Use of competitive DNA hybridization to identify differences in the genomes of bacteria.
Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we report the development and testing of a biochemical approach for targeted sequencing of only those chromosomal regions that differ between two DNA preparations. The method, designated GFE (genome fragment enrichment) uses competitive solution hybridization and positive selection to obtain genomic DNA fragments that are present in one pool of fragments but not another. Repeated comparisons of the genomes of Enterococcus faecalis and E. faecium led to the identification of 225 putative genome-specific DNA fragments. Species and strain variations within these fragments were confirmed by both experimental and bioinformatic analyses. The E. faecalis genome-specific sequences identified included both a preponderance of those predicted to encode surface-exposed proteins, as well as several previously described unique marker regions embedded within highly conserved rrn operons. The GFE strategy we describe efficiently identified genomic differences between two enterococcal genomes, and will be widely applicable for studying genetic variation among closely related bacterial species.
U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, OH, USA.
Orin C
OC
Shanks
Jorge W
JW
Santo Domingo
James E
JE
Graham
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D013293:Enterococcus faecalis / Q000235:genetics*; D016984:Enterococcus faecium / Q000235:genetics*; D014644:Genetic Variation; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial