Development of new positive-selection RIVET tools: detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)-gfp fusion.

LOZANO, M. J.; SALAS, M, E.; GIUSTI, M. A.; DRAGHI, W. O.; TORRES TEJERIZO, G. A.; MARTINI, C; DEL PAPA, M.F.; PISTORIO, M.; LAGARES, A.

JOURNAL OF BIOTECHNOLOGY

ELSEVIER SCIENCE BV

Año: 2011 vol. 155 p. 147 - 155

0168-1656

RIVET (Recombination Based in-vivo Expression Technology) is a powerful genetic tool originally conceived for the identification of genes induced in complex biological niches where conventional transcriptomics is difficult to use. With a broader application, genetic recombination-based technologies have also been used, in combination with regulatory proteins and specific transcriptional regulators, for the development of highly-sensitive biosensor systems. RIVET systems generally comprise two modules: a promoter-trap cassette generating genomic transcriptional fusions to the tnpR gene encoding the Tn-gd TnpR resolvase, and a reporter cassette carrying res-flanked selection markers that are excised upon expression of tnpR to produce an irreversible, inheritable phenotypic change. We report here the construction and validation of a new set of positive-selection RIVET systems that, upon induction of the promoter-trap module, generate the transcriptional activation of an antibiotic-resistant and a green-fluorescent phenotype. Two classes of promoter-trap tools were constructed to generate transcriptional fusions to tnpR: one based on the use of a narrow-host-range plasmid (pRIVET-I), integrative in several Gram-negative bacteria, and the other based on the use of a broad-host-range plasmid (pRIVET-R). The system was evaluated in the model soil bacteriumSinorhizobium meliloti, where a clear-cut phenotypic transition from NmR-GmS -GFP – toNmS-GmR -GFP+ occurred upon expression of tnpR. A S. meliloti integrative RIVET library was constructed in pRIVET-I and, as expected, changes in the extracellularconditions (e. g., salt stress) triggered a significant increase in the appearance of GmR - GFP + (excised) clones. The sacB-independent positive-selection RIVET systems here described provide suitable basic tools both for the construction of new recombinationbased biosensors and for the search of bacterial markers induced when microorganisms colonize and invade complex environments and eukaryotic hosts.