Sample records for haloarchaeon haloferax volcanii

  1. Transcriptional regulation of dimethyl sulfoxide respiration in a haloarchaeon, Haloferax volcanii.

    PubMed

    Qi, Qiuzi; Ito, Yoshiyasu; Yoshimatsu, Katsuhiko; Fujiwara, Taketomo

    2016-01-01

    The halophilic euryarchaeon Haloferax volcanii can grow anaerobically by DMSO respiration. DMSO reductase was induced by DMSO respiration not only under anaerobic growth conditions but also in denitrifying cells of H. volcanii. Deletion of the dmsR gene, encoding a putative regulator for the DMSO reductase, resulted in the loss of anaerobic growth by DMSO respiration. Reporter experiments revealed that only the anaerobic condition was essential for transcription of the dmsEABCD genes encoding DMSO reductase and that transcription was enhanced threefold by supplementation of DMSO. In the ∆dmsR mutant, transcription of the dmsEABCD genes induced by the anaerobic condition was not enhanced by DMSO, suggesting that DmsR is a DMSO-responsive regulator. Transcriptions of the dmsR and mgd genes for Mo-bisMGD biosynthesis were regulated in the same manner as the dmsEABCD genes. These results suggest that the genetic regulation of DMSO respiration in H. volcanii is controlled by at least two systems: one is the DMSO-responsive DmsR, and the other is an unknown anaerobic regulator.

  2. Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

    PubMed Central

    López-García, P; St Jean, A; Amils, R; Charlebois, R L

    1995-01-01

    Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution. PMID:7868620

  3. Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

    PubMed

    Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

    2016-08-15

    The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the

  4. N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function.

    PubMed

    Tamir, Adi; Eichler, Jerry

    2017-03-15

    N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeon Haloferax volcanii was considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surrounding H. volcanii cells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest that H. volcanii could adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function. IMPORTANCE Long held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent

  5. Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

    PubMed Central

    Sumper, M; Berg, E; Mengele, R; Strobel, I

    1990-01-01

    The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988). Images PMID:2123862

  6. Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

    PubMed

    Sumper, M; Berg, E; Mengele, R; Strobel, I

    1990-12-01

    The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).

  7. Biofilms formed by the archaeon Haloferax volcanii exhibit cellular differentiation and social motility, and facilitate horizontal gene transfer.

    PubMed

    Chimileski, Scott; Franklin, Michael J; Papke, R Thane

    2014-08-14

    Archaea share a similar microbial lifestyle with bacteria, and not surprisingly then, also exist within matrix-enclosed communities known as biofilms. Advances in biofilm biology have been made over decades for model bacterial species, and include characterizations of social behaviors and cellular differentiation during biofilm development. Like bacteria, archaea impact ecological and biogeochemical systems. However, the biology of archaeal biofilms is only now being explored. Here, we investigated the development, composition and dynamics of biofilms formed by the haloarchaeon Haloferax volcanii DS2. Biofilms were cultured in static liquid and visualized with fluorescent cell membrane dyes and by engineering cells to express green fluorescent protein (GFP). Analysis by confocal scanning laser microscopy showed that H. volcanii cells formed microcolonies within 24 h, which developed into larger clusters by 48 h and matured into flake-like towers often greater than 100 μm in height after 7 days. To visualize the extracellular matrix, biofilms formed by GFP-expressing cells were stained with concanavalin A, DAPI, Congo red and thioflavin T. Stains colocalized with larger cellular structures and indicated that the extracellular matrix may contain a combination of polysaccharides, extracellular DNA and amyloid protein. Following a switch to biofilm growth conditions, a sub-population of cells differentiated into chains of long rods sometimes exceeding 25 μm in length, compared to their planktonic disk-shaped morphology. Time-lapse photography of static liquid biofilms also revealed wave-like social motility. Finally, we quantified gene exchange between biofilm cells, and found that it was equivalent to the mating frequency of a classic filter-based experimental method. The developmental processes, functional properties and dynamics of H. volcanii biofilms provide insight on how haloarchaeal species might persist, interact and exchange DNA in natural communities. H

  8. Ionic strength-dependent conformations of a ubiquitin-like small archaeal modifier protein (SAMP1) from Haloferax volcanii

    PubMed Central

    Ye, Kaiqin; Liao, Shanhui; Zhang, Wen; Fan, Kai; Zhang, Xuecheng; Zhang, Jiahai; Xu, Chao; Tu, Xiaoming

    2013-01-01

    Eukaryotic ubiquitin and ubiquitin-like systems play crucial roles in various cellular biological processes. In this work, we determined the solution structure of SAMP1 from Haloferax volcanii by NMR spectroscopy. Under low ionic conditions, SAMP1 presented two distinct conformations, one folded β-grasp and the other disordered. Interestingly, SAMP1 underwent a conformational conversion from disorder to order with ion concentration increasing, indicating that the ordered conformation is the functional form of SAMP1 under the physiological condition of H. volcanii. Furthermore, SAMP1 could interact with proteasome-activating nucleotidase B, supposing a potential role of SAMP1 in the protein degradation pathway mediated by proteasome. PMID:23818097

  9. N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

    PubMed

    Yurist-Doutsch, Sophie; Magidovich, Hilla; Ventura, Valeria V; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

    2010-02-01

    Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

  10. Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morgunova, Ekaterina, E-mail: ekaterina.morgunova@ki.se; Gray, Fiona C.; MacNeill, Stuart A.

    2009-10-01

    The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs. The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from themore » halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R{sub free} = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.« less

  11. Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

    PubMed Central

    Pickl, Andreas; Johnsen, Ulrike

    2012-01-01

    The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

  12. Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor.

    PubMed

    Strillinger, Eva; Grötzinger, Stefan Wolfgang; Allers, Thorsten; Eppinger, Jörg; Weuster-Botz, Dirk

    2016-02-01

    The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

  13. Haloferax volcanii archaeosortase is required for motility, mating, and C-terminal processing of the S-layer glycoprotein: Haloferax volcanii archeosortase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abdul Halim, Mohd Farid; Pfeiffer, Friedhelm; Zou, James

    2013-05-28

    Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability.These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S-­layer glycoprotein, is processed and covalently linked tot he cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase-like system for proteolysis-coupled carboxy-terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide themore » first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low-salt conditions, (b) alterations in cell shape and the S-layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S-layer glycoprotein, using detailed proteomic analysis. While the carboxy-terminal region of S-layer glycoproteins, consisting of a threonine-rich O-glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild-type strain. This clearly shows that ArtA is involved in carboxy-terminal posttranslational processing of the S-layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy-terminal region of the S-layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis-coupled, covalent cell surface

  14. Comparative survival analysis of Deinococcus radiodurans and the haloarchaea Natrialba magadii and Haloferax volcanii exposed to vacuum ultraviolet irradiation.

    PubMed

    Abrevaya, Ximena C; Paulino-Lima, Ivan G; Galante, Douglas; Rodrigues, Fabio; Mauas, Pablo J D; Cortón, Eduardo; Lage, Claudia de Alencar Santos

    2011-12-01

    The haloarchaea Natrialba magadii and Haloferax volcanii, as well as the radiation-resistant bacterium Deinococcus radiodurans, were exposed to vacuum UV (VUV) radiation at the Brazilian Synchrotron Light Laboratory. Cell monolayers (containing 10(5) to 10(6) cells per sample) were prepared over polycarbonate filters and irradiated under high vacuum (10(-5) Pa) with polychromatic synchrotron radiation. N. magadii was remarkably resistant to high vacuum with a survival fraction of (3.77±0.76)×10(-2), which was larger than that of D. radiodurans (1.13±0.23)×10(-2). The survival fraction of the haloarchaea H. volcanii, of (3.60±1.80)×10(-4), was much smaller. Radiation resistance profiles were similar between the haloarchaea and D. radiodurans for fluences up to 150 J m(-2). For fluences larger than 150 J m(-2), there was a significant decrease in the survival of haloarchaea, and in particular H. volcanii did not survive. Survival for D. radiodurans was 1% after exposure to the higher VUV fluence (1350 J m(-2)), while N. magadii had a survival lower than 0.1%. Such survival fractions are discussed regarding the possibility of interplanetary transfer of viable microorganisms and the possible existence of microbial life in extraterrestrial salty environments such as the planet Mars and Jupiter's moon Europa. This is the first work to report survival of haloarchaea under simulated interplanetary conditions.

  15. Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

    PubMed Central

    Konrad, Zvia; Eichler, Jerry

    2002-01-01

    Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

  16. A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii*

    PubMed Central

    Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

    2014-01-01

    Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family. PMID:24596091

  17. A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii.

    PubMed

    Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

    2014-04-18

    Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

  18. Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

    DOE PAGES

    Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; ...

    2010-01-01

    Promore » teasomes are composed of 20S core particles (CPs) of α - and β -type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α 1 and α 2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α 1 Thr147, α 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α 1 , thus, revealing a new type of proteasomal modification. bing the biological role of α 1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α 1 variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α 1 . The α 1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.« less

  19. High salinity facilitates dolomite precipitation mediated by Haloferax volcanii DS52

    NASA Astrophysics Data System (ADS)

    Qiu, Xuan; Wang, Hongmei; Yao, Yanchen; Duan, Yong

    2017-08-01

    Although most modern dolomites occur in hypersaline environments, the effects of elevated salinity on the microbial mediation of dolomite precipitation have not been fully evaluated. Here we report results of dolomite precipitation in association with a batch culture of Haloferax volcanii DS52, a halophilic archaeon, under various salinities (from 120‰ to 360‰) and the impact of salinity on microbe-mediated dolomite formation. The mineral phases, morphology and atomic arrangement of the precipitates were analyzed by XRD, SEM and TEM, respectively. The amount of amino acids on the archaeal cell surface was quantified by HPLC/MS. The XRD analysis indicated that disordered dolomite formed successfully with the facilitation of cells harvested from cultures with relatively high salinities (200‰ and 280‰) but was not observed in association with cells harvested from cultures with lower salinity (120‰) or the lysates of cells harvested from extremely high salinity (360‰). The TEM analysis demonstrated that the crystals from cultures with a salinity of 200‰ closely matched that of dolomite. Importantly, we found that more carboxyl groups were presented on the cell surface under high salinity conditions to resist the high osmotic pressure, which may result in the subsequent promotion of dolomite formation. Our finding suggests a link between variations in the hydro-chemical conditions and the formation of dolomite via microbial metabolic activity and enhances our understanding about the mechanism of microbially mediated dolomite formation under high salinity conditions.

