Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Abstract

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

FIG. 1.

Detection of large tandem duplications. (A) Representation of the tandem genetic amplifications detected. The acrAB locus is shown as a black dot on the chromosome of E. coli. ISs are shown as black (IS3), dark gray (IS5), and white (IS186) arrows. The PCR primers used to detect the dupIS3, dupIS5, and dupIS186 events are shown at their hybridization sites along the chromosome. (B) PCR detection of the dupIS3, dupIS5, and dupIS186 mutations in AG100, M113, and the 13 mutants studied. Template DNA corresponded to suspensions of strains cultivated in LB broth, as described in Materials and Methods. For detection of dupIS3, the template DNA was diluted 1:5 to compensate for the high background of spontaneous dupIS3. (C) Detection of dupIS5 mutations in AG100 and MG1655. DNA from three independent growths of AG100 or MG1655 in LB medium with or without nalidixic acid (5 μg/ml) was extracted and used in comparative PCR of 35 cycles. A total of 150 ng (dilution 1), 30 ng (dilution 2), 0.24 ng (dilution 5), or 0.048 ng (dilution 6) of template DNA was used. Amplified bands were quantified under UV light (see Materials and Methods).

FIG. 2.

Isolation of drug-resistant mutants from wild-type AG100 compared with that from its lon mutant on two different media. Drug-resistant mutants of AG100 (wild-type) or M113R (lon3::IS186) were selected at 37°C on LB (A) or MacConkey (B) agar plates containing antibiotics for 6 days (see Materials and Methods). After each day of incubation, the new colonies that appeared were counted. Experiments were done in duplicate, except for the selection on LB medium with tetracycline at 4 μg/ml (four experiments) and on MacConkey medium with chloramphenicol at 10 μg/ml (single experiment). The curves (dots and thick line for AG100, rectangles and thin line for M113R) represent the number of drug-resistant mutants which appeared per bacterium initially plated on the selective medium since day 1 of the selection. The dotted line (triangles) represents the number of colonies with a lon::IS186 insertion isolated per AG100 bacterium initially plated since the beginning of the experiment. a, percentage of the new colonies of AG100 appearing on the specific day which carried a lon::IS186 insertion; b, percentage of AG100 colonies with a lon::IS186 insertion among all the colonies which appeared since the beginning of the selection; c, number of colonies isolated from AG100 after each day of incubation that were chosen randomly and analyzed by PCR for the presence of a lon::IS186 mutation (see Materials and Methods).

FIG. 3.

Genome instability of wild type versus that of lon mutant. (A) Schematic representation of the genomic inversion and tandem amplification detected. ISs are presented as black (IS3) or dark gray (IS5) arrows along the E. coli chromosome. The PCR primers used to detect the inversion and the duplication are shown on their hybridization sites along the chromosome. (B) Detection of genetic inversions and duplications in a wild-type strain and a lon mutant. DNA from three independent growths of AG100 (wild type) or M113R (lon3::IS186) in LB broth was extracted and quantified by determination of the A₂₆₀. Comparative PCR of 35 cycles was done with 150 ng (dilution 1), 50 ng (dilution 2), 5.5 ng (dilution 4), or 1.9 ng (dilution 5) of template DNA. The amplified bands were quantified (see Materials and Methods).