Resistance to topoisomerase cleavage complex induced lethality in Escherichia coli via titration of transcription regulators PurR and FNR
© Liu et al; licensee BioMed Central Ltd. 2011
Received: 29 September 2011
Accepted: 12 December 2011
Published: 12 December 2011
Accumulation of gyrase cleavage complex in Escherichia coli from the action of quinolone antibiotics induces an oxidative damage cell death pathway. The oxidative cell death pathway has also been shown to be involved in the lethality following accumulation of cleavage complex formed by bacterial topoisomerase I with mutations that result in defective DNA religation.
A high copy number plasmid clone spanning the upp-purMN region was isolated from screening of an E. coli genomic library and analyzed for conferring increased survival rates following accumulation of mutant topoisomerase I proteins as well as treatment with the gyrase inhibitor norfloxacin.
Analysis of the intergenic region upstream of purM demonstrated a novel mechanism of resistance to the covalent protein-DNA cleavage complex through titration of the cellular transcription regulators FNR and PurR responsible for oxygen sensing and repression of purine nucleotide synthesis respectively. Addition of adenine to defined growth medium had similar protective effect for survival following accumulation of topoisomerase cleavage complex, suggesting that increase in purine level can protect against cell death.
Perturbation of the global regulator FNR and PurR functions as well as increase in purine nucleotide availability could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex.
DNA topoisomerases catalyze topological transformations of DNA by concerted breaking and rejoining of DNA strands via the formation of a covalent complex between the enzyme and cleaved DNA . While the activities of topoisomerases are critical for vital cellular functions, topoisomerase enzymes are also vulnerable targets for cell killing because DNA rejoining by topoisomerases can often be inhibited by antibacterial or anticancer agents that are referred to as topoisomerase poisons [2, 3]. Quinolones are widely used antibacterial drugs that lead to the accumulation of covalent cleavage complex formed by the bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV [4, 5]. The accumulation of DNA gyrase covalent complex from the action of quinolones has been shown to induce an oxidative damage cell death pathway in E. coli as at least one of the potential mechanisms of cell killing [6–9]. The sequence of events following topoisomerase cleavage complex accumulation that leads to generation of reactive oxygen species remains unclear.
Although a specific poison for bacterial topoisomerase I remains to be identified, accumulation of topoisomerase I cleavage complex in E. coli has also been shown to lead to rapid cell death from the study of topoisomerase I mutants defective in DNA rejoining [10, 11]. Similar to gyrase cleavage complex, topoisomerase I cleavage complex accumulation in E. coli induces the SOS response via the RecBCD pathway . Increase in reactive oxygen species has been shown to also contribute to the cell death pathway initiated by accumulation of topoisomerase I cleavage complex . Recombinant E. coli and Yersinia pestis topoisomerase I mutants that accumulate the covalent cleavage complex due to deficiency in DNA rejoining provide useful model systems for studying the physiological effect of topoisomerase-DNA cleavage complex accumulation. Y. pestis topoisomerase I (YpTOP1) is highly homologous to E. coli topoisomerase I, with the advantage of its dominant lethal recombinant clones being more stable in E. coli than comparable E. coli topoisomerase I mutant clones. The Y. pestis mutant topoisomerase I model system has been utilized to screen for E. 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 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.
