JOURNAL OF BACTERIOLOGY, May 2005, p. 3255–3258
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 9
Random Insertional Mutagenesis of Leptospira interrogans, the Agent
of Leptospirosis, Using a mariner Transposon
Pascale Bourhy, He ´le `ne Louvel, Isabelle Saint Girons, and Mathieu Picardeau*
Laboratoire des Spiroche `tes, Institut Pasteur, Paris, France
Received 17 September 2004/Accepted 28 January 2005
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 mu-
tagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of
Leptospira, which remain largely unknown.
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. Lep-
tospirosis is also an important infectious disease in tropical and
subtropical countries and occurs in temperate countries (8).
The worldwide incidence rates of leptospirosis are underesti-
mated due to the difficulties of diagnosis, particularly in devel-
oping countries. Leptospira interrogans is the main species as-
sociated 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 incom-
plete protection against other serovars.
In comparison to other bacterial species, studies of the ge-
netics and the molecular basis of the pathogenesis of spiro-
chetes 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 repli-
cative vector in the saprophyte Leptospira biflexa (16). This
study was followed by the first gene knockouts by allelic ex-
change in saprophytes (2, 5, 11, 12, 18). Recently, the comple-
tion 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 pre-
dicted 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 construct-
ing mutants in pathogenic leptospires is developed, any func-
tion of these proteins, including virulence factors, remains
Random integration of Himar1 mariner into the L. interro-
gans genome. Research on Leptospira is now in the post-
genomic 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 ma-
chinery, 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 fac-
tors 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 sero-
var Lai strain Lai (National Reference Center of Leptospira,
Institut Pasteur, Paris, France) was performed by electropora-
tion as described for L. biflexa (8a). Briefly, cells were grown to
exponential phase, and pellets were washed in water and then
concentrated to 1011bacteria/ml in water at room temperature.
The competent cells were electroporated (1.8-kV, 200-?,
25-?F electric pulse in a prechilled 0.2-cm-diameter cuvette) in
the presence of 100 to 500 ng of plasmid DNA and then
* Corresponding author. Mailing address: Laboratoire des Spiro-
che `tes, 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:
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 ?g/ml).
Solid-medium plates were incubated at 30°C for 4 to 6 weeks.
Among several independent experiments, ?100 kanamycin-
resistant (Kmr) colonies per ?g 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 ?g of DNA (8a). Since the sui-
cide vector contains no sequences homologous to the genomic
DNA from Leptospira and as the transposase gene is adjacent
to Himar1, Kmrcolonies 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 Kmr
colonies was extracted, digested with DraI, separated by aga-
rose 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
Although transformation efficiency in L. interrogans is rela-
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 func-
tional 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. Trans-
posons MarKm and MarSp are bound by inverted repeats (IR-L and
TABLE 1. DNA sequence analysis of L. interrogans insertional mutants obtained with pSC189
Himar1 insertion site
ORF coordinates Potential function/comments
Sensory transduction histidine kinase
Peptidase family M23/M37
Ankyrin repeat protein
ATP-dependent RNA helicase
Fatty acid transport/FadL
Sugar transport protein
Phosphotransferase system, enzyme I
Conserved hypothetical protein
Unknown protein/FOG:HEAT repeat
Conserved hypothetical protein
Acyl-coenzyme A dehydrogenase/FadE2
Cytochrome c peroxidase
Conserved hypothetical protein
Cyclic nucleotide binding protein/Cgs
Peptidase family M48
aDirection of the ORF is indicated as (?) or (?).
bHimar1 insertion site is located in an intergenic sequence.
cHimar1 insertion site is located in the promoter region of the putative ORF (at least ?500 bp upstream of the start codon).
tively low, we obtained the first mutants in pathogenic Lepto-
spira 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 ?g/ml, compared to
?5 ?g/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 ?g 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 demon-
strated 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 com-
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 recombina-
tion machinery of the pathogens.
Characterization of the first mutants in L. interrogans. We
sequenced the Himar1 flanking sequences of 35 Kmrclones
(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 con-
sequently 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. inter-
rogans 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 chromo-
somal 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 un-
der 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 Hi-
mar1 insertion into genes encoding putative signal transduc-
tion proteins, of which 80 genes are present in the L. interro-
gans genome. Among the target genes that could give a
phenotype, mutant L2 exhibited an insertion into relA. RelA is
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).
VOL. 187, 2005 NOTES3257
a guanosine 3?-diphosphate 5?-diphosphate (ppGpp) syn-
thetase 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 perox-
ide sensitivity compared to the wild-type strain in solid media
(Fig. 3). Cytochrome c peroxidases are heme-dependent per-
oxidases usually found in the periplasm that catalyze reduction
of hydrogen peroxide to water and oxidation of ferrocyto-
chrome 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 com-
pensate for mutation in ccp.
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 pro-
viding 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 func-
We thank E. J. Rubin for the generous gift of plasmid pSC189 and
I. Old for critical reading of the manuscript.
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
2. Bauby, H., I. Saint Girons, and M. Picardeau. 2003. Construction and
complementation of the first auxotrophic mutant in the spirochaete Lepto-
spira meyeri. Microbiology 149:689–693.
3. Cashel, M., D. R. Gentry, V. J. Hernandez, and D. Vinella. 1996. The stress
response, p. 1458–1496. In F. C. Neidhart, R. Curtiss III, J. L. Ingraham,
E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella:
cellular and molecular biology. ASM Press, Washington, D.C.
4. Chiang, S. L., and E. J. Rubin. 2002. Construction of a mariner-based
transposon for epitope-tagging and genomic targeting. Gene 296:179–185.
