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Journal of Bacteriology, May 2005, p. 3255-3258, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3255-3258.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Random Insertional Mutagenesis of Leptospira interrogans, the Agent of Leptospirosis, Using a mariner Transposon

Pascale Bourhy, Hélène Louvel, Isabelle Saint Girons, and Mathieu Picardeau^*

Laboratoire des Spirochètes, Institut Pasteur, Paris, France

Received 17 September 2004/ Accepted 28 January 2005

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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.

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Spirochetes are the causative agents of several important animal and human diseases, such as syphilis, Lyme disease, and leptospirosis. Pathogenic Leptospira are the etiologic agents of leptospirosis, the most widespread zoonosis in the world. Leptospirosis is also an important infectious disease in tropical and subtropical countries and occurs in temperate countries (8). The worldwide incidence rates of leptospirosis are underestimated due to the difficulties of diagnosis, particularly in developing countries. Leptospira interrogans is the main species associated with human leptospirosis. Transmission to humans occurs through direct or indirect contacts with urine of infected animals, such as small rodents. Antileptospiral vaccines are available in some countries; however, these vaccines, usually killed bacteria of the prevalent serovar, suffer from numerous drawbacks, such as side effects, short-term efficacy, and incomplete protection against other serovars.

In comparison to other bacterial species, studies of the genetics and the molecular basis of the pathogenesis of spirochetes are in their infancy. Few laboratories have attempted to decipher the genetics of bacteria of the genus Leptospira, which is composed of both saprophyte and pathogen members. Their study is difficult due to their long generation times—from 4 h in saprophytes to 14 to 18 h in pathogens (8)—and the lack of efficient genetic tools. A breakthrough in leptospira genetics was the first report of genetic transformation by using a replicative vector in the saprophyte Leptospira biflexa (16). This study was followed by the first gene knockouts by allelic exchange in saprophytes (2, 5, 11, 12, 18). Recently, the completion of the genome sequences of L. interrogans serovar Lai and L. interrogans serovar Copenhageni has been achieved (10, 14). The L. interrogans genome consists of a 4.33-Mb large circular chromosome, a 0.35-Mb small circular chromosome, and no extrachromosomal elements. The complete genome sequences revealed an average G+C content of 36% and 4,500 predicted open reading frames (ORFs), among which >50% failed to exhibit similarities to proteins of known function or any protein in other organisms. Until a method for constructing mutants in pathogenic leptospires is developed, any function of these proteins, including virulence factors, remains speculative.

Random integration of Himar1 mariner into the L. interrogans genome. Research on Leptospira is now in the postgenomic era, but research on its genetics is still at a very early stage. In contrast to saprophytes, attempts at transformation in pathogens with either the L. biflexa-Escherichia coli shuttle vector (16) or a suicide vector containing L. interrogans DNA for homologous recombination with the chromosomal DNA have been unsuccessful (M. Picardeau, unpublished data). The failure to transform pathogenic Leptospira could be due to competence, selective marker expression, recombination machinery, and/or DNA restriction and modification systems that differ in pathogenic versus saprophytic strains.

Transposons have been widely used as genetic tools that can insert randomly into microbial genomes. Because transposons of the mariner family do not require species-specific host factors for efficient transposition (6), the Himar1 mariner element was tested in the pathogen L. interrogans. In this study, plasmid vector pSC189 (4), containing both the hyperactive transposase C9 (7) and transposon terminal inverted repeats flanking a kanamycin resistance gene, was used to deliver Himar1 in the L. interrogans genome. Transformation of L. interrogans serovar Lai strain Lai (National Reference Center of Leptospira, Institut Pasteur, Paris, France) was performed by electroporation as described for L. biflexa (8a). Briefly, cells were grown to exponential phase, and pellets were washed in water and then concentrated to 10¹¹ bacteria/ml in water at room temperature. The competent cells were electroporated (1.8-kV, 200-, 25-µF electric pulse in a prechilled 0.2-cm-diameter cuvette) in the presence of 100 to 500 ng of plasmid DNA and then transferred to 1 ml of EMJH liquid medium (4a, 6a), in which they were incubated for 24 h at 30°C. The bacteria were then plated on EMJH supplemented with kanamycin (25 µg/ml). Solid-medium plates were incubated at 30°C for 4 to 6 weeks. Among several independent experiments, 100 kanamycin-resistant (Km^r) colonies per µg of plasmid DNA were obtained in L. interrogans. In comparison, the saprophyte L. biflexa was transformable using the same plasmid, pSC189, at a higher rate: 5,000 transformants per µg of DNA (8a). Since the suicide vector contains no sequences homologous to the genomic DNA from Leptospira and as the transposase gene is adjacent to Himar1, Km^r colonies obtained after electroporation should result from transposition events into the L. interrogans genome, without subsequent transposase-mediated events. Genomic DNA from cultures inoculated from 50 randomly choosen Km^r colonies was extracted, digested with DraI, separated by agarose gel electrophoresis, transferred to nylon membranes, and probed with pSC189 as described previously (12). Since Himar1 contains a unique internal DraI site, a single random insertion will yield two Southern-hybridizing bands that are variable in size, and we demonstrated that to be the case (data not shown).