Isolation of clone pAQ5 containing the upp-purMN region in selection for resistance to topoisomerase I cleavage complex mediated cell death
Effect of high copy plasmid clones on survival following accumulation of mutant topoisomerase I cleavage complex
7.85 × 10-5 ± 1.19 × 10-5
4.95 × 10-3 ± 1.55 × 10-3
4.92 × 10-3 ± 1.20 × 10-3
1.25 × 10-2 ± 2.48 × 10-3
1.90 × 10-2 ± 4.12 × 10-3
4.22 × 10-3 ± 1.02 × 10-3
5.19 × 10-4 ± 1.73 × 10-4
Analysis of resistance to topoisomerase I cleavage complex conferred by upp-purMN intergentic region
To determine the basis of resistance from clone pAQ5, derivatives of pAQ5 were constructed by cloning of specific PCR amplified DNA into pCR-XL-TOPO vector. These include clones pAQ5-1with purM and the intergenic region, pAQ5-2 with uppA and the intergenic region, and pInter, with the intergenic region alone (Figure 1a). These clones were transformed into strain BW27784 containing pAYTOP128 expressing mutant Y. pestis topoisomerase I deficient in DNA religation due to the TOPRIM G122S mutation to investigate the effect of the clones on viability following induction of mutant topoisomerase I. The results (Table 1) showed that the intergenic region alone in clone pInter was sufficient to confer resistance to the mutant topoisomerase I. Western blot analysis confirmed that the protective effect of pInter was also not due to reduction in expression level of mutant topoisomerase I (Figure 2b).
Examination of this intergenic sequence showed that it includes the binding site sequences of two transcription factors, FNR and PurR (Figure 1b). The FNR binding sequence, TTGACTTTAGTCAA versus the TTGATN4ATCAA consensus sequence [18–20], is located 61.5 nucleotides upstream of the upp transcription start site. The PurR binding sequence, CGCAAACGTTTGCTT, versus the consensus PurR operator sequence of CGCAAACGTTTNCNT , is located 28 nucleotides upstream of the purM gene. FNR acts as a dual transcription regulator that activates certain genes required for anaerobic growth and represses many genes required for aerobic growth . Its interaction with the upp-purMN region has been reported previously . PurR negatively regulates the transcription of genes involved in purine and pyrimidine nucleotide synthesis including purMN [21, 23, 24]. We therefore hypothesize that the high copy number pInter could titrate these transcription factors to relieve the repression of other E. coli genes encoded on the chromosome. To test this hypothesis, these binding sites were eliminated individually by site-directed mutagenesis (Figure 1c). Nucleotides TGACTTTAGTCA were deleted from the FNR binding site to result in plasmid pInterD1. Nucleotides AAACGTTTGCTT were deleted from the PurR binding site to result in plasmid pInterD2. Measurement of cell viability following induction of mutant topoisomerase from pAYTOP128 showed that elimination of either of these two binding sites reduced the protective effect of pInter, (Table 1). Comparison of the growth curves of these strains (Figure 2c) showed that while cells transformed with pInter and pInterD1 grew to a lower density at saturation, the initial growth rates of these strains are similar. The slightly slower growth rate of cells transformed with pInterD1 was not statistically significant and since pInterD1 conferred a lesser degree of resistance than pInter, the difference in viability following accumulation of topoisomerase I cleavage complex cannot be accounted for simply as due to growth inhibition.
Effect of high copy plasmid clones on survival following treatment with norfloxacin
2.14 × 10-5 ± 4.1 × 10-6
7.57 × 10-4 ± 2.14 × 10-4
6.12 × 10-4 ± 1.28 × 10-4
8.41 × 10-5 ± 3.55 × 10-5
1.11 × 10-4 ± 2.01 × 10-5
Protective effect from adenine addition
The high copy number intergenic region clone decreases the level of hydroxyl radicals following norfloxacin treatment
Effect of chromosomal fnr and purR mutations on sensitivity to topoisomerase I cleavage complex accumulation
The protective effect of Δfnr mutation was greater under low oxygen conditions
Protective effect of Δfn r mutation for cell killing initiated by mutant topoisomerase I cleavage complex accumulation under aerobic and low oxygen conditions
1.18 × 10-4 ± 7.7 × 10-5
1.07 × 10-3 ± 4.7 × 10-4
1.30 × 10-3 ± 3.1 × 10-4
8.15 × 10-2 ± 3.1 × 10-3
However, chromosomal ΔpurR and Δfnr mutations were found to have little effect on the viable colony counts at 1 and 2 h after treatment with up to 250 ng/ml norfloxacin (data not shown). Greater than 1000-fold lower bactericidal rates were observed for BW27784 with oxygen limitation when compared to incubation with oxygen after treatment with norfloxacin, in agreement with previous report of decreased norfloxacin sensitivity under anaerobic conditions . It is therefore not feasible to investigate any potential protective effect from pInter or the Δfnr mutation under low oxygen conditions.
