title: Rationally rewiring the connectivity of the XylR/Pu regulatory node of the m-xylene degradation pathway in Pseudomonas putida.
abstract: The XylR/Pu regulatory node of the m-xylene biodegradation pathway of Pseudomonas putida mt-2 is one of the most intricate cases of processing internal and external cues into a single controlling element. Despite this complexity, the performance of the regulatory system is determined in vivo only by the occupation of Pu by m-xylene-activated XylR and σ(54)-RNAP. The stoichiometry between these three elements defines natural system boundaries that outline a specific functional space. This space can be expanded artificially following different strategies that involve either the increase of XylR or σ(54) or both elements at the same time (each using a different inducer). In this work we have designed a new regulatory architecture that drives the system to reach a maximum performance in response to one single input. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu node could be amended by simultaneously increasing σ(54) and XylR in response to only natural inducers. The exacerbation of Pu activity in vivo was tested in strains bearing synthetic transposons encoding xylR and rpoN (the σ(54) coding gene) controlled also by Pu, thereby generating a P. putida strain with the XylR/Pu output controlled by two intertwined feed forward loops (FFLs). The lack of a negative feedback loop in the expression node enables Pu activity to reach its physiological maximum in response to a single input. Only competition for cell resources might ultimately check the upper activity limit of such a rewired m-xylene sensing device.
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain. vdlorenzo@cnb.csic.es.
authors.forename: Aitor
authors.initials: A
authors.lastname: de Las Heras
authors.forename: Esteban
authors.initials: E
authors.lastname: Martínez-García
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Domingo-Sananes
authors.forename: Sofia
authors.initials: S
authors.lastname: Fraile
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D051877:RNA Polymerase Sigma 54 / Q000235:genetics; D012808:Sigma Factor / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014992:Xylenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; D014992:Xylenes; C497751:rpoN protein, E coli; D051877:RNA Polymerase Sigma 54; C031285:3-xylene