Thermophilic Cyanobacterium Phormidium Laminosum Research Articles

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties.

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.

Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode

Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0–90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV–Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands beta 5 and beta 6, loop L5 and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.

Lipid Fatty Acid Composition and Thermophilicity of Cyanobacteria

An analysis of lipid fatty acid composition in several unicellular and filamentous forms of mesophilic and thermophilic cyanobacteria was performed. At 47°C (the temperature of thermophilic cyanobacteria maintenance in the collection), the unicellular thermophilic Synechococcus strains were devoid of polyenoic acids as distinct from the mesophilic forms of this genus at the temperature of 20°C (the temperature of this cyanobacterial maintenance in the collection). In the thermophilic Synechococcus elongatusIPPAS B-267 strain, a decrease in temperature did not result in the occurrence of C18 polyenoic acids, but the quantitative relationship between the saturated and unsaturated fatty acids (S/U ratio) was decreased twofold. In contrast, the culturing of mesophilic strains at 25–32°C resulted in an increase in the S/U ratio due to an increase in the proportion of the 16:0 acid. In the Synechococcus IPPAS B-434 strain, this treatment resulted in a decrease in the relative content of monoenoic, mainly hexadecenoic, acids. The cyanobacterium Gloeobacter violaceus, which lacks thylakoids, and whose photosystems are formed in a cell membrane, contained polyenoic acids. The filamentous thermophilic cyanobacterium Phormidium laminosum, at the maintenance temperature of 47°C, did contain polyenoic acids, but their proportion was considerably lower than that in the filamentous mesophilic forms, such as Tolypothrix sp. and Spirulina platensis. A relative content of hexadecenoic acids in Ph. laminosum was higher than in the mesophilic forms. A possible role of hexadecenoic acids in the processes of adaptation of cyanobacteria to high temperatures is discussed. A relationship between the characteristics of fatty acid composition fixed by evolution and the changes caused by adaptation to a particular environment is considered.

Increased tolerance to thermal inactivation of oxygen evolution in spinach Photosystem II membranes by substitution of the extrinsic 33-kDa protein by its homologue from a thermophilic cyanobacterium

Photosynthetic oxygen evolution is an extremely heat-sensitive process and incubation of spinach Photosystem II (PSII) membranes at 40 °C for only several minutes leads to its complete inactivation. Substitution experiments of the spinach 33-kDa manganese stabilizing protein by a homologue protein, isolated either from the thermophilic cyanobacterium Phormidium laminosum, or from Escherichia coli as a recombinant thermophilic cyanobacterial protein, showed a significant increase in tolerance to heat inactivation of the oxygen-evolving activity. The results allow us to suggest that thermal inactivation of oxygen evolution in higher plant PSII membranes is due to dissociation of the 33-kDa protein as a consequence of temperature-induced conformational changes, and stabilization can be provided by substitution by a thermostable homologue whose secondary structure and binding to PSII remain unaltered at moderately high temperatures.

A comparative study of the thermal stability of plastocyanin, cytochrome c(6) and Photosystem I in thermophilic and mesophilic cyanobacteria.

Cytochrome c(6) (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems.

Nitrate and phosphate ion removal from water by Phormidium laminosum immobilized on hollow fibres in a photobioreactor

Removal of nitrate and phosphate ions from water, by using the thermophilic cyanobacterium Phormidium laminosum, immobilized on cellulose hollow fibres in the tubular photobioreactor at 43 °C, was studied by continuously supplying dilute growth medium for 7 days and then secondarily treated sewage (STS) for 12 days. The concentrations of NO− 3 and PO3− 4 in the effluent from the dilute growth medium decreased from 5.0 mg N/l to 3.1 mg N/l, and from 0.75 mg P/l to 0.05 mg P/l respectively, after a residence time of 12 h. The concentrations of NO− 3 and PO3− 4 in the effluent from STS decreased from 11.7 mg N/l to 2.0 mg N/l, and from 6.62 mg P/l to 0.02 mg P/l respectively, after a residence time of 48 h. The removal rates of nitrogenous␣and phosphate ions from STS were 0.24 and 0.11 mmol day−1 l reactor−1 respectively, under the same conditions. Although, among nitrogenous ions, nitrate and ammonium ions were efficiently removed by P.␣laminosum, the nitrite ion was released into the effluent when STS was used as influent. Treatment of water with thermophilic P. laminosum immobilized on hollow fibres thus appears to be an appropriate means for the removal of inorganic nitrogen and phosphorus from treated wastewater.

