Subtraction hybridisation and shot-gun sequencing: a new approach to identify symbiotic loci

• Published in: Nucleic acids research 1994-04, Vol.22 (8), p.1335-1341
• Main Authors: Perret, X, Fellay, R, Bjourson, A.J, Cooper, J.E, Brenner, S, Broughton, W.J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteriology; Base Sequence; biochemical techniques; Biological and medical sciences; Cloning, Molecular; DNA, Bacterial - analysis; Escherichia coli; Escherichia coli - genetics; Fabaceae - genetics; Fabaceae - microbiology; flavonoids; Fundamental and applied biological sciences. Psychology; genbank/x74134; gene expression; Genetic Complementation Test; genetic regulation; identification; loci; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; nodulation; Nucleic Acid Hybridization; nucleotide sequences; Open Reading Frames; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Phenotype; Plants, Medicinal; plasmids; Pseudomonas putida - genetics; Restriction Mapping; Rhizobium; Rhizobium - genetics; Rhizobium - physiology; sequence analysis; Sequence Analysis, DNA - methods; Sequence Homology, Amino Acid; Sinorhizobium fredii; symbiosis; Symbiosis - genetics; transcription (genetics)
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/22.8.1335

Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well 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, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.