title: Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.
abstract: Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
authors.affiliation: 1] State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China [2] Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3].
authors.forename: Bo
authors.initials: B
authors.lastname: Cao
authors.affiliation: 1] Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China [2].
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: 1] Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2].
authors.forename: Michael S
authors.initials: MS
authors.lastname: DeMott
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
authors.forename: Qiuxiang
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Tyson A
authors.initials: TA
authors.lastname: Clark
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Vincent
authors.initials: V
authors.lastname: Butty
authors.affiliation: Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Stuart S
authors.initials: SS
authors.lastname: Levine
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: George
authors.initials: G
authors.lastname: Yuan
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Boitano
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Khai
authors.initials: K
authors.lastname: Luong
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Song
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
authors.forename: Xiufen
authors.initials: X
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Turner
authors.affiliation: Pacific Biosciences, Menlo Park, California 94025, USA.
authors.forename: Jonas
authors.initials: J
authors.lastname: Korlach
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
authors.forename: Delin
authors.initials: D
authors.lastname: You
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
authors.forename: Lianrong
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
authors.forename: Shi
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D002874:Chromosome Mapping; D016384:Consensus Sequence / Q000235:genetics*; D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016680:Genome, Bacterial; D010710:Phosphates / Q000378:metabolism*; D014733:Vibrio / Q000235:genetics* / Q000378:metabolism
chemical_list: D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial; D010710:Phosphates; C035638:thiophosphoric acid