  20. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    PubMed Central

    2011-01-01

    Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H

  1. d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

    PubMed Central

    Johnsen, Ulrike; Dambeck, Michael; Zaiss, Henning; Fuhrer, Tobias; Soppa, Jörg; Sauer, Uwe; Schönheit, Peter

    2009-01-01

    The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following 13C-labeling patterns of proteinogenic amino acids after growth on [13C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus. PMID:19584053

  2. GlpR is a direct transcriptional repressor of fructose metabolic genes in Haloferax volcanii.

    PubMed

    Martin, Jonathan H; Rawls, Katie Sherwood; Chan, Jou Chin; Hwang, Sungmin; Martinez-Pastor, Mar; McMillan, Lana J; Prunetti, Laurence; Schmid, Amy K; Maupin-Furlow, Julie A

    2018-06-18

    DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon Haloferax volcanii , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase ( pfkB ) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that Hfx. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators. IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in

  3. Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

    PubMed Central

    St Jean, A; Charlebois, R L

    1996-01-01

    Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed. PMID:8682791

  4. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    PubMed

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  5. RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes

    PubMed Central

    Grosjean, Henri; Gaspin, Christine; Marck, Christian; Decatur, Wayne A; de Crécy-Lagard, Valérie

    2008-01-01

    Background Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes. Results By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions. Conclusion The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation. PMID:18844986

  6. Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

    PubMed

    Abdul Halim, Mohd Farid; Karch, Kelly R; Zhou, Yitian; Haft, Daniel H; Garcia, Benjamin A; Pohlschroder, Mechthild

    2015-12-28

    For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood

  7. Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

    PubMed

    Mazguene, Souhila; Rossi, Mosè; Gogliettino, Marta; Palmieri, Gianna; Cocca, Ennio; Mirino, Sara; Imadalou-Idres, Nacera; Benallaoua, Said

    2018-03-01

    Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

  8. The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

    PubMed Central

    Winter, Jody A; Christofi, Panayiotis; Morroll, Shaun; Bunting, Karen A

    2009-01-01

    Background The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å. Results The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in

  9. A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.

    PubMed

    Brendel, Jutta; Stoll, Britta; Lange, Sita J; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

    2014-03-07

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

  10. A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii*

    PubMed Central

    Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

    2014-01-01

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA. PMID:24459147

  11. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

    PubMed

    Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

    2014-01-01

    The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

  12. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    PubMed Central

    Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

    2014-01-01

    The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

  13. Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei

    PubMed Central

    Liu, Guiming; Cai, Shuangfeng; Hou, Jing; Zhao, Dahe; Han, Jing; Zhou, Jian; Xiang, Hua

    2016-01-01

    Although polyhydroxyalkanoate (PHA) accumulation and mobilization are one of the most general mechanisms for haloarchaea to adapt to the hypersaline environments with changeable carbon sources, the PHA mobilization pathways are still not clear for any haloarchaea. In this study, the functions of five putative (R)-specific enoyl-CoA hydratases (R-ECHs) in Haloferax mediterranei, named PhaJ1 to PhaJ5, respectively, were thoroughly investigated. Through gene deletion and complementation, we demonstrated that only certain of these ECHs had a slight contribution to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis. But significantly, PhaJ1, the only R-ECH that is associated with PHA granules, was shown to be involved in PHA mobilization in this haloarchaeon. PhaJ1 catalyzes the dehydration of (R)-3-hydroxyacyl-CoA, the common product of PHA degradation, to enoyl-CoA, the intermediate of the β-oxidation cycle, thus could link PHA mobilization to β-oxidation pathway in H. mediterranei. This linkage was further indicated from the up-regulation of the key genes of β-oxidation under the PHA mobilization condition, as well as the obvious inhibition of PHA degradation upon inhibition of the β-oxidation pathway. Interestingly, 96% of phaJ-containing haloarchaeal species possess both phaC (encoding PHA synthase) and the full set genes of β-oxidation, implying that the mobilization of carbon storage in PHA through the β-oxidation cycle would be general in haloarchaea. PMID:27052994

  14. Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.

    PubMed

    Liu, Guiming; Cai, Shuangfeng; Hou, Jing; Zhao, Dahe; Han, Jing; Zhou, Jian; Xiang, Hua

    2016-04-07

    Although polyhydroxyalkanoate (PHA) accumulation and mobilization are one of the most general mechanisms for haloarchaea to adapt to the hypersaline environments with changeable carbon sources, the PHA mobilization pathways are still not clear for any haloarchaea. In this study, the functions of five putative (R)-specific enoyl-CoA hydratases (R-ECHs) in Haloferax mediterranei, named PhaJ1 to PhaJ5, respectively, were thoroughly investigated. Through gene deletion and complementation, we demonstrated that only certain of these ECHs had a slight contribution to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis. But significantly, PhaJ1, the only R-ECH that is associated with PHA granules, was shown to be involved in PHA mobilization in this haloarchaeon. PhaJ1 catalyzes the dehydration of (R)-3-hydroxyacyl-CoA, the common product of PHA degradation, to enoyl-CoA, the intermediate of the β-oxidation cycle, thus could link PHA mobilization to β-oxidation pathway in H. mediterranei. This linkage was further indicated from the up-regulation of the key genes of β-oxidation under the PHA mobilization condition, as well as the obvious inhibition of PHA degradation upon inhibition of the β-oxidation pathway. Interestingly, 96% of phaJ-containing haloarchaeal species possess both phaC (encoding PHA synthase) and the full set genes of β-oxidation, implying that the mobilization of carbon storage in PHA through the β-oxidation cycle would be general in haloarchaea.

  15. Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria.

    PubMed Central

    Scoarughi, G L; Cimmino, C; Donini, P

    1995-01-01

    The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates [(p)pp Gpp] in eubacteria via the relA-independent (spoT) pathway. The conditions used were temperature upshift, treatment with cyanide, and total starvation. Under none of these conditions were detectable levels of (p)ppGpp observed. This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp. During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed. The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used. The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae. PMID:7798153

  16. LccA, an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii▿ †

    PubMed Central

    Uthandi, Sivakumar; Saad, Boutaiba; Humbard, Matthew A.; Maupin-Furlow, Julie A.

    2010-01-01

    Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ± 10 mU·ml−1) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U·mg−1 with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO4. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent Km values for SGZ, bilirubin, and ABTS were 35, 236, and 670 μM, with corresponding kcat values of 22, 29, and 10 s−1, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h. PMID:19966030

  17. Halophilic archaebacteria from the Kalamkass oil field

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zvyagintseva, I.S.; Belyaev, S.S.; Borzenkov, I.A.

    1995-01-01

    Two strains of halophilic archaebacteria, growing in a medium containing from 10 to 25% NaCl, were isolated from the brines of the Kalamkass (Mangyshlak) oil field. Both strains are extremely halophilic archaebacteria according to the complex of their phenotypic properties. Strain M-11 was identified as Haloferax mediterranei on the basis of the composition of polar lipids and DNA-DNA homology. The composition of polar lipids and 16S rRNA sequence of strain M-18 allowed us to assign it to the genus Haloferax. This strain differs from the approved species of the genus Haloferax, H. volcanii, and H. mediterranei. However, to describe itmore » as a new species, additional investigations are necessary. 13 refs., 3 figs.« less

  18. Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei

    PubMed Central

    Cai, Shuangfeng; Cai, Lei; Liu, Hailong; Liu, Xiaoqing; Han, Jing; Zhou, Jian

    2012-01-01

    The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The ΔphaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the ΔphaP mutant cells was significantly lower, and in most of the ΔphaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin. PMID:22247127

  19. Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

    PubMed

    Maier, Lisa-Katharina; Benz, Juliane; Fischer, Susan; Alstetter, Martina; Jaschinski, Katharina; Hilker, Rolf; Becker, Anke; Allers, Thorsten; Soppa, Jörg; Marchfelder, Anita

    2015-10-01

    Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

    PubMed Central

    Graf, Michael; Blaby, Ian K.; Makkay, Andrea M.; Starosta, Agata L.; Papke, R. Thane; Oshima, Tairo; Wilson, Daniel N.

    2016-01-01

    Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies. PMID:28053595

  1. Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

    PubMed

    Mengele, R; Sumper, M

    1992-04-25

    The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

  2. A simple laser-based device for simultaneous microbial culture and absorbance measurement

    NASA Astrophysics Data System (ADS)

    Abrevaya, X. C.; Cortón, E.; Areso, O.; Mauas, P. J. D.

    2013-07-01

    In this work we present a device specifically designed to study microbial growth with several applications related to environmental microbiology and other areas of research as astrobiology. The Automated Measuring and Cultivation device (AMC-d) enables semi-continuous absorbance measurements directly during cultivation. It can measure simultaneously up to 16 samples. Growth curves using low and fast growing microorganism were plotted, including Escherichia coli and Haloferax volcanii, a halophilic archaeon.

  3. Analysis of the Transcriptional Regulator GlpR, Promoter Elements, and Posttranscriptional Processing Involved in Fructose-Induced Activation of the Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System in Haloferax mediterranei

    PubMed Central

    Cai, Lei; Cai, Shuangfeng; Zhao, Dahe; Wu, Jinhua; Wang, Lei; Liu, Xiaoqing; Li, Ming; Hou, Jing; Zhou, Jian; Liu, Jingfang; Han, Jing

    2014-01-01

    Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5′ untranslated region (UTR) were observed, and promoter or 5′ UTR swap experiments indicated that the shorter 5′ UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5′ UTR was significantly higher and the ratio of T8 (with the shorter 5′ UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose. PMID:24334671

  4. Biology and survival of extremely halophilic archaeon Haloarcula marismortui RR12 isolated from Mumbai salterns, India in response to salinity stress.

    PubMed

    Thombre, Rebecca S; Shinde, Vinaya D; Oke, Radhika S; Dhar, Sunil Kumar; Shouche, Yogesh S

    2016-05-27

    Haloarchaea are unique microorganism's resistant to environmental and osmotic stresses and thrive in their habitats despite extreme fluctuating salinities. In the present study, haloarchaea were isolated from hypersaline thalossohaline salterns of Bhandup, Mumbai, India and were identified as Haloferax prahovense, Haloferax alexandrines, Haloferax lucentense, Haloarcula tradensis, Haloarcula marismortui and Haloarcula argentinensis. The mechanism of adaptation to contrasting salinities (1.5 M and 4.5 M) was investigated in the extreme haloarchaeon, Hal. marismortui RR12. Hal. marismortui RR12 increased the intracellular sequestration of K(+) and Cl(-) ions in hypo salinity and hyper salinity respectively as detected by Energy-dispersive X-ray spectroscopy microanalysis (EDAX) and Inductively Coupled Plasma- atomic Emission Spectroscopy (ICP-AES) indicating the presence of 'salt-in' strategy of osmoadaptation. As a cellular response to salinity stress, it produced small heat shock like proteins (sHSP) identified using MALDI-TOF MS and increased the production of protective red carotenoid pigment. This is the first report on the study of the concomitant cellular, molecular and physiological mechanism adapted by Hal. marismortui RR12 when exposed to contrasting salinities in external environment.

  5. Post-translational Modification of Extremophilic Proteins: N-glycosylation in Archaea

    DTIC Science & Technology

    2014-12-02

    Kaminski, Z. Guan, S. Yurist-Doutsch, J. Eichler. Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes...Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans, Applied and Environmental Microbiology, (11 2013...Archaea,  N-­‐linked   oligosaccharides  are   assembled  on  dolichol  phosphate  prior  to  transfer  of  the  glycan

  6. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tatmore » pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.« less

  7. Archaea-based microbial fuel cell operating at high ionic strength conditions.

    PubMed

    Abrevaya, Ximena C; Sacco, Natalia; Mauas, Pablo J D; Cortón, Eduardo

    2011-11-01

    In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC's performance. Without any added redox mediator, maximum power (P (max)) and current at P (max) were 11.87/4.57/0.12 μW cm(-2) and 49.67/22.03/0.59 μA cm(-2) for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P (max) was 50.98 and 5.39 μW cm(-2) for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.

  8. Draft Genome Sequence of Haloferax sp. Strain ATB1, Isolated from a Semi-Arid Region in the Brazilian Caatinga.

    PubMed

    Castro, Wendel de Oliveira; Torres-Ballesteros, Adriana Maria; Nakayama, Cristina Rossi; Melo, Itamar Soares; Pellizari, Vivian Helena; Silva, Artur; Ramos, Rommel Thiago Jucá

    2014-08-14

    Organisms in the Haloferax genus are extreme halophiles that grow in environments with pH values between 4 and 12, and temperatures between 0°C and 60°C. In the present study, a draft of the first Haloferax sp. strain ATB1 genome isolated from the region of Cariri (in Paraíba State, Brazil) is presented. Copyright © 2014 Castro et al.

  9. Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents.

