Novel approach to mapping of resistance mutations in whole genomes by using restriction enzyme modulation of transformation efficiency

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.

FIG. 1.

Diagram of the basic principle of REMOTE. Genomic DNA from a mutant strain is digested individually with restriction enzymes (e.g., A, B, C, and D). The frequency with which the digests transform a nonmutant strain to the mutant phenotype is assessed. Digests that transform with low frequency indicate the restriction sites are in close proximity to the mutation (★). Conversely, digests that transform with high frequency indicate the sites are far from the mutation (denoted by ). The mutation is indicated by an inverse white circle (). For a hypothetical genomic segment containing 10 genes, the data fit best to gene 3, which contains sites for both low-transforming digests (C and D) in close proximity and the sites for the high-transforming digests (A and B), which are further away. The other nine genes do not have clusters of the sites for both low-transforming digests that are also far from sites for the high-transforming digests.

FIG. 2.

Relationship between transformation frequency and the distance of a resistance mutation from the end of a DNA restriction fragment in H. influenzae. Ciprofloxacin-sensitive NP200 host cells were transformed to ciprofloxacin resistance with 1-kb PCR products containing a ciprofloxacin resistance gyrA mutation located at various distances from the end of the fragments. The PCR products were amplified from the ciprofloxacin-resistant H. influenzae gyrA strain Super8. To roughly approximate the conditions used in transformations with genomic digests, the transformation contained 1 ng of PCR product and 200 ng of digested NP200 genomic DNA.

FIG. 3.

Dependence of transformation frequency on the length of restriction fragments carrying a resistance mutation. H. influenzae NP200 host cells were transformed to A-344583 resistance with 200 ng of restriction enzyme digests of H. influenzae strain FLUSKO genomic DNA. To minimize the impact of end effects, data are shown only for enzymes that contain the mutation at least 200 bp from the ends of the restriction fragments.

FIG. 4.

Transformation frequencies obtained with H. influenzae NP200 host cells using restriction enzyme digests of H. influenzae strain Super8, which contains a Cip^r mutation in gyrA, a Nov^r mutation in gyrB, and a Spt^r mutation in rpsE. Restriction enzyme names are indicated as labels above the bars, whose heights correspond to the observed transformation frequencies. The ordinate indicates the background-corrected number of transformants obtained with the digests. Enzyme classifications determined by method A or B are indicated at the top of the graphs (F, full-effect enzymes; M, moderate-effect enzymes; N, no-effect enzymes). Asterisks indicate which classification method correctly identified the mutation as the top coordinate.

FIG. 5.

Locations of restriction enzyme cleavage sites surrounding the Cip^r mutation in gyrA, the Nov^r mutation in gyrB, and the Spt^r mutation in rpsE. For each mutation, groups of maps are shown for enzymes from the transformation effect categories, no-effect (N), moderate-effect (M), and full-effect enzymes (F). Classification method A (for details, see Materials and Methods) was used to assign the enzymes into the categories for the Cip^r and Spt^r transformation data, and method B was used for the Nov^r data, as depicted in Fig. 4. Vertical bars represent locations of restriction endonuclease cleavage sites. Horizontal lines represent the genome sequence, and vertical dotted lines represent locations of USS DNA uptake sequences. The distances (bp) from the mutations are indicated at the bottom of the panels. Only the two restriction sites that are nearest to the mutation (one on each side) are shown. Note that clustering of full-effect enzymes will not appear to be perfect, since only one of the two sites shown needs to be near enough to the mutation to significantly the transformation efficiency.

FIG. 6.

Transformation frequencies obtained with B. subtilis BD170 host cells using restriction enzyme digests of a rifampin-resistant derivative, BD170-R5, containing a Rif^r mutation in the rpoB gene. Restriction enzyme names are indicated as labels above the bars, whose heights correspond to the observed transformation frequencies.