Extremophiles (Extremophiles)

Publications in this journal

• Article: Evidence for surfactant production by the haloarchaeon Haloferax sp. MSNC14 in hydrocarbon-containing media.

  Ikram Djeridi, Cécile Militon, Vincent Grossi, Philippe Cuny
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  ABSTRACT: The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60-70 %) and pristane (by 30-50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration.
  Extremophiles 06/2013;
• Article: Halomonas socia sp. nov., isolated from high salt culture of Dunaliella salina.

  Jiao Cao, Huai-Yuan Ma, Hai-Yu Li, Kui-Rong Wang, Kun Ruan, Lin-Han Bai
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  ABSTRACT: A moderately halophilic bacteria designed strain NY-011(T) was isolated from the high salt culture of Dunaliella salina in Chengdu of Sichuan Province, China. The isolate was Gram-negative, nonmotile, rod-shaped and 12.5-21.6 μm in length. Colonies on solid media are circular, wet, smooth and cream. The strain grew optimally at 37 °C, pH 7.0 and in the presence of 8 % NaCl. Acid was produced from glycerol, D-arabinose, glucose, trehalose, inositol, mannose, mannitol, sucrose, maltose and sorbitol. Catalase is produced but not oxidase. The major fatty acids are C18: 1ω7c (37.59 %), C19: 0 cyclo ω8c (18.29 %), C16: 0 (16.05 %) and C6: 0 (12.43 %). The predominant respiratory lipoquinone found in strain NY-011(T) is ubiquinone with nine isoprene units (Q-9). The genomic DNA G + C content of strain NY-011(T) was 62.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NY-011(T) belonged to the genus Halomonas. The highest levels of 16S rRNA gene sequence similarity were found between the strain NY-011(T) and H. pantelleriensis (sequence similarity 98.43 %). However, the levels of DNA-DNA relatedness between them were only 23.1 %. In addition, the strain NY-011(T) had a phenotypic profile that readily distinguished it from H. pantelleriensis. The strain NY-011(T) therefore represents a new species of the genus Halomonas, for which the name Halomonas socia sp. nov. is proposed, with NY-011(T) (=CCTCC AB 2011033(T) = KCTC 23671(T)) as the type strain.
  Extremophiles 05/2013;
• Article: The HOG signal transduction pathway in the halophilic fungus Wallemia ichthyophaga: identification and characterisation of MAP kinases WiHog1A and WiHog1B.

  Tilen Konte, Ana Plemenitas
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  ABSTRACT: The high-osmolarity glycerol (HOG) pathway is one of the several MAP kinase cascades in fungi. It is the main signal transduction system that is responsible for cellular stress responses, and has primarily been studied in the context of osmotic stress. In the present study, we provide the first insights into the HOG pathway of the obligatory halophilic basidiomycetous fungus Wallemia ichthyophaga, with the characterisation of its two Hog1-like kinases: WiHog1A and WiHog1B. These share high similarity to Hog1 kinase from Saccharomyces cerevisiae (ScHog1) at the level of amino-acid sequence. While WiHog1A could not optimally complement the function of ScHog1, WiHog1B was a fully functional Hog1-like kinase and could improve the halotolerance of the yeast, compared to the wild-type or the ScHog1-expressing hog1Δ strain. In W. ichthyophaga cells, Hog1 was constitutively phosphorylated under optimal osmotic conditions and dephosphorylated when the cells were challenged with hypo-osmolar or hyperosmolar stress. This pattern of phosphorylation kinetics is opposite to that of yeast. Transcriptional analysis of these two kinases in W. ichthyophaga shows that WiHOG1B is more responsive to changes in NaCl concentrations than WiHOG1A. Our identification and characterisation of these Hog1-like kinases from W. ichthyophaga confirm the existence of the HOG signalling pathway and its role in osmosensing in this halophilic fungus.
  Extremophiles 05/2013;
• Article: How hyperthermophiles adapt to change their lives: DNA exchange in extreme conditions.

  Marleen van Wolferen, Małgorzata Ajon, Arnold J M Driessen, Sonja-Verena Albers
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  ABSTRACT: Transfer of DNA has been shown to be involved in genome evolution. In particular with respect to the adaptation of bacterial species to high temperatures, DNA transfer between the domains of bacteria and archaea seems to have played a major role. In addition, DNA exchange between similar species likely plays a role in repair of DNA via homologous recombination, a process that is crucial under DNA damaging conditions such as high temperatures. Several mechanisms for the transfer of DNA have been described in prokaryotes, emphasizing its general importance. However, until recently, not much was known about this process in prokaryotes growing in highly thermophilic environments. This review describes the different mechanisms of DNA transfer in hyperthermophiles, and how this may contribute to the survival and adaptation of hyperthermophilic archaea and bacteria to extreme environments.
  Extremophiles 05/2013;
• Article: The DNA uptake ATPase PilF of Thermus thermophilus: a reexamination of the zinc content.