4a.Ellinghausen, H. C., and W. G. McCullough. 1965. Nutrition of Leptospira
pomona and growth of 13 other serotypes: fractionation of oleic albumin
complex and a medium of bovine albumin and polysorbate 80. Am. J. Vet.
5. Gue ´gan, R., J. M. Camadro, I. Saint Girons, and M. Picardeau. 2003.
Leptospira spp. possess a complete heme biosynthetic pathway and are able
to use exogenous heme sources. Mol. Microbiol. 49:745–754.
6. Hayes, F. 2003. Transposon-based strategies for microbial functional genom-
ics and proteomics. Annu. Rev. Genet. 37:3–29.
6a.Johnson, R. C., and V. G. Harris. 1967. Differentiation of pathogenic and
saprophytic leptospires. J. Bacteriol. 94:27–31.
7. Lampe, D. J., M. E. Churchill, and H. M. Robertson. 1996. A purified
mariner transposase is sufficient to mediate transposition in vitro. EMBO J.
8. Levett, P. N. 2001. Leptospirosis. Clin. Microbiol. Rev. 14:296–326.
8a.Louvel, H., I. Saint Girons, and M. Picardeau. 2005. Isolation and charac-
terization of FecA- and FeoB-mediated iron acquisition systems of the
spirochete Leptospira biflexa by random insertional mutagenesis. J. Bacteriol.
9. Moszer, I., L. M. M. Jones, S. C. Fabry, and A. Danchin. 2002. SubtiList: the
reference database for the Bacillus subtilis genome. Nucleic Acids Res.
10. Nascimento, A. L., A. I. Ko, E. A. Martins, C. B. Monteiro-Vitorello, P. L.
Ho, D. A. Haake, S. Verjovski-Almeida, R. A. Hartskeerl, M. V. Marques,
M. C. Oliveira, C. F. Menck, L. C. Leite, H. Carrer, L. L. Coutinho, W. M.
Degrave, O. A. Dellagostin, H. El-Dorry, E. S. Ferro, M. I. Ferro, L. R.
Furlan, M. Gamberini, E. A. Giglioti, A. Goes-Neto, G. H. Goldman, M. H.
Goldman, R. Harakava, S. M. Jeronimo, I. L. Junqueira-de-Azevedo, E. T.
Kimura, E. E. Kuramae, E. G. Lemos, M. V. Lemos, C. L. Marino, L. R.
Nunes, R. C. de Oliveira, G. G. Pereira, M. S. Reis, A. Schriefer, W. J.
Siqueira, P. Sommer, S. M. Tsai, A. J. Simpson, J. A. Ferro, L. E. Camargo,
J. P. Kitajima, J. C. Setubal, and M. A. Van Sluys. 2004. Comparative
genomics of two Leptospira interrogans serovars reveals novel insights into
physiology and pathogenesis. J. Bacteriol. 186:2164–2172.
11. Picardeau, M., H. Bauby, and I. Saint Girons. 2003. Genetic evidence for the
existence of two pathways for the biosynthesis of methionine in Leptospira
spp. FEMS Microbiol. Lett. 225:257–262.
12. Picardeau, M., A. Brenot, and I. Saint Girons. 2001. First evidence for gene
replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-
motile mutants deficient in endoflagella. Mol. Microbiol. 40:189–199.
13. Prod’hom, G., B. Lagier, V. Pelicic, A. J. Hance, B. Gicquel, and C. Guilhot.
1998. A reliable amplification technique for the characterization of genomic
DNA sequences flanking insertion sequences. FEMS Microbiol. Lett. 158:
14. Ren, S., G. Fu, X. Jiang, R. Zeng, H. Xiong, G. Lu, H. Q. Jiang, Y. Miao, H.
Xu, Y. Zhang, X. Guo, Y. Shen, B. Q. Qiang, X. K. Guo, A. Danchin, I. Saint
Girons, R. L. Somerville, Y. M. Weng, M. Shi, Z. Chen, J. G. Xu, and G. P.
Zhao. 2003. Unique physiological and pathogenic features of Leptospira
interrogans revealed by whole-genome sequencing. Nature 422:888–893.
15. Rubin, E. J., B. J. Akerley, V. N. Novik, D. J. Lampe, R. N. Husson, and J. J.
Mekalanos. 1999. In vivo transposition of mariner-based elements in enteric
bacteria and mycobacteria. Proc. Natl. Acad. Sci. USA 96:1645–1650.
16. Saint Girons, I., P. Bourhy, C. Ottone, M. Picardeau, D. Yelton, R. W.
Hendrix, P. Glaser, and N. Charon. 2000. The LE1 bacteriophage replicates
as a plasmid within Leptospira biflexa: construction of an L. biflexa-Esche-
richia coli shuttle vector. J. Bacteriol. 182:5700–5705.
17. Stewart, P. E., J. Hoff, E. Fischer, J. G. Krum, and P. A. Rosa. 2004.
Genome-wide transposon mutagenesis of Borrelia burgdorferi for identifica-
tion of phenotypic mutants. Appl. Environ. Microbiol. 70:5973–5979.
18. Tchamedeu-Kameni, A. P., E. Couture-Tosi, I. Saint-Girons, and M.
Picardeau. 2002. Inactivation of the spirochete recA gene results in a mutant
with low viability and irregular nucleoid morphology. J. Bacteriol. 184:452–
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 ?l 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 stan-
dard deviation of at least five independent observations.
3258 NOTESJ. BACTERIOL.