FIG 4 .

Epistatic interactions between multiple domains of mtrD contribute to elevated azithromycin MICs. (A) GCGS0276 mtrD in the 28Bl background elevated the azithromycin MIC from 0.125 µg/ml to 0.5 µg/ml (red). (B) Primer pairs designed to amplify ~300-bp fragments over the length of mtrD (see Table S4) resulted in no observed transformants on 0.38-µg/ml azithromycin selection plates, suggesting that multiple mutations across mtrD are needed for resistance. (C and D) To determine the regions that contributed to resistance, multiple fragment sizes were constructed by holding the rightmost forward primer (black) constant while adding different reverse primers (gray) (C) and by holding the leftmost reverse primer (black) constant while adding forward primers (gray) to separate reactions (D). The red lines in panels C and D indicate the PCR products that generated transformants on azithromycin selection plates. (E) At a minimum, SNPs at base pair positions 18 to 356 coupled with SNPs at positions 2356 to 2724 (indicated by yellow background and an asterisk) were required to raise the MIC from the 0.125 µg/ml of the recipient 28Bl strain to 0.5 µg/ml, though we are unable to exclude the possibility that additional SNPs between these two regions are also involved.