Analyzing Possible Native Functions of the Quinolone Resistance Gene qnr in Vibrio vulnificus

Abstract

The worldwide distribution of qnr genes found on plasmids and their presence on the chromosomes of aquatic bacteria, such as Vibrio vulnificus, one of the suspected sources, suggests an origin before the development of synthetic quinolones. However, their native function remains unknown. Previous work indicated that expression of qnrVv in V. vulnificus was induced by cold shock. To investigate its role further, we constructed single in-frame deletion mutants in qnrVv and cspA (the gene for cold shock protein) and a double mutant in qnrVv and cspA in V. vulnificus ATCC 17562 to evaluate the response to different environmental conditions and stresses and to exposure to various DNA-damaging agents. We found that qnrVv is involved in resistance to ciprofloxacin, levofloxacin, and mitomycin C and in the cold shock response in V. vulnificus Moreover, ΔqnrVv and ΔcspA mutants showed slower growth when they were treated with bile salts at 37°C and then shifted to 15°C (cold shock) without bile salts in the medium, with the effect being stronger in the double mutant. This transition may mimic what happens when V. vulnificus is ingested into the gastrointestinal tract and released in its natural environment. Cold shock and bile salts induced the expression of cspA and DNA gyrase and topoisomerase IV genes. However, no induction was found in the ΔqnrVv mutant, suggesting that the qnrVv gene is involved in the response to DNA damage and nucleic acid secondary structure.

Keywords: Qnr; Vibrio vulnificus; bile salts; cold shock; quinolones.

FIG 1

Growth curves of V. vulnificus ATCC 17562, the ΔqnrVv mutant, ΔcspA mutant, and ΔqnrVv ΔcspA mutant in 3% NaCl LB under different temperatures. (A) Growth curves at 42°C. AUC ΔqnrVv = 10.57 Log₁₀ CFU·h/ml; AUC ΔcspA = 10.54 Log₁₀ CFU·h/ml; AUC ΔqnrVv ΔcspA = 10.67 Log₁₀ CFU·h/ml; versus AUC wild type (WT) = 10.92 Log₁₀ CFU·h/ml. (B) Growth curves at 15°C. AUC ΔqnrVv = 7.88 Log₁₀ CFU·h/ml; AUC ΔqnrVv ΔcspA = 7.88 Log₁₀ CFU·h/ml; AUC WT = 9.20 Log₁₀ CFU·h/ml. (C) Growth curves in response to cold shock (growth at 37°C followed by a quick change to 15°C). AUC ΔqnrVv = 10.20 Log₁₀ CFU·h/ml; AUC ΔcspA = 10.06 Log₁₀ CFU·h/ml; AUC ΔqnrVv ΔcspA = 9.37 Log₁₀ CFU·h/ml; AUC WT = 10.32 Log₁₀ CFU·h/ml. (D) Complementation of the phenotype shown in panel C. (E) Growth curves at 37°C after growing strains at 37°C and applying cold shock at 15°C. (F) Growth curves at 37°C after growing strains at 15°C.

FIG 2

Growth curves of V. vulnificus ATCC 17562, the ΔqnrVv mutant, the ΔcspA mutant, and the ΔqnrVv ΔcspA mutant in LB broth under different conditions in the presence of bile salts. (A) Growth curves with 0.16% bile salts. (B) Growth curves with 0.08% bile salts. (C) Growth curves at 37°C after growing the strains at 37°C with 0.08% bile salts and applying cold shock at 15°C. (D) Growth curves at 37°C with 0.08% bile salts after growing the strains at 15°C. AUC ΔqnrVv = 9.93 Log₁₀ CFU·h/ml; AUC ΔqnrVv ΔcspA = 9.99 Log₁₀ CFU·h/ml; AUC WT = 10.01 Log₁₀ CFU·h/ml. (E) Growth curves at 15°C after growing the strains at 37°C with 0.08% bile salts and applying cold shock at 15°C. AUC ΔqnrVv = 9.52 Log₁₀ CFU·h/ml; AUC ΔcspA= 10.69 Log₁₀ CFU·h/ml; AUC ΔqnrVv ΔcspA = 8.01 Log₁₀ CFU·h/ml; AUC WT = 10.91 Log₁₀ CFU·h/ml. (F) Complementation of the phenotype shown in panel E.

FIG 3

Effect of environmental stresses on the relative expression levels of gyrA, gyrB, parC, parE, qnrVv, cspA, and recA of V. vulnificus ATCC 17562, the ΔqnrVv mutant, the ΔcspA mutant, and the ΔqnrVv ΔcspA mutant under different conditions. (A) Relative gene expression after treatment with 0.08% bile salts for 30 min. LB cultures were used as the control. (B) Relative gene expression in response to 30-min cold shock treatment. LB cultures at 37°C were used as the controls. (C) Relative gene expression in response to 30-min cold shock treatment (with no bile salts) after growth with 0.08% bile salts. LB cultures with 0.08% of bile salts at 37°C were used as the controls. (D) Relative gene expression in response to 30-min cold shock treatment (with 0.08% of bile salts) after a growth with 0.08% of bile salts. LB cultures with 0.08% of bile salts at 37°C were used as the controls. (E) Relative gene expression after a treatment with 0.5 μg/ml of novobiocin for 30 min. LB cultures were used as the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.