- A.G. Livingston (1)
- V. Lorenzo de (3)
- A. Mantalaris (2)
- V.A.P. Martins Dos Santos (3)
- E.N. Pistikopoulos (2)
- K. Plufger-Grau (1)
- J. Puchalka (1)
- R. Silva-Rocha (3)
Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida
Kremling, A. ; Plufger-Grau, K. ; Silva-Rocha, R. ; Puchalka, J. ; Martins Dos Santos, V.A.P. ; Lorenzo, V. de - \ 2012
BMC Systems Biology 6 (2012). - ISSN 1752-0509 - 25 p.
escherichia-coli - catabolite repression - pu promoter - metabolism - iia(ntr) - protein - pathways - phosphorylation - bacteria - nitrogen
BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterialphosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents asignaling unit that acts as a sensory element and measures the activity of the central metabolism.Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr),which communicate with each other by phosphate exchange. Recent experimental results showed a cross talkbetween the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one ofthe PTS proteins, for different environmental conditions and different strain variants. Additionally, data fromflux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based onthe calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructosecould be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major partof the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a newrole of the second branch of the PTS and will serve as a basis for further experimental studies
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach
Koutinas, M. ; Kiparissides, A. ; Silva-Rocha, R. ; Lam, M.C. ; Martins Dos Santos, V.A.P. ; Lorenzo, V. de; Pistikopoulos, E.N. ; Mantalaris, A. - \ 2011
Metabolic Engineering 13 (2011)4. - ISSN 1096-7176 - p. 401 - 413.
pseudomonas tol plasmid - escherichia-coli - rna-polymerase - regulatory networks - pu promoter - putida mt-2 - host factor - in-vivo - transcription - activation
The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses
The regulatory logic of m-xylene biodegradation by Pseudomonas putida mt-2 exposed by dynamic modelling of the principal node Ps/Pr of the TOL plasmid
Koutinas, M. ; Lam, M.C. ; Kiparissides, A. ; Silva-Rocha, R. ; Godinho, M. ; Livingston, A.G. ; Pistikopoulos, E.N. ; Lorenzo, V. de; Martins Dos Santos, V.A.P. ; Mantalaris, A. - \ 2010
Environmental Microbiology 12 (2010)6. - ISSN 1462-2912 - p. 1705 - 1718.
global sensitivity-analysis - escherichia-coli - rna-polymerase - pu promoter - catabolite repression - transcription - xylr - activation - pathway - translation
P>The structure of the extant transcriptional control network of the TOL plasmid pWW0 born by Pseudomonas putida mt-2 for biodegradation of m-xylene is far more complex than one would consider necessary from a mere engineering point of view. In order to penetrate the underlying logic of such a network, which controls a major environmental cleanup bioprocess, we have developed a dynamic model of the key regulatory node formed by the Ps/Pr promoters of pWW0, where the clustering of control elements is maximal. The model layout was validated with batch cultures estimating parameter values and its predictive capability was confirmed with independent sets of experimental data. The model revealed how regulatory outputs originated in the divergent and overlapping Ps/Pr segment, which expresses the transcription factors XylS and XylR respectively, are computed into distinct instructions to the upper and lower catabolic xyl operons for either simultaneous or stepwise consumption of m-xylene and/or succinate. In this respect, the model reveals that the architecture of the Ps/Pr is poised to discriminate the abundance of alternative and competing C sources, in particular m-xylene versus succinate. The proposed framework provides a first systemic understanding of the causality and connectivity of the regulatory elements that shape this exemplary regulatory network, facilitating the use of model analysis towards genetic circuit optimization.