  Ralf Salzer, Martin Herzberg, Dietrich H Nies, Goran Biuković, Gerhard Grüber, Volker Müller, Beate Averhoff
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  ABSTRACT: The DNA-translocator ATPase PilF of Thermus thermophilus HB27 is a hexamer built by six identical subunits. Despite the presence of a conserved zinc-binding site in every subunit, only one zinc atom per hexamer was found. Re-examination of the zinc content of PilF purified from cells grown in complex media with different lots of yeast extract revealed six zinc atoms per hexamer. These data demonstrate that the low zinc content reported before was most likely a result of zinc depletion of the yeast extract used.
  Extremophiles 05/2013;
• Article: Response of Acidithiobacillus caldus toward suboptimal pH conditions.

  Stefanie Mangold, Venkateswara Rao Jonna, Mark Dopson
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  ABSTRACT: Maintenance of a circumneutral intracellular pH is important for any organism. Acidophilic microorganisms thrive at low pH while maintaining their intracellular pH around 6.5. However, the mechanisms contributing to acidophile pH homeostasis are not well characterized. The authors investigated the proteomic response and cytoplasmic membrane fatty acid profiles of Acidithiobacillus caldus toward three pH values: 1.1, 2.5, and 4.0. Major rearrangements were observed but lower pH elicited larger changes. Differentially expressed transcription factors suggested tight transcriptional control of pH induced genes. Enzymes involved in sulfur metabolism were up-regulated at pH 1.1 suggesting either that: (1) cells required more energy for maintenance or (2) increased metabolic activity was a specific acid stress response to export intracellular protons via 1° electron transport proton pumps. Furthermore, glutamate decarboxylase, an important enzyme in Escherichia coli acid resistance, was uniquely expressed at pH 1.1. Other proteins previously shown to be involved in neutrophilic acid response, such as spermidine synthase, PspA, and toluene tolerance protein, were differentially expressed in At. caldus but require further investigation to show a direct link to pH homeostasis. Their roles in acidophilic organisms are discussed. Active modulation of fatty acid profiles was detected and suggested a more rigid membrane at low pH.
  Extremophiles 05/2013;
• Article: Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a.

  Praveen Kumar Mehta, Shashi Kant Bhatia, Ravi Kant Bhatia, Tek Chand Bhalla
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  ABSTRACT: A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg(-1). The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5-11.5) and temperature (40-90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K m and V max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min(-1) mg(-1) protein by analyzing Michaelis-Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.
  Extremophiles 05/2013;
• Article: High abundance of heterotrophic prokaryotes in hydrothermal springs of the Azores as revealed by a network of 16S rRNA gene-based methods.

  Kerstin Sahm, Patrick John, Heiko Nacke, Bernd Wemheuer, Ralf Grote, Rolf Daniel, Garabed Antranikian
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  ABSTRACT: Two hydrothermal springs (AI: 51 °C, pH 3; AIV: 92 °C, pH 8) were analysed to determine prokaryotic community composition. Using pyrosequencing, 93,576 partial 16S rRNA gene sequences amplified with V2/V3-specific primers for Bacteria and Archaea were investigated and compared to 16S rRNA gene sequences from direct metagenome sequencing without prior amplification. The results were evaluated by fluorescence in situ hybridization (FISH). While in site AIV Bacteria and Archaea were detected in similar relative abundances (Bacteria 40 %, Archaea 35 %), the acidic spring AI was dominated by Bacteria (68 %). In spring AIV the combination of 16S rRNA gene sequence analysis and FISH revealed high abundance (>50 %) of heterotrophic bacterial genera like Caldicellulosiruptor, Dictyoglomus, and Fervidobacterium. In addition, chemolithoautotrophic Aquificales were detected in the bacterial community with Sulfurihydrogenibium being the dominant genus. Regarding Archaea, only Crenarchaeota, were detected, dominated by the family Desulfurococcaceae (>50 %). In addition, Thermoproteaceae made up almost 25 %. In the acidic spring (AI) prokaryotic diversity was lower than in the hot, slightly alkaline spring AIV. The bacterial community of site AI was dominated by organisms related to the chemolithoautotrophic genus Acidithiobacillus (43 %), to the heterotrophic Acidicaldus (38 %) and to Anoxybacillus (7.8 %). This study reveals differences in the relative abundance of heterotrophic versus autotrophic microorganisms as compared to other hydrothermal habitats. Furthermore, it shows how different methods to analyse prokaryotic communities in complex ecosystems can complement each other to obtain an in-depth picture of the taxonomic composition and diversity within these hydrothermal springs.
  Extremophiles 05/2013;
• Article: Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4.

