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Due to an improved understanding of past climatological conditions, it has now become possible to study the potential concordance between former climatological models and present-day genetic structure. Genetic variability was assessed in 26 samples from different rivers of Atlantic salmon in Iceland (total of 2,352 individuals), using 15 microsatellite loci. F-statistics revealed significant differences between the majority of the populations that were sampled. Bayesian cluster analyses using both prior information and no prior information on sampling location revealed the presence of two distinguishable genetic pools - namely, the Northern (Group 1) and Southern (Group 2) regions of Iceland. Furthermore, the random permutation of different allele sizes among allelic states revealed a significant mutational component to the genetic differentiation at four microsatellite loci (SsaD144, Ssa171, SSsp2201 and SsaF3), and supported the proposition of a historical origin behind the observed variation. The estimated time of divergence, using two different ABC methods, suggested that the observed genetic pattern originated from between the Last Glacial Maximum to the Younger Dryas, which serves as additional evidence of the relative immaturity of Icelandic fish populations, on account of the re-colonisation of this young environment following the Last Glacial Maximum. Additional analyses suggested the presence of several genetic entities which were likely to originate from the original groups detected. [ABSTRACT FROM AUTHOR]

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus has been extensively studied. In this study the isolation and characterization of a third oxygen reductase expressed in this organism are described. This newly isolated enzyme is a typical member of the type B family of haem-copper oxygen reductases, showing 43% amino acid sequence identity and 63% similarity with the ba3 oxygen reductase from Thermus thermophilus. It constitutes two subunits with apparent molecular masses of 42 and 38 kDa. It contains a low-spin B-type haem and a high-spin A-type haem. A stoichiometry of 1B: 1A haem per protein was obtained by spectral integration of UV-visible spectra. Metal analysis showed the presence of two iron and three copper ions, which is in agreement with the existence of a CuA centre. Taking advantage of having two spectroscopically distinct haems, the redox behaviour of the ba3 oxygen reductase was analysed and discussed in the framework of a model with interacting centres. Both haems, B and A, present two transitions, have unusually low reduction potentials of −65 mV and an interaction potential of −52.5 mV. [ABSTRACT FROM AUTHOR]

Abstract: Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes. [Copyright &y& Elsevier]

An a-l-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum ofThermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned intoEscherichia coliand its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 °C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 °C. AraF had aKm value of 0.6 mm andVmax value of 122 U mg-1 onp-nitrophenyl-a-l-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in a-l-arabinofuranosidases in GH family 51. [ABSTRACT FROM AUTHOR]

Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry. Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 °C, the structure of which is presented here to 1.8 A˚ resolution. The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved. The β-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously. A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor. The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S. lividans CelB2. Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity. [Copyright &y& Elsevier]

Abstract: The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo 1 to nqo 7 (nqoA) and nqo 10 to nqo 14 (nqoB)] and two independent genes (nqo 8 and nqo 9 ). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo 13 and nqo 14 . Eight conserved motives to harbour iron–sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed. [Copyright &y& Elsevier]

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (β1→3) linkages preferably and to a lesser extent (β1→6) or (β1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (β1→3)(β1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic β-glucans in B. diazoefficiens., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.

Strains PRI 2268 and PRI 3838(T) were isolated from two separate hot springs in the Torfajokull geothermal area of South Iceland. The cells were non-motile rods, approximately 0.3 microm in width and 1.5-2.5 microm in length. Electron microscopy revealed a Gram-negative cell-wall structure. The strains grew at 45-79 degrees C (optimum, 65 degrees C) and pH 5.5-10.5 (optimum, pH 6.0-7.0). 16S rRNA gene sequence analysis indicated that they formed a separate branch within the genus Thermus with 'Thermus kawarayensis' KW11 as their closest cultured relative (96.5 % similarity). The gene sequence similarities of both new isolates to Thermus aquaticus YT-1(T) and Thermus igniterrae RF-4(T) were 96.1 % and 95.5 %, respectively. DNA-DNA relatedness between strain PRI 3838(T) and 'T. kawarayensis' was 46.1 %. The DNA G+C content of strain PRI 3838(T) was 69.0 mol%. The predominant menaquinones, pigmentation, fatty acid profiles and phospholipid profiles of the novel strains were similar to those of other members of the genus Thermus. However, the new strains could be differentiated from the type strains of all other species of the genus Thermus by their lack of catalase activity and their utilization of only a few carbon sources. Furthermore, the novel strains exhibited mixotrophic growth with sulfur oxidation. On the basis of 16S rRNA gene sequence comparisons, DNA-DNA hybridization and physiological and biochemical characteristics, the new isolates represent a novel species. Since the species appears to be ubiquitous in Icelandic hot springs, the name Thermus islandicus sp. nov. is proposed. The type strain is PRI 3838(T) (=DSM 21543(T)=ATCC BAA-1677(T)).

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus has been extensively studied. In this study the isolation and characterization of a third oxygen reductase expressed in this organism are described. This newly isolated enzyme is a typical member of the type B family of haem-copper oxygen reductases, showing 43% amino acid sequence identity and 63% similarity with the ba3 oxygen reductase from Thermus thermophilus. It constitutes two subunits with apparent molecular masses of 42 and 38 kDa. It contains a low-spin B-type haem and a high-spin A-type haem. A stoichiometry of 1B: 1A haem per protein was obtained by spectral integration of UV-visible spectra. Metal analysis showed the presence of two iron and three copper ions, which is in agreement with the existence of a CuA centre. Taking advantage of having two spectroscopically distinct haems, the redox behaviour of the ba3 oxygen reductase was analysed and discussed in the framework of a model with interacting centres. Both haems, B and A, present two transitions, have unusually low reduction potentials of -65 mV and an interaction potential of -52.5 mV.

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88 degrees C and pH 6.5. The recombinant trehalase exhibited a K(m) of 0.16 mM and a V(max) of 81 micromol of trehalose (min)(-1) (mg of protein)(-1) at the optimal temperature for growth of R. marinus (65 degrees C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K(i) of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADeltasp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.