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Hua Xiang - One of the best experts on this subject based on the ideXlab platform.
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halolysin r4 of haloferax mediterranei confers its host antagonistic and defensive activities
Applied and Environmental Microbiology, 2021Co-Authors: Shaoxing Chen, Siqi Sun, Rui Wang, Hongli Feng, Hua XiangAbstract:Halolysins, which are subtilisin-like serine proteases of Haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident abd potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax mediterranei HlyR4 shows significant antagonistic activity against other Haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. mediterranei towards other Haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in milieu, and this function of hlyR4 is reproducible in Haloarcula hispanica The versatility of HlyR4 enables its host to outcompete other Haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independent of growth phase. This study provides a new peptide antibiotics candidate in Haloarchaea, as well as new insight towards a better understanding of the ecological roles of halolysins.Importance: This study shows that halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other Haloarchaea, which expands our knowledge on the traditional function of Haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth-phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from Haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective towards understanding the intricate interactions between Haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells living in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.
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unusual phosphoenolpyruvate pep synthetase like protein crucial to enhancement of polyhydroxyalkanoate accumulation in haloferax mediterranei revealed by dissection of pep pyruvate interconversion mechanism
Applied and Environmental Microbiology, 2019Co-Authors: Junyu Chen, Hua Xiang, Dahe Zhao, Ruchira Mitra, Shengjie Zhang, Zhenqiang Zuo, Lin Lin, Jing HanAbstract:ABSTRACT Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei. The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pykHm gene and ppsHm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in Haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the Haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials. IMPORTANCEArchaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea. However, this process is still uncharacterized in the Haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in Haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.
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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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Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.
Scientific reports, 2016Co-Authors: Guiming Liu, Jian Zhou, Jing Han, Jing Hou, Shuangfeng Cai, Dahe Zhao, Hua XiangAbstract: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.
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a patatin like protein associated with the polyhydroxyalkanoate pha granules of haloferax mediterranei acts as an efficient depolymerase in the degradation of native pha
Applied and Environmental Microbiology, 2015Co-Authors: Guiming Liu, Jing Han, Jing Hou, Shuangfeng Cai, Lei Cai, Dahe Zhao, Hua XiangAbstract: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.
Jing Han - One of the best experts on this subject based on the ideXlab platform.
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unusual phosphoenolpyruvate pep synthetase like protein crucial to enhancement of polyhydroxyalkanoate accumulation in haloferax mediterranei revealed by dissection of pep pyruvate interconversion mechanism
Applied and Environmental Microbiology, 2019Co-Authors: Junyu Chen, Hua Xiang, Dahe Zhao, Ruchira Mitra, Shengjie Zhang, Zhenqiang Zuo, Lin Lin, Jing HanAbstract:ABSTRACT Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei. The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pykHm gene and ppsHm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in Haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the Haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials. IMPORTANCEArchaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea. However, this process is still uncharacterized in the Haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in Haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.
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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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Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.
Scientific reports, 2016Co-Authors: Guiming Liu, Jian Zhou, Jing Han, Jing Hou, Shuangfeng Cai, Dahe Zhao, Hua XiangAbstract: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.
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a patatin like protein associated with the polyhydroxyalkanoate pha granules of haloferax mediterranei acts as an efficient depolymerase in the degradation of native pha
Applied and Environmental Microbiology, 2015Co-Authors: Guiming Liu, Jing Han, Jing Hou, Shuangfeng Cai, Lei Cai, Dahe Zhao, Hua XiangAbstract: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.
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a novel dna binding protein phar plays a central role in the regulation of polyhydroxyalkanoate accumulation and granule formation in the haloarchaeon haloferax mediterranei
Applied and Environmental Microbiology, 2015Co-Authors: Shuangfeng Cai, Jing Han, Lei Cai, Dahe Zhao, Guiming Liu, Hua XiangAbstract:Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in Haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The Haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the Haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.
Jing Hou - One of the best experts on this subject based on the ideXlab platform.
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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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Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.
Scientific reports, 2016Co-Authors: Guiming Liu, Jian Zhou, Jing Han, Jing Hou, Shuangfeng Cai, Dahe Zhao, Hua XiangAbstract: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.
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a patatin like protein associated with the polyhydroxyalkanoate pha granules of haloferax mediterranei acts as an efficient depolymerase in the degradation of native pha
Applied and Environmental Microbiology, 2015Co-Authors: Guiming Liu, Jing Han, Jing Hou, Shuangfeng Cai, Lei Cai, Dahe Zhao, Hua XiangAbstract: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.
