title: Two pathways for glutamate biosynthesis in the syntrophic bacterium Syntrophus aciditrophicus.
abstract: The anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate by Syntrophus aciditrophicus grown syntrophically with Methanospirillum hungatei provides a model to study syntrophic cooperation. Recent studies revealed that S. aciditrophicus contains Re-citrate synthase but lacks the common Si-citrate synthase. To establish whether the Re-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-(13)C]acetate and [1-(14)C]acetate as well as [(13)C]bicarbonate as additional carbon sources during axenic growth of S. aciditrophicus on crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and (13)C nuclear magnetic resonance (NMR) spectroscopy of the obtained [(13)C]glutamate, as well as decarboxylation of [(14)C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route used Re-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium of S. aciditrophicus when grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. Besides Clostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany Fachbereich Biologie and Synmikro, Philipps-Universität, Marburg, Germany.
authors.forename: Marie
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
authors.forename: Huynh M
authors.initials: HM
authors.lastname: Le
authors.affiliation: Fachbereich Chemie, Philipps-Universität, Marburg, Germany.
authors.forename: Xiulan
authors.initials: X
authors.lastname: Xie
authors.affiliation: Energy, Environmental and Chemical Engineering Department, Washington University, St. Louis, Missouri, USA.
authors.forename: Xueyang
authors.initials: X
authors.lastname: Feng
authors.affiliation: Energy, Environmental and Chemical Engineering Department, Washington University, St. Louis, Missouri, USA.
authors.forename: Yinjie J
authors.initials: YJ
authors.lastname: Tang
authors.affiliation: Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
authors.forename: Housna
authors.initials: H
authors.lastname: Mouttaki
authors.affiliation: Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McInerney
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany Fachbereich Biologie and Synmikro, Philipps-Universität, Marburg, Germany buckel@staff.uni-marburg.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
mesh_terms: D000085:Acetates / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000235:genetics; D020564:Deltaproteobacteria / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D019639:Methanospirillum / Q000378:metabolism; D056265:Microbial Interactions / Q000502:physiology*; D010084:Oxidation-Reduction; D019802:Succinic Acid / Q000737:chemistry
chemical_list: C101347:4-hydroxybutyryl-coenzyme A; D000085:Acetates; D000214:Acyl Coenzyme A; D006885:Hydroxybutyrates; C111420:4-hydroxybutyric acid; D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; C010701:crotonyl-coenzyme A; D019802:Succinic Acid; D002950:Citrate (si)-Synthase; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases