SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary Table 1 and Supplementary Figures 1–4. ACKNOWLEDGEMENTS The authors are grateful to Dr Daniel Blanchard from UC Berkeley and Dr Tobias Kieser for editing this article. FUNDING The National Science Foundation of China, the Ministry of Science and Technology [973 (2012CB721004) and 863 programs]; Ministry of Education of China, the Shanghai Municipal Council of Science and Technology and Shanghai Leading Academic Discipline Project B203; State Key Laboratory of Bio-organic and Natural Products Chemistry (CAS); National Program of Development of Transgenic New Species of China. Funding for open access charge: the Ministry of Science and Technology [973 (2012CB721004)]. Conﬂict of interest statement. None declared. REFERENCES 1. Liang,J.D., Wang,Z.J., He,X.Y., Li,J.L., Zhou,X.F. and Deng,Z.X. (2007) DNA modiﬁcation by sulfur: analysis of the sequence recognition speciﬁcity surrounding the modiﬁcation sites. Nucleic Acids Res., 35, 2944–2954. 2. Wang,L., Chen,S., Xu,T., Taghizadeh,K., Wishnok,J.S., Zhou,X., You,D., Deng,Z. and Dedon,P.C. (2007) Phosphorothioation of DNA in bacteria by dnd genes. Nat. Chem. Biol., 3, 709–710. 3. Xu,T.G., Liang,J.D., Chen,S., Wang,L.R., He,X.Y., You,D.L., Wang,Z.J., Li,A.Y., Xu,Z.L., Zhou,X.F. et al. (2009) DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus. BMC Microbiol., 9, 41. 4. Zhou,X.F., Deng,Z.X., Firmin,J.L., Hopwood,D.A. and Kieser,T. (1988) Site-speciﬁc degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron. Nucleic Acids Res., 16, 4341–4352. 5. Zhou,X., He,X., Liang,J., Li,A., Xu,T., Kieser,T., Helmann,J.D. and Deng,Z. (2005) A novel DNA modiﬁcation by sulphur. Mol. Microbiol., 57, 1428–1438. 6. Wang,L., Chen,S., Vergin,K.L., Giovannoni,S.J., Chan,S.W., DeMott,M.S., Taghizadeh,K., Cordero,O.X., Cutler,M., Timberlake,S. et al. (2011) DNA phosphorothioation is widespread and quantized in bacterial genomes. Proc. Natl Acad. Sci. USA, 108, 2963–2968. 7. He,X., Ou,H.Y., Yu,Q., Zhou,X., Wu,J., Liang,J., Zhang,W., Rajakumar,K. and Deng,Z. (2007) Analysis of a genomic island housing genes for DNA S-modiﬁcation system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria. Mol. Microbiol., 65, 1034–1048. 8. Ou,H.Y., He,X., Shao,Y., Tai,C., Rajakumar,K. and Deng,Z. (2009) dndDB: a database focused on phosphorothioation of the DNA backbone. PLoS One, 4, e5132. 9. Liu,G.A., Ou,H.Y., Wang,T., Li,L., Tan,H.R., Zhou,X.F., Rajakumar,K., Deng,Z.X. and He,X.Y. (2010) Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA. PLos Genet., 6., e1001253. 10. Xu,T.G., Yao,F., Zhou,X.F., Deng,Z.X. and You,D.L. (2010) A novel host-speciﬁc restriction system associated with DNA backbone S-modiﬁcation in Salmonella. Nucleic Acids Res., 38, 7133–7141. Nucleic Acids Research, 2012, Vol. 40, No. 18 9123 11. Ray,T., Weaden,J. and Dyson,P. (1992) Tris-dependent site-speciﬁc cleavage of Streptomyces lividans DNA. FEMS Microbiol. Lett., 96, 247–252. 12. Ray,T., Mills,A. and Dyson,P. (1995) Tris-dependent oxidative DNA strand scission during electrophoresis. Electrophoresis, 16, 888–894. 13. Sambrook,J. (2001) Molecular Cloning: A Laboratory Manual. 