title: Nitrogen and carbon status are integrated at the transcriptional level by the nitrogen regulator NtrC in vivo.
abstract: Nitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency.
  
  
  OBJECTIVE
  Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling--sensed by the UTase (glnD) and PII (glnB), respectively--and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.
authors.affiliation: Division of Cell & Molecular Biology, Imperial College London, London, United Kingdom.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schumacher
authors.forename: Volker
authors.initials: V
authors.lastname: Behrends
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Pan
authors.forename: Dan R
authors.initials: DR
authors.lastname: Brown
authors.forename: Franziska
authors.initials: F
authors.lastname: Heydenreich
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Lewis
authors.forename: Mark H
authors.initials: MH
authors.lastname: Bennett
authors.forename: Banafsheh
authors.initials: B
authors.lastname: Razzaghi
authors.forename: Michal
authors.initials: M
authors.lastname: Komorowski
authors.forename: Mauricio
authors.initials: M
authors.lastname: Barahona
authors.forename: Michael P H
authors.initials: MP
authors.lastname: Stumpf
authors.forename: Sivaramesh
authors.initials: S
authors.lastname: Wigneshweraraj
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Bundy
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D064751:Ammonium Compounds / Q000378:metabolism; D002244:Carbon / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005973:Glutamine / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D064751:Ammonium Compounds; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D014157:Transcription Factors; C497876:glnG protein, E coli; D005973:Glutamine; D002244:Carbon; D009584:Nitrogen