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Propionyl coenzyme A (propionyl-CoA) carboxylase in Haloferax mediterranei: Indispensability for propionyl-CoA assimilation and impacts on global metabolism.
Applied and environmental microbiology, 2014Co-Authors: Jing Hou, Hua Xiang, Jing HanAbstract:Propionyl coenzyme A (propionyl-CoA) is an important intermediate during the biosynthesis and catabolism of intracellular carbon storage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in Haloarchaea. However, the Haloarchaeal propionyl-CoA carboxylase (PCC) and its physiological significance remain unclear. In this study, we identified a PCC that catalyzed propionyl-CoA carboxylation with an acetyl-CoA carboxylation side activity in Haloferax mediterranei. Gene knockout/complementation demonstrated that the PCC enzyme consisted of a fusion protein of a biotin carboxylase and a biotin-carboxyl carrier protein (PccA [HFX_2490]), a carboxyltransferase component (PccB [HFX_2478]), and an essential small subunit (PccX [HFX_2479]). Knockout of pccBX led to an inability to utilize propionate and a higher intracellular propionyl-CoA level, indicating that the PCC enzyme is indispensable for propionyl-CoA utilization. Interestingly, H. mediterranei DBX (pccBX-deleted strain) displayed multiple phenotypic changes, including retarded cell growth, decreased glucose consumption, impaired PHBV biosynthesis, and wrinkled cells. A propionyl-CoA concentration equivalent to the concentration that accumulated in DBX cells was demonstrated to inhibit succinyl-CoA synthetase of the tricarboxylic acid cycle in vitro. Genome-wide microarray analysis showed that many genes for glycolysis, pyruvate oxidation, PHBV accumulation, electron transport, and stress responses were affected in DBX. This study not only identified the Haloarchaeal PCC for the metabolism of propionyl-CoA, an important intermediate in Haloarchaea, but also demonstrated that impaired propionyl-CoA metabolism affected global metabolism in H. mediterranei.
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Haloarchaeal type β ketothiolases involved in poly 3 hydroxybutyrate co 3 hydroxyvalerate synthesis in haloferax mediterranei
Applied and Environmental Microbiology, 2013Co-Authors: Jing Hou, Hailong Liu, Jian Zhou, Jing Han, Bo Feng, Dahe Zhao, Hua XiangAbstract:The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in Haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the Haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (phaAα) and HFX_1022 (phaAβ) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (bktBα) and HFX_6003 (bktBβ). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad "Ser-His-His" was distinct from the bacterial "Cys-His-Cys." BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in Haloarchaea. This is the first report of the Haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.
Shuangfeng Cai - One of the best experts on this subject based on the ideXlab platform.
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Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei.
Scientific reports, 2016Co-Authors: Guiming Liu, Jian Zhou, Jing Han, Jing Hou, Shuangfeng Cai, Dahe Zhao, Hua XiangAbstract: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.
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a patatin like protein associated with the polyhydroxyalkanoate pha granules of haloferax mediterranei acts as an efficient depolymerase in the degradation of native pha
Applied and Environmental Microbiology, 2015Co-Authors: Guiming Liu, Jing Han, Jing Hou, Shuangfeng Cai, Lei Cai, Dahe Zhao, Hua XiangAbstract: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.
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a novel dna binding protein phar plays a central role in the regulation of polyhydroxyalkanoate accumulation and granule formation in the haloarchaeon haloferax mediterranei
Applied and Environmental Microbiology, 2015Co-Authors: Shuangfeng Cai, Jing Han, Lei Cai, Dahe Zhao, Guiming Liu, Hua XiangAbstract:Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in Haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The Haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the Haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.
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identification of the Haloarchaeal phasin phap that functions in polyhydroxyalkanoate accumulation and granule formation in haloferax mediterranei
Applied and Environmental Microbiology, 2012Co-Authors: Shuangfeng Cai, Hailong Liu, Jian Zhou, Xiaoqing Liu, Jing Han, Lei Cai, Hua XiangAbstract: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.
