Víctor de Lorenzo (Madrid, 1957) is a Chemist by training and he holds a position of Research Professor in the Spanish National Research Council (CSIC), where he currently heads the Laboratory of Environmental Molecular Microbiology at the National Center for Biotechnology. After his PhD at the CSIC Institute of Enzymology (1983), he worked at the Pasteur Institute (1984), the University of California at Berkeley (1985-1987), the University of Geneva (1988) and the Federal Center for Biotechnology in Braunschweig until 1991, the year in which he joined the CSIC in Madrid. He specializes in Molecular Biology and Biotechnology of soil bacteria (particularly Pseudomonas putida) as agents for the decontamination of sites damaged by industrial waste. At present, his work explores the interface between the Synthetic Biology and Environmental Biotechnology.
View http://www.cnb.csic.es/~meml for details.
Abstract
Reshaping the architecture of the regulatory circuit of the TOL plasmid of Pseudomonas putida for an increased response to environmental pollutants
V. de Lorenzo and Aitor de las Heras. Centro Nacional de Biotecnología, CSIC Madrid 28049. E-mail: vdlorenzo@cnb.cnb.es
The TOL plasmid pWW0 of Pseudomonas putida mt-2 encodes two catabolic operons for biodegradation of m-xylene. These operons are subject to an intricate regulatory circuit that involves the interplay between various plasmid-encoded and chromosome encoded transcription factors. The master regulatory element of the system is the m-xylene responsive s54-dependent regulator XylR, which acts on the Pu promoter. This, in turn, drives expression of the upper TOL operon for expression of the early catabolic enzymes. Examination of the extant architecture of the regulatory network reveals that the transcriptional output of Pu is limited by the intracellular concentrations of XylR (which represses its own expression) and the sigma factor s54. This specific configuration leads to a quick response to m-xylene when cells enter stationary phase, but also restricts the performance of the Pu promoter to a low-capacity regime. In the natural system, this probably reflects a tradeoff between transcriptional efficiency and physiological burden of the plasmid-encoded functions. Yet, we aim to use the Pu/XylR system as the basis for a whole-cell biosensor for aromatic chemicals that goes much beyond the natural response in terms of robustness and sensitivity. To this end we have examined whether such an innate regulatory scenario can be reshaped into a high-capacity regime by refactoring the connectivity of the sensor protein and the sigma factor within the rest of the circuit. For this, we first eliminated the auto-regulatory, negative feedback loop that operates on xylR transcription and replaced it by a positive feedback in which XylR was transcribed from either Pu or from another XylR-dependent promoter of the TOL plasmid, Ps. In the first case (xylR under Pu), we observed a substantial increase of Pu promoter capacity (measured as eventual emission of light by a reporter Pu-luxCDABE fusion) along with a noticeable slower response to aromatic effectors. In the second case (xylR expressed under the Ps promoter) the system developed an unexpected ability to sharply discriminate optimal (m-xylene) and suboptimal (3-methylbenzylalcohol) inducers. In a subsequent step, we also eliminated the bottleneck on Pu activity imposed by the naturally low levels of s54. This was made by increasing its intracellular concentration either with a heterologous expression system or by co-transcription with xylR from a Pu promoter. This architecture resulted in a considerably higher capacity of the promoter and a much-increased signal-to-noise ratio. These results show that the working regimes of given promoters can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops. On a more practical side, the designed circuits are being implemented for area wide detection of predetermined chemicals (e.g. explosives) in soil.
de las Heras, A., Carreño, CA, de Lorenzo, V. (2008) Stable implantation of orthogonal sensor circuits in Gram-negative bacteria for environmental release. Env Microbiol 10: 3305-3316. Silva-Rocha, R. and de Lorenzo (2008) Mining logic gates in prokaryotic transcriptional regulation networks. FEBS Lett 582: 1237-44. Garmendia, J., de las Heras, A., Calcagno Galvão T. and de Lorenzo V. (2008) Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes. Microb Biotech. 1: 236–246
