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At least 19 recordsLinked to original sources

PubMed2002

Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis.

The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta-xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic; beta-xylanase activity exhibiting two optima at 55 degrees and 70 degrees C, while beta-xylosidase activity was optimal at 65 degrees C. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon.

Endo-1,4-beta Xylanases↗

PubMed2003

Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei.

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Animals↗

PubMed2002

Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C. Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions.

Adenosine Triphosphatases↗

PubMed2000

Haloterrigena thermotolerans sp. nov., a halophilic archaeon from Puerto Rico.

An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism, designated strain PR5T, is rod-shaped, non-motile and requires at least 12% (w/v) NaCl to grow. The strain is highly thermotolerant: its temperature optimum is 50 degrees C and growth is possible up to 60 degrees C. Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S2-DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. Both C20,C20 and C20,C25 core lipids are present. The G+C content of the DNA is 63.3 mol%. According to 16S rDNA sequence data, strain PR5T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5T cannot be assigned to any of the recognized species within these genera. On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena. The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate. The type strain is strain PR5T (= DSM 11552T = ATCC 700275T).

Base Composition↗

PubMed1996

Halolysin R4, a serine proteinase from the halophilic archaeon Haloferax mediterranei; gene cloning, expression and structural studies.

A gene encoding a halophilic serine proteinase, halolysin R4, from a halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11. The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica). Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively. Removal of this "tail' region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function. Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).

Amino Acid Sequence↗

PubMed2005

Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica.

Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted alpha-amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.

Amino Acid Sequence↗

PubMed1998

Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui.

The genome of the halophilic archaeon Haloarcula marismortui contains two rRNA operons designated rrnA and rrnB. Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized. The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated. The two operons exhibit other profound differences that include (i) the presence in rrnA and the absence in rrnB of tRNAAla and tRNACys genes in the intergenic and distal regions, respectively, (ii) divergent 5' flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA from rrnA operon transcripts and of 23S rRNA from rrnB operon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA from rrnB operon transcripts follows an unusual and different pathway that is apparently devoid of any 5' processing intermediate.

Base Sequence↗

PubMed1998

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico.

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Base Composition↗

PubMed2004

Novel xylose dehydrogenase in the halophilic archaeon Haloarcula marismortui.

During growth of the halophilic archaeon Haloarcula marismortui on D-xylose, a specific D-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to D-xylose, D-ribose was oxidized at similar kinetic constants, whereas D-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)-coding for a 39.9-kDA protein-was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui.

Alcohol Oxidoreductases↗

PubMed1995

Absolute requirement of ammonium sulfate for reconstitution of active 70S ribosomes from the extreme halophilic archaeon Haloferax mediterranei.

Active 70S ribosomes from the halophilic archaeon Haloferax mediterranei have been reconstituted from their isolated rRNAs and proteins. The reconstitution procedure consists of a two-step incubation; first with 1 M ammonium sulfate and 100 mM magnesium acetate for 1 h at 42 degrees C, followed by a 90-min incubation at 50 degrees C after increasing the ammonium sulfate to 2 M final concentration. The total reconstitution of halophilic 70S ribosomes is a process with its own identity, which does not correspond to the conditions required for the reconstitution of the isolated subunits. Ammonium sulfate is the only salt capable of promoting the assembly of active ribosomes. The increase of ammonium sulfate salts in the second incubation step is obligatory for the isolation of functional particles.

Ammonium Sulfate↗

PubMed1996

The structure of the exopolysaccharide produced by the halophilic Archaeon Haloferax mediterranei strain R4 (ATCC 33500).

The halophilic Archaeon Haloferax mediterranei exudes into the growth medium a high molecular weight sulfated polysaccharide. The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulfate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidised/reduced polysaccharides. The location of the sulfate group was established from the 1H and 13C NMR data. The structure of the repeating unit of the polysaccharide may be written as [formula: see text]

Acetylglucosamine↗

PubMed1993

Ch2, a novel halophilic archaeon from an Australian solar saltern.