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wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type iii synthases
Applied and Environmental Microbiology, 2010Co-Authors: Jing Han, Hailong Liu, Jing Hou, Shuangfeng Cai, Bo Feng, Hua XiangAbstract:Haloarchaea are a distinct evolutionary branch of the domain Archaea, and they usually comprise the majority of the prokaryotic population in hypersaline environments (31). Most Haloarchaea are able to utilize glucose as a carbon source. However, Halobacterium (15) and some Natrialba (42) and Natronomonas (6, 7) strains cannot. In the presence of excess carbon substrates, certain Haloarchaea synthesize polyhydroxyalkanoates (PHAs) and deposit them as intracellular granules (27), which has been proposed as an optional standard for describing new Haloarchaeal species (32). Compared with members of the domain Bacteria, Haloarchaea have several advantages as PHA producers; e.g., they utilize unrelated cheap carbon sources, strict sterilization is not needed, and isolation of PHAs from Haloarchaea is much easier (11, 13, 27, 34). Thus, Haloarchaea have regained attention in biotechnology lately, especially as an alternative source for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production (11, 20, 28). Although the family Halobacteriaceae includes 30 genera, currently, only a few Haloarchaeal strains belonging to the genera Haloferax, Haloarcula, Haloquadratum, Haloterrigena, Halorhabdus, Halobiforma, and Halopiger are found to accumulate short-chain-length PHAs (scl-PHAs), such as polyhydroxybutyrate (PHB) and PHBV (2, 11-14, 22, 27, 36, 41). In the pathway of PHA biosynthesis, PHA synthases play a key role by catalyzing the polymerization of (R)-3-hydroxyalkanoyl coenzyme A into PHAs. In bacteria, four types of PHA synthases have been distinguished according to their primary structures, substrate specificities, and subunit compositions (35). Types I and II are composed of only one subunit, whereas types III and IV consist of two subunits. The PHA synthases from Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei have been identified and characterized genetically and biochemically, and all need a high salt concentration for enzyme activity (11, 28). The known Haloarchaeal PHA synthases are composed of two subunits (PhaE and PhaC) and share the closest identities with bacterial type III PHA synthases (28, 34). However, these Haloarchaeal PHA synthases are still quite different from their bacterial counterparts in molecular weight and amino acid sequence, especially the level of identity of PhaEs between Haloarchaea and bacteria, which is quite low (∼20%) (11, 28). Whether these differences are universal for Haloarchaeal strains remains to be answered. Moreover, it is still unknown whether other Haloarchaeal strains are able to synthesize PHAs and whether there are other types of PHA synthases. Thus, it is necessary to perform large-scale screening for the PHA synthases in Haloarchaea. In this work, the PHA accumulation ability of 28 Haloarchaeal strains from 15 genera were assessed, and the types of their PHA synthases were also characterized. We propose that the PHA synthases widespread in Haloarchaea be clustered as a novel subtype (IIIA; A for halophilic Archaea) of type III PHA synthases, whereas their counterparts from bacteria might be assigned to subtype IIIB (B for Bacteria).
Hailong Liu - One of the best experts on this subject based on the ideXlab platform.
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Haloarchaeal type β ketothiolases involved in poly 3 hydroxybutyrate co 3 hydroxyvalerate synthesis in haloferax mediterranei
Applied and Environmental Microbiology, 2013Co-Authors: Jing Hou, Hailong Liu, Jian Zhou, Jing Han, Bo Feng, Dahe Zhao, Hua XiangAbstract:The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in Haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the Haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (phaAα) and HFX_1022 (phaAβ) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (bktBα) and HFX_6003 (bktBβ). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad "Ser-His-His" was distinct from the bacterial "Cys-His-Cys." BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in Haloarchaea. This is the first report of the Haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.
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Diversity and evolution of multiple orc/cdc6-adjacent replication origins in Haloarchaea
BMC Genomics, 2012Co-Authors: Zhenfang Wu, Jingfang Liu, Hailong Liu, Xiaoqing Liu, Hua XiangAbstract:BackgroundWhile multiple replication origins have been observed in archaea, considerably less is known about their evolutionary processes. Here, we performed a comparative analysis of the predicted (proved in part) orc/cdc6 -associated replication origins in 15 completely sequenced Haloarchaeal genomes to investigate the diversity and evolution of replication origins in halophilic Archaea.ResultsMultiple orc/cdc6 -associated replication origins were predicted in all of the analyzed Haloarchaeal genomes following the identification of putative ORBs (origin recognition boxes) that are associated with orc/cdc6 genes. Five of these predicted replication origins in Haloarcula hispanica were experimentally confirmed via autonomous replication activities. Strikingly, several predicted replication origins in H. hispanica and Haloarcula marismortui are located in the distinct regions of their highly homologous chromosomes, suggesting that these replication origins might have been introduced as parts of new genomic content. A comparison of the origin-associated Orc/Cdc6 homologs and the corresponding predicted ORB elements revealed that the replication origins in a given haloarchaeon are quite diverse, while different Haloarchaea can share a few conserved origins. Phylogenetic and genomic context analyses suggested that there is an original replication origin ( oriC1 ) that was inherited from the ancestor of archaea, and several other origins were likely evolved and/or translocated within the Haloarchaeal species.ConclusionThis study provides detailed information about the diversity of multiple orc/cdc6 -associated replication origins in Haloarchaeal genomes, and provides novel insight into the evolution of multiple replication origins in Archaea.
