate ecological diversity in these bacteria, we directly isolated laboratory strains of Synechococcus B' from samples collected along the thermal gradients of two geothermal environments in Yellowstone National Park. Extensive recombination was evident for a multi-locus sequence data set, and, consequently, our sample did not exhibit the sequence clustering expected for distinct ecotypes evolving by periodic clonal selection. Evidence for local selective sweeps at specific loci suggests that sweeps may be common but that recombination is effective for maintaining diversity of unlinked genomic regions. Thermal performance for strain growth was positively associated with the temperature of the environment, indicating that Synechococcus B' populations consist of locally adapted ecological specialists that occupy specific thermal niches. Because this ecological differentiation is observed despite the absence of dispersal barriers among sites, we conclude that these bacteria may freely exchange much of the genome but that barriers to gene flow exist for loci under direct temperature selection.

Photosynthetic temperature adaptation during niche diversification of the thermophilic cyanobacterium Synechococcus A/B clade.

We take an in vivo fluorescence approach to investigate photosynthetic adaptation by ecologically divergent members of the A/B clade of the hot spring cyanobacterium Synechococcus, the most thermotolerant of which defines the upper thermal limit for photosynthesis. During Synechococcus diversification, both photosystem II and the light-harvesting phycobilisome have evolved greater thermostability as the group has invaded higher temperature habitats, particularly for the most thermotolerant lineage. This enhanced function at higher temperatures has come at the cost of reduced performance at lower temperatures, and these trade-offs contribute to niche specialization in the clade. Molecular evolutionary analyses revealed specific adaptive protein changes in the most thermotolerant lineage. Our study advances our understanding of the origins of Synechococcus diversity.

Production of Hydrogen by Thermophilic Cyanobacterium Synechococcus sp. strain H-1

The production of molecular hydrogen (H2) from water by hydrogenase of thermophilic cyanobacterium Synechococcus sp. strain H-1 has been studied by following the time courses of the hydrogen concentration in the gas-phase, cell mass concentration and cellular content of D-glucose in dark batch reactions at temperatures ranging from 313 to 333 K. An anaerobic, dark, NO3− starvation, neutral or alkaline pH and shaking condition was found to be a prerequisite for H2 production. Strain H-1 could multiply and produce hydrogen at all temperatures studied. A linear relation between hydrogen production and glucose consumption was observed and the yield coefficient of hydrogen in terms of D-glucose consumption was 71, 115 and 146 at initial concentrations of cell mass of 0.75, 2.5 and 9.5 g · L−1, respectively. The activation energies for the maximum specific rates of cell mass growth, D-glucose consumption and hydrogen production were found to be 62, 12.6 and 122 kJ · mol−1, respectively. The maximum specific hydrogen production rate was found to be 0.09 μmol · mg-chl a−1 · h−1 at 328 K, which is comparable to that reported in non-nitrogen-fixing unicellular cyanobacteria. It is concluded that utilization of thermophilic Synechococcus sp. strain H-1 for hydrogen production is beneficial since the contamination risk of mesophilic bacteria is low.

In situ dynamics of O2, pH and cyanobacterial transcripts associated with CCM, photosynthesis and detoxification of ROS

The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B' was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O(2) and pH. Fluctuations in pH and O(2) in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O(2) levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O(2) and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark-light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O(2) concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle.

Equal-quantum Action Spectra Indicate Fluence-rate-selective Action of Multiple Photoreceptors for Photomovement of the Thermophilic Cyanobacterium Synechococcus elongatus¶

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55°C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 μmol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 μmol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30–90 μmol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700–740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).

Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from Synechococcus vulcanus at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.

Specific features of oxidative stress in the chilled tobacco plants following transformation with the desC gene for acyl-lipid Δ9-desaturase from Synechococcus vulcanus

Tobacco plants (Nicotiana tabacum L.) transformed with the desC gene for acyl-lipid Δ9-desaturase from a thermophilic cyanobacterium Synechococcus vulcanus were cultivated on the agarized Murashige and Skoog medium at 22°C and a 16-h photoperiod. Tobacco plants transformed with an empty binary vector pGA482 served as the control. The investigations showed that, in contrast to the control, transgenic plants maintained a higher activity of antioxidant enzymes during 2-h incubation at 2°C; as a result, these plants resisted more efficiently the accumulation of reactive oxygen species and reduced the rate of the lipid peroxidation. The activity of antioxidant enzymes in the transformed plants is apparently related to the operation of the introduced desC gene for acyl-lipid Δ9-desaturase because the enhanced activity of the latter increased the relative content of polyunsaturated FAs in membrane lipids and in this way promoted the liquid state of membranes during the chilling period. These changes helped preserve the cellular homeostasis and thereby maintain the steady synthesis of antioxidant enzymes at hypothermic conditions; as a result, cold resistance of transformed tobacco plants increased.

The Effect of Tobacco Plant Transformation with a Gene for Acyl-Lipid Δ9-Desaturase from Synechococcus vulcanus on Plant Chilling Tolerance

Tobacco plants with the introduced desC gene for acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were cultivated on agar-solidified Murashige and Skoog nutrient medium supplemented with ferulic acid and antibiotics at 22°C and a 16-h photoperiod. Control plants were transformed with an empty pGA482 vector. The analysis of fatty acids (FAs) showed that, in transgenic plants, the level of 16:0 and 18:0 FAs decreased substantially, whereas the levels of di- and trienoic FAs increased. Transformed plants were more cold-tolerant. The tolerance to chilling was evaluated from electrolyte leakage from tissues damaged by cold treatments and from the accumulation of a product of lipid peroxidation, malondialdehyde. It was concluded that acyl-lipid Δ9-desaturase was actively expressed in transgenic tobacco plants and converted stearic acid into oleic acid, thus producing a substrate for further synthesis of di- and trienoic FAs. An increased proportion of polyunsaturated FAs in membrane lipids resulted in improved tobacco plant tolerance to chilling.

