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Dennis H. Bamford - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for assembly of vertical single β-barrel viruses.
Nature communications, 2019Co-Authors: Isaac Santos-pérez, Hanna M. Oksanen, Dennis H. Bamford, David Gil-carton, Diego Charro, Mikel Azkargorta, Felix Elortza, Nicola G. A. AbresciaAbstract:The vertical double β-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 A resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single β-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double β-barrel MCPs. Here, the authors present the cryo-EM structures of two archaeal, halophilic, internal membrane-containing icosahedral viruses at 3.7 and 3.8 A resolution, providing insights into the assembly process of these and related PRD1-adeno lineage viruses.
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The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules.
Genes, 2018Co-Authors: Nina S. Atanasova, Tatiana A. Demina, Dennis H. Bamford, Camilla Heiniö, Hanna M. OksanenAbstract:Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.
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Vesicle-like virion of Haloarcula hispanica pleomorphic virus 3 preserves high infectivity in saturated salt.
Virology, 2016Co-Authors: Tatiana A. Demina, Nina S. Atanasova, Maija K. Pietilä, Hanna M. Oksanen, Dennis H. BamfordAbstract:Hypersaline environments that are subject to salinity changes are particularly rich in viruses. Here we report a newly isolated archaeal halovirus, Haloarcula hispanica pleomorphic virus 3 (HHPV3). Its reproduction significantly retards host growth and decreases cell viability without causing lysis. HHPV3 particles require a minimum of 3M NaCl for stability and maintain high infectivity even in saturated salt. Notably, virions are irreversibly inactivated at ~1.5M NaCl in neutral pH, but tolerate this salinity at alkaline pH. The HHPV3 virion is a pleomorphic membrane vesicle containing two major protein species and lipids acquired nonselectively from the host membrane. The circular double-stranded DNA genome contains a conserved gene block characteristic of pleolipoviruses. We propose that HHPV3 is a member of the Betapleolipovirus genus (family Pleolipoviridae). Our findings add insights into the diversity observed among the pleolipoviruses found in hypersaline environments.
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Monitoring physiological changes in haloarchaeal cell during virus release
Viruses, 2016Co-Authors: Julija Svirskaitė, Hanna M. Oksanen, Rimantas Daugelavičius, Dennis H. BamfordAbstract:The slow rate of adsorption and non-synchronous release of some archaeal viruses have hindered more thorough analyses of the mechanisms of archaeal virus release. To address this deficit, we utilized four viruses that infect Haloarcula hispanica that represent the four virion morphotypes currently known for halophilic euryarchaeal viruses: (1) icosahedral internal membrane-containing SH1; (2) icosahedral tailed HHTV-1; (3) spindle-shaped His1; and (4) pleomorphic His2. To discern the events occurring as the progeny viruses exit, we monitored culture turbidity, as well as viable cell and progeny virus counts of infected and uninfected cultures. In addition to these traditional metrics, we measured three parameters associated with membrane integrity: the binding of the lipophilic anion phenyldicarbaundecaborane, oxygen consumption, and both intra- and extra-cellular ATP levels.
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Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.
Structure (London England : 1993), 2015Co-Authors: David Gil-carton, Hanna M. Oksanen, Dennis H. Bamford, Salla T. Jaakkola, Diego Charro, Bibiana Peralta, Daniel Castaño-díez, Nicola G. A. AbresciaAbstract:Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly.
Hua Xiang - One of the best experts on this subject based on the ideXlab platform.
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Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.
Molecular microbiology, 2020Co-Authors: Caixia Pei, Hua Xiang, Jing Han, Hui Zhou, Jerry Eichler, Cheng JinAbstract:Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.
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CTP synthase forms cytoophidia in archaea.
Journal of genetics and genomics = Yi chuan xue bao, 2020Co-Authors: Shuang Zhou, Hua Xiang, Ji-long LiuAbstract:CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.