Some characteristics of cytochrome f in the cyanobacterium Phormidium laminosum: its sequence and charge properties in the reaction with plastocyanin

Part of the petCA operon was cloned and the sequence of the cytochrome f gene from the moderately thermophilic cyanobacterium Phormidium laminosum determined. A partial sequence of the petC gene encoding the Rieske iron-sulphur protein was also obtained. The cytochrome f gene encodes a mature protein of 385 residues and a leader sequence of 45 residues. The mature protein contains several acidic or neutral residues corresponding to basic residues in the turnip protein. Some of the latter are thought to be important for the interaction with plastocyanin via its ‘eastern’ face. Many of the corresponding residues on the eastern face of P. laminosum plastocyanin are either basic or neutral instead of acidic. These comparisons suggested that the local charges on P. laminosum cytochrome f that are important for its interaction with the homologous plastocyanin may be negative rather than positive. The importance of acidic groups was confirmed by measuring the rates of reduction of horse heart cytochrome c and P. laminosum and spinach plastocyanins by the cytochrome bf complex isolated from P. laminosum. P. laminosum plastocyanin gave the highest rates, which decreased at high ionic strength, confirming the importance of positive local charges on this protein. When extrapolated to infinite ionic strength the rates observed with the two kinds of plastocyanin were similar, but cytochrome c became unreactive. An optimum was observed in the ionic strength response with P. laminosum plastocyanin.

Stabilization of photosystem II particles isolated from the thermophilic cyanobacterium Phormidium laminosum with glycinebetaine and glycerol

Photosystem II particles isolated from the thermophilic cyanobacterium Phormidium laminosum have their O 2 evolution reversibly inhibited by thermally-induced (Rolfe, S.A. and Bendall, D.S. (1989) Biochim. Biophys. Acta 973, 220–226) or NaCl-induced (Stewart, A.C., Ljungberg, U., Akerlund, H.E. and Andersson, B. (1985) Biochim. Biophys. Acta 808, 353–362) dissociation of an extrinsic 9 kDa protein. In the present paper, we show that, with regard to the thermal stress, glycinebetaine, an osmoregulant of halophilic organisms, was effective as glycerol in preventing the dissociation of the 9 kDa protein and the inhibition of O 2 evolution, whereas with regard to the NaCl stress only glycinebetaine was effective. In addition, it is shown that Photosystem II particles which were rendered inactive as a result of NaCl-induced depletion of the 9 kDa protein acquire O 2-evolution competence if resuspended in the presence glycinebetaine but not if resuspended in the presence of glycerol.

Ordered arrays of the photosystem I reaction centre after reconstitution: projections and surface reliefs of the complex at 2 nm resolution.

We present an electron microscopical analysis of the photosystem I reaction centre, the membrane complex involved in the second light-driven step of photosynthetic electron transfer in plants and cyanobacteria. To this end, ordered two-dimensional arrays were reconstituted from detergent solubilized photosystem I reaction centres and phospholipids, and studied by electron microscopy and digital image processing. Small (P1) and large (P3) hexagonal lattices obtained with reaction centres of the thermophilic cyanobacterium Phormidium laminosum had unit cell sizes of a = b = 8.8 nm and 15.8 nm, respectively. Reaction centres of a second thermophilic strain, Synechococcus sp. OD24, gave square lattices (a = b = 14.5 nm; P2(1)). Irrespective of the packing arrangement, projections of negatively stained photosystem I complexes showed elongated asymmetric shapes with a large domain at one end which was tilted with respect to a small domain forming the tip of the other end. Such features were also found in averaged projections of solubilized reaction centre trimers. Surface reliefs reconstructed from freeze-dried metal-shadowed P2(1) lattices revealed that reaction centres had a ridge of 2.5 nm height projecting from one side of the membrane while their other side was rather flat and exhibited a shallow, central indentation.

