Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon

Abstract

The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.

FIG. 1.

Physical maps of the mariner delivery plasmids pMKL and pMSL. The promoterless hyperactive transposase C9 was linked to the spirochetal promoter of B. burgdorferi flgB as previously described (17). The plasmids are derivatives of pSC189 (4) bearing the native C9 transposase. Plasmids pMKL (A) and pMSL (B) include the origin of replication from plasmid pGEM-7Zf+ (Promega). Plasmid pMKL also includes the origin of replication of plasmid R6K which is functional in E. coli λ pir⁺. Plasmids pMKL and pMSL do not replicate in Leptospira spp. Kanamycin and spectinomycin resistance cassettes are derivatives from pGKLep4 (16) and pGKLS (2), respectively. Transposons MarKm and MarSp are bound by inverted repeats (IR-L and IR-R).

FIG. 2.

Positions of the 35 insertion sites of Himar1 in the L. interrogans genome. The loci with genes encoding the components of lipopolysaccharide (rfb), 103 kb in size, and heme biosynthetic genes (hem) are indicated in CI and CII, respectively. Himar1 insertion sites were mapped onto the genome of L. interrogans 56601 using LeptoList (http://bioinfo.hku.hk/genochore.html).

FIG. 3.

Effects of hydroperoxide and cumene hydroperoxide on L. interrogans wild-type strain (wt) and ccp mutant strain (L37). Bacterial cells were spread onto EMJH plates, and 6-mm-diameter filter disks containing 10 μl of 10 mM hydrogen peroxide (top of the plate) and 10 mM cumene peroxide (bottom of the plate) were placed on the plates. The plates were incubated for 15 days at 30°C. For hydrogen peroxide, the diameter of the inhibition zone was 19 ± 1 mm and 11 ± 2 mm in L37 and wt strains, respectively. For cumene peroxide, the diameter of the inhibition zone was 44 ± 1 mm and 37 ± 3 mm in L37 and wt strains, respectively. The results are indicated as the average and standard deviation of at least five independent observations.