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CTP synthase forms cytoophidia in archaea
2019Co-Authors: Shuang Zhou, Hua Xiang, Ji-long LiuAbstract:Abstract CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of de novo synthesis of the nucleotide CTP. Since 2010, a series studies have demonstrated that CTPS can form filamentous structures termed cytoophidia in bacteria and eukaryotes. However, it remains unknown whether cytoophidia exist in archaea, the third domain of life. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaeal cells. Under stimulated emission depletion (STED) microscopy, we find that those HhCTPS compartments are elongated filamentous structure, resembling cytoophidia in bacteria and eukaryotes. When Haloarcula cells cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. Moreover, overexpression CTPS or glutamine analog treatment promote cytoophidium assembly in H. hispanica. Our study reveals that CTPS forms cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS. Summary Author CTP synthase (CTPS), as a textbook molecule, has been studied biochemically for almost 70 years. It catalyses the last step of making up the nucleotide CTP. Since biochemical properties and the regulatory mechanisms of CTPS were thoroughly studied in the past decades, it came as a surprise when a new feature of CTPS was revealed in 2010. Multiple studies reveal that CTPS can form snakeshaped structures termed cytoophidia in bacteria and eukaryotes. However, it is not clear whether cytoophidia exist in archaea, the third domain of life. Here we use halophilic archaeon Haloarcula hispanica as a model and show that CTPS can form defined structures in archaea. Using super resolution microscopy, we confirm that those CTPS-containing structures are elongated filaments, similar to cytoophidia described in bacteria and eukaryotic cells. Therefore, this study demonstrates that CTPS forms cytoophidia not only in bacteria and eukaryotes, but also in archaea.
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primed adaptation tolerates extensive structural and size variations of the crispr rna guide in Haloarcula hispanica
Nucleic Acids Research, 2019Co-Authors: Luyao Gong, Dahe Zhao, Ming Li, Feiyue Cheng, Yihua Chen, Hua XiangAbstract:: Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3' handle, which seriously impaired crRNA stability and interference effects. With 3' handle deletion, further truncation of 5' handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5' handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.
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Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon.
Journal of genetics and genomics = Yi chuan xue bao, 2017Co-Authors: Feiyue Cheng, Jian Zhou, Dahe Zhao, Haibo Yang, Luyao Gong, Hua XiangAbstract:Abstract 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.
Hanna M. Oksanen - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for assembly of vertical single β-barrel viruses.
Nature communications, 2019Co-Authors: Isaac Santos-pérez, Hanna M. Oksanen, Dennis H. Bamford, David Gil-carton, Diego Charro, Mikel Azkargorta, Felix Elortza, Nicola G. A. AbresciaAbstract:The vertical double β-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 A resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single β-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double β-barrel MCPs. Here, the authors present the cryo-EM structures of two archaeal, halophilic, internal membrane-containing icosahedral viruses at 3.7 and 3.8 A resolution, providing insights into the assembly process of these and related PRD1-adeno lineage viruses.
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The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules.
Genes, 2018Co-Authors: Nina S. Atanasova, Tatiana A. Demina, Dennis H. Bamford, Camilla Heiniö, Hanna M. OksanenAbstract:Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.
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Vesicle-like virion of Haloarcula hispanica pleomorphic virus 3 preserves high infectivity in saturated salt.
Virology, 2016Co-Authors: Tatiana A. Demina, Nina S. Atanasova, Maija K. Pietilä, Hanna M. Oksanen, Dennis H. BamfordAbstract:Hypersaline environments that are subject to salinity changes are particularly rich in viruses. Here we report a newly isolated archaeal halovirus, Haloarcula hispanica pleomorphic virus 3 (HHPV3). Its reproduction significantly retards host growth and decreases cell viability without causing lysis. HHPV3 particles require a minimum of 3M NaCl for stability and maintain high infectivity even in saturated salt. Notably, virions are irreversibly inactivated at ~1.5M NaCl in neutral pH, but tolerate this salinity at alkaline pH. The HHPV3 virion is a pleomorphic membrane vesicle containing two major protein species and lipids acquired nonselectively from the host membrane. The circular double-stranded DNA genome contains a conserved gene block characteristic of pleolipoviruses. We propose that HHPV3 is a member of the Betapleolipovirus genus (family Pleolipoviridae). Our findings add insights into the diversity observed among the pleolipoviruses found in hypersaline environments.
