Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool

Abstract

Our understanding of leptospiral pathogenesis, which remains poorly understood, depends on reliable genetic tools for functional analysis of genes in pathogenic strains. In this study, we report the first demonstration of conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids. The DNA transfer described here was due to authentic conjugation, as shown by the requirement for cell-to-cell contact and the resistance of DNA transfers to the addition of DNase I. Transposition via conjugation of a plasmid delivering Himar1 yielded frequencies ranging from 1 x 10(-6) to 8.5 x 10(-8) transconjugants/recipient cell in the saprophyte L. biflexa and the pathogen L. interrogans, respectively. Analysis of mutants indicated that transposition occurs randomly, and at single sites in the genome of these strains, allowing the utilization of this system to generate libraries of transposon mutants.

FIG. 1.

Schematic representation of the construction of conjugal plasmids vectors. Plasmid pCjSpLe94 was generated by cloning the LE1 replication origin (17), which carries the rep and parAB genes, in the NotI-BamHI restriction sites of pSW29T (3). The spectinomycin resistance cassette (Spc^r) was then ligated to the plasmid digested by SmaI, leading to the construction of pCJSpLe94. Plasmid pCjTKS1 was generated by replacement of both the kanamycin resistance cassette (Km^r) and the ori R6K (SacI-PvuII fragment) of pSW29T by the Himar1 transposon, which carries an ori R6K and a kanamycin resistance cassette (Km^r). A BamHI-SpeI fragment carrying the C9 transposase gene under the control of the promoter of L. interrogans hsp10 was then ligated into the plasmid, leading to the construction of pCjTKS1. Black arrowheads on pCjTK and pCjTKS1 represent the borders of the transposons. The spectinomycin resistance cassette (Spc^r) was also ligated into the ClaI-digested pSW29T to generate pSW29TSp. Plasmid DNAs were purified using a plasmid miniprep kit (QIAGEN GmbH, Hilden, Germany).