Figure 6
Wild type C. jejuni gyrase A and gyrases possessing all known FQR point mutations were expressed and their ability to supercoil relaxed plasmid DNA over time was determined via in vitro supercoiling assay. Relaxed 0.3 μg pBR322 was incubated in the presence of 36 nM each of WT GyrB and WT GyrA or GyrA possessing Thr86Ile, Thr86Lys, Thr86Ala, Asp90Tyr and Asp90Asn. The reactions were then stopped at intervals from time: 0 to time: 150 mins and the reaction products were analysed via electrophoresis on 1% agarose. (A) Representative images of supercoiling assay for WT GyrA and Thr86Ile GyrA visualising he proportion relaxed (R) pBR322 plasmid converted to supercoiled DNA (SC) at each timepoint (t). Lane C contains relaxed pBR322 plasmid, lanes t0–t150 contain reaction mixture in which the reaction was stopped at the timepoint denoted by t. (B) Supercoiled plasmid (SC) was quantified via densitometry analysis using DNA standards of known concentration with t60 being represented in this graph. The predominant mutant Thr86Ile exhibited statistically lower supercoiling activity (p ≤ 0.05) indicating this common mutant could shift the supercoiling profile of FQR strains, conferring a phenotype promoting virulence.
