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  1. Article ; Online: Complete Biochemical Characterization of Pantaphos Biosynthesis Highlights an Unusual Role for a SAM-Dependent Methyltransferase.

    Polidore, Alexander L A / Caserio, Angelica D / Zhu, Lingyang / Metcalf, William W

    Angewandte Chemie (International ed. in English)

    2024  Volume 63, Issue 7, Page(s) e202317262

    Abstract: Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its ... ...

    Abstract Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its production have yet to be established. Herein, we determine the complete biosynthetic pathway using a combination of bioinformatics, in vitro biochemistry and in vivo heterologous expression. Only 6 of the 11 hvr genes are needed to produce pantaphos, while a seventh is likely to be required for export. Surprisingly, the pathway involves a series of O-methylated intermediates, which are then hydrolyzed to produce the final product. The methylated intermediates are produced by an irreversible S-adenosylmethione (SAM)-dependent methyltransferase that is required to drive a thermodynamically unfavorable dehydration in the preceding step, a function not previously attributed to members of this enzyme class. Methylation of pantaphos by the same enzyme is also likely to limit its toxicity in the producing organism. The pathway also involves a novel flavin-dependent monooxygenase that differs from homologous proteins due to its endogenous flavin-reductase activity. Heterologous production of pantaphos by Escherichia coli strains expressing the minimal gene set strongly supports the in vitro biochemical data.
    MeSH term(s) Methyltransferases/metabolism ; Methylation ; Biosynthetic Pathways ; Plants/metabolism ; Flavins/metabolism
    Chemical Substances Methyltransferases (EC 2.1.1.-) ; Flavins
    Language English
    Publishing date 2024-01-15
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 2011836-3
    ISSN 1521-3773 ; 1433-7851
    ISSN (online) 1521-3773
    ISSN 1433-7851
    DOI 10.1002/anie.202317262
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  2. Article ; Online: Classic Spotlight: What's on (in) Your Plate Today?

    Metcalf, William W

    Journal of bacteriology

    2016  Volume 198, Issue 21, Page(s) 2897–2898

    Language English
    Publishing date 2016-11-01
    Publishing country United States
    Document type Editorial
    ZDB-ID 2968-3
    ISSN 1098-5530 ; 0021-9193
    ISSN (online) 1098-5530
    ISSN 0021-9193
    DOI 10.1128/JB.00609-16
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  3. Article ; Online: Classic Spotlight: Metabolic Flux-Which Way To Go?

    Metcalf, William W

    Journal of bacteriology

    2016  Volume 198, Issue 24, Page(s) 3248–3249

    Language English
    Publishing date 2016-12-15
    Publishing country United States
    Document type Editorial
    ZDB-ID 2968-3
    ISSN 1098-5530 ; 0021-9193
    ISSN (online) 1098-5530
    ISSN 0021-9193
    DOI 10.1128/JB.00731-16
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  4. Article ; Online: Classic Spotlight: Electron Bifurcation, a Unifying Concept for Energy Conservation in Anaerobes.

    Metcalf, William W

    Journal of bacteriology

    2016  Volume 198, Issue 9, Page(s) 1358

    MeSH term(s) Acyl Coenzyme A/metabolism ; Butyryl-CoA Dehydrogenase/metabolism ; Clostridium kluyveri/enzymology ; Ferredoxins/metabolism ; NAD/metabolism
    Chemical Substances Acyl Coenzyme A ; Ferredoxins ; NAD (0U46U6E8UK) ; Butyryl-CoA Dehydrogenase (EC 1.3.8.1)
    Language English
    Publishing date 2016-05
    Publishing country United States
    Document type Comment ; Editorial
    ZDB-ID 2968-3
    ISSN 1098-5530 ; 0021-9193
    ISSN (online) 1098-5530
    ISSN 0021-9193
    DOI 10.1128/JB.00185-16
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  5. Article ; Online: Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea.

    Thomsen, Johanna / Weidenbach, Katrin / Metcalf, William W / Schmitz, Ruth A

    Methods in molecular biology (Clifton, N.J.)

    2022  Volume 2522, Page(s) 105–117

    Abstract: Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable ... ...