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identification of the Haloarchaeal phasin phap that functions in polyhydroxyalkanoate accumulation and granule formation in haloferax mediterranei
Applied and Environmental Microbiology, 2012Co-Authors: Shuangfeng Cai, Hailong Liu, Jian Zhou, Xiaoqing Liu, Jing Han, Lei Cai, Hua XiangAbstract: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.
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wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type iii synthases
Applied and Environmental Microbiology, 2010Co-Authors: Jing Han, Hailong Liu, Jing Hou, Shuangfeng Cai, Bo Feng, Hua XiangAbstract:Haloarchaea are a distinct evolutionary branch of the domain Archaea, and they usually comprise the majority of the prokaryotic population in hypersaline environments (31). Most Haloarchaea are able to utilize glucose as a carbon source. However, Halobacterium (15) and some Natrialba (42) and Natronomonas (6, 7) strains cannot. In the presence of excess carbon substrates, certain Haloarchaea synthesize polyhydroxyalkanoates (PHAs) and deposit them as intracellular granules (27), which has been proposed as an optional standard for describing new Haloarchaeal species (32). Compared with members of the domain Bacteria, Haloarchaea have several advantages as PHA producers; e.g., they utilize unrelated cheap carbon sources, strict sterilization is not needed, and isolation of PHAs from Haloarchaea is much easier (11, 13, 27, 34). Thus, Haloarchaea have regained attention in biotechnology lately, especially as an alternative source for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production (11, 20, 28). Although the family Halobacteriaceae includes 30 genera, currently, only a few Haloarchaeal strains belonging to the genera Haloferax, Haloarcula, Haloquadratum, Haloterrigena, Halorhabdus, Halobiforma, and Halopiger are found to accumulate short-chain-length PHAs (scl-PHAs), such as polyhydroxybutyrate (PHB) and PHBV (2, 11-14, 22, 27, 36, 41). In the pathway of PHA biosynthesis, PHA synthases play a key role by catalyzing the polymerization of (R)-3-hydroxyalkanoyl coenzyme A into PHAs. In bacteria, four types of PHA synthases have been distinguished according to their primary structures, substrate specificities, and subunit compositions (35). Types I and II are composed of only one subunit, whereas types III and IV consist of two subunits. The PHA synthases from Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei have been identified and characterized genetically and biochemically, and all need a high salt concentration for enzyme activity (11, 28). The known Haloarchaeal PHA synthases are composed of two subunits (PhaE and PhaC) and share the closest identities with bacterial type III PHA synthases (28, 34). However, these Haloarchaeal PHA synthases are still quite different from their bacterial counterparts in molecular weight and amino acid sequence, especially the level of identity of PhaEs between Haloarchaea and bacteria, which is quite low (∼20%) (11, 28). Whether these differences are universal for Haloarchaeal strains remains to be answered. Moreover, it is still unknown whether other Haloarchaeal strains are able to synthesize PHAs and whether there are other types of PHA synthases. Thus, it is necessary to perform large-scale screening for the PHA synthases in Haloarchaea. In this work, the PHA accumulation ability of 28 Haloarchaeal strains from 15 genera were assessed, and the types of their PHA synthases were also characterized. We propose that the PHA synthases widespread in Haloarchaea be clustered as a novel subtype (IIIA; A for halophilic Archaea) of type III PHA synthases, whereas their counterparts from bacteria might be assigned to subtype IIIB (B for Bacteria).
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wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type iii synthases
Applied and Environmental Microbiology, 2010Co-Authors: Jing Han, Hailong Liu, Jian Zhou, Jing Hou, Shuangfeng Cai, Bo Feng, Hua XiangAbstract:Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few Haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these Haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using Haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that Haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of Haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in Haloarchaea and appears to be involved in PHA biosynthesis.