A segment of E. coli chromosomal DNA spanning the upp-purMN region was selected from a high copy number plasmid library of E. coli genomic DNA fragments based on its ability to confer resistance to cell killing mediated by accumulation of topoisomerase I cleavage complex. The intergenic region of upp-purMN was found to protect against bacterial cell death initiated by both type I and type II covalent topoisomerase-DNA cleavage complex. Deletion of the binding sites for FNR and PurR decreased the protective effect, suggesting that the protective effect we observed for pInter resulted from titration of the transcription regulators FNR and PurR.
PurR is a repressor of purine biosynthesis in E. coli . The hypothesis that the protective effects observed from the high copy number plasmid pInter is related to purine nucleotide pool availability is supported by the increased viability when adenine was added to defined medium. The ΔpurR mutation resulted in up to 475-fold higher survival rate following topoisomerase I covalent cleavage complex accumulation. Although pInter could increase survival rate following norfloxacin treatment, the ΔpurR chromosomal mutation did not affect norfloxacin sensitivity. Deletion mutation of a global transcription regulator is likely to affect the many metabolic genes under its regulation differently than titration of the global transcription regulator by the presence of its binding site on a high copy number plasmid. Chromosomal PurR recognition sites with the strongest binding affinity for PurR might still be repressed by PurR even in the presence of pInter but they would be depressed in the ΔpurR background. The cell death pathways initiated by type IA and type IIA topoisomerases may be affected to different degrees by the change in metabolic gene expression resulting from ΔpurR mutation. The level of cellular ATP and NAD+/NADH ratio are factors that could influence the induction of the reactive oxygen species following accumulation of the topoisomerase cleavage complex [7, 8].
FNR is a global regulator for the response of many genes to oxygen level [22, 28]. It can activate or repress different genes directly by binding to the upstream regulatory region . FNR also activates the transcription of the small non-coding RNA FnrS which negatively regulates the expression of multiple genes, including many that encode enzymes with functions linked to oxidative stress [26, 27]. The presence of its binding site on pInter was responsible for part of the resistance to topoisomerase I cleavage complex mediated cell killing conferred by this high copy number plasmid. The oxygen level in the culture decreased as cell growth approached stationary phase even with shaking, probably resulting in partial activity of the FNR protein. Regulatory effect of FNR on transcription of acetyl coenzyme A synthetase gene in E. coli has been previously observed under conditions that are not strictly anaerobic . We showed that the protective effect of the Δfnr mutation on cell death following topoisomerase I cleavage complex accumulation was more prominent under low oxygen condition, consistent with the increased activity of FNR expected when oxygen is limiting. FNR may influence cell death pathway initiated by topoisomerase cleavage complex by suppressing the genes that can enhance the response to reactive oxygen species implicated in the cell death pathway. Alternatively, decrease in FNR activity may alter the metabolic state of the cell, so that it is less susceptible to the oxidative damage cell death pathway.
In future studies, it would be informative to express FNR and/or PurR in the corresponding deletion mutants under the control of an inducible promoter. This would allow examination of promoter occupation across the genome and correlate global gene expression pattern with sensitivity to the oxidative damage cell death pathway.