    Abstract Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.
    MeSH term(s) Adenosine Diphosphate ; Archaea ; Hypoxanthine Phosphoribosyltransferase/genetics ; Mutation ; Purines
    Chemical Substances Purines ; Adenosine Diphosphate (61D2G4IYVH) ; Hypoxanthine Phosphoribosyltransferase (EC 2.4.2.8)
    Language English
    Publishing date 2022-09-20
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-2445-6_6
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  6. Article ; Online: Energy Conservation and Hydrogenase Function in Methanogenic Archaea, in Particular the Genus

    Mand, Thomas D / Metcalf, William W

    Microbiology and molecular biology reviews : MMBR

    2019  Volume 83, Issue 4

    Abstract: The biological production of methane is vital to the global carbon cycle and accounts for ca. 74% of total methane emissions. The organisms that facilitate this process, methanogenic archaea, belong to a large and phylogenetically diverse group that ... ...

    Abstract The biological production of methane is vital to the global carbon cycle and accounts for ca. 74% of total methane emissions. The organisms that facilitate this process, methanogenic archaea, belong to a large and phylogenetically diverse group that thrives in a wide range of anaerobic environments. Two main subgroups exist within methanogenic archaea: those with and those without cytochromes. Although a variety of metabolisms exist within this group, the reduction of growth substrates to methane using electrons from molecular hydrogen is, in a phylogenetic sense, the most widespread methanogenic pathway. Methanogens without cytochromes typically generate methane by the reduction of CO
    MeSH term(s) Cytochromes/metabolism ; Energy Metabolism ; Hydrogen/metabolism ; Hydrogenase/metabolism ; Methane/metabolism ; Methanosarcina/enzymology ; Phylogeny
    Chemical Substances Cytochromes ; Hydrogen (7YNJ3PO35Z) ; Hydrogenase (EC 1.12.7.2) ; Methane (OP0UW79H66)
    Language English
    Publishing date 2019-09-18
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
    ZDB-ID 1376131-6
    ISSN 1098-5557 ; 1070-6275 ; 1092-2172
    ISSN (online) 1098-5557 ; 1070-6275
    ISSN 1092-2172
    DOI 10.1128/MMBR.00020-19
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  7. Article ; Online: The streptothricin acetyltransferase (sat) gene as a positive selectable marker for methanogenic archaea.

    Farley, Kristen R / Metcalf, William W

    FEMS microbiology letters

    2019  Volume 366, Issue 17

    Abstract: A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive selectable markers has limited the types of genetic experiments that can be performed. In this study, we report the ...

    Abstract A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive selectable markers has limited the types of genetic experiments that can be performed. In this study, we report the development of an additional positive selection system for Methanosarcina that utilizes the antibiotic nourseothricin and the Streptomyces rochei streptothricin acetyltransferase (sat) gene, which may be broadly applicable to other groups of methanogenic archaea. Nourseothricin was found to inhibit growth of four different methanogen species at concentrations ≤300 μg/ml in liquid or on solid media. Selection of nourseothricin resistant transformants was possible in two genetically tractable Methanosarcina species, M. acetivorans and M. barkeri, using the sat gene as a positive selectable marker. Additionally, the sat marker was useful for constructing a gene deletion mutant strain of M. acetivorans, emphasizing its utility as a second positive selectable marker for genetic analyses of Methanosarcina genera. Interestingly, two human gut-associated methanogens Methanobrevibacter smithii and Methanomassillicoccus luminyensis were more sensitive to nourseothricin than either Methanosarcina species, suggesting the nourseothricin-sat gene pair may provide a robust positive selection system for development of genetic tools in these and other methanogens.
    MeSH term(s) Acetyltransferases/genetics ; Anti-Infective Agents/pharmacology ; Archaea/drug effects ; Archaea/genetics ; Drug Resistance, Microbial ; Evolution, Molecular ; Genes, Archaeal ; Humans ; Microbial Sensitivity Tests ; Mutation ; Selection, Genetic ; Sequence Deletion
    Chemical Substances Anti-Infective Agents ; Acetyltransferases (EC 2.3.1.-) ; streptothricin acetyltransferase (EC 2.3.1.-)
    Language English
    Publishing date 2019-10-08
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 752343-9
    ISSN 1574-6968 ; 0378-1097
    ISSN (online) 1574-6968
    ISSN 0378-1097
    DOI 10.1093/femsle/fnz216
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  8. Article ; Online: Methylamine-specific methyltransferase paralogs in Methanosarcina are functionally distinct despite frequent gene conversion.

