An Integrated Approach to Functional Genomics: Construction of a Novel Reporter Gene Fusion Library forSinorhizobium melilotiJournal Articles
Overview
Research
Identity
Additional Document Info
View All
Overview
abstract
ABSTRACTAs a means of investigating gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for theSinorhizobium melilotigenome. pTH1522 replicates inEscherichia coliand can be transferred to, but cannot replicate in,S. meliloti. Homologous recombination of the DNA fragments cloned in pTH1522 into theS. melilotigenome generates transcriptional fusions to either the reporter genesgfp+andlacZorgusAandrfp, depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis, and the plasmid clones were recombined intoS. meliloti. Reporter enzyme activities following growth of these recombinants in complex medium (LBmc) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those expressed at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an inability to recover recombinants from pTH1522 clones that carried fragments internal to gene or operon transcripts. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed (www.sinorhizobium.org).