  Zhangwei Xue, Yi Hu, Shujing Xu, Kouhei Ohnishi, Yanhe Ma, Jiansong Ju, Baohua Zhao
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  ABSTRACT: A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His6-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70-75 °C and 11.0, respectively. The kinetic parameters K m and V max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min(-1) for L-alanine, and 9.95 mM and 702.6 μmol min(-1) for D-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.
  Extremophiles 05/2013;
• Article: Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense.

  Mahsa Moshfegh, Ahmad Reza Shahverdi, Gholamreza Zarrini, Mohammad Ali Faramarzi
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  ABSTRACT: An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K m and V max of the enzyme were determined as 3.8 mg ml(-1) and 12.4 U mg(-1), respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.
  Extremophiles 05/2013;
• Article: An inter-order horizontal gene transfer event enables the catabolism of compatible solutes by Colwellia psychrerythraea 34H.

  R Eric Collins, Jody W Deming
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  ABSTRACT: Colwellia is a genus of mostly psychrophilic halophilic Gammaproteobacteria frequently isolated from polar marine sediments and sea ice. In exploring the capacity of Colwellia psychrerythraea 34H to survive and grow in the liquid brines of sea ice, we detected a duplicated 37 kbp genomic island in its genome based on the abnormally high G + C content. This island contains an operon encoding for heterotetrameric sarcosine oxidase and is located adjacent to several genes used in the serial demethylation of glycine betaine, a compatible solute commonly used for osmoregulation, to dimethylglycine, sarcosine, and glycine. Molecular clock inferences of important events in the adaptation of C. psychrerythraea 34H to compatible solute utilization reflect the geological evolution of the polar regions. Validating genomic predictions, C. psychrerythraea 34H was shown to grow on defined media containing either choline or glycine betaine, and on a medium with sarcosine as the sole organic source of carbon and nitrogen. Growth by 8 of 9 tested Colwellia species on a newly developed sarcosine-based defined medium suggested that the ability to catabolize glycine betaine (the catabolic precursor of sarcosine) is likely widespread in the genus Colwellia. This capacity likely provides a selective advantage to Colwellia species in cold, salty environments like sea ice, and may have contributed to the ability of Colwellia to invade these extreme niches.
  Extremophiles 05/2013;
• Article: The role of disulfide bond in hyperthermophilic endocellulase.

  Han-Woo Kim, Kazuhiko Ishikawa
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  ABSTRACT: The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.
  Extremophiles 04/2013;
• Article: Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.

  Matsujiro Ishibashi, Tomoe Hayashi, Chiho Yoshida, Masao Tokunaga
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  ABSTRACT: Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations.
  Extremophiles 04/2013;
• Article: Planktonic bacterial community composition of an extremely shallow soda pond during a phytoplankton bloom revealed by cultivation and molecular cloning.

  Andrea K Borsodi, Mónika Knáb, Katalin Czeibert, Károly Márialigeti, Lajos Vörös, Boglárka Somogyi
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  ABSTRACT: Böddi-szék is one of the shallow soda ponds located in the Kiskunság National Park, Hungary. In June 2008, immediately prior to drying out, an extensive algal bloom dominated by a green alga (Oocystis submarina Lagerheim) was observed in the extremely saline and alkaline water of the pond. The aim of the present study was to reveal the phylogenetic diversity of the bacterial communities inhabiting the water of Böddi-szék during the blooming event. Using two different selective media, altogether 110 aerobic bacterial strains were cultivated. According to the sequence analysis of the 16S rRNA gene, most of the strains belonged to alkaliphilic or alkalitolerant and moderately halophilic species of the genera Bacillus and Gracilibacillus (Firmicutes), Algoriphagus and Aquiflexum (Bacteroidetes), Alkalimonas and Halomonas (Gammaproteobacteria). Other strains were closely related to alkaliphilic and phototrophic purple non-sulfur bacteria of the genera Erythrobacter and Rhodobaca (Alphaproteobacteria). Analysis of the 16S rRNA gene-based clone library indicated that most of the total of 157 clone sequences affiliated with the anoxic phototrophic bacterial genera of Rhodobaca and Rhodobacter (Alphaproteobacteria), Ectothiorhodospira (Gammaproteobacteria) and Heliorestis (Firmicutes). Phylotypes related to the phylum Bacteroidetes formed the second most abundant group. Clones related to the mainly anaerobic and alkaliphilic bacterial genera of Anoxynatronum (Firmicutes), Spirochaeta (Spirochaetes) and Desulfonatronum (Deltaproteobacteria) were also abundant. Further clone sequences showed less than 95 % similarity values to cultivated species of the phyla Actinobacteria, Cyanobacteria, Deinococcus-Thermus, Fibrobacteres, Gemmatimonadetes and Lentisphaerae.
  Extremophiles 04/2013;
• Article: Characterization of culturable Paenibacillus spp. from the snow surface on the high Antarctic Plateau (DOME C) and their dissemination in the Concordia research station.