    PubMed

    McMillan, Lana J; Hepowit, Nathaniel L; Maupin-Furlow, Julie A

    2016-01-15

    Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

    PubMed Central

    McMillan, Lana J.; Hepowit, Nathaniel L.

    2015-01-01

    Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg−1 for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg2+. Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a “salt-loving” noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies. PMID:26546423

  11. A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

    PubMed Central

    Liu, Guiming; Hou, Jing; Cai, Shuangfeng; Zhao, Dahe; Cai, Lei; Han, Jing; Zhou, Jian

    2015-01-01

    The key enzymes and pathways involved in polyhydroxyalkanoate (PHA) biosynthesis in haloarchaea have been identified in recent years, but the haloarchaeal enzymes for PHA degradation remain unknown. In this study, a patatin-like PHA depolymerase, PhaZh1, was determined to be located on the PHA granules in the haloarchaeon Haloferax mediterranei. PhaZh1 hydrolyzed the native PHA (nPHA) [including native polyhydroxybutyrate (nPHB) and native poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (nPHBV) in this study] granules in vitro with 3-hydroxybutyrate (3HB) monomer as the primary product. The site-directed mutagenesis of PhaZh1 indicated that Gly16, Ser47 (in a classical lipase box, G-X-S47-X-G), and Asp195 of this depolymerase were essential for its activity in nPHA granule hydrolysis. Notably, phaZh1 and bdhA (encoding putative 3HB dehydrogenase) form a gene cluster (HFX_6463 to _6464) in H. mediterranei. The 3HB monomer generated from nPHA degradation by PhaZh1 could be further converted into acetoacetate by BdhA, indicating that PhaZh1-BdhA may constitute the first part of a PHA degradation pathway in vivo. Interestingly, although PhaZh1 showed efficient activity and was most likely the key enzyme in nPHA granule hydrolysis in vitro, the knockout of phaZh1 had no significant effect on the intracellular PHA mobilization, implying the existence of an alternative PHA mobilization pathway(s) that functions effectively within the cells of H. mediterranei. Therefore, identification of this patatin-like depolymerase of haloarchaea may provide a new strategy for producing the high-value-added chiral compound (R)-3HB and may also shed light on the PHA mobilization in haloarchaea. PMID:25710370

  12. Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome

    PubMed Central

    2017-01-01

    Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts. PMID:29154755

  13. Transcription-coupled repair of UV damage in the halophilic archaea.

    PubMed

    Stantial, Nicole; Dumpe, Jarrod; Pietrosimone, Kathryn; Baltazar, Felicia; Crowley, David J

    2016-05-01

    Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) in which excision repair proteins are targeted to RNA polymerase-arresting lesions located in the transcribed strand of active genes. TCR has been documented in a variety of bacterial and eukaryotic organisms but has yet to be observed in the Archaea. We used Halobacterium sp. NRC-1 and Haloferax volcanii to determine if TCR occurs in the halophilic archaea. Following UV irradiation of exponentially growing cultures, we quantified the rate of repair of cyclobutane pyrimidine dimers in the two strands of the rpoB2B1A1A2 and the trpDFEG operons of Halobacterium sp. NRC-1 and the pts operon of H. volcanii through the use of a Southern blot assay and strand-specific probes. TCR was observed in all three operons and was dependent on the NER gene uvrA in Halobacterium sp. NRC-1, but not in H. volcanii. The halophilic archaea likely employ a novel mechanism for TCR in which an as yet unknown coupling factor recognizes the arrested archaeal RNA polymerase complex and recruits certain NER proteins to complete the process. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA*

    PubMed Central

    Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

    2012-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum. PMID:22767603

  15. FtsZ ring: the eubacterial division apparatus conserved in archaebacteria.

    PubMed

    Wang, X; Lutkenhaus, J

    1996-07-01

    FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.

  16. Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples.

    PubMed

    Arahal, D R; Gutiérrez, M C; Volcani, B E; Ventosa, A

    2000-10-01

    A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.

  17. Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Lang, F.

    1991-01-01

    A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

  18. Draft genome sequence of a human-associated isolate of Haloferax alexandrinus strain Arc-hr, an extremely halophilic archaea.

    PubMed

    Khelaifia, S; Caputo, A; Djossou, F; Raoult, D

    2017-01-01

    We report the draft genome sequence of Haloferax alexandrinus strain Arc-hr (CSUR P798), isolated from the human gut of a 10-year-old Amazonian individual. Its 3 893 626 bp genome exhibits a 66.00% GC content. The genome of the strain Arc-hr contains 37 genes identified as ORFans, seven genes associated to halocin and 11 genes associated with polyketide synthases or nonribosomal peptide synthetases.

  19. Construction of a Specialized Cloning Strain of E. Coli for the Nitrate Reductase Genes of Haloferax Denitrificans

    NASA Technical Reports Server (NTRS)

    Johnson, Emmett

    1999-01-01

    This is the final report on Joint Research Interchange (NCC2-5011) "Construction of a Specialized Cloning Strain of E.. coli for the Nitrate Reductase Genes of Haloferax denitrificans." Originally the award was 11/l/93-10/31/95, but there were no-cost extensions made, because of a year Sabbatical at the Pasteur Institute in Paris and other leaves of 3 months each at the Pasteur Institute, during which work could not be done on this project, which extended the closing date to 10/30/98.

  20. Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome, Translation, and RNA Processing System Homologs

    PubMed Central

    Chavarria, Nikita E.; Hwang, Sungmin; Cao, Shiyun; Fu, Xian; Holman, Mary; Elbanna, Dina; Rodriguez, Suzanne; Arrington, Deanna; Englert, Markus; Uthandi, Sivakumar; Söll, Dieter; Maupin-Furlow, Julie A.

    2014-01-01

    While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNALys UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNALys UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems. PMID:24906001

  1. Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

    PubMed

    Chavarria, Nikita E; Hwang, Sungmin; Cao, Shiyun; Fu, Xian; Holman, Mary; Elbanna, Dina; Rodriguez, Suzanne; Arrington, Deanna; Englert, Markus; Uthandi, Sivakumar; Söll, Dieter; Maupin-Furlow, Julie A

    2014-01-01

    While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

  2. Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica.

    PubMed

    Santorelli, Marco; Maurelli, Luisa; Pocsfalvi, Gabriella; Fiume, Immacolata; Squillaci, Giuseppe; La Cara, Francesco; Del Monaco, Giovanni; Morana, Alessandra

    2016-11-01

    An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage.

    PubMed

    Evans, Jessica J; Gygli, Patrick E; McCaskill, Julienne; DeVeaux, Linda C

    2018-04-20

    The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii , causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

  4. The role of Cas8 in type I CRISPR interference.

    PubMed

    Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

    2015-05-05

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

  5. Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

    PubMed

    Cassidy, Jennifer; Bruen, Larah; Rosini, Elena; Molla, Gianluca; Pollegioni, Loredano; Paradisi, Francesca

    2017-01-01

    An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

  6. Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

    PubMed

    Munawar, Nayla; Engel, Paul C

    2013-01-01

    Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

  7. Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei.

    PubMed

    Oren, Aharon; Hallsworth, John E

    2014-10-01

    Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as 'microbial weeds' as defined by Cray et al. (Microb Biotechnol 6: 453-492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a 'microbial weed' than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. Nitrogen metabolism in haloarchaea

    PubMed Central

    Bonete, María José; Martínez-Espinosa, Rosa María; Pire, Carmen; Zafrilla, Basilio; Richardson, David J

    2008-01-01

    The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments. The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3-, NO2- or NH4+ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils. PMID:18593475

  9. Carotenoids from Haloarchaea and Their Potential in Biotechnology

    PubMed Central

    Rodrigo-Baños, Montserrat; Garbayo, Inés; Vílchez, Carlos; Bonete, María José; Martínez-Espinosa, Rosa María

    2015-01-01

    The production of pigments by halophilic archaea has been analysed during the last half a century. The main reasons that sustains this research are: (i) many haloarchaeal species possess high carotenoids production availability; (ii) downstream processes related to carotenoid isolation from haloarchaea is relatively quick, easy and cheap; (iii) carotenoids production by haloarchaea can be improved by genetic modification or even by modifying several cultivation aspects such as nutrition, growth pH, temperature, etc.; (iv) carotenoids are needed to support plant and animal life and human well-being; and (v) carotenoids are compounds highly demanded by pharmaceutical, cosmetic and food markets. Several studies about carotenoid production by haloarchaea have been reported so far, most of them focused on pigments isolation or carotenoids production under different culture conditions. However, the understanding of carotenoid metabolism, regulation, and roles of carotenoid derivatives in this group of extreme microorganisms remains mostly unrevealed. The uses of those haloarchaeal pigments have also been poorly explored. This work summarises what has been described so far about carotenoids production by haloarchaea and their potential uses in biotechnology and biomedicine. In particular, new scientific evidence of improved carotenoid production by one of the better known haloarchaeon (Haloferax mediterranei) is also discussed. PMID:26308012

  10. Carotenoids from Haloarchaea and Their Potential in Biotechnology.

    PubMed

    Rodrigo-Baños, Montserrat; Garbayo, Inés; Vílchez, Carlos; Bonete, María José; Martínez-Espinosa, Rosa María

    2015-08-25

    The production of pigments by halophilic archaea has been analysed during the last half a century. The main reasons that sustains this research are: (i) many haloarchaeal species possess high carotenoids production availability; (ii) downstream processes related to carotenoid isolation from haloarchaea is relatively quick, easy and cheap; (iii) carotenoids production by haloarchaea can be improved by genetic modification or even by modifying several cultivation aspects such as nutrition, growth pH, temperature, etc.; (iv) carotenoids are needed to support plant and animal life and human well-being; and (v) carotenoids are compounds highly demanded by pharmaceutical, cosmetic and food markets. Several studies about carotenoid production by haloarchaea have been reported so far, most of them focused on pigments isolation or carotenoids production under different culture conditions. However, the understanding of carotenoid metabolism, regulation, and roles of carotenoid derivatives in this group of extreme microorganisms remains mostly unrevealed. The uses of those haloarchaeal pigments have also been poorly explored. This work summarises what has been described so far about carotenoids production by haloarchaea and their potential uses in biotechnology and biomedicine. In particular, new scientific evidence of improved carotenoid production by one of the better known haloarchaeon (Haloferax mediterranei) is also discussed.

  11. Extreme halophilic alcohol dehydrogenase mediated highly efficient syntheses of enantiopure aromatic alcohols.

    PubMed

    Alsafadi, Diya; Alsalman, Safaa; Paradisi, Francesca

    2017-11-07

    Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

  12. In Vitro Antioxidant, Antihemolytic, and Anticancer Activity of the Carotenoids from Halophilic Archaea.

    PubMed

    Hou, Jing; Cui, Heng-Lin

    2018-03-01

    Halophilic archaea represent a promising natural source of carotenoids. However, little information is available about the biological effects of carotenoids from halophilic archaea. In this study, the carotenoids produced by seven halophilic archaeal strains Halogeometricum rufum, Halogeometricum limi, Haladaptatus litoreus, Haloplanus vescus, Halopelagius inordinatus, Halogranum rubrum, and Haloferax volcanii were identified by ultraviolet/visible spectroscopy, thin-layer chromatography, and high-performance liquid chromatography-tandem mass spectrometry. The C 50 carotenoids bacterioruberin and its derivatives monoanhydrobacterioruberin and bisanhydrobacterioruberin were found to be the predominant carotenoids. The antioxidant capacities of the carotenoids from these strains were significantly higher than β-carotene as determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. The antihemolytic activities of these carotenoid extracts against H 2 O 2 -induced hemolysis in mouse erythrocytes were 3.9-6.3 times higher than β-carotene. A dose-dependent in vitro antiproliferative activity against HepG2 cells was observed for the extract from Hgm. limi, while that from Hpn. vescus exhibited a relatively high activity in a dose-independent manner. These results suggested that halophilic archaea could be considered as an alternative source of natural carotenoids with high antioxidant, antihemolytic, and anticancer activity.

