SUBTRACTION HYBRIDIZATION AND SHOT-GUN SEQUENCING - A NEW APPROACH TOIDENTIFY SYMBIOTIC LOCI

Traditionally, new loci involved in the Rhizobium-legume symbiosis hav e been identified by transposon mutagenesis and/or complementation. Wi de dispersal of the symbiotic loci in Rhizobium species NGR234, as wel l as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, su btractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of mast nodulation genes is triggered by compound s released by the host-plant, we identified, in the ordered cosmid lib rary of the large symbiotic plasmid pNGR234a, restriction fragments th at carry transcripts induced by flavonoids. To target genes not presen t in the closely related strain R. fredii USDA257, we selected fragmen ts that also carried sequences purified by subtractive DNA hybridisati on. Shot-gun sequencing of this subset of fragments lead to the identi fication of sequences with strong homology to diverse prokaryotic gene s/proteins. Amongst these, a symbiotically active ORF from pNGR234a, i s highly homologous to the leucine responsive regulatory protein of Es cherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.