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A role for the bacterial GATC methylome in antibiotic stress survival

Nature Genetics volume 48, pages 581–586 (2016)Cite this article

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Abstract

Antibiotic resistance is an increasingly serious public health threat1. Understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets2,3,4,5,6,7,8. 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. Although 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 Pol IV, 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 β-lactam and quinolone classes. This work indicates that the GATC methylome provides structural support for bacterial survival during antibiotic stress and suggests targeting bacterial DNA methylation as a viable approach to enhancing antibiotic activity.

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Figure 1: Increased sensitivity to β-lactams in the absence of Dam methylation.
Figure 2: Kinetics of the Dam methylome during normal growth and under antibiotic stress.
Figure 3: Pol IV–dependent mutagenesis fuels MMR-mediated DNA damage in β-lactam–stressed Δdam E. coli.
Figure 4: Quinolone toxicity is potentiated in laboratory and pathogenic Δdam E. coli.

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Acknowledgements

We thank T. Ferrante, E. Cameron, K. Allison, J. Winkler, J. Way, C. Gruber, P. Bhargava, M. Painter, N. Sherpa, T. Lieberman, L. Certain and Y. Furuta for critical feedback and technical support over the course of this study. We also thank M. Conover and S. Hultgren (Washington University School of Medicine in St. Louis) for generously providing the UTI89 strain, as well as related reagents and protocols. Additionally, we are grateful for SMRT sequencing support from M. Zapp and E. Kittler at the University of Massachusetts Medical School Sequencing Core and from S. Mark, K. Luong, M. Weiand and O. Banerjee at Pacific Biosciences. This work was supported by funding from Defense Threat Reduction Agency grant HDTRA1-15-1-0051, US National Institutes of Health grant 1U54GM114838-01, the Mayo Clinic Center for Individualized Medicine and Donors Cure Foundation, the Howard Hughes Medical Institute, the Banting Postdoctoral Fellowship from the Canadian Institutes of Health and Research, and the Wyss Institute for Biologically Inspired Engineering, Harvard University.

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  1. Christian A Ross and Saloni Jain: These authors contributed equally to this work.

Authors and Affiliations

  1. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    Nadia R Cohen & James J Collins

  2. Department of Biological Engineering, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Nadia R Cohen, Saloni Jain, Rebecca S Shapiro, Arnaud Gutierrez & James J Collins

  3. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Nadia R Cohen & James J Collins

  4. Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Information Technology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA

    Christian A Ross & Hu Li

  5. Boston University, Boston, Massachusetts, USA

    Saloni Jain & James J Collins

  6. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Rebecca S Shapiro, Arnaud Gutierrez & James J Collins

  7. Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, USA

    Peter Belenky

  8. Harvard-MIT Program in Health Sciences and Technology, Boston, Massachusetts, USA

    James J Collins

  9. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    James J Collins

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Contributions

N.R.C. conceived of the project, designed and performed experiments, and wrote the manuscript. C.A.R. performed bioinformatics analyses. S.J. performed experiments. R.S.S. performed experiments and bioinformatics analyses. A.G. contributed intellectually to the project and helped design experiments. P.B. contributed intellectually to the project. H.L. performed bioinformatics analyses and provided mentorship. J.J.C. oversaw the project and provided mentorship.

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Integrated supplementary information

Supplementary Figure 1 Susceptibility of MTase-deficient E. coli K12 to sublethal ampicillin treatment.

(a,b) Wild-type (wt) or MTase-deficient E. coli BW25113 (a) or MG1655 (b) were grown in LB to an OD of 0.3 and then treated with ampicillin (2.5 μg/ml) or left untreated. CFUs and/or OD were monitored hourly. (c) Determination of ampicillin MIC (left) and MBC90 (right) for E. coli BW25113 by broth microdilution in LB. Wild-type and Δdam MIC values were 6 μg/ml and 4 μg/ml, respectively; wild-type and Δdam MBC90 values were 2.7 μg/ml and 1.5 μg/ml, respectively. Dotted lines indicate cutoff values for MIC (OD < 0.1) or MBC90 (10% survival). MBC90 values were interpolated using a sigmoidal curve fit model as shown. In a–c, data are shown as means ± s.e.m. of n = 2–3 independent experiments.

Supplementary Figure 2 Complementation of Δdam E. coli ampicillin sensitivity with wild-type or catalytically inactive mutant Dam.

Wild-type or Δdam E. coli BW25113 were transformed with the indicated Cmr plasmid expressing gfp, dam or mutant dam in which the catalytic DPPY motif is disrupted by a single amino acid change (D181S, D181N or P183R). Plasmid-bearing strains were grown in LB supplemented with chloramphenicol (15 μg/ml). (a) Genomic DNA extracted from the indicated strains at stationary phase was either digested with DpnII, which cleaves at GATC sites only in the absence of methylation, or left undigested. Digests were run on a 0.8% agarose gel containing ethidium bromide. (b) OD kinetics of wild-type or Δdam E. coli BW25113 harboring the indicated Cmr plasmids cultured in LB supplemented with chloramphenicol (15 μg/ml) with or without ampicillin (2.5 μg/ml) at log phase. (c) The indicated E. coli BW25113 strains were grown in LB to an OD of 0.2 and then treated with 2 μg/ml ampicillin or left untreated. In b and c, CFUs and/or OD were monitored hourly to assess survival. Data are shown as means ± s.e.m. of n = 3 independent experiments.

Supplementary Figure 3 Stability of GATC methylome kinetics during growth in the presence or absence of ampicillin.

Genome-wide GATC methylation kinetics displayed as in Figure 2d during growth in LB (top) or in LB supplemented with 2.5 μg/ml ampicillin (bottom). The top panel is the same as the plot displayed in Figure 2d. The empty green arrowhead highlights the only GATC site displaying statistically significant differential methylation between untreated and ampicillin-treated samples (Supplementary Table 2 and Supplementary Data Set 1).

Supplementary Figure 4 dinB, mutS and mutH dependence of Δdam E. coli sensitivity to ampicillin.

The indicated E. coli BW25113 strains were grown in LB to an OD of 0.3 and then treated with 2.5 μg/ml of ampicillin or left untreated. OD was monitored hourly to assess survival. Data are shown as means ± s.e.m. of n = 2 independent experiments.

Supplementary Figure 5 dinB, mutS and mutH dependence of Δdam sensitivity to ofloxacin.

The indicated E. coli BW25113 strains were grown in LB to an OD of 0.3 and then treated with ofloxacin (50 ng/ml) or left untreated. CFUs in bacterial cultures were monitored hourly to assess survival. Data are shown as mean percent survival ± s.e.m. of n = 2 independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3. (PDF 2670 kb)

Supplementary Data Set 1

Raw methylation data and statistical analyses. (XLSX 10603 kb)

Supplementary Data Set 2

SMRT sequence coverage. (XLSX 6317 kb)

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Cohen, N., Ross, C., Jain, S. et al. A role for the bacterial GATC methylome in antibiotic stress survival. Nat Genet 48, 581–586 (2016). https://doi.org/10.1038/ng.3530

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