It should be noted that pInterD1 conferred more protection than pInterD2 to mutant topoisomerase I killing (Table 1) and the opposite was true for norfloxacin killing (Table 2). Table

2 Effect of high copy plasmid clones on survival following treatment with norfloxacin Plasmid Survival Ratio pCRII vector 2.14 × 10-5 ± 4.1 × 10-6 pAQ5 7.57 × 10-4 ± 2.14 × 10-4 pInter 6.12 × 10-4 ± 1.28 PF299 datasheet × 10-4 pInterD1 8.41 × 10-5 ± 3.55 × 10-5 pInterD2 1.11 × 10-4 ± 2.01 × 10-5 E. coli BW27784 transformed with high copy number plasmid was grown to exponential phase with shaking. Cultures were treated with 250 ng/ml norfloxacin for 2 h before serial dilution and plating on LB plates with kanamycin Survival ratio was determined by calculating the ratio of the viable colony counts obtained from the treated cultures versus the viable counts from untreated culture. The results represent the average and standard errors from at least three experiments Protective effect from adenine addition The protective effect from titration of PurR could be due to increased availability of purine nucleotides. This was tested by check details growth of BW27784 transformed with pAYTOP128 in minimal media. Greater than 3 logs of loss of viability could be measured at 2 h after induction of mutant topoisomerase I expression

by 0.0002% arabinose (Figure 3a). The presence of 100 μg/ml adenine in the growth medium increased the number of viable colonies by 30-fold at 2 h after arabinose addition. The presence of adenine did not affect expression level of mutant topoisomerase I as determined by western blot (Figure 3B). Bucladesine mouse Figure 3 Addition of adenine to minimal medium increases survival following induction of mutant topoisomerase I cleavage complex BW27784 transformed with pAYTOP128 was grown overnight SPTLC1 in RM minimal medium with 2% glucose to suppress mutant topoisomerase I expression, then diluted 1:100 into RM medium with 0.2% glycerol. When OD600 reached

0.4, 0.00008% or 0.0002% arabinose was added with or without 100 μg/ml adenine included. Viable colony counts were determined at 1 h and 2 h after arabinose addition (a). The presence of adenine did not affect expression of mutant YpTOP after induction of 0.0002% arabinose for 2 h as analyzed by Western blot (b). To determine if addition of adenine affects sensitivity to norfloxacin, BW27784 cells grown in minimal medium with different adenine concentrations were first evaluated by examining growth inhibition by norfloxacin. Increased resistance to growth inhibition by norfloxacin was observed in the presence of 250 μg/ml adenine (Figure 4a). Growth of BW27784 in the absence of norfloxacin was not affected significantly by the presence of adenine. Viable colony counts at 3 h after norfloxacin treatment were then measured and found to be increased 24-fold by the presence of adenine (Figure 4b).