  13. Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Esclapez, Julia; Britton, K. Linda; Baker, Patrick J.

    2005-08-01

    Single crystals of binary and ternary complexes of wild-type and D38C mutant H. mediterranei glucose dehydrogenase have been obtained by the hanging-drop vapour-diffusion method. Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse themore » significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.« less

  14. Structure and regulation of an archaebacterial promoter: An in vivo study. Progress report, August 1, 1991--March 31, 1993

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Daniels, C.J.

    1993-06-01

    We have established that a 100 bp DNA fragment from the Haloferax volcanii tRNALys gene directs transcription in vivo. This element served as the starting point for a detailed analysis of the requirements for in vivo transcription. Among several gene tentatively identified as reporter elements, we selected a eukaryotic intron-containing tRNAPro gene for when it is driven by the H. volcanii tRNALys promoter fragment, produces a single small transcript. Transcript analysis, by Sl mapping and primer extension, showed that this RNA initiated at the expected tRNALys BoxB sequence and terminated in the tRNAPro RNA Pol III termination element present onmore » the DNA fragment. In initial studies we determined that the 3 inches proximal region of this tRNALys promoter element was sufficient for transcription initiation in vivo. This 40 bp region contains only the BoxA and BoxB regions and short purine rich regions 5 inches to the BoxA and BoxB sequence. Using the tRNAPro gene as the reporter and this minimal promoter, we performed a comprehensive analysis of the BoxA region. Each position of the BoxA region was converted to an four possible nucleotides and the transcription of 36 mutants was quantitated. Among the sites analyzed, only five of the positions showed high levels of discrimination; the preferred BoxA element was 5 inches-TT({sub T}/A)({sup A}/T) ANNNN-3 inches. Mutational analysis demonstrated that a transition from T-rich to A-rich sequences in the BoxA element is essential and that there is some flexibility in the location of the ``TA`` sequence. Additionally the TA sequence appears to determine the location of the transcription start site. The BoxA element defined in this study is similar to those observed for Sulfolobus and the methanogen promoters, and supports the hypothesis that a similar core promoter element is used by all archaeal RNA polymerases.« less

  15. Chaperones and protein folding in the archaea.

    PubMed

    Large, Andrew T; Goldberg, Martin D; Lund, Peter A

    2009-02-01

    A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.

  16. A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T

    PubMed Central

    2012-01-01

    Background Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab

  17. Halocin C8: an antimicrobial peptide distributed among four halophilic archaeal genera: Natrinema, Haloterrigena, Haloferax, and Halobacterium.

    PubMed

    Besse, Alison; Vandervennet, Manon; Goulard, Christophe; Peduzzi, Jean; Isaac, Stéphanie; Rebuffat, Sylvie; Carré-Mlouka, Alyssa

    2017-05-01

    Halophilic archaea thrive in hypersaline ecosystems and produce antimicrobial peptides (AMPs) named halocins. AMPs are essential effectors of microbial interactions in natural ecosystems. Halocin C8 is a 7.4 kDa peptide produced by Natrinema sp. AS7092. Surrounded by genes involved in regulation and transport, the halC8 gene encodes a precursor, processed into the mature halocin and an immunity protein, protecting the producing strain against its halocin. This feature constitutes a unique property of halocin C8, as known AMPs and their immunity proteins are generally encoded on distinct ORFs in an operon. By complementary in silico and PCR-based approaches, the presence of halC8 in halophilic archaea collected from various parts of the world was evidenced. The full-length halC8 gene is restricted and consistently found in the genomes of strains belonging to the phylogenetically related genera Natrinema and Haloterrigena, along with transport and regulation genes. Functional expression of halC8 was demonstrated by RT-PCR and antimicrobial assays. Active halocin C8 was shown to contain five disulphide bridges, presumably conferring a compact structure resistant to harsh environmental conditions. In other archaeal genera, Haloferax and Halobacterium, genes encoding halocin C8 with diverging immunity protein moiety were evidenced. A phylogenetic analysis of halocin C8 sequences was conducted.

  18. Complete genome sequence of 'Halanaeroarchaeum sulfurireducens' M27-SA2, a sulfur-reducing and acetate-oxidizing haloarchaeon from the deep-sea hypersaline anoxic lake Medee.

    PubMed

    Messina, Enzo; Sorokin, Dimitry Y; Kublanov, Ilya V; Toshchakov, Stepan; Lopatina, Anna; Arcadi, Erika; Smedile, Francesco; La Spada, Gina; La Cono, Violetta; Yakimov, Michail M

    2016-01-01

    Strain M27-SA2 was isolated from the deep-sea salt-saturated anoxic lake Medee, which represents one of the most hostile extreme environments on our planet. On the basis of physiological studies and phylogenetic positioning this extremely halophilic euryarchaeon belongs to a novel genus 'Halanaeroarchaeum' within the family Halobacteriaceae. All members of this genus cultivated so far are strict anaerobes using acetate as the sole carbon and energy source and elemental sulfur as electron acceptor. Here we report the complete genome sequence of the strain M27-SA2 which is composed of a 2,129,244-bp chromosome and a 124,256-bp plasmid. This is the second complete genome sequence within the genus Halanaeroarchaeum. We demonstrate that genome of 'Halanaeroarchaeum sulfurireducens' M27-SA2 harbors complete metabolic pathways for acetate and sulfur catabolism and for de novo biosynthesis of 19 amino acids. The genomic analysis also reveals that 'Halanaeroarchaeum sulfurireducens' M27-SA2 harbors two prophage loci and one CRISPR locus, highly similar to that of Kulunda Steppe (Altai, Russia) isolate 'H. sulfurireducens' HSR2(T). The discovery of sulfur-respiring acetate-utilizing haloarchaeon in deep-sea hypersaline anoxic lakes has certain significance for understanding the biogeochemical functioning of these harsh ecosystems, which are incompatible with life for common organisms. Moreover, isolations of Halanaeroarchaeum members from geographically distant salt-saturated sites of different origin suggest a high degree of evolutionary success in their adaptation to this type of extreme biotopes around the world.

  19. Activity-guided separation and characterization of new halocin HA3 from fermented broth of Haloferax larsenii HA3.

    PubMed

    Kumar, Vijay; Tiwari, Santosh Kumar

    2017-05-01

    Haloferax larsenii HA3 was able to grow optimally in HS medium containing 15% NaCl, at pH 7.2 and 42 °C in aerobic conditions. Strain HA3 was found to be round shape, Gram-negative, catalase-positive, sensitive to bile acid, and resistant to chloramphenicol, and could not utilize arginine. The lipid profile revealed the presence of glycerol diether moiety (GDEM) suggesting Haloarchaea characteristics. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that it was closely related to H. larsenii ZJ206. Interestingly, strain HA3 was found to produce halocin HA3 which was purified using ultrafiltration and chromatography. It was found to be stable up to 80 °C, pH 2.0-10.0, organic solvents, surfactants, and detergents tested. However, the activity of halocin HA3 was completely reduced in the presence of proteinase K and trypsin. It was found to be halocidal against H. larsenii HA10, rupturing cell boundary and leading to cell death. The molecular weight of halocin HA3 was found to be ~13 kDa and MALDI-TOF MS/MS analysis suggested no homology with known halocins. The N-terminal ten amino-acid residues, NH 2 MNLGIILETN-COOH, suggested a new/novel halocin. These properties of halocin HA3 may be applicable for control of Haloarchaea in environments and salted foods.

  20. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

    PubMed

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-07-15

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

    PubMed Central

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

  2. Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine

    PubMed Central

    Guymon, Rebecca; Pomerantz, Steven C.; Ison, J. Nicholas; Crain, Pamela F.; McCloskey, James A.

    2007-01-01

    Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80°C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of ∼14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (M r 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii. PMID:17255199

  3. Evolutionary consequences of polyploidy in prokaryotes and the origin of mitosis and meiosis.

    PubMed

    Markov, Alexander V; Kaznacheev, Ilya S

    2016-06-08

    The origin of eukaryote-specific traits such as mitosis and sexual reproduction remains disputable. There is growing evidence that both mitosis and eukaryotic sex (i.e., the alternation of syngamy and meiosis) may have already existed in the basal eukaryotes. The mating system of the halophilic archaeon Haloferax volcanii probably represents an intermediate stage between typical prokaryotic and eukaryotic sex. H. volcanii is highly polyploid, as well as many other Archaea. Here, we use computer simulation to explore genetic and evolutionary outcomes of polyploidy in amitotic prokaryotes and its possible role in the origin of mitosis, meiosis and eukaryotic sex. Modeling suggests that polyploidy can confer strong short-term evolutionary advantage to amitotic prokaryotes. However, it also promotes the accumulation of recessive deleterious mutations and the risk of extinction in the long term, especially in highly mutagenic environment. There are several possible strategies that amitotic polyploids can use in order to reduce the genetic costs of polyploidy while retaining its benefits. Interestingly, most of these strategies resemble different components or aspects of eukaryotic sex. They include asexual ploidy cycles, equalization of genome copies by gene conversion, high-frequency lateral gene transfer between relatives, chromosome exchange coupled with homologous recombination, and the evolution of more accurate chromosome distribution during cell division (mitosis). Acquisition of mitosis by an amitotic polyploid results in chromosome diversification and specialization. Ultimately, it transforms a polyploid cell into a functionally monoploid one with multiple unique, highly redundant chromosomes. Specialization of chromosomes makes the previously evolved modes of promiscuous chromosome shuffling deleterious. This can result in selective pressure to develop accurate mechanisms of homolog pairing, and, ultimately, meiosis. Emergence of mitosis and the first

  4. Mass production of C50 carotenoids by Haloferax mediterranei in using extruded rice bran and starch under optimal conductivity of brined medium.

    PubMed

    Chen, C Will; Hsu, Shu-hui; Lin, Ming-Tse; Hsu, Yi-hui

    2015-12-01

    Microbial carotenoids have potentially healthcare or medical applications. Haloferax mediterranei was difficult to economically grow into a large quantities as well as producing a valuable pigment of carotenoids. This study reports a novel investigation into the optimal conductivity on the mass production of carotenoids from H. mediterranei. The major component at about 52.4% in the extracted red pigment has been confirmed as bacterioruberin, a C50 carotenoids, by liquid chromatography separation and mass spectrometry analysis. By maintaining higher conductivity of 40 S/m in the brined medium, the cell concentration attained to 7.73 × 10(9) cells/L with low pigments concentration of 125 mg/L. When the conductivity was controlled at about 30 S/m, we obtained the highest cell concentration to 1.29 × 10(10) cells/L with pigments of 361.4 mg/L. When the conductivity was maintained at optimal 25 S/m, the pigments can be increased to maximum value of 555.6 mg/L at lower cell concentration of 9.22 × 10(9) cells/L. But conductivity below 20 S/m will cause the significant decrease in cell concentration as well as pigments due to the osmotic stress around the cells. Red pigment of carotenoids from an extremely halophilic archaebacterium could be efficiently produced to a high concentration by applying optimal conductivity control in the brined medium with extruded low-cost rice bran and corn starch.

  5. Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

    PubMed

    Guy, Colin P; Haldenby, Sam; Brindley, Amanda; Walsh, David A; Briggs, Geoffrey S; Warren, Martin J; Allers, Thorsten; Bolt, Edward L

    2006-04-21

    The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.

  6. Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

    PubMed Central

    Mandal, Debabrata; Köhrer, Caroline; Su, Dan; Babu, I. Ramesh; Chan, Clement T.Y.; Liu, Yuchen; Söll, Dieter; Blum, Paul; Kuwahara, Masayasu; Dedon, Peter C.; RajBhandary, Uttam L.