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Monitoring physiological changes in haloarchaeal cell during virus release
Viruses, 2016Co-Authors: Julija Svirskaitė, Hanna M. Oksanen, Rimantas Daugelavičius, Dennis H. BamfordAbstract:The slow rate of adsorption and non-synchronous release of some archaeal viruses have hindered more thorough analyses of the mechanisms of archaeal virus release. To address this deficit, we utilized four viruses that infect Haloarcula hispanica that represent the four virion morphotypes currently known for halophilic euryarchaeal viruses: (1) icosahedral internal membrane-containing SH1; (2) icosahedral tailed HHTV-1; (3) spindle-shaped His1; and (4) pleomorphic His2. To discern the events occurring as the progeny viruses exit, we monitored culture turbidity, as well as viable cell and progeny virus counts of infected and uninfected cultures. In addition to these traditional metrics, we measured three parameters associated with membrane integrity: the binding of the lipophilic anion phenyldicarbaundecaborane, oxygen consumption, and both intra- and extra-cellular ATP levels.
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Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.
Structure (London England : 1993), 2015Co-Authors: David Gil-carton, Hanna M. Oksanen, Dennis H. Bamford, Salla T. Jaakkola, Diego Charro, Bibiana Peralta, Daniel Castaño-díez, Nicola G. A. AbresciaAbstract:Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly.
Jing Han - One of the best experts on this subject based on the ideXlab platform.
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Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.
Molecular microbiology, 2020Co-Authors: Caixia Pei, Hua Xiang, Jing Han, Hui Zhou, Jerry Eichler, Cheng JinAbstract:Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.
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An Acidic Exopolysaccharide from Haloarcula hispanica ATCC33960 and Two Genes Responsible for Its Synthesis
Archaea (Vancouver B.C.), 2017Co-Authors: Shiwei Wang, Hua Xiang, Jing Han, Cheng JinAbstract:A 1.1 × 106 Da acidic exopolysaccharide (EPS) was purified from an extremely halophilic archaeon Haloarcula hispanica ATCC33960 with a production of 30 mg L-1 when grown in AS-168 medium, which mainly composed of mannose and galactose with a small amount of glucose in a molar ratio of 55.9 : 43.2 : 0.9. Two glycosyltransferase genes (HAH_1662 and HAH_1667) were identified to be responsible for synthesis of the acidic EPS. Deletion of either HAH_1662 or HAH_1667 led to loss of the acidic EPS. The mutants displayed a different cell surface morphology, retarded growth in low salty environment, an increased adhesion, and swimming ability. Our results suggest that biosynthesis of the acidic EPS might act as an adaptable mechanism to protect the cells against harsh environments.
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Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica.
Journal of bacteriology, 2017Co-Authors: Farshad Borjian, Hua Xiang, Jing Han, Jing Hou, Jan Zarzycki, Ivan A. BergAbstract:Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.
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Identification of the S-layer glycoproteins and their covalently linked glycans in the halophilic archaeon Haloarcula hispanica
Glycobiology, 2015Co-Authors: Jinwei Ren, Hua Xiang, Jing Han, Yuanming Luo, Zhongfu Wang, Qian Wang, Cheng JinAbstract:Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research. Although the Har. hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet. In this study, the S-layer proteins of Har. hispanica were extracted and characterized. The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences. The genes coding for these two S-layer glycoproteins were found next to each other in the genome. Moreover, the N- and O-linked glycans were released from these two S-layer glycoproteins for structural determination. Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified. Besides, the O-glycan was characterized as a Glcα-1,4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment. The determination of the N- and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing H. hispanica as a new model.
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Xiang H: Identification of the polyhydroxyalkanoate (PHA)-specific acetoacetyl-CoA reductase from multiple FabG paralogs in Haloarcula hispanica and reconstruction of PHA biosynthetic pathway in Haloferax volcanii
2013Co-Authors: Jing Han, Hailong Liu, Ligang Zhou, Hua XiangAbstract:Genome-wide analysis has revealed abundant FabG (�-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the �fabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea. Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteri
Hailong Liu - One of the best experts on this subject based on the ideXlab platform.
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Multiple replication origins with diverse control mechanisms in Haloarcula hispanica
Nucleic acids research, 2013Co-Authors: Jingfang Liu, Hailong Liu, Haibo Yang, Hua XiangAbstract:The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea.
