PMID:15980348

Citation	Lerner, CG, Kakavas, SJ, Wagner, C, Chang, RT, Merta, PJ, Ruan, X, Metzger, RE and Beutel, BA (2005) Novel approach to mapping of resistance mutations in whole genomes by using restriction enzyme modulation of transformation efficiency. Antimicrob. Agents Chemother. 49:2767-77
Abstract	Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.
Links	PubMed PMC1168657 Online version:10.1128/AAC.49.7.2767-2777.2005
Keywords	Anti-Bacterial Agents/chemistry; Anti-Bacterial Agents/pharmacology; Bacterial Outer Membrane Proteins/genetics; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Carrier Proteins/genetics; DNA Restriction Enzymes/metabolism; Drug Resistance, Bacterial/genetics; Escherichia coli Proteins/genetics; Fatty Acid Transport Proteins; Genome, Bacterial; Haemophilus influenzae/drug effects; Haemophilus influenzae/genetics; Membrane Transport Proteins; Microbial Sensitivity Tests/methods; Mutation; Restriction Mapping/methods; Transformation, Bacterial/genetics
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