    2014-01-01

    Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes. PMID:24344322

  7. Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts.

    PubMed

    Bhattacharyya, Anirban; Saha, Jayeeta; Haldar, Saubhik; Bhowmic, Asit; Mukhopadhyay, Ujjal Kumar; Mukherjee, Joydeep

    2014-03-01

    Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2% (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD5 and COD values of stillage by 83% was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96% of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.

  8. Integration of poly-3-(hydroxybutyrate-co-hydroxyvalerate) production by Haloferax mediterranei through utilization of stillage from rice-based ethanol manufacture in India and its techno-economic analysis.

    PubMed

    Bhattacharyya, Anirban; Jana, Kuntal; Haldar, Saubhik; Bhowmic, Asit; Mukhopadhyay, Ujjal Kumar; De, Sudipta; Mukherjee, Joydeep

    2015-05-01

    Haloferax mediterranei has potential for economical industrial-scale production of polyhydroxyalkanoate (PHA) as it can utilize cheap carbon sources, has capacity for nonsterile cultivation and allows simple product recovery. Molasses-based Indian distilleries are converting themselves to cereal-based distilleries. Waste stillage (14 l) of rice-based ethanol industry was used for the production of PHA by H. mediterranei in the simple plug-flow reactor configuration of the activated sludge process. Cells utilized stillage and accumulated 63 ± 3 % PHA of dry cell weight and produced 13.12 ± 0.05 g PHA/l. The product yield coefficient was 0.27 while 0.14 g/l h volumetric productivity was reached. Simultaneous lowering of 5-day biochemical oxygen demand and chemical oxygen demand values of stillage by 82 % was attained. The biopolymer was characterized as poly-3-(hydroxybutyrate-co-17.9 mol%-hydroxyvalerate) (PHBV). Directional properties of decanoic acid jointly with temperature-dependent water solubility in decanoic acid were employed for two-step desalination of the spent stillage medium in a cylindrical baffled-tank with an immersed heater and a stirrer holding axial and radial impellers. 99.3 % of the medium salts were recovered and re-used for PHA production. The cost of PHBV was estimated as US$2.05/kg when the annual production was simulated as 1890 tons. Desalination contributed maximally to the overall cost. Technology and cost-analysis demonstrate that PHA production integrated with ethanol manufacture is feasible in India. This study could be the basis for construction of a pilot plant.

  9. Resistance of extremely halophilic archaea to zinc and zinc oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Salgaonkar, Bhakti B.; Das, Deepthi; Bragança, Judith Maria

    2016-02-01

    Industrialization as well as other anthropogenic activities have resulted in addition of high loads of metal and/or metal nanoparticles to the environment. In this study, the effect of one of the widely used heavy metal, zinc (Zn) and zinc oxide nanoparticles (ZnO NPs) on extremely halophilic archaea was evaluated. One representative member from four genera namely Halococcus, Haloferax, Halorubrum and Haloarcula of the family Halobacteriaceae was taken as the model organism. All the haloarchaeal genera investigated were resistant to both ZnCl2 and ZnO NPs at varying concentrations. Halococcus strain BK6 and Haloferax strain BBK2 showed the highest resistance in complex/minimal medium of up to 2.0/1.0 mM ZnCl2 and 2.0/1.0-0.5 mM ZnO NP. Accumulation of ZnCl2/ZnO NPs was seen as Haloferax strain BBK2 (287.2/549.6 mg g-1) > Halococcus strain BK6 (165.9/388.5 mg g-1) > Haloarcula strain BS2 (93.2/28.5 mg g-1) > Halorubrum strain BS17 (29.9/16.2 mg g-1). Scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDX) analysis revealed that bulk ZnCl2 was sorbed at a higher concentration (21.77 %) on the cell surface of Haloferax strain BBK2 as compared to the ZnO NPs (14.89 %).

  10. Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars.

    PubMed

    Oren, Aharon; Elevi Bardavid, Rahel; Mana, Lily

    2014-01-01

    In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.

  11. Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

    PubMed

    Borjian, Farshad; Johnsen, Ulrike; Schönheit, Peter; Berg, Ivan A

    2017-01-01

    Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria ) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C 4 -dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica , succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii , and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as

  12. The Role of Multiple Transcription Factors In Archaeal Gene Expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Charles J. Daniels

    2008-09-23

    Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcaniimore » was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of

  13. Response of Haloalkaliphilic Archaeon Natronococcus Jeotgali RR17 to Hypergravity

    NASA Astrophysics Data System (ADS)

    Thombre, Rebecca S.; Bhalerao, Aniruddha R.; Shinde, Vinaya D.; Dhar, Sunil Kumar; Shouche, Yogesh S.

    2017-06-01

    The survival of archaeabacteria in extreme inhabitable environments on earth that challenge organismic survival is ubiquitously known. However, the studies related to the effect of hypergravity on the growth and proliferation of archaea are unprecedented. The survival of organisms in hypergravity and rocks in addition to resistance to cosmic radiations, pressure and other extremities is imperative to study the possibilities of microbial travel between planets and endurance in hyperaccelerative forces faced during ejection of rocks from planets. The current investigation highlights the growth of an extremophilic archaeon isolated from a rocky substrate in hypergravity environment. The haloalkaliphilic archaeon, Natronococcus jeotgali RR17 was isolated from an Indian laterite rock, submerged in the Arabian sea lining Coastal Maharashtra, India. The endolithic haloarchaeon was subjected to hypergravity from 56 - 893 X gusing acceleration generated by centrifugal rotation. The cells of N. jeotgali RR17 proliferated and demonstrated good growth in hypergravity (223 X g). This is the first report on isolation of endolithic haloarchaeon N. jeotgali RR17 from an Indian laterite rock and its ability to proliferate in hypergravity. The present study demonstrates the ability of microbial life to survive and proliferate in hypergravity. Thus the inability of organismic growth in hypergravity may no longer be a limitation for astrobiology studies related to habitability of substellar objects, brown dwarfs and other planetary bodies in the universe besides planet earth.

  14. Folic acid and pteroylpolyglutamate contents of archaebacteria.

    PubMed Central

    Worrell, V E; Nagle, D P

    1988-01-01

    Cell extracts of methanogens and the thermoacidophile Sulfolobus solfataricus contained little or no folic acid (pteroylglutamate) or pteroylpolyglutamate activity (less than 0.1 nmol/g [dry weight]). However, the halophile Halobacterium salinarum contained pteroylmono- or pteroyldiglutamates, and Halobacterium volcanii and Halobacterium halobium contained pteroyltriglutamates at levels equivalent to those in eubacteria (greater than 1 nmol/g [dry weight]). PMID:3137217

  15. Isolation of hydrocarbon-degrading extremely halophilic archaea from an uncontaminated hypersaline pond (Camargue, France).

    PubMed

    Tapilatu, Yosmina H; Grossi, Vincent; Acquaviva, Monique; Militon, Cécile; Bertrand, Jean-Claude; Cuny, Philippe

    2010-03-01

    Little information exists about the ability of halophilic archaea present in hypersaline environments to degrade hydrocarbons. In order to identify the potential actors of hydrocarbon degradation in these environments, enrichment cultures were prepared using samples collected from a shallow crystallizer pond with no known contamination history in Camargue, France, with n-alkanes provided as source of carbon and energy. Five alkane-degrading halophilic archaeal strains were isolated: one (strain MSNC 2) was closely related to Haloarcula and three (strains MSNC 4, MSNC 14, and MSNC 16) to Haloferax. Biodegradation assays showed that depending on the strain, 32 to 95% (0.5 g/l) of heptadecane was degraded after 30 days of incubation at 40 degrees C in 225 g/l NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This work clearly shows for the first time the potential role of halophilic archaea belonging to the genera Haloarcula and Haloferax in the degradation of hydrocarbons in both pristine and hydrocarbon-contaminated hypersaline environments.

  16. Development of Shuttle Vectors for Halobacteria

    DTIC Science & Technology

    1989-02-01

    the time of application, we had completed a study of transfection, using Halobacterium halobiun and its phage ,DH. We showed (Cline and Doolittle... phage particles), producing plaques titratable on H. halobium lawns. Uptake of DNA by both species appears to be about equally efficient, but DNA from H...halobium-grown phage is restricted in H. volcanii, which shows a 104 - 105 reduction in transfection efficiency (400 plaques per pg) but no reduction

  17. Halosiccatus urmianus gen. nov., sp. nov., a haloarchaeon from a salt lake.

    PubMed

    Mehrshad, Maliheh; Amoozegar, Mohammad Ali; Makhdoumi, Ali; Fazeli, Seyed Abolhassan Shahzadeh; Farahani, Homa; Asadi, Basaer; Schumann, Peter; Ventosa, Antonio

    2016-02-01

    A novel, orange-pigmented, halophilic archaeon, strain DC8 T , was isolated from Urmia salt lake in north-west Iran. The cells of strain DC8 T were non-motile and pleomorphic, from small rods to triangular or disc shaped. The novel strain needed at least 2.5 M NaCl and 0.02 M MgCl 2 for growth. Optimal growth was achieved at 4.0 M NaCl and 0.1 M MgCl 2 . The optimum pH and temperature for growth were pH 7.5 and 45 °C, respectively, and it was able to grow over a pH range of 7.0 to 8.5 and a temperature range of 25 to 55 °C. Analysis of the 16S rRNA gene sequence showed that strain DC8 T was a member of the family Halobacteriaceae ; however, its similarity was as low as 90.1 %, 89.3 % and 89.1 % to the most closely related haloarchaeal taxa, including type species of members of the genera Halosimplex , Halobaculum and Halomicrobium , respectively. The G+C content of its DNA was 68.1 mol%. Polar lipid analyses revealed that strain DC8 T contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and phosphatidic acid. One unknown phospholipid, two major glycolipids and one minor glycolipid were also detected. The only quinone present was MK-8 (II-H 2 ). The physiological, biochemical and phylogenetic differences between strain DC8 T and other extremely halophilic archaeal genera with validly published names supported that this strain represents a novel species of a new genus within the family Halobacteriaceae , for which the name Halosiccatus urmianus gen. nov., sp. nov. is proposed. The type strain is strain DC8 T ( = IBRC-M 10911 T  = CECT 8793 T ).

  18. Halopiger thermotolerans sp. nov., a thermo-tolerant haloarchaeon isolated from commercial salt.

    PubMed

    Minegishi, Hiroaki; Shimogaki, Ryuta; Enomoto, Shigeaki; Echigo, Akinobu; Kondo, Yusuke; Nagaoka, Shuhei; Shimane, Yasuhiro; Kamekura, Masahiro; Itoh, Takashi; Ohkuma, Moriya; Nunoura, Takuro; Takai, Ken; Usami, Ron

    2016-12-01

    Three thermo-tolerant halophilic archaeal strains, SR-441T, SR-412 and SR-188, were isolated from commercial salt samples. Cells were non-motile pleomorphic rod-shaped, and stained Gram-negative. Colonies were pink-pigmented. The three strains were able to grow with 1.7-4.6 M NaCl (optimum, 2.5 M), at pH 6.5-9.0 (optimum, pH 8.0) and at 35-60 °C (optimum, 45 °C). The orthologous 16S rRNA gene sequence similarities amongst the three strains were 98.8-99.3 %, and the level of DNA-DNA relatedness was 71-74 and 72-75 % (reciprocally). The closest relative was Halopiger aswanensis JCM 11628T with 98.6 %-99.1 % similarity in the orthologous 16S rRNA gene sequences, followed by two more Halopiger species, Halopiger xanaduensis JCM 14033T (98.5 %-99.1 %) and Halopiger salifodinae JCM 9578T (95.5 %-95.6 %). DNA-DNA relatednesses between the three strains and H. aswanensis JCM 11628T and H. xanaduensis JCM 14033T were 61 and 54 %, respectively. The polar lipids of the three novel strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and bis-sulfated diglycosyl archaeol-1. The most distinctive feature of the three strains was the ability to grow at 60 °C, while the maximum growth temperature of H. aswanensis is 55 °C. Based on phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halopiger, for which the name Halopiger thermotolerans sp. nov. is proposed. The type strain is SR-441T (=JCM 19583T=KCTC 4248T) isolated from solar salt produced in Australia. SR-412 (=JCM 19582) and SR-188 (=JCM 19581) isolated from commercial salt samples are additional strains of the species.