Bacterial strains and plasmids
E. coli strains and plasmids used in this study
Source or construction
E. coli strains
Yale E. coli Genetic Stock Center
BW27784 with chromosomally integrated YpTOP1-D117N gene
Δara leu7697 NBRP
NBRP-E. coli at NIG
U. Wisconsin 
P1(FB20344) × AQ4335, Kanr
Yale E. coli Genetic Stock Center 
Yale E. coli Genetic Stock Center 
BW27784 Δ fnr771::kan
P1(JW1328-1) × BW27784, Kanr
BW27784 Δ purR746::kan
P1(JW1650-1) × BW27784, Kanr
Mutant derivative of pAYTOP encoding YpTOP1 with G122S, M326V and A383P mutations
High copy number cloning vector
pCR-XL-TOPO cloning product of E. coli chromosome fragment 2618398-2620765
pCR-XL-TOPO carrying upp gene and the intergenic region of upp-purMN
pCR-XL-TOPO carrying purM gene and the intergenic region of upp-purMN
pCR-XL-TOPO carrying the intergenic region of upp-purMN
pInter with the FNR binding site deleted
pInter with the PurR binding site deleted
Screening of clones conferring resistance to topoisomerase I cleavage complex
E. coli YT103 chromosomal fragments, with sizes between 2.5 and 4.5 kbp, generated from partial Sau3A1 digestion and sonication were gel purified and used to generate a high copy number plasmid library with the pCR-XL-TOPO cloning system (Invitrogen). The pooled plasmid library with >10,000 genomic DNA clones was used to transform E. coli BW117N by electroporation. Transformants that were resistant to the dominant lethal effect of YpTOP1-D117N were selected by plating on LB plates with antibiotics and 0.002% arabinose. Plasmid was isolated from viable colonies and confirmed in subsequent transformation of BW117N to confer resistance to cell killing mediated by topoisomerase I cleavage complex accumulation.
Cell viability assays
Transformants of BW27784 or BW117N were grown in LB medium with antibiotics to exponential phase (OD600 = 0.4). The cultures were treated with either arabinose to induce recombinant mutant topoisomerase I or the gyrase inhibitor norfloxacin for the stated length of time at 37°C with shaking at 215 rpm unless otherwise stated. Serial dilutions of the cultures were then plated on LB plates with antibiotics with 2% glucose added for BW117N or BW27784 transformed with pAYTOP128, and incubated overnight. The viable colony counts from the treated cultures were normalized against the untreated culture to calculate the survival ratio. The results shown represent the average and standard errors of at least three experiments.
Western blot analysis of recombinant Y. pestis topoisomerase I expression
Exponential phase cultures were treated with indicated concentration of arabinose for 2 or 2.5 h. Cells were collected by centrifugation from volumes based on OD600 and resuspended in SDS gel sample buffer before boiling for 5 min and SDS page for total protein analysis. The coomassie blue stained gel was examined to confirm equal loading. For improved control of equal loading in experiments using minimal media, total soluble proteins were prepared and quantitated by the BioRad Dc protein assay. Mouse monoclonal antibodies against E. coli topoisomerase I were used in Western blot analysis to detect the highly homologous Y. pestis topoisomerase I. Partially degraded Y. pestis topoisomerase I (YpTOP*) was also detected.
Hydroxyl radicals formation assay
BW27784 transformed with vector or pInter was grown to exponential phase in LB before treatment with 250 ng/ml norfloxacin, or left untreated as control. After the indicated time, hydroxyl radicals were measured with the fluorescent reporter dye, 3'(p-hydroxyphenyl) fluorescence (HPF) in a FACScan flow cytometer (Becton Dickinson) .
We demonstrated that titration of the E. coli transcription factors FNR and PurR by plasmid clones with the transcription factor binding sites can confer resistance to cell killing mediated by mutant topoisomerase I cleavage complex and norfloxacin acting on DNA gyrase. Our study showed that perturbation of the global regulator FNR and PurR function as well as increase in purine nucleotide availability, could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex. The metabolic state of the cell is likely to be an important factor for the bactericidal outcome in this cell death pathway.
We acknowledge NBRP-E. coli at NIG and the Yale E. coli Genetic Stock Center for providing strains. This study was funded by NIH grant R01AI069313 to Yuk-Ching Tse-Dinh.
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