Photoactive yellow protein from the halophilic bacterium Salinibacter ruber

A gene for photoactive yellow protein (PYP) was identified from the genome sequence of the extremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to the prototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acid residues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosynthetic genes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, we cloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis from Rhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPs that appears to be important for dimerization; however, truncation of these extra residues did not change the spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is at shorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversibly bleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPs reported to date (4.27 x 10(-4) s(-1) at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 and I2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperature and alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nm form, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizes the 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated 6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropic series. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature is increased from 74 degrees C in buffer alone to 92 degrees C in 2 M KCl. Sr accumulates KCl in the cytoplasm, like Halobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP is an example of a halophilic protein that requires KCl to electrostatically screen the excess negative charge and stabilize the tertiary structure.     

Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by a small number of sugars (glucose, maltose, starch at 1 g l(-1)). Glucose consumption starts after other substrates have been depleted. Glucose metabolism proceeds via a constitutive, salt-inhibited hexokinase and a constitutive salt-dependent nicotinamide adenine dinucleotide phosphate (NADP)-linked glucose-6-phosphate dehydrogenase. Glucose dehydrogenase and fructose-1,6-bisphosphate aldolase activity could not be detected. It is therefore suggested that Salinibacter metabolizes glucose by the classic Entner-Doudoroff pathway and not by the Embden-Meyerhof glycolytic pathway or by the modified Entner-Doudoroff pathway present in halophilic Archaea of the family Halobacteriaceae, in which the phosphorylation step is postponed. However, activity of 2-keto-3-deoxy-6-phosphogluconate aldolase could not be detected in extracts of Salinibacter cells, whether or not grown in the presence of glucose.     

Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber

Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S. ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]- of m/z 660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13'-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol. 153:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta 528:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.     

Glycerol metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by glycerol. In contrast to glucose consumption, which starts only after more easily degradable substrates present in yeast extract have been depleted, glycerol is consumed during the earliest growth phases. When U-(14)C-labeled glycerol was added to the culture, up to 25% of the radioactivity was incorporated by the cells. Glycerol kinase activity was detected only in cells grown in the presence of glycerol (up to 90 nmol mg protein(-1) min(-1)). This enzyme functioned over salt concentrations from 0.6 to 2.8 M KCl. No significant activity of NAD-dependent glycerol dehydrogenase was found. It is suggested that Salinibacter may use glycerol as one of its principal substrates in its habitat, the saltern crystallizer ponds.     

Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber

Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Delta9cis and C18:1 Delta11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

A new dehydrogenase specific towards aromatic aldehydes from a halophilic bacterium

A new enzyme showing a dehydrogenase activity towards aromatic aldehydes was isolated, purified and characterized from a halophilic strain isolated from saline environment. The enzyme is a monomer of 54 kDa; it is rather thermostable (optimal temperature: 50 degrees C) showing a broad spectrum of activity in a large pH range with the maximum at pH 9.5. The substrate specificity and the effect of ions were evaluated and compared with analogous described proteins.     

Purification and properties of malate dehydrogenase from the extreme thermophileBacillus caldolyticus

The enzyme malate dehydrogenase (EC 1.1.1.37) from an extreme thermophileB. Caldolyticus was purified to about 91% homogeneity. The molar mass of the enzyme was determined as 73 000 daltons and it is composed of two subunits, each with a molar mass of 37 000. Initial velocity studies with oxaloacetic acid and NADH as substrates at pH 8.1, over a range of temperatures, indicate that the enzyme operates via a sequential type mechanism. Van't Hoff plots of the kinetic parameters displayed sharp changes in slope at characteristic temperatures, whereas the Arrhenius plot exhibited no such breaks over the temperature interval investigated. The enzyme was found to be stable at 41°C and lower temperatures. At 51°C and 59°C an almost immediate 20% reduction in activity was obtained, but no further inactivation occurred during the 60 min of incubation. At 59°C the enzyme lost 50% of its initial activity in about 38 s. High concentration of NADH was observed to greatly stabilize the enzyme at that temperature.It is suggested that the slope changes in the Van't Hoff plots and the stability profies at 51°C and 59°C are representative of a temperature induced conformational change in the enzyme.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

High-pressure adaptation by salt stress in a moderately halophilic bacterium obtained from open seawater

High-pressure adaptation was examined using a moderately halophilic bacterium (Micrococcus roseus), which was isolated from open seawater and capable of growing in 15% w/v NaCl (optimum NaCl concentration: 3% w/v). After treatment at 207 MPa, colony-forming units (CFUs) significantly decreased; however, the loss of integral cells after pressure was only 30% when direct cell count was performed microscopically. In order to investigate the piezotolerance of M. roseus under high pressure without morphological change, the survival of cells was examined under pressure at 138 MPa for 2 h. M. roseus in 3% NaCl was still sensitive to pressure at 138 MPa. However, the cells in the third generations showed remarkably increased pressure resistance, and no significant loss of viability was confirmed. Furthermore, when M. roseus was cultured in 1, 3, 5, 10 and 15% NaCl, the survival ratio proportionally increased at increased NaCl concentration. M. roseus cultured in 15% NaCl was remarkably resistant (94.7% viability) to pressure at 138 MPa, even when suspended in lower concentration of NaCl. This suggests that NaCl concentrations in growth culture affect the piezotolerance of M. roseus and that this species has an ability to adapt to high pressure.     

Influence of an anion-binding site in the stabilization of halophilic malate dehydrogenase from Haloarcula marismortui

Halophilic proteins have evolved to be soluble, stable and active in high salt concentration. Crystallographic studies have shown that surface enrichment by acidic amino acids is a common structural feature of halophilic proteins. In addition, ion-binding sites have also been observed in most of the cases. The role of chloride-binding sites in halophilic adaptation was addressed in a site-directed mutagenesis study of tetrameric malate dehydrogenase from Haloarcula marismortui. The mutation of K 205, which is involved in an inter-subunit chloride-binding site, drastically modified the enzyme stability in the presence of KCl, but not in the presence of KF. The oligomeric state of the [K205A] mutant changes with the nature of the anion. At high salt concentration, the [K205A] mutant is a dimer when the anion is a chloride ion, whereas it is a tetramer when the fluoride ion is used. The results highlight the role of anion-binding sites in protein adaptation to high salt conditions.     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

Purification and characterization of malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB

Abstract Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold. The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa. The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 °C. The K _m for oxaloacetate was 50 μM and for NADH 30 μM. The K _m values for l-malate and NAD were 4 and 1.1 mM, respectively. Substrate inhibition was found at oxaloacetate concentrations higher than 250 μM. The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms.     

Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation

The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NACl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively.     

Physicochemical properties of malate dehydrogenase from the bacterium Rhodopseudomonas palustris strain f8pt

Electrophoretically homogenous isoforms of malate dehydrogenase with different quaternary structure were prepared from Rhodopseudomonas palustris strain f8pt cultured photolithoheterotrophically on malate and acetate. By selective inhibition of the tricarboxylic acid cycle or glyoxylate cycle, it was shown that the dimeric isoform of the enzyme is responsible for Krebs cycle functioning and the tetrameric isoform is involved in functioning of the glyoxylate cycle.     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.     

Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI ( approximately 40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.     

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.     

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD⁺ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.