title: Development of new positive-selection RIVET tools: detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)-gfp fusion.
abstract: 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-γδ 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 bacterium Sinorhizobium meliloti, where a clear-cut phenotypic transition from Nm(R)-Gm(S)-GFP(-) to Nm(S)-Gm(R)-GFP(+) occurred upon expression of tnpR. A S. meliloti integrative RIVET library was constructed in pRIVET-I and, as expected, changes in the extracellular conditions (e.g., salt stress) triggered a significant increase in the appearance of Gm(R)-GFP(+) (excised) clones. The sacB-independent positive-selection RIVET systems here described provide suitable basic tools both for the construction of new recombination-based biosensors and for the search of bacterial markers induced when microorganisms colonize and invade complex environments and eukaryotic hosts.
authors: IBBM - Instituto de Biotecnología y Biología Molecular, CONICET - Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calles 47 y 115, 1900 La Plata, Argentina.
authors: M J
authors: MJ
authors: Lozano
authors: M E
authors: ME
authors: Salas
authors: M A
authors: MA
authors: Giusti
authors: W O
authors: WO
authors: Draghi
authors: G A
authors: GA
authors: Torres Tejerizo
authors: M C
authors: MC
authors: Martini
authors: M F
authors: MF
authors: Del Papa
authors: M
authors: M
authors: Pistorio
authors: A
authors: A
authors: Lagares
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015723:Gene Library; D049452:Green Fluorescent Proteins; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D045564:Transposon Resolvases / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D045564:Transposon Resolvases