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the "thylakoid-transfer domain" of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.

The role of an extrinsic 9 kDa polypeptide in oxygen evolution by Photosystem II particles from Phormidium laminosum

Photosystem II particles from the thermophilic cyanobacterium Phormidium laminosum have been reported to contain an extrinsic 9 kDa polypeptide that can be dissociated by exposure to a glycerol-free medium, with concomitant loss of activity (Stewart, A.C., Ljungberg, U., kerlund, H.E. and Andersson, B. (1985) Biochim. Biophys. Acta 808, 353–362; Stewart, A.C., Siczkowski, M. and Ljungberg, U. (1985) FEBS Lett. 193, 175–179). In the present paper it is shown that a partial recovery of oxygen-evolution activity could be achieved by reconcentrating the particles in the presence of glycerol and an excess of the purified polypeptide. This was shown to be a specific effect of the 9 kDa polypeptide which rebound stoichiometrically. Rapid lowering of the glycerol concentration by dilution gave less inactivation but nearly complete reversibility. When diphenylcarbazide was substituted for water as electron donor no loss of activity was observed. It was concluded that dissociation of the 9 kDa polypeptide caused a reversible inactivation of the oxygen-evolution machinery. Evidence was presented that this could be explained by a slowing, but not complete inhibition, of a dark step in the S-statetransitions. No evidence could be obtained for Cl− or Ca2+ concentrating functions of the kind proposed for certain extrinsic polypeptides associated with Photosystem II in chloroplasts.

Structural studies on improved crystals of the photosystem I reaction centre from Phormidium laminosum

Improved crystallization methods led to the growth of large single crystals of the photosystem I reaction centre isolated from the thermophilic cyanobacterium Phormidium laminosum. Although the crystals exhibited a high degree of mosaicity, some preliminary X-ray diffraction studies were performed. The highest order reflections found correspond to interplanar spacings of approx. 0.8 nm. In conjunction with electron microscopy of thin three-dimensional crystals, a triclinic or monoclinic crystal system with unit cell dimensions 30 × 18 × 18 nm was assigned. In addition, our data suggest the presence of four trimeric complexes per unit cell, which may be organised as two face-to-face pairs.

Investigation of the structure of trimeric and monomeric photosystem I reaction centre complexes

Electron microscopy of monomeric and trimeric forms of the reaction centre of photosystem I from the thermophilic cyanobacterium Phormidium laminosum has allowed the construction of a three-dimensional model describing the shape of the complex. The trimeric form of the Photosystem I reaction centre complex was found to have a very regular shape corresponding to a rounded equilateral triangle with edges 18 nm long and a thickness of 6 nm. A distinctive chiral arrangement of the three reaction centres in the trimer could be observed on one face of the complex, whereas the opposing face appeared to be smooth with no distinctive internal features. The monomeric reaction centre is roughly pearshaped, with a length of 15 nm and a width of 9 nm. A thickness of 6 nm is assumed from comparison with the trimer. It is predicted to lie with its shortest axis spanning the membrane. A double-lobed structure, with one lobe larger than the other, was occasionally observed for the monomeric reaction centre. No experimental evidence could be obtained for the existence of the trimeric form in the membrane. The formation of the trimeric form after detergent extraction is suggested. The trimeric form was found to be more stable than the monomeric form in solutions containing anionic and non-ionic detergents.