Although transformation efficiency in L. interrogans is relatively low, we obtained the first mutants in pathogenic Leptospira spp. The replacement of the native kanamycin resistance gene of pSC189 with the gram-positive cassette for kanamycin or spectinomycin resistance used in the E. coli-L. biflexa shuttle vectors (2, 16) did not improve transformation efficiency. For both antibiotics, kanamycin and spectinomycin, the MIC of transformants in liquid medium was >500 µg/ml, compared to <5 µg/ml for the wild-type strain. The presence of specific restriction and modification systems in pathogenic leptospira can also reduce transformation efficiencies using plasmid DNA extracted from wild-type E. coli. No significant differences were observed if plasmid DNA was isolated from a methylation-free E. coli strain (data not shown). To improve expression of the Himar1 transposase, the hyperactive transposase C9 was fused to a spirochetal promoter (Fig. 1). Approximately fivefold more colonies, i.e., 500 transformants per µg of DNA, were obtained with plasmids pMKL and pMSL than with plasmid pSC189 expressing transposase from its native promoter. In the spirochete Borrelia burgdorferi, a recent study demonstrated that a high number of mutants could only be obtained when the Himar1 transposase was expressed from this flgB promoter (17). It has to be noted that due to the presence of a hyperactive transposase in plasmid pSC189 and derivatives, these plasmids may not be stable in E. coli (4). Each plasmid preparation should therefore be done with fresh E. coli competent cells.

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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 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).

In conclusion, our results show that (i) foreign DNA can enter pathogenic species of leptospira, (ii) the transposase C9 is both expressed and functional in L. interrogans, and (iii) the selective markers are appropriate. Since mariner-mediated events do not require host accessory factors, the failure of previous gene transfer attempts may be due to the recombination machinery of the pathogens.

Characterization of the first mutants in L. interrogans. We sequenced the Himar1 flanking sequences of 35 Km^r clones (containing a unique and randomly inserted transposon, as demonstrated by Southern analysis) obtained with pSC189 using ligation-mediated PCR as described previously (8a, 13). PCR products were directly sequenced using the linker-specific primer LKgd (5'-TAGAGTATTCCTCAAGGCACGAGC-3') at Genome Express (Meylan, France). The DNA sequence data were then analyzed with the LeptoList World Wide Web server (http://bioinfo.hku.hk/genochore.html) (9) and the BLAST program (1). Sequence analysis indicated that each of the insertions occurred after a TA dinucleotide that was consequently duplicated, indicating that all insertions arose by transposition (15). The majority of the insertions were located within putative ORFs (29/35) and in the large chromosome CI (31/35) (Table 1 and Fig. 2), which is in agreement with the proportions of the protein-coding genome and chromosome sizes, respectively (10, 14). Apart from a 650-kb region of the chromosome CI where Himar1 was integrated in 11 out of 35 mutants, the distribution of the insertion sites in the L. interrogans genome showed that there was no obvious preferential spot for transposition (Fig. 2). Representative clones were further tested for the insertion of the Himar1 transposon in the target gene by PCR using primers flanking the putative site of insertion. In each case, an increase in size of the PCR product by 2.2 kb is due to the insertion of Himar1 into the chromosomal locus (data not shown). Among the 35 mutant strains (Table 1), no obvious phenotype was observed by microscopic observation and growth analysis in liquid and solid media under the conditions tested. In 11 mutants, Himar1 was inserted into putative ORFs encoding hypothetical proteins or proteins with unknown functions. In two other mutants, the insertion mapped into the tranposase genes of insertion sequences; >50 of these insertion sequences are scattered throughout the L. interrogans genome (14). Mutants L5 and L14 exhibited Himar1 insertion into genes encoding putative signal transduction proteins, of which 80 genes are present in the L. interrogans genome. Among the target genes that could give a phenotype, mutant L2 exhibited an insertion into relA. RelA is a guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetase that plays a major role in the stringent response and/or entry into the stationary phase (3). Mutations affecting ppGpp metabolism result in pleiotropic phenotypes (3). The effects of temperature and medium osmolarity on mutant L2 were found to be equivalent to those of the wild-type strain (data not shown). The transposon insertion was located near the 3' end of relA, removing only 65 amino acids of the carboxy terminus of the protein (680 amino acids in length). This insertion, therefore, may not disrupt the ppGpp synthetase activity, as previously observed for some truncated RelA proteins (3). Mutant L37, with an insertion in the start codon of ccp, which encodes a cytochrome c peroxidase, showed increased peroxide sensitivity compared to the wild-type strain in solid media (Fig. 3). Cytochrome c peroxidases are heme-dependent peroxidases usually found in the periplasm that catalyze reduction of hydrogen peroxide to water and oxidation of ferrocytochrome c. Putative genes encoding products that could be involved in oxidative defenses, such as glutathione peroxidase, methionine sulfoxide reductase, and catalase, are present in the L. interrogans genome and may therefore partially compensate for mutation in ccp.

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TABLE 1. DNA sequence analysis of L. interrogans insertional mutants obtained with pSC189

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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).

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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.

Conclusion. To the best of our knowledge, these mutants are the first isolated in pathogenic Leptospira. We demonstrated that gene transfer is feasible in pathogenic strains, thus providing a starting point for the improvement of transformation efficiency in pathogenic strains. This could allow large-scale mutagenesis studies, such as the screening of mutant libraries in search of motility, amino acid biosynthesis, and virulence mutants. A random-mutagenesis system will be particularly useful for discovering new genes and studying protein functions.

 
We thank E. J. Rubin for the generous gift of plasmid pSC189 and I. Old for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Laboratoire des Spirochètes, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 (1) 45 68 83 68. Fax: 33 (1) 40 61 30 01. E-mail: mpicard{at}pasteur.fr.

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Journal of Bacteriology, May 2005, p. 3255-3258, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3255-3258.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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