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Xiang H: Identification of the polyhydroxyalkanoate (PHA)-specific acetoacetyl-CoA reductase from multiple FabG paralogs in Haloarcula hispanica and reconstruction of PHA biosynthetic pathway in Haloferax volcanii
2013Co-Authors: Jing Han, Hailong Liu, Ligang Zhou, Hua XiangAbstract:Genome-wide analysis has revealed abundant FabG (�-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the �fabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea. Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteri
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Proteome reference map of Haloarcula hispanica and comparative proteomic and transcriptomic analysis of polyhydroxyalkanoate biosynthesis under genetic and environmental perturbations.
Journal of proteome research, 2013Co-Authors: Hailong Liu, Jingfang Liu, Jing Han, Yuanming Luo, Deqin Feng, Shuangfeng Cai, Jian ZhouAbstract:Many haloarchaea are known as polyhydroxyalkanoates (PHAs) producers, but a global and integrated view of the PHA biosynthesis is still lacking in this group of archaea. In this study, a combined proteomic and transcriptomic approach was employed in Haloarcula hispanica, a model haloarchaeon that accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under nutrient-limiting conditions with excess carbon source. First, a comprehensive proteome reference map was established for H. hispanica. A total of 936 spots representing 839 unique proteins (21.7% of the predicted proteome) were identified by MALDI-TOF/TOF PMF and MS/MS. The map was further utilized to reconstruct central metabolic pathways to facilitate functional genomic analysis in H. hispanica. The results from the proteomic and transcriptomic analysis indicated that active PHA production coordinated with the TCA cycle to maintain balanced growth in wild-type H. hispanica, which was grown in nutrient-limited medium (PHA-accumulating conditi...
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Multiple replication origins with diverse control mechanisms in Haloarcula hispanica,” Nucleic Acids Research
2013Co-Authors: Jingfang Liu, Hailong Liu, Haibo Yang, Hua XiangAbstract:The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is es-sential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differ-ently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompat-ible with the wild-type (but not the "oriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea
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Proteome Reference Map of Haloarcula hispanica and Comparative Proteomic and Transcriptomic Analysis of Polyhydroxyalkanoate Biosynthesis under Genetic and Environmental Perturbations
2013Co-Authors: Hailong Liu, Jingfang Liu, Jing Han, Yuanming Luo, Deqin Feng, Shuangfeng Cai, Jian ZhouAbstract:Many haloarchaea are known as polyhydroxyalkanoates (PHAs) producers, but a global and integrated view of the PHA biosynthesis is still lacking in this group of archaea. In this study, a combined proteomic and transcriptomic approach was employed in Haloarcula hispanica, a model haloarchaeon that accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under nutrient-limiting conditions with excess carbon source. First, a comprehensive proteome reference map was established for H. hispanica. A total of 936 spots representing 839 unique proteins (21.7% of the predicted proteome) were identified by MALDI-TOF/TOF PMF and MS/MS. The map was further utilized to reconstruct central metabolic pathways to facilitate functional genomic analysis in H. hispanica. The results from the proteomic and transcriptomic analysis indicated that active PHA production coordinated with the TCA cycle to maintain balanced growth in wild-type H. hispanica, which was grown in nutrient-limited medium (PHA-accumulating conditions) versus nutrient-rich medium (non-PHA-accumulating conditions). Under nutrient-limiting conditions with excess carbon source, the PHA biosynthetic genes including phaEC, phaB, and phaP were upregulated at the transcriptional level, whereas the TCA cycle and respiratory chain were downregulated. Thus, acetyl-CoA could be fed into the PHA biosynthetic pathway, leading to the accumulation of PHA granules in the cell. Simultaneously, the large amount of NADPH required during PHA accumulation was likely supplied by the C3 (pyruvate) and C4 (malate) pathway coupled with the urea cycle. When PHA biosynthesis was blocked, that is, in the PHA synthase mutant (ΔphaEC) versus wild type grown in nutrient-limited medium, the mutant might direct additional carbon and energy to the TCA cycle, but without obvious contribution to biomass accumulation. The combined approaches of proteomic and transcriptomic analysis were highly complementary, extending the physiological understanding of PHA biosynthesis and its regulation. This is the first integrated proteome and transcriptome investigation of PHA biosynthesis and regulation in haloarchaea. It has provided basic information for future systemic engineering of haloarchaea to meet industrial needs