A novel halophilic archaeon, strain Ch2, was isolated from a marine solar saltern in Geelong, Australia. The fact that this organism had a dam-methylated genome suggested that it is closely related to the taxon that includes Halobacterium saccharovorum, Halobacterium sodomense, and Halobacterium trapanicum. A sequence analysis of the 16S rRNA gene (Ch2 has three copies of this gene) showed that Ch2 is phylogenetically equidistant from the genera Haloarcula and Haloferax and closely related to H. saccharovorum. The susceptibility of both Ch2 and H. saccharovorum to the recently isolated halophage HF2 supported the hypothesis that these two organisms are closely related.

Bacterial Typing Techniques↗

PubMed1995

Halobaculum gomorrense gen. nov., sp. nov., a novel extremely halophilic archaeon from the Dead Sea.

A novel extremely halophilic archaeon was isolated from the Dead Sea. This isolate is rod shaped and, like Halobacterium sodomense, requires a relatively low level of sodium ions for growth and a very high level of magnesium; optimal growth occurs in the presence of 0.6 to 1.0 M Mg2+. The new strain resembles members of the Halobacterium saccharovorum-Halobacterium sodomense-Halobacterium trapanicum group in many physiological properties. However, the polar lipid composition of this organism is characteristic of representatives of the genus Haloferax; a sulfated diglycosyl diether is present, and the glycerol diether analog of phosphatidylglycerosulfate is absent. The G+C content of the DNA is 70 mol%. We found that on the basis of 16S rRNA sequence data our new isolate occupies a position intermediate between the position of the Halobacterium saccharovorum group and the position of the genus Haloferax and is sufficiently different from the previously described members of the Halobacteriaceae to justify classification in a new species and a new genus. We propose the name Halobaculum gomorrense gen. nov., sp. nov. for this organism; the type strain is strain DSM 9297.

Archaea↗

PubMed2004

Facilitated folding and subunit assembly in Escherichia coli and in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag.

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 degrees C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.

Archaea↗

PubMed1996

Evidence for farnesol-mediated isoprenoid synthesis regulation in a halophilic archaeon, Haloferax volcanii.

Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 microM (0.4 microgram/ml) in rich medium and 50 nM (0.01 microgram/ml) in minimal medium without lysis. Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 microM did not affect its growth. Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H. volcanii, but acetate had no such effect. Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction. These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl-CoA) to mevalonate. Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon. Moreover, the cell-free extracts from H. volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP. We conclude that farnesol-mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H. volcanii.

Acetates↗

PubMed1996

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei.

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36 degrees C in the presence of 1.5 M of (NH4)2SO4, 100 mM of MgAc2, 20 mM Tris-HCl (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K+ and NH4+ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg2+ concentration present in the system.

Halobacteriaceae↗

PubMed2006

Halorubrum ezzemoulense sp. nov., a halophilic archaeon isolated from Ezzemoul sabkha, Algeria.

A novel extremely halophilic archaeon was isolated from Ezzemoul sabkha, Algeria. The strain, designated 5.1(T), was neutrophilic, motile and Gram-negative. At least 15 % (w/v) NaCl was required for growth. The isolate grew at pH 6.5-9.0, with optimum growth at pH 7.0-7.5. Mg(2+) was required for growth. Polar lipids were C(20)C(20) derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate and sulfated diglycosyl diether. The genomic DNA G+C content of strain 5.1(T) was 61.9 mol% (T(m)). Phylogenetic analysis based on comparison of 16S rRNA gene sequences revealed that strain 5.1(T) clustered with Halorubrum species. The results of DNA-DNA hybridization and biochemical tests allowed genotypic and phenotypic differentiation of strain 5.1(T) from other Halorubrum species. The name Halorubrum ezzemoulense sp. nov. (type strain 5.1(T)=CECT 7099(T)=DSM 17463(T)) is proposed.

Algeria↗

PubMed2001

Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei.

The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised. H. mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source. The overall purification was 46-fold with about 4% recovery of activity. The enzyme is a monomeric protein of approximately 66 kDa. A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity. Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin. NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H. mediterranei.

Ferredoxins↗