laboratory bacterial strains used are derived from e. coli k12 ( bw25113 obtained from the coli genetic stock center or mg1655 obtained from atcc ) . the uropathogenic e. coli strain uti89 was kindly provided to us from matt conover and scott hultgren . the ciprofloxacin - resistant uropathogenic e. coli isolate ( upec cipro ) was collected from the brigham and women s hospital specimen bank ( sup . table 3 ) . deletion mutants on the bw25113 background were derived from the keio collection following kan cassette removal . deletion mutants on the mg1655 background were constructed by allelic transduction from keio collection strains using classical p1 phage transduction , followed by kan cassette excision . the dam null phenotype was confirmed by pcr alone or with electrophoresis of genomic dna digested with dpnii , which cleaves unmethylated gatc sites only . for construction of the dam uti89 and dam upec cipo strains , the parent strain bearing a km208 plasmid - based red - recombinase system was electroporated with a pcr amplicon encoding the dam::kan allele . the genotype of each deletion strain was verified by colony pcr . the plasmid used in the dam complementation studies , namely pzs*31 ( fig . pzs*31 has a psc101 * origin of replication ( which yields a low copy number of 35 plasmids per cell ) and a chloramphenicol - resistance marker . genes encoding either dam ( with 500bp upstream flanking region ) or gfp were inserted into the multiple cloning site . for complementation experiments using mutated versions of dam , the plasmid containing the dam insert was engineered using either gibson cloning or site - directed mutagenesis ( neb , q5 site - directed mutagenesis kit ) . quinolone resistance - conferring mutations in the cipo upec clinical isolate were identified though whole - genome illumina sequencing of genomic dna ( purelink pro-96 genomic purification kit ; life technologies ) . raw sequencing reads were processed by trimming adapter sequences and discarding reads shorter then 28bp . the genome alignments were searched for known quinolone resistance - conferring mutations in acra , acrr , beas , cpxa , cpxb , envz , gyra , gyrb , mara , marr , mdta , mdtb , mdtc , ompc , ompf , ompr , parc , pare , soxr , soxs and tolc genes and their regulatory regions . for timecourse kill curves and mbc assays , stationary - phase bacterial cultures were diluted at 1:1,000 in 25mls of lb medium in 250ml baffled flasks . cultures were transferred to 24-well plates at 500 l per well , or to 96-well plates at a final volume of 150ul per well , and either left untreated or treated with the indicated drugs at specified doses . plates were sealed using breathable membranes ( breatheeasy , cat # : bem-1 ) and incubated at 37c and 900rpm for the remainder of the experiment . cfus were enumerated at desired time points ( 4 hours for mbc determination ) by spot plating 5 l of ten - fold serially diluted culture onto lb agar and counting colonies after overnight growth at 37c . percent survival at each timepoint was calculated in relation to the cfu immediately before treatment ( 0h ) . for mic determination , antibiotics were serially diluted in a 96-well plate and mixed with stationary - phase bacterial cultures diluted at 1:10,000 in a final volume of 150 l lb per well . genomic dna ( gdna ) was extracted from e. coli k12 mg1655 lb cultures grown in the presence or absence of ampicillin using the genelute bacterial genomic dna extraction kit ( sigma ) . to assess genomic methylation status , gdna extracted from stationary - phase cultures was quantified , digested using dpnii ( neb ) and run on an 0.8% agarose gel containing ethidium bromide . for methylome analyses , samples were sent to umass medical school deep sequencing core , where methylome data were obtained by pacbio core enterprise instrument smrt . smrtbell dna template libraries for smrt sequencing were prepared according to the instructions described in the  procedure & checklist for 10 kb template preparation and sequencing document ( pacific biosciences ) . briefly , genomic dna samples were first sheared to a target shear size of 10 kb using g - tube devices ( covaris , inc . ) , treated with dna damage repair mix , end - repaired and ligated to hairpin adapters . the smrtbell libraries were prepared using the dna template prep kit 2.0 ( 310 kb ) fro pacific biosciences . incompletely formed smrtbell templates were digested using exonuclease iii ( new england biolabs ) and exonuclease vii ( affymetrix ) . the prepared smrtbell libraries were sequenced using a 120-min movie acquisition time and p4 polyerase - c2 dna sequencing reagent kits following standard instructions for a pacbio rs ii instrument ( pacific biosciences ) . each e. coli sample was sequenced on four or more smrt cells yielding a total of approximately 200-fold double - stranded coverage of the bacterial genome , and two or three biological replicates were sequenced for each antibiotic treatment condition ( sup . sequencing coverage was comparable between methylated and non - methylated sites ( sup . table 1 , sup . data set 2 ) , ruling out coverage loss as an explanation for the absence of methylation . genome - wide detection of base modification and the affected motifs was performed using the standard ( default ) settings in the the fasta reference genome sequence ( e. coli k12 mg1655 , ncbi nc_000913.2 ) used for the base modification detection analyses was obtained from pacific biosciences . for motif identification , the base modification quality value ( qv ) threshold setting was left at the default value of 30 . interpulse duration ( ipd ) values were measured for all nucleotide positions in the genome and compared with expected durations in an in silico kinetic model of the polymerase for significant associations . frac values were calculated in smrt analysis using a standard mixture model analysis of the pooled kinetic data for a given sample . the frac output value provides information about the fraction of individual molecules displaying a methylation signal at each identified motif site within the genome ( sup . methylation frac values were derived from ipd data within the smrt pipeline using the single site mixture model . the values from two or three experimental replicates were compared by student s t - test and fdr adjusted p - values were obtained by the method of benjamini and hochberg ( sup . circular graphs were generated using the circos software package   e. coli log - phase cultures were transferred to a 96-well plate ( 200 l / well ) and treated with ampicillin ( 2.5 g / ml ) or hydrogen peroxide ( 100 mm ) for 30 minutes to 2 hours at 37c and 900rpm . cell pellets were resuspended vigorously in 200 l of cold 4% paraformaldehyde / pbs and incubated at room temperature for 30 minutes to allow fixation . bacteria were centrifuged again , then resuspended in 200 l of cold permeabilization buffer ( 0.1%tritonx-100 in 0.1% sodium citrate ) . after pelleting the cells and discarding the supernatant , cells were resuspended in 50 l of tunel labeling mix ( dutp - fitc and tdt enzyme ) or 50 l tunel labeling reagent ( dutp - fitc ) according to manufacturer s instructions ( roche ; in situ cell death detection kit , fluorescein ) . cells were then washed twice with pbs , resuspended in 1 g / ml pi / pbs and analyzed by flow cytometry ( bd lsr fortessa ) . pi negative cells , which lack genomic material , were excluded from the analysis . 3d and statistical analysis , background staining with labeling reagent only was subtracted for each sample to account for treatment dependent shifts in auto - fluorescence or stain retention . statistical analysis performed on log10-transformed data ( for survival experiments ) or on untransformed data ( for tunel assay ) using a two - way anova followed by a post - hoc t - test using sidak s multiple comparison test correction . in all cases , laboratory bacterial strains used are derived from e. coli k12 ( bw25113 obtained from the coli genetic stock center or mg1655 obtained from atcc ) . the uropathogenic e. coli strain uti89 was kindly provided to us from matt conover and scott hultgren . the ciprofloxacin - resistant uropathogenic e. coli isolate ( upec cipro ) was collected from the brigham and women s hospital specimen bank ( sup . table 3 ) . deletion mutants on the bw25113 background were derived from the keio collection following kan cassette removal . deletion mutants on the mg1655 background were constructed by allelic transduction from keio collection strains using classical p1 phage transduction , followed by kan cassette excision . the dam null phenotype was confirmed by pcr alone or with electrophoresis of genomic dna digested with dpnii , which cleaves unmethylated gatc sites only . for construction of the dam uti89 and dam upec cipo strains , the parent strain bearing a km208 plasmid - based red - recombinase system was electroporated with a pcr amplicon encoding the dam::kan allele . the genotype of each deletion strain was verified by colony pcr . the plasmid used in the dam complementation studies , namely pzs*31 ( fig . pzs*31 has a psc101 * origin of replication ( which yields a low copy number of 35 plasmids per cell ) and a chloramphenicol - resistance marker . genes encoding either dam ( with 500bp upstream flanking region ) or gfp were inserted into the multiple cloning site . for complementation experiments using mutated versions of dam , the plasmid containing the dam insert was engineered using either gibson cloning or site - directed mutagenesis ( neb , q5 site - directed mutagenesis kit ) . quinolone resistance - conferring mutations in the cipo upec clinical isolate were identified though whole - genome illumina sequencing of genomic dna ( purelink pro-96 genomic purification kit ; life technologies ) . raw sequencing reads were processed by trimming adapter sequences and discarding reads shorter then 28bp . the genome alignments were searched for known quinolone resistance - conferring mutations in acra , acrr , beas , cpxa , cpxb , envz , gyra , gyrb , mara , marr , mdta , mdtb , mdtc , ompc , ompf , ompr , parc , pare , soxr , soxs and tolc genes and their regulatory regions . for timecourse kill curves and mbc assays , stationary - phase bacterial cultures were diluted at 1:1,000 in 25mls of lb medium in 250ml baffled flasks . cultures were transferred to 24-well plates at 500 l per well , or to 96-well plates at a final volume of 150ul per well , and either left untreated or treated with the indicated drugs at specified doses . plates were sealed using