title: Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors.
abstract: BACKGROUND
  The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.
  
  
  RESULTS
  Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans-Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.
  
  
  CONCLUSIONS
  Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. zhuweinan@sjtu.edu.cn.
authors.forename: Weinan
authors.initials: W
authors.lastname: Zhu
authors.affiliation: CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. jinwang@sibs.ac.cn.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. yzhzhu@hotmail.com.
authors.forename: Yongzhang
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China. tb_411@163.com.
authors.forename: Biao
authors.initials: B
authors.lastname: Tang
authors.affiliation: CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. zhangyunyi2003@163.com.
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. hpatsh@sjtu.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: He
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. zhangyan007236@126.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. centian2004@163.com.
authors.forename: Boyu
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. xkguo@shsmu.edu.cn.
authors.forename: Xiaokui
authors.initials: X
authors.lastname: Guo
authors.affiliation: CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. gpzhao@sibs.ac.cn.|State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China. gpzhao@sibs.ac.cn.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Microbiology and Immunology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. jinhongqin@sjtu.edu.cn.
authors.forename: Jinhong
authors.initials: J
authors.lastname: Qin
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D007921:Leptospira interrogans / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012093:Replicon / Q000235:genetics*; D017422:Sequence Analysis, DNA