Post-transcriptional control of the cyanobacterial hspA heat-shock induction

The hspA gene encodes a small heat-shock protein in the thermophilic cyanobacterium Synechococcus vulcanus. To gain insight into the post-transcriptional regulation, hspA was expressed in Escherichia coli. HspA was induced upon a temperature upshift from 30 to 42 °C, although the hspA transcription in E. coli occurred constitutively at both 30 and 42 °C. Neither replacement of the native hspA promoter with the lacZ promoter nor the addition of rifampin abolished the heat induction. Thus, the primary form of regulation of the heat induction is at the post-transcriptional level. Analyses of expression of a series of the transcriptional and translational hspA–lacZ fusions confirmed the constitutive transcription, and demonstrated that the heat induction occurred only in the translational fusions. They further indicated the presence of regulatory elements involved in the translational regulation. An element in the 5′-untranslated region of the hspA mRNA suppressed the translation, while that in the hspA coding region was required for the de-repression of the translation and for thermal regulation.

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.

Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2 12 12 1, with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll ( D Z ) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D X and D Y components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants.

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.

Functional Role of C α–H⋯O Hydrogen Bonds Between Transmembrane α-Helices in Photosystem I

The crystal structure at 2.5 Å resolution of the membrane-intrinsic, homotrimeric photosystem I (PSI) isolated from the thermophilic cyanobacterium Synechococcus elongatus shows that each monomer is composed of 12 protein subunits of which nine are embedded in the membrane and feature a total of 34 transmembrane α-helices (TMH). Hence, PSI provides an ideal case to study “conventional” and C α–H⋯O hydrogen bonds between TMH engaged in intra- and intersubunit interactions. Of the total of 75 C α–H⋯O hydrogen bonds between TMHs, 72 are intrasubunit and only three are intersubunit. The two largest subunits PsaA and PsaB are each folded into 11 TMHs showing 29 and 24 intrasubunit C α–H⋯O hydrogen bonds, respectively, that are not distributed randomly but many of them flank chlorophyll a (Chl a) co-ordinating amino acids, suggesting stabilisation of these structural segments. As major constituent of the trimerisation domain, subunit PsaL is located next to the 3-fold axis relating the three monomers of PSI. PsaL features a unique number of 19 intrasubunit C α–H⋯O hydrogen bonds that connect two of its three TMHs but there are no intersubunit C α–H⋯O hydrogen bonds between the three PsaL. Of the three intersubunit C α–H⋯O hydrogen bonds, two are formed between PsaA and PsaB and one between PsaB and PsaM. The large number of 75 C α–H⋯O hydrogen bonds contrasts the 49 conventional hydrogen bonds, indicating that the former and van der Waals contacts determine association and orientation of TMHs in PSI.

The 1.45 Å three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 Å resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7 h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.

Immobilisation of engineered molecules on electrodes and optical surfaces

Monolayers of genetically modified proteins with an hexahistidine tag, (His) 6, were obtained by using a Ni–NTA chelator synthesized on gold-sputtered surfaces (via sulphide bonds), or on gold and graphite (via sililating agents) working electrodes of screen-printed devices. Two kinds of proteins were produced and purified for this study: (a) a recombinant antibody, derived from the ‘single-chain Fv’ (scFv) format, and (b) a photosystem II (PSII) core complex isolated from the mutant strain CP43-H of the thermophilic cyanobacterium Synechococcus elongatus. An scFv previously isolated from a synthetic ‘phage display’ library was further engineered with an alkaline phosphatase activity genetically added between the carboxy-terminal of the scFvs and the (His) 6 to allow direct measurement of immobilisation. Renewable specific binding of (His) 6 proteins to gold and graphite surfaces and fast and sensitive electrochemical or optical detection of analytes were obtained. Additionally, “on chip” protein preconcentration was conveniently achieved for biosensing purposes, starting from crude unpurified extracts and avoiding protein purification steps.

Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 Å: insights into the role of solvent molecules in thermal stability and co-factor structure

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 Å resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

Specific binding of a protein to a novel DNA element in the cyanobacterial small heat-shock protein gene

Previously, it was shown that transcription of the small heat-shock protein gene, hspA, from the thermophilic cyanobacterium Synechococcus vulcanus is transiently heat-inducible at a vegetative promoter that lacks any known regulatory DNA elements. A novel regulatory mechanism that suppresses the expression of hspA under non-heat-shock condition has been postulated. In this study, it is demonstrated that a protein(s) in the extract of unstressed cells of the thermophilic cyanobacterium Thermosynechococcus elongatus, a cyanobacterium closely related to S. vulcanus, specifically binds to a 5 ′-untranslated region of the hspA gene. An AT-rich imperfect inverted-repeat sequence (ACAAgcAAA-TTTagTTGT) as a target for a putative DNA-binding protein has been identified. The DNA-binding activity in the cell as well as in the cell extract was lost much more quickly at a heat-shock temperature than a normal growth temperature. In a cell, the activity was restored within 45 min after a heat-shock by the heat-induced synthesis and stabilization of a DNA-binding protein. It is proposed that the inverted repeat is a specific target for a DNA-binding protein and that it plays a role in the regulation of the cyanobacterial hspA gene expression.