coli genomic clones,

that when present in high copy number on a plasmid, can confer resistance to topoisomerase cleavage complex induced cell killing. Additional experiments on an isolated clone demonstrated a novel mechanism of increased resistance to topoisomerase cleavage complex via titration of the transcription factors FNR and PurR by a high copy number plasmid clone of the intergenic region between upp and purM. This plasmid clone also increased bacterial resistance to norfloxacin that induces click here the accumulation of the type IIA topoisomerase covalent cleavage complex. FNR regulates transition between anaerobic and aerobic conditions [14, 15]. Genome-wide expression analysis has previously shown that FNR contributes to the repression of a number of genes induced by oxidative stress conditions [16, 17]. PurR is a suppressor of purine biosynthesis. Titration of the FNR and PurR transcription factors by

the high copy number clone is expected to increase the expression level of genes normally suppressed by these two regulators. These results provide further insights into the oxidative cell death pathways initiated by topoisomerase cleavage complex accumulation. Results Isolation of clone pAQ5 containing AR-13324 in vivo the upp-purMN region in selection for resistance to topoisomerase I cleavage complex mediated cell death After transformation of E. coli strain BW117N with the E. coli genomic DNA library generated with the pCR-XL-TOPO cloning system, four different plasmid clones isolated from colonies obtained on LB plates with 0.002% arabinose were confirmed to increase resistance to the dominant lethal effect of the mutant Y. pestis topoisomerase I, YpTOP1-D117N [10]. Detailed analysis of the clone pAQ5 containing the upp-purMN region of E. coli chromosome (corresponding

to nucleotides 2618398-2620765 of E. coli MG1655 sequence, Figure 1a) is described here. Strain BW117N is under strong selective pressure to eliminate expression of the dominant lethal mutant YpTOP1-D117N. Subsequent analysis of the effect of clone pAQ5 or its derivatives was therefore carried out with strain BW27784 carrying plasmid pAYTOP128 expressing YpTOP1 with tuclazepam the less lethal G122S mutation that also leads to accumulation of the topoisomerase I cleavage complex [11]. Clone pAQ5 was found to increase survival following arabinose induction of this mutant YpTOP1 by 63-fold compared to the control empty vector (Table 1). This clone (Figure 1a) contains the entire purM (5′-phosphoribosyl-5-aminoimidazole synthetase) coding sequence (2619219-2620256), part of the purN (phosphoribosylglycinamide formyltransferase) coding sequence (2620256-2620894), and part of the upp (uracil phosphoribosyltransferase) coding sequence (2618268-2618894), plus the intergenic regulatory region between the upp and purMN genes (2618946-2619178).