Published online 5 July 2012 Nucleic Acids Research, 2012, Vol. 40, No. 18 9115–9124 doi:10.1093/nar/gks650 Phosphorothioate DNA as an antioxidant in bacteria Xinqiang Xie, Jingdan Liang, Tianning Pu, Fei Xu, Fen Yao, Yan Yang, Yi-Lei Zhao, Delin You, Xiufen Zhou, Zixin Deng* and Zhijun Wang* State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, People’s Republic of China Received March 3, 2012; Revised May 24, 2012; Accepted June 7, 2012 ABSTRACT Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioa- tion). We found that in vitro oxidation of phosphoro- thioate (PT) DNA by hydrogen peroxide (H2O2) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physio- logical relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H2O2. DNA phospho- rothioation was found to correlate with increasing resistance to the growth inhibition by H2O2. Resistance to H2O2 was abolished when each of the three dnd genes, required for phosphorothioa- tion, was inactivated. In vivo, PT DNA is more resist- ant to the double-strand break damage caused by H2O2 than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was con- verted to PT-free state when the bacteria were incubated with H2O2. These findings are consistent with a hypothesis that phosphorothioation modifica- tion endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is main- tained by diverse bacteria. INTRODUCTION Many bacteria contain DNA that is stereo-speciﬁcally phosphorothioate (PT) modiﬁed at speciﬁc sequences (1–6). DNA phosphorothioation is controlled by ﬁve clus- tered genes, dndA–E, which are located on genetic islands (7,8). Four of the genes, dndA and dndC–E, are essential for the PT modiﬁcation, while inactivation of dndB, resulted in increased phosphorothioation and altered sequence preference (1,3 and personal communication). The biological function(s) of DNA phosphorothioation remained elusive until the recent discoveries of a Streptomyces coelicolor gene that speciﬁcally restricts PT DNA from Streptomyces lividans, and a restriction system in Salmonella enterica serovar Cerro 87 against PT-free heterologous DNA (9,10). The extent of phosphorothioation varies between differ- ent host strains, ranging from 300 to 3000 modiﬁcations per 106 nt (6). In the case of Escherichia coli B7A and S. enterica 87, the amount is around 750 per 106 nt, and in S. lividans 474 per 106 nt. The study of 63 Vibrio strains revealed that seven of them possess from six per 104 nt to as high as two to three PT modiﬁcations per 103 nt (6). PTs modiﬁcation are also sequence speciﬁc (1,6). A high frequency of GA was found in Bermanella marisrubri RED65 and Hahella chejuensis KCTC2396, using high pressure (or high performance) liquid chromatography (HPLC)/mass method. While the dinucleotide sequence of GG was found in Pseudomonas ﬂuorescens Pf0-1, S. lividans 1326 and Geobacter uraniumreducens Rf4. In the case of E. coli B7A and S. enterica 87, the most commonly PT modiﬁed dinucleotide sequences are GA and GT. A high frequency of PT modiﬁcations at GA, GT, GG and CC were found in the seven Vibrio strains. Using cleavage and ligation methods, a conserved modiﬁcation sequence of -cGGCCgccg- (including a highly conserved 4-bp central core -GGCC-) was identiﬁed in S. lividans, indicating that the modiﬁcation is double stranded (ds) (1). Phosphorothioation across the bacterial genome DNA leads to the often-observed Dnd phenotype: DNA degradation during electrophoresis (4). It was proposed that the DNA degradation comes from speciﬁc double- strand cleavage at the site of the modiﬁcation by Tris-peracetic acid, which is generated at the anode during gel electrophoresis (11,12). *To whom correspondence should be addressed. Tel: +86 21 62933751 2061; Fax: +86 21 62932418; Email: wangzhijun@sjtu.edu.cn Correspondence may also be addressed to Zixin Deng. Tel: +86 21 62933751 2061; Fax: +86 21 62932418; Email: zxdeng@sjtu.edu.cn The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.