  19. Halococcus agarilyticus sp. nov., an agar-degrading haloarchaeon isolated from commercial salt.

    PubMed

    Minegishi, Hiroaki; Echigo, Akinobu; Shimane, Yasuhiro; Kamekura, Masahiro; Itoh, Takashi; Ohkuma, Moriya; Usami, Ron

    2015-05-01

    Two agar-degrading halophilic archaeal strains, 62 E(T) and 197 A, were isolated from commercial salt samples. Cells were non-motile cocci, approximately 1.2-2.0 µm in diameter and stained Gram-negative. Colonies were pink-pigmented. Strain 62 E(T) was able to grow with 24-30% (w/v) NaCl (optimum, 27%), at pH 6.5-8.5 (optimum, pH 7.5) and at 22-47 °C (optimum, 42 °C). The 16S rRNA gene sequences of strains 62 E(T) and 197 A were identical, and the level of DNA-DNA relatedness between them was 90 and 90% (reciprocally). The closest relative was Halococcus saccharolyticus JCM 8878(T) with 99.7% similarity in 16S rRNA orthologous gene sequences, followed by Halococcus salifodinae JCM 9578(T) (99.6%), while similarities with other species of the genus Halococcus were equal to or lower than 95.1%. The rpoB' gene tree strongly supported that the two strains were members of the genus Halococcus . Mean DNA-DNA relatedness between strain 62 E(T) and H. saccharolyticus JCM 8878(T) and H. salifodinae JCM 9578(T) was 46 and 44%, respectively. The major polar lipids were archaeol derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 archaeol, and sulfated diglycosyl archaeol-1. Several unidentified glycolipids were present. Based on the phenotypic and phylogenetic analyses, the isolates are considered to represent a novel species of the genus Halococcus , for which the name Halococcus agarilyticus sp. nov. is proposed. The type strain is 62 E(T) ( = JCM 19592(T) =KCTC 4143(T)). © 2015 IUMS.

  20. Susceptibility of Halobacteria to Heavy Metals

    PubMed Central

    Nieto, J. J.; Ventosa, A.; Ruiz-Berraquero, F.

    1987-01-01

    Sixty-eight halobacteria, including both culture collection strains and fresh isolates from widely differing geographical areas, were tested for susceptibility to arsenate, cadmium, chromium, cobalt, copper, lead, mercury, nickel, silver, and zinc ions by an agar dilution technique. The culture collection strains showed different susceptibilities, clustering into five groups. Halobacterium mediterranei and Halobacterium volcanii were the most metal tolerant, whereas Haloarcula californiae and Haloarcula sinaiiensis had the highest susceptibilities of the culture collection strains. Different patterns of metal susceptibility were found for all the halobacteria tested, and there was a uniform susceptibility to mercury and silver. All strains tested were multiply metal tolerant. PMID:16347350

  1. Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon

    PubMed Central

    Sorokin, Dimitry Y; Kublanov, Ilya V; Gavrilov, Sergei N; Rojo, David; Roman, Pawel; Golyshin, Peter N; Slepak, Vladlen Z; Smedile, Francesco; Ferrer, Manuel; Messina, Enzo; La Cono, Violetta; Yakimov, Michail M

    2016-01-01

    Archaea domain is comprised of many versatile taxa that often colonize extreme habitats. Here, we report the discovery of strictly anaerobic extremely halophilic euryarchaeon, capable of obtaining energy by dissimilatory reduction of elemental sulfur using acetate as the only electron donor and forming sulfide and CO2 as the only products. This type of respiration has never been observed in hypersaline anoxic habitats and is the first example of such metabolic capability in the entire Archaea domain. We isolated and cultivated these unusual organisms, selecting one representative strain, HSR2, for detailed characterization. Our studies including physiological tests, genome sequencing, gene expression, metabolomics and [14C]-bicarbonate assimilation assays revealed that HSR2 oxidized acetate completely via the tricarboxylic acid cycle. Anabolic assimilation of acetate occurred via activated glyoxylate bypass and anaplerotic carboxylation. HSR2 possessed sulfurtransferase and an array of membrane-bound polysulfide reductase genes, all of which were expressed during the growth. Our findings suggest the biogeochemical contribution of haloarchaea in hypersaline anoxic environments must be reconsidered. PMID:25978546

  2. Antimicrobial Activity and Mechanism of Inhibition of Silver Nanoparticles against Extreme Halophilic Archaea.

    PubMed

    Thombre, Rebecca S; Shinde, Vinaya; Thaiparambil, Elvina; Zende, Samruddhi; Mehta, Sourabh

    2016-01-01

    Haloarchaea are salt-loving halophilic microorganisms that inhabit marine environments, sea water, salterns, and lakes. The resistance of haloarchaea to physical extremities that challenge organismic survival is ubiquitous. Metal and antibiotic resistance of haloarchaea has been on an upsurge due to the exposure of these organisms to metal sinks and drug resistance genes augmented in their natural habitats due to anthropogenic activities and environmental pollution. The efficacy of silver nanoparticles (SNPs) as a potent and broad spectrum inhibitory agent is known, however, there are no reports on the inhibitory activity of SNPs against haloarchaea. In the present study, we have investigated the antimicrobial potentials of SNPs synthesized using aqueous leaf extract of Cinnamomum tamala against antibiotic resistant haloarchaeal isolates Haloferax prahovense RR8, Haloferax lucentense RR15, Haloarcula argentinensis RR10 and Haloarcula tradensis RR13. The synthesized SNPs were characterized by UV-Vis spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, X-ray diffraction and Fourier transform infrared spectroscopy. The SNPs demonstrated potent antimicrobial activity against the haloarchaea with a minimum inhibitory concentration of 300-400 μg/ml. Growth kinetics of haloarchaea in the presence of SNPs was studied by employing the Baranyi mathematical model for microbial growth using the DMFit curve fitting program. The C. tamala SNPs also demonstrated cytotoxic activity against human lung adenocarcinoma epithelial cell line (A540) and human breast adenocarcinoma cell line (MCF-7). The mechanism of inhibition of haloarchaea by the SNPs was investigated. The plausible mechanism proposed is the alterations and disruption of haloarchaeal membrane permeability by turbulence, inhibition of respiratory dehydrogenases and lipid peroxidation causing cellular and DNA damage resulting in cell death.

  3. In vivo synthesis of selenium nanoparticles by Halococcus salifodinae BK18 and their anti-proliferative properties against HeLa cell line.

    PubMed

    Srivastava, Pallavee; Braganca, Judith M; Kowshik, Meenal

    2014-01-01

    Nanoparticles synthesis by bacteria and yeasts has been widely reported, however, synthesis using halophilic archaea is still in a nascent stage. This study aimed at the intracellular synthesis of selenium nanoparticles (SeNPs) by the haloarchaeon Halococcus salifodinae BK18 when grown in the presence of sodium selenite. Crystallographic characterization of SeNPs by X-ray diffraction, Selected area electron diffraction, and transmission electron microscopy exhibited rod shaped nanoparticles with hexagonal crystal lattice, a crystallite domain size of 28 nm and an aspect ratio (length:diameter) of 13:1. Energy disruptive analysis of X-ray analysis confirmed the presence of selenium in the nano-preparation. The nitrate reductase enzyme assay and the inhibitor studies indicated the involvement of NADH-dependent nitrate reductase in SeNPs synthesis and metal tolerance. The SeNPs exhibited good anti-proliferative properties against HeLa cell lines while being non-cytotoxic to normal cell line model HaCat, suggesting the use of these SeNPs as cancer chemotherapeutic agent. This is the first study on selenium nanoparticles synthesis by haloarchaea. © 2014 American Institute of Chemical Engineers.

  4. Survival and death of the haloarchaeon Natronorubrum strain HG-1 in a simulated martian environment

    NASA Astrophysics Data System (ADS)

    Peeters, Z.; Vos, D.; ten Kate, I. L.; Selch, F.; van Sluis, C. A.; Sorokin, D. Yu.; Muijzer, G.; Stan-Lotter, H.; van Loosdrecht, M. C. M.; Ehrenfreund, P.

    2010-11-01

    Halophilic archaea are of interest to astrobiology due to their survival capabilities in desiccated and high salt environments. The detection of remnants of salty pools on Mars stimulated investigations into the response of haloarchaea to martian conditions. Natronorubrum sp. strain HG-1 is an extremely halophilic archaeon with unusual metabolic pathways, growing on acetate and stimulated by tetrathionate. We exposed Natronorubrum strain HG-1 to ultraviolet (UV) radiation, similar to levels currently prevalent on Mars. In addition, the effects of low temperature (4, -20, and -80 °C), desiccation, and exposure to a Mars soil analogue from the Atacama desert on the viability of Natronorubrum strain HG-1 cultures were investigated. The results show that Natronorubrum strain HG-1 cannot survive for more than several hours when exposed to UV radiation equivalent to that at the martian equator. Even when protected from UV radiation, viability is impaired by a combination of desiccation and low temperature. Desiccating Natronorubrum strain HG-1 cells when mixed with a Mars soil analogue impaired growth of the culture to below the detection limit. Overall, we conclude that Natronorubrum strain HG-1 cannot survive the environment currently present on Mars. Since other halophilic microorganisms were reported to survive simulated martian conditions, our results imply that survival capabilities are not necessarily shared between phylogenetically related species.

  5. Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon.

    PubMed

    Cheng, Feiyue; Gong, Luyao; Zhao, Dahe; Yang, Haibo; Zhou, Jian; Li, Ming; Xiang, Hua

    2017-11-20

    Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600-800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  6. Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

    PubMed

    Fairley, D J; Wang, G; Rensing, C; Pepper, I L; Larkin, M J

    2006-12-01

    Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

  7. Diversity of halophilic archaea from six hypersaline environments in Turkey.

    PubMed

    Ozcan, Birgul; Ozcengiz, Gulay; Coleri, Arzu; Cokmus, Cumhur

    2007-06-01

    The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.

  8. Sequencing of Seven Haloarchaeal Genomes Reveals Patterns of Genomic Flux

    PubMed Central

    Lynch, Erin A.; Langille, Morgan G. I.; Darling, Aaron; Wilbanks, Elizabeth G.; Haltiner, Caitlin; Shao, Katie S. Y.; Starr, Michael O.; Teiling, Clotilde; Harkins, Timothy T.; Edwards, Robert A.; Eisen, Jonathan A.; Facciotti, Marc T.

    2012-01-01

    We report the sequencing of seven genomes from two haloarchaeal genera, Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ∼20×coverage and assembled to an average of 50 contigs (range 5 scaffolds - 168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology. PMID:22848480

  9. Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece

    PubMed Central

    Mancinelli, Rocco L.; Landheim, Ragnhild; Sanchez-Porro, Cristina; Dornmayr-Pfaffenhuemer, Marion; Gruber, Claudia; Legat, Andrea; Ventosa, Antonio; Radax, Christian; Ihara, Kunio; White, Melisa R.; Stan-Lotter, Helga

    2011-01-01

    Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2–97.1 %. The DNA–DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75 %, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2 %, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5–66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T =NCIMB 14426T =ATCC BAA-1602T). PMID:19567575

  10. Precise determination, cross-recognition, and functional analysis of the double-strand origins of the rolling-circle replication plasmids in haloarchaea.