  Rob Van Houdt, Marie Deghorain, Marjorie Vermeersch, Ann Provoost, Angelina Lo Giudice, Natalie Leys, David Perez-Morga, Laurence Van Melderen, Luigi Michaud
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  ABSTRACT: Culturable psychrotolerant bacteria were isolated from the top snow on the high Antarctic Plateau surrounding the research station Concordia. A total of 80 isolates were recovered, by enrichment cultures, from two different isolation sites (a distant pristine site [75° S 123° E] and a site near the secondary runway of Concordia). All isolates were classified to the genus Paenibacillus by 16S rRNA gene phylogenetic analysis and belonged to two different species (based on threshold of 97 % similarity in 16S rRNA gene sequence). ERIC-PCR fingerprinting indicated that the isolates from the two different sites were not all clonal. All isolates grew well from 4 to 37 °C and were resistant to ampicillin and streptomycin. In addition, the isolates from the secondary runway were resistant to chromate and sensitive to chloramphenicol, contrary to those from the pristine site. The isolates were compared to 29 Paenibacillus isolates, which were previously recovered from inside the Concordia research station. One of these inside isolates showed ERIC- and REP-PCR fingerprinting profiles identical to those of the runway isolates and was the only inside isolate that was resistant to chromate and sensitive to chloramphenicol. The latter suggested that dissemination of culturable Paenibacillus strains between the harsh Antarctic environment and the inside of the Concordia research station occurred. In addition, inducible prophages, which are potentially involved in horizontal dissemination of genes, were detected in Paenibacillus isolates recovered from outside and inside the station. The highest lysogeny was observed in strains harvested from the hostile environment outside the station.
  Extremophiles 04/2013;
• Article: Archaeal and bacterial diversity in two hot spring microbial mats from a geothermal region in Romania.

  Cristian Coman, Bogdan Drugă, Adriana Hegedus, Cosmin Sicora, Nicolae Dragoş
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  ABSTRACT: The diversity of archaea and bacteria was investigated in two slightly alkaline, mesophilic hot springs from the Western Plain of Romania. Phylogenetic analysis showed a low diversity of Archaea, only three Euryarchaeota taxa being detected: Methanomethylovorans thermophila, Methanomassiliicoccus luminyensis and Methanococcus aeolicus. Twelve major bacterial groups were identified, both springs being dominated by Cyanobacteria, Chloroflexi and Proteobacteria. While at the phylum/class-level the microbial mats share a similar biodiversity; at the species level the geothermal springs investigated seem to be colonized by specific consortia. The dominant taxa were filamentous heterocyst-containing Fischerella, at 45 °C and non-heterocyst Leptolyngbya and Geitlerinema, at 55 °C. Other bacterial taxa (Thauera sp., Methyloversatilis universalis, Pannonibacter phragmitetus, Polymorphum gilvum, Metallibacterium sp. and Spartobacteria) were observed for the first time in association with a geothermal habitat. Based on their bacterial diversity the two mats were clustered together with other similar habitats from Europe and part of Asia, most likely the water temperature playing a major role in the formation of specific microbial communities that colonize the investigated thermal springs.
  Extremophiles 04/2013;
• Article: Isolation and characterization of two novel alkalitolerant sulfidogens from a Thiopaq bioreactor, Desulfonatronum alkalitolerans sp. nov., and Sulfurospirillum alkalitolerans sp. nov.

