The construction of new integrative and replicative plasmid-borne RIVET tools

LOZANO MJ., M E SALAS , M DE LOS Á GIUSTI , M C MARTINI , G A. TORRES TEJERIZO, M PISTORIO , M F DEL PAPA , AND A LAGARES

San Carlos de Bariloche

Conferencia; International Plasmid Biology Conference 2010; 2010

Recombination-based In Vivo Expression Technology is a promoter- trap technology that has been devised for the identification of bacterial genes specifically induced in natural niches where the location and/or the low number of microorganisms limit the application of a conventional transcriptome analysis. All RIVET systems are conformed by two different DNA cassettes: a promoter trap module, and a target module that changes by action of a recombinase upon transcription of the promoter trap (1). We present here the construction of three new promoter-trap modules, and a new target module to mediate the positive selection of genes differentially expressed under specific conditions. The promoter-trap modules include the possibility of using as carrier vector a suicide (intergrative) plasmid, a replicative (broad host-range) plasmid, or a miniTn5. All of these configurations were designed to generate libraries of transcriptional fusions to tnpR the recombinase from transposon Tn-gd. When tnpR is expressed from a bacterial promoter the target cassette is resolved at specific res sites leading as a consequence to the expression of a gentamycin resistance gene (aacC1, positive selection) and a green-fluorescent phenotype under light blue illumination (gfp marker). The functional elements used in the new RIVET tools (promoters and resistance genes) were chosen to be applicable in diverse gram-negative bacteria. The system was validated by using the integrative RIVET-plasmid and following the time-course and tissue-specific expression of the nifH promoter from the N2-fixing legume-symbiont Sinorhizobium meliloti. As expected, p-nifH was only expressed in planta within root nodules, and late in symbiosis. The new collection of RIVET plasmids and the target module provide a powerful alternative to the conventional transcriptomics for the positive selection of bacterial promoters differentially expressed during the colonization of hidden/recalcitrant natural niches.