    PubMed

    Zhou, Ligang; Zhou, Meixian; Sun, Chaomin; Han, Jing; Lu, Qiuhe; Zhou, Jian; Xiang, Hua

    2008-08-01

    The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO(Z)) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

  11. Utilization of Virus ϕCh1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii

    PubMed Central

    Mayrhofer-Iro, M.; Ladurner, A.; Meissner, C.; Derntl, C.; Reiter, M.; Haider, F.; Dimmel, K.; Rössler, N.; Klein, R.; Baranyi, U.; Scholz, H.

    2013-01-01

    In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus ϕCh1. The shuttle vector pRo-5, based on the repH-containing region of ϕCh1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The ϕCh1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of ϕCh1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus ϕCh1 in their extreme environments. PMID:23416999

  12. Haloarchaea and the Formation of Gas Vesicles

    PubMed Central

    Pfeifer, Felicitas

    2015-01-01

    Halophilic Archaea (Haloarchaea) thrive in salterns containing sodium chloride concentrations up to saturation. Many Haloarchaea possess genes encoding gas vesicles, but only a few species, such as Halobacterium salinarum and Haloferax mediterranei, produce these gas-filled, proteinaceous nanocompartments. Gas vesicles increase the buoyancy of cells and enable them to migrate vertically in the water body to regions with optimal conditions. Their synthesis depends on environmental factors, such as light, oxygen supply, temperature and salt concentration. Fourteen gas vesicle protein (gvp) genes are involved in their formation, and regulation of gvp gene expression occurs at the level of transcription, including the two regulatory proteins, GvpD and GvpE, but also at the level of translation. The gas vesicle wall is solely formed of proteins with the two major components, GvpA and GvpC, and seven additional accessory proteins are also involved. Except for GvpI and GvpH, all of these are required to form the gas permeable wall. The applications of gas vesicles include their use as an antigen presenter for viral or pathogen proteins, but also as a stable ultrasonic reporter for biomedical purposes. PMID:25648404

  13. Characterization and antimicrobial potential of extremely halophilic archaea isolated from hypersaline environments of the Algerian Sahara.

    PubMed

    Quadri, Inès; Hassani, Imene Ikrame; l'Haridon, Stéphane; Chalopin, Morgane; Hacène, Hocine; Jebbar, Mohamed

    2016-01-01

    Halophilic archaea were isolated from different chotts and sebkha, dry salt lakes and salt flat respectively, of the Algerian Sahara and characterized using phenotypic and phylogenetic approaches. From 102 extremely halophilic strains isolated, forty three were selected and studied. These strains were also screened for their antagonistic potential and the production of hydrolytic enzymes. Sequencing of the 16S rRNA genes and phylogenetic analysis allowed the identification of 10 archaeal genera within the class Halobacteria: Natrinema (13 strains), Natrialba (12 strains), Haloarcula (4 strains), Halopiger (4 strains), Haloterrigena (3 strains), Halorubrum (2 strains), Halostagnicola (2 strains), Natronococcus, Halogeometricum and Haloferax (1 strain each). The most common producers of antimicrobial compounds belong to the genus Natrinema while the most hydrolytic isolates, with combined production of several enzymes, belong to the genus Natrialba. The strain affiliated to Halopiger djelfamassilliensis was found to produce some substances of interest (halocins, anti-Candida, enzymes). After partial purification and characterization of one of the strains Natrinema gari QI1, we found similarities between the antimicrobial compound and the halocin C8. Therefore, the gene encoding halocin C8 was amplified and sequenced. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

    PubMed Central

    Ding, Yan; Meyer, Benjamin H.; Albers, Sonja-Verena; Kaminski, Lina; Eichler, Jerry

    2014-01-01

    SUMMARY N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus. PMID:24847024

  15. The Function of Gas Vesicles in Halophilic Archaeaand Bacteria: Theories and Experimental Evidence

    PubMed Central

    Oren, Aharon

    2012-01-01

    A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms. PMID:25371329

  16. Haloarchaea Endowed with Phosphorus Solubilization Attribute Implicated in Phosphorus Cycle

    PubMed Central

    Yadav, Ajar Nath; Sharma, Divya; Gulati, Sneha; Singh, Surender; Dey, Rinku; Pal, Kamal Krishna; Kaushik, Rajeev; Saxena, Anil Kumar

    2015-01-01

    Archaea are unique microorganisms that are present in ecological niches of high temperature, pH and salinity. A total of 157 archaea were obtained from thirteen sediment, water and rhizospheric soil samples collected from Rann of Kutch, Gujarat, India. With an aim to screen phosphate solubilizing archaea, a new medium was designed as Haloarchaea P Solubilization (HPS) medium. The medium supported the growth and P solubilization activity of archaea. Employing the HPS medium, twenty isolates showed the P-solubilization. Phosphate solubilizing archaea were identified as seventeen distinct species of eleven genera namely Haloarcula, Halobacterium, Halococcus, Haloferax, Halolamina, Halosarcina, Halostagnicola, Haloterrigena, Natrialba, Natrinema and Natronoarchaeum. Natrinema sp. strain IARI-WRAB2 was identified as the most efficient P-solubilizer (134.61 mg/L) followed by Halococcus hamelinensis strain IARI-SNS2 (112.56 mg/L). HPLC analysis detected seven different kinds of organic acids, namely: gluconic acid, citric acid, formic acid, fumaric acid succinic acid, propionic acid and tartaric acid from the cultures of these isolates. These phosphate solubilizing halophilic archaea may play a role in P nutrition to vegetation growing in these hypersaline soils. This is the first report for these haloarchaea to solubilize considerable amount of P by production of organic acids and lowering of pH. PMID:26216440

  17. Expression, purification, crystallization and preliminary X-ray analysis of the Met244Ala variant of catalase–peroxidase (KatG) from the haloarchaeon Haloarcula marismortui

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ten-i, Tomomi; Kumasaka, Takashi; Higuchi, Wataru

    2007-11-01

    The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. The covalent modification of the side chains of Trp95, Tyr218 and Met244 within the active site of Haloarcula marismortui catalase–peroxidase (KatG) appears to be common to all KatGs and has been demonstrated to be particularly significant formore » its bifunctionality [Smulevich et al. (2006 ▶), J. Inorg. Biochem.100, 568–585; Jakopitsch, Kolarich et al. (2003 ▶), FEBS Lett.552, 135–140; Jakopitsch, Auer et al. (2003 ▶), J. Biol. Chem.278, 20185–20191; Jakopitsch et al. (2004 ▶), J. Biol. Chem.279, 46082–46095; Regelsberger et al. (2001 ▶), Biochem. Soc. Trans.29, 99–105; Ghiladi, Knudsen et al. (2005 ▶), J. Biol. Chem.280, 22651–22663; Ghiladi, Medzihradzky et al. (2005 ▶), Biochemistry, 44, 15093–15105]. The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant showed a complete loss of catalase activity, whereas the peroxidase activity of this mutant was highly enhanced owing to an increase in its affinity for the peroxidatic substrate. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. A crystal frozen using lithium sulfate as the cryoprotectant diffracted to beyond 2.0 Å resolution. Preliminary X-ray analysis suggests the presence of a dimer in the asymmetric unit.« less

  18. An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

    PubMed

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

    2015-02-13

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

    PubMed Central

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J.; Backofen, Rolf; Marchfelder, Anita

    2015-01-01

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

  20. Membrane-acting bacteriocin purified from a soil isolate Pediococcus pentosaceus LB44 shows broad host-range.

    PubMed

    Kaur, Ramanjeet; Tiwari, Santosh Kumar

    2018-04-15

    Bacteriocin LB44 was purified from cell-free supernatant (CFS) of Pediococcus pentosaceus LB44 using activity-guided chromatography techniques. It was stable up to 121 °C, pH 2.0-6.0, sensitive to proteinase K, papain and trypsin, and retained complete activity in the presence of organic solvents tested. The molecular weight of bacteriocin was ∼6 kDa and initial ten amino acid residues (GECGMCXECG) suggested a new compound. The loss in viable cell count and K + ion efflux of target cells of Micrococcus luteus suggested bactericidal activity. The cell membrane of bacteriocin-treated cells was found to be ruptured which was further confirmed by Fourier Transform Infrared (FTIR) analysis suggesting interaction of bacteriocin with phospholipids in cell membrane. It showed broad host-range and inhibited the growth of Lactobacillus delbrueckii NRRL B-4525, L. plantarum NRRL B-4496, L. acidophilus NRRL B-4495, Enterococcus hirae LD3, Weissella confusa LM85, Staphylococcus aureus, Salmonella typhi ATCC 13311, Serratia marcescens ATCC 27137, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 29905, Haloferax larsenii HA1, HA3, HA8, HA9 and HA10. These properties suggested a new bacteriocin from soil isolate P. pentosaceus LB44 which may offers possible applications in food-safety and therapeutics. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Responses of Haloarchaea to Simulated Microgravity

    NASA Astrophysics Data System (ADS)

    Dornmayr-Pfaffenhuemer, Marion; Legat, Andrea; Schwimbersky, Karin; Fendrihan, Sergiu; Stan-Lotter, Helga

    2011-04-01

    Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites).

  2. Cleanroom Maintenance Significantly Reduces Abundance but Not Diversity of Indoor Microbiomes.

    PubMed

    Mahnert, Alexander; Vaishampayan, Parag; Probst, Alexander J; Auerbach, Anna; Moissl-Eichinger, Christine; Venkateswaran, Kasthuri; Berg, Gabriele

    2015-01-01

    Cleanrooms have been considered microbially-reduced environments and are used to protect human health and industrial product assembly. However, recent analyses have deciphered a rather broad diversity of microbes in cleanrooms, whose origin as well as physiological status has not been fully understood. Here, we examined the input of intact microbial cells from a surrounding built environment into a spacecraft assembly cleanroom by applying a molecular viability assay based on propidium monoazide (PMA). The controlled cleanroom (CCR) was characterized by ~6.2*103 16S rRNA gene copies of intact bacterial cells per m2 floor surface, which only represented 1% of the total community that could be captured via molecular assays without viability marker. This was in contrast to the uncontrolled adjoining facility (UAF) that had 12 times more living bacteria. Regarding diversity measures retrieved from 16S rRNA Illumina-tag analyzes, we observed, however, only a minor drop in the cleanroom facility allowing the conclusion that the number but not the diversity of microbes is strongly affected by cleaning procedures. Network analyses allowed tracking a substantial input of living microbes to the cleanroom and a potential enrichment of survival specialists like bacterial spore formers and archaeal halophiles and mesophiles. Moreover, the cleanroom harbored a unique community including 11 exclusive genera, e.g., Haloferax and Sporosarcina, which are herein suggested as indicators of cleanroom environments. In sum, our findings provide evidence that archaea are alive in cleanrooms and that cleaning efforts and cleanroom maintenance substantially decrease the number but not the diversity of indoor microbiomes.