breathable membranes ( breatheeasy , cat # : bem-1 ) and incubated at 37c and 900rpm for the remainder of the experiment . cfus were enumerated at desired time points ( 4 hours for mbc determination ) by spot plating 5 l of ten - fold serially diluted culture onto lb agar and counting colonies after overnight growth at 37c . percent survival at each timepoint was calculated in relation to the cfu immediately before treatment ( 0h ) . for mic determination , antibiotics were serially diluted in a 96-well plate and mixed with stationary - phase bacterial cultures diluted at 1:10,000 in a final volume of 150 l lb per well . genomic dna ( gdna ) was extracted from e. coli k12 mg1655 lb cultures grown in the presence or absence of ampicillin using the genelute bacterial genomic dna extraction kit ( sigma ) . to assess genomic methylation status , gdna extracted from stationary - phase cultures was quantified , digested using dpnii ( neb ) and run on an 0.8% agarose gel containing ethidium bromide . for methylome analyses , samples were sent to umass medical school deep sequencing core , where methylome data were obtained by pacbio core enterprise instrument smrt . smrtbell dna template libraries for smrt sequencing were prepared according to the instructions described in the  procedure & checklist for 10 kb template preparation and sequencing document ( pacific biosciences ) . briefly , genomic dna samples were first sheared to a target shear size of 10 kb using g - tube devices ( covaris , inc . ) , treated with dna damage repair mix , end - repaired and ligated to hairpin adapters . the smrtbell libraries were prepared using the dna template prep kit 2.0 ( 310 kb ) fro pacific biosciences . incompletely formed smrtbell templates were digested using exonuclease iii ( new england biolabs ) and exonuclease vii ( affymetrix ) . the prepared smrtbell libraries were sequenced using a 120-min movie acquisition time and p4 polyerase - c2 dna sequencing reagent kits following standard instructions for a pacbio rs ii instrument ( pacific biosciences ) . each e. coli sample was sequenced on four or more smrt cells yielding a total of approximately 200-fold double - stranded coverage of the bacterial genome , and two or three biological replicates were sequenced for each antibiotic treatment condition ( sup . sequencing coverage was comparable between methylated and non - methylated sites ( sup . table 1 , sup . data set 2 ) , ruling out coverage loss as an explanation for the absence of methylation . genome - wide detection of base modification and the affected motifs was performed using the standard ( default ) settings in the  rs_modification_and_motif_analysis.1 protocol included in smrt analysis version 2.3.0 patch 5 . the fasta reference genome sequence ( e. coli k12 mg1655 , ncbi nc_000913.2 ) used for the base modification detection analyses was obtained from pacific biosciences . for motif identification , the base modification quality value ( qv ) threshold setting was left at the default value of 30 . interpulse duration ( ipd ) values were measured for all nucleotide positions in the genome and compared with expected durations in an in silico kinetic model of the polymerase for significant associations . frac values were calculated in smrt analysis using a standard mixture model analysis of the pooled kinetic data for a given sample . the frac output value provides information about the fraction of individual molecules displaying a methylation signal at each identified motif site within the genome ( sup . methylation frac values were derived from ipd data within the smrt pipeline using the single site mixture model . the values from two or three experimental replicates were compared by student s t - test and fdr adjusted p - values were obtained by the method of benjamini and hochberg ( sup . e. coli log - phase cultures were transferred to a 96-well plate ( 200 l / well ) and treated with ampicillin ( 2.5 g / ml ) or hydrogen peroxide ( 100 mm ) for 30 minutes to 2 hours at 37c and 900rpm . cell pellets were resuspended vigorously in 200 l of cold 4% paraformaldehyde / pbs and incubated at room temperature for 30 minutes to allow fixation . bacteria were centrifuged again , then resuspended in 200 l of cold permeabilization buffer ( 0.1%tritonx-100 in 0.1% sodium citrate ) . after 2 minutes at room temperature after pelleting the cells and discarding the supernatant , cells were resuspended in 50 l of tunel labeling mix ( dutp - fitc and tdt enzyme ) or 50 l tunel labeling reagent ( dutp - fitc ) according to manufacturer s instructions ( roche ; in situ cell death detection kit , fluorescein ) . cells were then washed twice with pbs , resuspended in 1 g / ml pi / pbs and analyzed by flow cytometry ( bd lsr fortessa ) . pi negative cells , which lack genomic material , were excluded from the analysis . 3d and statistical analysis , background staining with labeling reagent only was subtracted for each sample to account for treatment dependent shifts in auto - fluorescence or stain retention . statistical analysis performed on log10-transformed data ( for survival experiments ) or on untransformed data ( for tunel assay ) using a two - way anova followed by a post - hoc t - test using sidak s multiple comparison test correction . in all cases ,