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 14. O’Reilly,L.C. (2011) A method for overcoming DNA degradation during PFGE for Serratia marcescens. J. Microbiol. Methods, 85, 173–174. 15. Miled-Bennour,R., Ells,T.C., Pagotto,F.J., Farber,J.M., Kerouanton,A., Meheut,T., Colin,P., Joosten,H., Leclercq,A. and Besse,N.G. (2010) Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates. Int. J. Food Microbiol., 139, 116–125. 16. Ribeiro,R.L., Machry,L., Brazil,J.M., Ramos,T.M., Avelar,K.E. and Pereira,M.M. (2009) Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed ﬁeld gel electrophoresis. Lett. Appl. Microbiol., 49, 289–291. 17. Bosilevac,J.M., Guerini,M.N., Kalchayanand,N. and Koohmaraie,M. (2009) Prevalence and characterization of salmonellae in commercial ground beef in the United States. Appl. Environ. Microbiol., 75, 1892–1900. 18. Banerjee,S.K. and Farber,J.M. (2009) Susceptibility of Vibrio parahaemolyticus to tris-dependent DNA degradation during pulsed-ﬁeld gel electrophoresis. J. Clin. Microbiol., 47, 870–871. 19. Leclair,D., Pacyotto,F., Farber,J.M., Cadieux,B. and Austin,J.W. (2006) Comparison of DNA ﬁngerprinting methods for use in investigation of type E botulism outbreaks in the Canadian arctic. J. Clin. Microbiol., 44, 1635–1644. 20. Ciofu,O., Riis,B., Pressler,T., Poulsen,H.E. and Hoiby,N. (2005) Occurrence of hypermutable Pseudomonas aeruginosa in cystic ﬁbrosis patients is associated with the oxidative stress caused by chronic lung inﬂammation. Antimicrobial Agents Chemother., 49, 2276–2282. 21. Zhang,Y.S., Yakrus,M.A., Graviss,E.A., Williams-Bouyer,N., Turenne,C., Kabani,A. and Wallace,R.J. (2004) Pulsed-ﬁeld gel electrophoresis study of Mycobacterium abscessus isolates previously affected by DNA degradation. J. Clin. Microbiol., 42, 5582–5587. 22. Murase,T., Nagato,M., Shirota,K., Katoh,H. and Otsuki,K. (2004) Pulsed-ﬁeld gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm. Vet. Microbiol., 99, 139–143. 23. Silbert,S., Boyken,L., Hollis,R.J. and Pfaller,M.A. (2003) Improving typeability of multiple bacterial species using pulsed-ﬁeld gel electrophoresis and thiourea. Diagn. Micr. Infec. Dis., 47, 619–621. 24. Hata,M., Suzuki,M., Matsumoto,M., Takahashi,M., Yamazaki,M., Hiramatsu,R., Matsui,H., Sakae,K., Suzuki,Y. and Miyazaki,Y. (2003) A new clonal line of Salmonella Saintpaul having emerged and prevailed since 1999 in Aichi, Japan. Jpn J. Infect. Dis., 56, 77–79. 25. Liesegang,A. and Tschape,H. (2002) Modiﬁed pulsed-ﬁeld gel electrophoresis method for DNA degradation-sensitive Salmonella enterica and Escherichia coli strains. IJMM, 291, 645–648. 26. Koort,J.M.K., Lukinmaa,S., Rantala,M., Unkila,E. and Siitonen,A. (2002) Technical improvement to prevent DNA degradation of enteric pathogens in pulsed-ﬁeld gel electrophoresis. J. Clin. Microbiol., 40, 3497–3498. 27. Klaassen,C.H.W., de Valk-Van Haren,H.A. and Horrevorts,A.M. (2002) Pulsed-ﬁeld gel electrophoresis can yield DNA ﬁngerprints of degradation-susceptible Clostridium difﬁcile strains—Reply. J. Clin. Microbiol., 40, 3547–3547. 28. Inglis,T.J.J., O’Reilly,L., Foster,N., Clair,A. and Sampson,J. (2002) Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei. J. Clin. Microbiol., 40, 3198–3203. 29. Fawley,W.N. and Wilcox,M.H. (2002) Pulsed-ﬁeld gel electrophoresis can yield DNA ﬁngerprints of