Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC In Vivo

Jörg Schumacher,Michael P H Stumpf,Dan R Brown,Sivaramesh Wigneshweraraj,Michal Komorowski,Banafsheh Razzaghi,Matthew R Lewis,Franziska Heydenreich,Jacob G Bundy,Mauricio Barahona,Mark H Bennett,Martin Buck,Volker Behrends,Zhensheng Pan,Caroline S Harwood

doi:10.1128/mbio.00881-13

Jörg Schumacher, Michael P H Stumpf + Show 13 more

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https://doi.org/10.1128/mbio.00881-13

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Abstract

ABSTRACTNitrogen 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.

Highlights

Integrative systems biology approaches provide comprehensive data on the physiological state of the cell that can reveal control parameters and limits of regulatory networks, inform predictive models, and guide metabolic engineering approaches
NCM3722 is prototrophic and a close reconstruction of the originally sequenced but genetically corrupted MG1655 E. coli K-12 strain, which suffers from various growth defects [23]
To capture changes in the physiological state of cells as they pass from nitrogen-replete to nitrogen-poor growth conditions, we carried out ammonium run-out experiments from an initial 3 mM NH4Cl

Summary

Introduction

Integrative systems biology approaches provide comprehensive data on the physiological state of the cell that can reveal control parameters and limits of regulatory networks, inform predictive models, and guide metabolic engineering approaches. PII controls the activity of the bifunctional enzyme NtrB, reducing its histidine kinase and stimulating its regulated phosphatase activity [6]. These PII activities were shown to be modulated in vitro through direct binding of ␣-KG, ATP, and ADP, presumably to coordinate carbon and energy with nitrogen assimilation [7]. NtrC can act as a transcriptional repressor, and the role of its phosphorylation at repressed promoters is unclear One consequence of this alternative regulatory mechanism is the strict requirement of an enhancer binding protein for the activation of ␴54-dependent transcription [12]. The cAMP receptor protein (CRP) regulates transcription of glnAp1 and glnAp2 in response to C source availability [14]

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