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 ,
 antibiotic resistance is an increasingly serious public health threat1 . 
 understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets28 . 
 we explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single - molecule real - time sequencing to characterize genomic methylation kinetics . 
 we find that escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at gatc sites . while the adenine methylome remains stable during drug stress , without gatc methylation , methyl - dependent mismatch repair ( mmr ) is deleterious , and fueled by the drug - induced error - prone polymerase poliv , overwhelms cells with toxic dna breaks . in multiple 
 e. coli strains , including pathogenic and drug - resistant clinical isolates , dna adenine methyltransferase deficiency potentiates antibiotics from the -lactam and quinolone classes . 
 this work indicates that the gatc methylome provides structural support for bacterial survival during antibiotics stress and suggests targeting bacterial dna methylation as a viable approach to enhancing antibiotic activity . 
Online methods
Bacterial strains and plasmids
Bacterial kill curves, MBC and MIC determination
Genomic DNA extraction and PacBio sequencing
Bioinformatics analyses of SMRT sequencing data
Flow cytometric assessment of DNA damage
Statistical analyses
Supplementary Material