Investigation of highly stable Photosystem I chlorophyll-protein complexes from the thermophilic cyanobacterium Phormidium laminosum

The photosynthetic complex, Photosystem I, of the thermophilic cyanobacterium Phormidium laminosum was found to be highly resistant to denaturation by ionic and non-ionic detergents including SDS. This property was exploited to allow the preparation by sucrose density gradient centrifugation and by SDS-poly-acrylamide gel electrophoresis of several protein complexes containing the PS I reaction centre components, chlorophyll a and carotenoid. In this report I have focused attention on the three most abundant complexes of estimated molecular masses of 450, 150 and 105 kDa. An initial description of the complexes is presented. The 105 kDa complex resembles plant CP-1 with a very low quantum efficiency for stable charge separation when compared with the 150 and 450 kDa complexes. This deficiency was associated with the loss of two low-molecular-weight polypeptides from the 105 kDa complex. The 450 kDa complex can be separated from the other complexes by centrifugation in a sucrose density gradient, and was found to be the most stable of the complexes in the presence of SDS. All three complexes contain roughly equivalent amounts of the reaction centre component P-700, suggesting that the 450 kDa complex is a multimer of the 150 kDa complex.

Induction of protein synthesis in response to ultraviolet light, nalidixic acid and heat shock in the cyanobacterium Phormidium laminosum

Synthesis of three polypeptides (43, 34 and 26 kDa) was enhanced or induced in the thermophilic cyanobacterium Phormidium laminosum following irradiation with UV light. Synthesis of several polypeptides was also enhanced/induced following treatment with nalidixic acid. Three had similar molecular masses to those induced by UV. One of these (43 kDa) was also apparently enhanced following prolonged heat shock, as were a number of other polypeptides with a wide range of molecular masses. The specific induction of the 34 and 26 kDa polypeptides following DNA damage and inhibition of replication suggests that they may be involved in DNA repair and, possibly, also in recombination.

Crystallization of the photosystem I reaction centre

The reaction centre of the photosynthetic membrane complex photosystem I (PSI) from the thermophilic cyanobacterium Phormidium laminosum was found to crystallize under a range of conditions. The crystallization method, which can occur in the presence of larger detergent molecules than those used previously for the crystallization of membrane proteins, is presented in this report. Several crystal forms have been observed, and some of these show birefringence and linear dichroism. Optical measurements on crystals thicker than 5 microm were severely restricted because of the very high chlorophyll density within the crystals, but linear dichroism measurements on thin single crystals were possible and the results are presented here. By comparing the data with earlier measurements on oriented PSI complexes, a working model for the orientation of the PSI complexes within the crystal could be proposed. The PSI reaction centre is one of the largest and most complex membrane protein units that have been crystallized to date.

Freeze-fracture analysis of thylakoid membranes and photosystem I and II enriched fractions from Phormidium laminosum.

Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have been fractionated into photosystem II and photosystem I particles. These fractions have been characterized by their partial electron transport activities, and biochemical and spectral properties. Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoid membranes were shown to correspond in size to particles in freeze-fractured photosystem II and photosystem I fractions, respectively. Differences between the histograms of the thylakoid membrane protoplasmic fracture face particles and the isolated photosystem I particles suggest that in addition to photosystem I complexes some of the particles on the thylakoid protoplasmic fracture face may be related to cytochrome b/f complexes, the hydrophobic component of the coupling factor, or respiratory complexes.

Electron acceptors of photosystem 2 in the cyanobacterium Phormidium laminosum

Photosystem 2 preparations with very high rates of oxygen evolution from the thermophilic cyanobacterium Phormidium laminosum have been studied by EPR spectrometry. In the presence of DCMU the g = 1.82 signal of the iron—quinone electron acceptor (Q) can be observed. It is proposed that DCMU is necessary to disrupt a magnetic interaction, between the semiquinone forms of Q and the secondary acceptor B, which otherwise prevents detection of the Q −Fe signal. A doublet EPR signal arising from magnetic interaction between Q −Fe and the reduced intermediary electron acceptor pheophytin (I −), and a spin-polarized triplet signal assumed to arise from the back reaction between I − and P680 + can also be seen. Preliminary redox titrations of Q reduction have been carried out, indicating E m ⋍ 0 mV.