J. SCHUMACHER - Systems and synthetic approaches to understand, model and manipulate nitrogen regulation in bacteria

The regulation of bacterial nitrogen metabolism is of fundamental importance for the global ecosystem and agriculture. The application of chemical fertilisers has dramatically increased food production and the human population but also led to global nitrogen imbalances that pose severe ecological challenges for human development (1). With the emergence of systems and synthetic biology new strategies are being explored to harness the metabolic capacity of bacteria to produce nitrogen sources where it is lacking or consume nitrogen compounds where they are in excess.

Most bacteria rely on conserved regulatory proteins to adequately respond to nitrogen availability and needs, but the regulatory network is complex, where metabolic, proteomic and transcriptomic molecules dynamically interdependent. In E.coli, we studied the dynamic changes of transcripts, proteins and metabolites quantitatively upon entry into nitrogen limitation stress using RNA-seq transcriptomics, MRM-MS proteomics and NMR, LC-MS metabolomics. We derived a comprehensive description and models of the multi-level interdependencies of nitrogen regulation, revealing in vivo kinetic parameters of nitrogen regulation and limits of system robustness that challenge the long held assumption that glutamine is the dominant signal for nitrogen availability (e.g (2)).

Regulation of ammonium consumption (assimilation), and in the case of diazotrophs ammonium production (fixation), commonly relies on transcriptional regulators of the bacterial s⁵⁴ RNA polymerase, involved in various adaptations to nutrient stress and plant pathogenesis (3, 4). We engineered chimeric transcription activators of the s⁵⁴ RNA polymerase as novel perturbation tool to study and manipulate nitrogen regulation (5). First, we used such synthetically rewired transcription control proteins to further study intra and inter systems level interdependencies. In relation to N regulation, results revealed regulon wide and inter-systems level control hierarchies and optimal signalling properties through post-translational modifications. Second, synthetic activators of s⁵⁴ dependent transcription can serve as broad systems metabolic engineering tools that could more generally be employed to rebalance global carbon/nitrogen metabolism towards the latter and enhance biological nitrogen fixation in diazotrophs.

1. Rockstrom, J., et al. (2009) A safe operating space for humanity, Nature 461, 472-475.

2. Schumacher, J., et al. (2013) Nitrogen and carbon status are integrated at the transcriptional level by the     nitrogen regulator NtrC in vivo, mBio 4, e00881-00813.

3. Schumacher, J., Joly, N., Rappas, M., Zhang, X., and Buck, M. (2006) Structures and organisation of AAA+ enhancer binding proteins in transcriptional activation, J Struct Biol 156, 190-199.

4. Jovanovic, M., James, E. H., Rego, F. G., Buck, M., and Schumacher, J. (2011) Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity, Nat Commun 2, 177.

5. Wang, B., Barahona, M., Buck, M., and Schumacher, J. (2013) Rewiring cell signalling through chimaeric regulatory protein engineering, Biochem Soc Trans 41, 1195-1200.

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