    Nayak, Dipti D / Metcalf, William W

    The ISME journal

    2019  Volume 13, Issue 9, Page(s) 2173–2182

    Abstract: Sequenced archaeal genomes are mostly smaller and more streamlined than typical bacterial genomes; however, members of the Methanosarcina genus within the Euryarchaeaota are a significant exception, with M. acetivorans being the largest archaeal genome ( ... ...

    Abstract Sequenced archaeal genomes are mostly smaller and more streamlined than typical bacterial genomes; however, members of the Methanosarcina genus within the Euryarchaeaota are a significant exception, with M. acetivorans being the largest archaeal genome (5.8 Mbp) sequenced thus far. This finding is partially explained by extensive gene duplication within Methanosarcina spp. Significantly, the evolutionary pressures leading to gene duplication and subsequent genome expansion have not been well investigated, especially with respect to biological methane production (methanogenesis), which is the key biological trait of these environmentally important organisms. In this study, we address this question by specifically probing the functional evolution of two methylamine-specific methyltransferase paralogs in members of the Methanosarcina genus. Using the genetically tractable strain, M. acetivorans, we first show that the two paralogs have distinct cellular functions: one being required for methanogenesis from methylamine, the other for use of methylamine as a nitrogen source. Subsequently, through comparative sequence analyses, we show that functional divergence of paralogs is primarily mediated by divergent evolution of the 5' regulatory region, despite frequent gene conversion within the coding sequence. This unique evolutionary paradigm for functional divergence of genes post-duplication underscores a divergent role for an enzyme singularly associated with methanogenic metabolism in other aspects of cell physiology.
    MeSH term(s) Archaeal Proteins/genetics ; Archaeal Proteins/metabolism ; Gene Conversion ; Genome, Archaeal ; Methanosarcina/enzymology ; Methanosarcina/genetics ; Methanosarcina/metabolism ; Methylamines/metabolism ; Methyltransferases/genetics ; Methyltransferases/metabolism
    Chemical Substances Archaeal Proteins ; Methylamines ; methylamine (BSF23SJ79E) ; Methyltransferases (EC 2.1.1.-)
    Language English
    Publishing date 2019-05-03
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2406536-5
    ISSN 1751-7370 ; 1751-7362
    ISSN (online) 1751-7370
    ISSN 1751-7362
    DOI 10.1038/s41396-019-0428-6
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    Z 7773: Show issues

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  9. Article: Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction.

    Spietz, Rachel L / Payne, Devon / Kulkarni, Gargi / Metcalf, William W / Roden, Eric E / Boyd, Eric S

    Frontiers in microbiology

    2022  Volume 13, Page(s) 878387

    Abstract: ... Pyrite ( ... ...

    Abstract Pyrite (FeS
    Language English
    Publishing date 2022-05-09
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2587354-4
    ISSN 1664-302X
    ISSN 1664-302X
    DOI 10.3389/fmicb.2022.878387
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  10. Article ; Online: Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

    Nayak, Dipti D / Metcalf, William W

    Methods in enzymology

    2018  Volume 613, Page(s) 325–347

    Abstract: Methanogenic archaea generate methane as a by-product of anaerobic respiration using ... ...

    Abstract Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO
    MeSH term(s) Gene Editing ; Methanosarcina/enzymology ; Oxidoreductases/genetics ; Oxidoreductases/metabolism ; Plasmids/genetics
    Chemical Substances Oxidoreductases (EC 1.-) ; methyl coenzyme M reductase (EC 2.8.4.1)
    Language English
    Publishing date 2018-11-23
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1557-7988 ; 0076-6879
    ISSN (online) 1557-7988
    ISSN 0076-6879
    DOI 10.1016/bs.mie.2018.10.012
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