title: Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate.
abstract: Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2was used to understand how O2concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2(pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2and cell death.
  
  
  
  Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2tensions during growth in soil and N2fixation in symbiosis with legumes.
authors: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.
authors: Rachel M
authors: RM
authors: Wheatley
authors: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.
authors: Vinoy K
authors: VK
authors: Ramachandran
authors: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.
authors: Barney A
authors: BA
authors: Geddes
authors: Department of Biology, University of Regina, Regina, SK, Canada.
authors: Benjamin J
authors: BJ
authors: Perry
authors: Department of Biology, University of Regina, Regina, SK, Canada.
authors: Chris K
authors: CK
authors: Yost
authors: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom philip.poole@plants.ox.ac.uk.
authors: Philip S
authors: PS
authors: Poole
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques; D010100:Oxygen / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D016961:Rhizobium leguminosarum / Q000254:growth & development* / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D019802:Succinic Acid; D005947:Glucose; D010100:Oxygen
keywords: INSeq; Pisum sativum; Rhizobium leguminosarum; legumes; nitrogen fixation; nodules