Amplification of the gene for isoleucyl-tRNA synthetase facilitates adaptation to the fitness cost of mupirocin resistance in Salmonella enterica

Abstract

Mutations that cause resistance to antibiotics in bacteria often reduce growth rate by impairing some essential cellular function. This growth impairment is expected to counterselect resistant organisms from natural populations following discontinuation of antibiotic therapy. Unfortunately (for disease control) bacteria adapt and improve their growth rate, often without losing antibiotic resistance. This adaptation process was studied in mupirocin-resistant (Mup(R)) strains of Salmonella enterica. Mupirocin (Mup) is an isoleucyl-adenylate analog that inhibits the essential enzyme, isoleucyl-tRNA synthetase (IleRS). Mutations causing Mup(R) alter IleRS and reduce growth rate. Fitness is restored by any of 23 secondary IleRS amino acid substitutions, 60% of which leave resistance unaffected. Evidence that increased expression of the original mutant ileS gene (Mup(R)) also improves fitness while maintaining resistance is presented. Expression can be increased by amplification of the ileS gene (more copies) or mutations that improve the ileS promoter (more transcription). Some adapted strains show both ileS amplification and an improved promoter. This suggests a process of adaptation initiated by common amplifications and followed by later acquisition of rare point mutations. Finally, a point mutation in one copy relaxes selection and allows loss of defective ileS copies. This sequence of events is demonstrated experimentally. A better understanding of adaptation can explain why antibiotic resistance persists in bacterial populations and may help identify drugs that are least subject to this problem.

Figure 1.—

(A) Copy number of ileS gene and levels of mRNA ileS in wild-type (WT), mupirocin-resistant (R1, R2, and R3), and compensated strains (C1–C12). The amino acid substitutions in IleRS that confer mupirocin resistance are indicated under the x-axis (H594Y, F596L, and WV631L). Wild-type level was set to 1.0. (B) Improvement of fitness as a function of the relative level of ileS expression. Fitness corresponds to the relative growth rate measured in LB (solid circles) and LB containing 100 μg/ml of mupirocin (open circles). Wild-type fitness in LB was set to 1.0 and the relative fitness of the strains was calculated as the ratio of t_gen(wt)/t_gen(mutant).

Figure 2.—

Map of the S. typhimurium LT2 chromosome near ileS. Mutations in the evolved lineages C1 (C-591 → A), C2 (C-1036 → T), C4 (C-577 → T), C5 (G-22 → T), C6 (C-1036 → T), C7 (T-421 → C), C8 (T-405 → C), C9 (T-405 → C), and C11 (C-567 → T) found upstream of the ileS gene are indicated in boldface type above the sequence; the promoter sequences previously characterized by Miller et al. (1987) are underlined. The RBS region is indicated by a dotted line.

Figure 3.—

(A) Localization of the markers a–j used as probes for Southern blot. A gray rectangle indicates the amplified regions determined from Southern blot done with several set of probes. (B) Marker frequency hybridization with a set of the probes (ileS, j, g, b, d, i, and recA used as standard) and targets chosen so that each probe lights up one band of distinct size (see materials and methods for details). (C) Size of the amplified arrays and sequence at the joint points in the lineages C3 and C12.

Figure 4.—

Fitness and copy number of ileS in lineages of C12 evolved in LB (A) or LB containing 100 μg/ml of mupirocin (B). Wild-type fitness in LB was set to 1.0 and the relative fitness of the strains was calculated as the ratio of t_gen(wt)/t_gen(mutant). Bars represent relative ileS DNA levels; circles are relative growth rates in LB (solid) or LB containing 100 μg/ml of mupirocin (open).