Versatile RIVET variants for the identification of transiently expressed genes in bacteria

LOZANO, M. GIUSTI, M.A., DRAGHI, W. O., TORRES TEJERIZO, G., PISTORIO, M. Y LAGARES, A.

Rosario, Sante fe, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The availability of the complete genomic sequence of several microorganisms made possible genome-wide high throughput methods, like transcriptomics and proteomics. However, these approaches can be poorly effective in identifying transiently/low expressed genes, or when the collection of the target sample is difficult and/or the amount limiting. In such cases, alternative approaches like RIVET (Recombination-based In Vivo Expression Technology can be attempted to identify specific genes induced under particular conditions. We have constructed three RIVET systems, each one consisting on two components: 1) a promoter-trap plasmid/transposon, consisting on a promoter-less tnpR (resolvase)-lacZ transcriptional fusion; and 2) an independent DNA cassette carrying positive (sacB) and negative (nptII) selectable markers which are excised only upon expression of TnpR. We constructed three variants of the promoter trap element supported in: a) a suicide R6K derivative plasmid, b) a broad host-range stable RK2 derivative, and c) a mini-Tn5 variant for the generation of promoter fusions without the need of a plasmid library as in the first two cases. The three configurations of our RIVET system represent a versatile set of tool for the analysis of transiently expressed transcriptomes during the in vivo/ex vivo interaction of diverse bacteria with their natural environments.