  3. Cleanroom Maintenance Significantly Reduces Abundance but Not Diversity of Indoor Microbiomes

    PubMed Central

    Mahnert, Alexander; Vaishampayan, Parag; Probst, Alexander J.; Auerbach, Anna; Moissl-Eichinger, Christine; Venkateswaran, Kasthuri; Berg, Gabriele

    2015-01-01

    Cleanrooms have been considered microbially-reduced environments and are used to protect human health and industrial product assembly. However, recent analyses have deciphered a rather broad diversity of microbes in cleanrooms, whose origin as well as physiological status has not been fully understood. Here, we examined the input of intact microbial cells from a surrounding built environment into a spacecraft assembly cleanroom by applying a molecular viability assay based on propidium monoazide (PMA). The controlled cleanroom (CCR) was characterized by ~6.2*103 16S rRNA gene copies of intact bacterial cells per m2 floor surface, which only represented 1% of the total community that could be captured via molecular assays without viability marker. This was in contrast to the uncontrolled adjoining facility (UAF) that had 12 times more living bacteria. Regarding diversity measures retrieved from 16S rRNA Illumina-tag analyzes, we observed, however, only a minor drop in the cleanroom facility allowing the conclusion that the number but not the diversity of microbes is strongly affected by cleaning procedures. Network analyses allowed tracking a substantial input of living microbes to the cleanroom and a potential enrichment of survival specialists like bacterial spore formers and archaeal halophiles and mesophiles. Moreover, the cleanroom harbored a unique community including 11 exclusive genera, e.g., Haloferax and Sporosarcina, which are herein suggested as indicators of cleanroom environments. In sum, our findings provide evidence that archaea are alive in cleanrooms and that cleaning efforts and cleanroom maintenance substantially decrease the number but not the diversity of indoor microbiomes. PMID:26273838

  4. Archaeal Production of Polyhydroxyalkanoate (PHA) Co- and Terpolyesters from Biodiesel Industry-Derived By-Products

    PubMed Central

    Hermann-Krauss, Carmen; Koller, Martin; Stelzer, Franz; Braunegg, Gerhart

    2013-01-01

    The archaeon Haloferax mediterranei was selected for production of PHA co- and terpolyesters using inexpensive crude glycerol phase (CGP) from biodiesel production as carbon source. CGP was assessed by comparison with the application of pure glycerol. Applying pure glycerol, a copolyester with a molar fraction of 3-hydroxybutyrate (3HB) of 0.90 mol/mol and 3-hydroxyvalerate (3HV) of 0.10 mol/mol, was produced at a volumetric productivity of 0.12 g/Lh and an intracellular PHA content of 75.4 wt.-% in the sum of biomass protein plus PHA. Application of CGP resulted in the same polyester composition and volumetric productivity, indicating the feasibility of applying CGP as feedstock. Analysis of molar mass distribution revealed a weight average molar mass M w of 150 kDa and polydispersity P i of 2.1 for pure glycerol and 253 kDa and 2.7 for CGP, respectively; melting temperatures ranged between 130 and 140°C in both setups. Supplying γ-butyrolactone as 4-hydroxybutyrate (4HB) precursor resulted in a poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate-co-4-hydroxybutyrate] (PHBHV4HB) terpolyester containing 3HV (0.12 mol/mol) and 4HB (0.05 mol/mol) in the poly[(R)-3-hydroxybutyrate] (PHB) matrix; in addition, this process runs without sterilization of the bioreactor. The terpolyester displayed reduced melting (melting endotherms at 122 and 137°C) and glass transition temperature (2.5°C), increased molar mass (391 kDa), and a polydispersity similar to the copolyesters. PMID:24453697

  5. Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. nov. and description of Natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid).

    PubMed

    Hezayen, F F; Rehm, B H; Tindall, B J; Steinbüchel, A

    2001-05-01

    A novel extremely halophilic member of the Archaea, strain 40T, was isolated from Egypt (Aswan). This isolate requires at least 1.6 M sodium chloride for growth and exhibits optimal growth between 37 and 42 degrees C. Determination of the entire 16S rRNA gene sequence revealed the highest similarity to the type strain of Natrialba asiatica (> 99%). Polar lipid analysis indicated that strain 40T and Natrialba asiatica have essentially identical compositions, indicating that the former is a member of genus Natrialba. However, physiological and biochemical data provided evidence that Natrialba asiatica strains B1T and 172P1T, as well as strain 40T, are sufficiently different to be divided in three different species. The G+C content of strain 40T was 61.5+/-0.6 mol%. In addition, DNA-DNA hybridization data supported the placement of the isolate in a new species in the genus Natrialba, Natrialba aegyptiaca sp. nov., and indicated that Natrialba asiatica strain B1T should also be placed in a separate species, Natrialba taiwanensis sp. nov. Morphological studies of strain 40T indicated clearly that this isolate appears in three completely different cell shapes (cocci, rods, tetrads) under different conditions of growth, including different sodium chloride concentrations and different growth temperatures. Another interesting property of strain 40T is the ability to produce an extracellular polymer, which was found to be composed predominantly of glutamic acid (85% w/w), representing poly(glutamic acid), carbohydrates (12.5% w/w) and unidentified compounds (2.5% w/w). Among the Archaea, production of an extracellular polysaccharide has been described for some members of the genera Haloferax and Haloarcula.

  6. Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India.

    PubMed

    Singh, Aparna; Singh, Anil Kumar

    2018-01-01

    Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl 3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

  7. Recent studies in microbial degradation of petroleum hydrocarbons in hypersaline environments

    PubMed Central

    Fathepure, Babu Z.

    2014-01-01

    Many hypersaline environments are often contaminated with petroleum compounds. Among these, oil and natural gas production sites all over the world and hundreds of kilometers of coastlines in the more arid regions of Gulf countries are of major concern due to the extent and magnitude of contamination. Because conventional microbiological processes do not function well at elevated salinities, bioremediation of hypersaline environments can only be accomplished using high salt-tolerant microorganisms capable of degrading petroleum compounds. In the last two decades, there have been many reports on the biodegradation of hydrocarbons in moderate to high salinity environments. Numerous microorganisms belonging to the domain Bacteria and Archaea have been isolated and their phylogeny and metabolic capacity to degrade a variety of aliphatic and aromatic hydrocarbons in varying salinities have been demonstrated. This article focuses on our growing understanding of bacteria and archaea responsible for the degradation of hydrocarbons under aerobic conditions in moderate to high salinity conditions. Even though organisms belonging to various genera have been shown to degrade hydrocarbons, members of the genera Halomonas Alcanivorax, Marinobacter, Haloferax, Haloarcula, and Halobacterium dominate the published literature. Despite rapid advances in understanding microbial taxa that degrade hydrocarbons under aerobic conditions, not much is known about organisms that carry out similar processes in anaerobic conditions. Also, information on molecular mechanisms and pathways of hydrocarbon degradation in high salinity is scarce and only recently there have been a few reports describing genes, enzymes and breakdown steps for some hydrocarbons. These limited studies have clearly revealed that degradation of oxygenated and non-oxygenated hydrocarbons by halophilic and halotolerant microorganisms occur by pathways similar to those found in non-halophiles. PMID:24795705

  8. Stoichiometric and kinetic analysis of extreme halophilic Archaea on various substrates in a corrosion resistant bioreactor.

    PubMed

    Lorantfy, Bettina; Seyer, Bernhard; Herwig, Christoph

    2014-01-25

    Extreme halophilic Archaea are extremophile species which can thrive in hypersaline environments of up to 3-5 M sodium chloride concentration. Although their ecology and physiology are widely identified on the microbiological level, little emphasis has been laid on quantitative bioprocess development with extreme halophiles. The goal of this study was to establish, on the one hand, a methodological basis for quantitative bioprocess analysis of extreme halophilic Archaea with an extreme halophilic strain as an example. Firstly, as a novel usage, a corrosion resistant bioreactor setup for extreme halophiles has been implemented. Then, paying special attention to total bioprocess quantification approaches, an indirect method for biomass quantification using on-line process signals was introduced. Subsequently, robust quantitative data evaluation methods for halophiles could be developed, providing defined and controlled cultivation conditions in the bioreactor and therefore obtaining suitable quality of on-line as well as off-line datasets. On the other hand, new physiological results of extreme halophiles in bioreactor have also been obtained based on the quantitative methodological tools. For the first time, quantitative data on stoichiometry and kinetics were collected and evaluated on different carbon sources. The results on various substrates were interpreted, with proposed metabolic mechanisms, by linking to the reported primary carbon metabolism of extreme halophilic Archaea. Moreover, results of chemostat cultures demonstrated that extreme halophilic organisms show Monod-kinetics on different sole carbon sources. A diauxic growth pattern was described on a mixture of substrates in batch cultivations. In addition, the methodologies presented here enable one to characterize the utilized strain Haloferax mediterranei (HFX) as a potential new host organism. Thus, this study offers a strong methodological basis as well as a fundamental physiological assessment for

  9. Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring.

    PubMed

    Elshahed, Mostafa S; Najar, Fares Z; Roe, Bruce A; Oren, Aharon; Dewers, Thomas A; Krumholz, Lee R

    2004-04-01

    The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via

  10. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Swanson, Juliet S.; Reed, Donald T.; Ams, David A.

    . Although these were most likely introduced into the WIPP as contaminants from above-ground, their survival and potential role in the WIPP (e.g., cellulose degradation) is under investigation. WIPP groundwaters comprise the far-field microbial environment. Bacteria cultivated and identified from the overlying Culebra and nearby borehole groundwater are capable of aerobic respiration, denitrification, fermentation, metal reduction, and sulfate reduction and are distributed across many different phyla. Two of the Bacteria found in groundwater were also found in WIPP halite (Chromohalobacter sp. and Virgibacillus sp.). Archaea identified in groundwater include Halococcus saccharolyticus, Haloferax sp., and Natrinema sp. The differences in the microbial communities detected thus far in halite and groundwater suggest that there will be significant differences in the associated metabolic potential of the near- and far-field environments. Whereas the near-field is dominated by Archaea with more limited metabolic capabilities, the far-field is dominated by Bacteria with extremely broad capabilities. Because the majority of the repository's lifetime will be anoxic, ongoing and future work focuses on the presence and role of anaerobic organisms in WIPP. Further tasks on biosorption, cellulose degradation, and bioreduction are being performed using organisms obtained from this characterization work.« less

  11. Astrobiological Significance of Microbial Extremophiles

    NASA Technical Reports Server (NTRS)

    Pikuta, Elena V.; Hoover, Richard B.

    2007-01-01

    The microflora of the cryosphere of planet Earth provides the best analogs for life forms that might be found in the permafrost or polar ice caps of Mars, near the surface of the cometary nuclei, or in the liquid water beneath and the ice crusts of icy moons of Jupiter and Saturn. The importance of study alkaliphilic microorganisms for astrobiology was enhanced by the findings of abundant carbonates and carbonate globules rimmed with possibly biogenic magnetites in association with the putative microfossils in the ALH84001 meteorite. Although the ALH84001 "nanofossils" were to small and simple to be unambiguously recognized as biogenic, they stimulated Astrobiology research and studies of microbial extremophiles and biomarkers in ancient rocks and meteorites. Recent studies of CI and CM carbonaceous meteorites have resulted in the detection of the well-preserved mineralized remains of coccoidal and filamentous microorganisms in cyanobacterial mats. Energy Dispersive X-ray Analysis has shown anomalous biogenic element ratios clearly indicating they are not recent biological contaminants. This paper reviews microbial extremophiles in context of their significance to Astrobiology. The study of halophilic microorganisms was started from work with saline soils and lakes, and one of the record of good growth for Haloferax mediterranei was shown at 30 percent NaC1. Although alkali-tolerant nitrifying bacteria had previously been reported, the first described alkaliphilic microorganism was the bacterium Streptococcus faecalis. Halophilic and alkaliphilic forms are relevant to conditions that might be found in closed impact basins and craters on Mars filled with evaporite deposits. The first obligately acidophilic bacterium described was Acidithiobacillus ferrooxydans (formally Thiobacillus ferrooxidans). Later thermophilic lithotrophic acidophiles were found, and the hyperacidophilic moderately thermophilic species of the genus Picrophilus were found to grow at negative p