  D Y Sorokin, T P Tourova, G Muyzer
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  ABSTRACT: Two obligately anaerobic sulfidogenic bacterial strains were isolated from the full-scale Thiopaq bioreactor in Lelystad (The Netherlands) removing H2S from biogas under oxygen-limiting and moderately haloalkaline conditions. Strain HSRB-L represents a dominant culturable sulfate-reducing bacterium in the reactor. It utilizes formate, H2 (with acetate as C-source) and lactate as e-donors, and sulfate, thiosulfate and sulfite as e-acceptors. It is haloalkalitolerant, with a pH range for lithotrophic growth from 7.5 to 9.7 (optimum at 8.5-9) and a salt range from 0.1 to 1.75 M total Na(+) (optimum at 0.6 M). The strain is a member of the genus Desulfonatronum and is proposed as a novel species D. alkalitolerans. The second strain, strain HTRB-L1, represents a dominant thiosulfate/sulfur reducer in the reactor. It is an obligate anaerobe utilizing formate and H2 (with acetate as C-source), lactate, pyruvate and fumarate as e-donors, and thiosulfate (incomplete reduction), sulfur, arsenate and fumarate as e-acceptors. With lactate as e-donor it also grows as an ammonifyer in the presence of nitrate and nitrite. HTRB-L1 is haloalkalitolerant, with a pH range for lithotrophic growth from 7.1 to 9.7 (optimum at 8.5) and a salt range from 0.6 to 1.5 M total Na(+) (optimum at 0.6 M). Phylogenetic analysis showed that strain HTRB-L1 is a novel species within the genus Sulfurospirillum (Epsilonproteobacteria) for which a name Sulfurospirillum alkalitolerans is proposed.
  Extremophiles 04/2013;
• Article: Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

  Jun-Hwan Kim, Kang-Keun Lee, Younguk Sun, Gang-Jin Seo, Sung Suk Cho, Suk Hyung Kwon, Suk-Tae Kwon
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  ABSTRACT: The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.
  Extremophiles 04/2013;
• Article: Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus.

  Fumiaki Tomoike, Seiki Kuramitsu, Ryoji Masui
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  ABSTRACT: The degradation of purine nucleoside is the first step of purine nucleoside uptake. This degradation is catalyzed by purine nucleoside phosphorylase, which is categorized into two classes: hexameric purine nucleoside phosphorylase (6PNP) and trimeric purine nucleoside phosphorylase (3PNP). Generally, 6PNP and 3PNP degrade adenosine and guanosine, respectively. However, the substrate specificity of 6PNP and 3PNP of Thermus thermophilus (tt6PNP and tt3PNP, respectively) is the reverse of that anticipated based on comparison to other phosphorylases. Specifically, in this paper we reveal by gene disruption that tt6PNP and tt3PNP are discrete enzymes responsible for the degradation of guanosine and adenosine, respectively, in T. thermophilus HB8 cells. Sequence comparison combined with structural information suggested that Asn204 in tt6PNP and Ala196/Asp238 in tt3PNP are key residues for defining their substrate specificity. Replacement of Asn204 in tt6PNP with Asp changed the substrate specificity of tt6PNP to that of a general 6PNP. Similarly, substitution of Ala196 by Glu and Asp238 by Asn changed the substrate specificity of tt3PNP to that of a general 3PNP. Our results indicate that the residues at these positions determine substrate specificity of PNPs in general. Sequence analysis further suggested most 6PNP and 3PNP enzymes in thermophilic species belonging to the Deinococcus-Thermus phylum share the same critical residues as tt6PNP and tt3PNP, respectively.
  Extremophiles 04/2013;
• Article: Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulf coasts.

  D M Al-Mailem, M Eliyas, S S Radwan
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  ABSTRACT: Two halophilic, hydrocarbonoclastics bacteria, Marinobacter sedimentarum and M. flavimaris, with diazotrophic potential occured in hypersaline waters and soils in southern and northern coasts of Kuwait. Their numbers were in the magnitude of 10(3) colony forming units g(-1). The ambient salinity in the hypersaline environments was between 3.2 and 3.5 M NaCl. The partial 16S rRNA gene sequences of the two strains showed, respectively, 99 and 100 % similarities to the sequences in the GenBank. The two strains failed to grow in the absence of NaCl, exhibited best growth and hydrocarbon biodegradation in the presence of 1 to 1.5 M NaCl, and still grew and maintained their hydrocarbonoclastic activity at salinities up to 5 M NaCl. Both species utilized Tween 80, a wide range of individual aliphatic hydrocarbons (C9-C40) and the aromatics benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole sources of carbon and energy. Experimental evidence was provided for their nitrogen-fixation potential. The two halophilic Marinobacter strains successfully mineralized crude oil in nutrient media as well as in hypersaline soil and water microcosms without the use of any nitrogen fertilizers.
  Extremophiles 03/2013;