INFECTION AND IMMUNITY, Feb. 2009, p. 810–816
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 2
Genome-Wide Transposon Mutagenesis in Pathogenic
Gerald L. Murray,1Viviane Morel,2Gustavo M. Cerqueira,2,3Julio Croda,2,4Amporn Srikram,5
Rebekah Henry,1Albert I. Ko,4,6Odir A. Dellagostin,3Dieter M. Bulach,1†
Rasana W. Sermswan,7Ben Adler,1,8* and Mathieu Picardeau2*
Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, VIC 3800, Australia1; Institut Pasteur,
Unite ´ de Biologie des Spiroche `tes, Paris, France2; Centro de Biotecnologia, Universidade Federal de Pelotas, P.O. Box 354,
96010-900, Pelotas, RS, Brazil3; Gonc ¸alo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health,
Salvador, Brazil4; Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand5; Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University,
New York, New York6; Department of Biochemistry, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand7; and Australian Research Council Centre of
Excellence in Structural and Functional Microbial Genomics, Department of
Microbiology, Monash University, Clayton, VIC 3800, Australia8
Received 21 October 2008/Returned for modification 11 November 2008/Accepted 23 November 2008
Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis
of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed
the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a
total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these
mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of
transposon insertion sites indicated that transposition occurred in an essentially random fashion in the
genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs,
suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing
notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon
insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated
using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were
identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in
L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans.
Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that
contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.
Leptospira interrogans is a spirochete that is the main caus-
ative agent of leptospirosis. This zoonosis has emerged as a
major public health problem in much of the developing world,
with more than 500,000 cases of severe leptospirosis reported
each year, for which the mortality rate is more than 10% (17).
The genus Leptospira is composed of both saprophytic and
pathogenic species. The genome sequences of two epidemic
strains of L. interrogans serovars Lai and Copenhageni have
been determined (20, 25). More recently a human and an
animal L. borgpetersenii isolate were sequenced (3), and this
year, we determined the genome sequence of the saprophyte
L. biflexa (22). The resulting sequences provide an invaluable
source of information for identification of genetic determi-
nants involved in the pathogenicity and environmental biology
of the organism. For example, the host-adapted L. borgpeterse-
nii genome is 16% smaller and has many more pseudogenes
than the L. interrogans genome. These findings suggest that
genome reduction has resulted in a reduced environmental
transmission potential (3). L. interrogans has 627 genes that are
absent in the L. biflexa genome, and more than 500 of these
genes have unknown functions, suggesting the presence of
novel virulence mechanisms (22). However, the lack of tools
for L. interrogans genetics has hindered elucidation of the role
of these genes in pathogenesis.
Pathogenic leptospires are difficult to propagate under in
vitro conditions. L. interrogans is a slow-growing organism with
a generation time of ?20 h, and colonies take up to 4 weeks to
appear on solid medium. Furthermore, unlike saprophytic lep-
tospires, these bacteria are genetically intractable, with no rep-
licating vectors (21, 27), and only one mutant has recently been
obtained by homologous recombination (5). The lack of ge-
netic systems has hampered molecular analyses of pathogenic
leptospires, with no method to assess directly the role of L.
interrogans genes in virulence. Recently we demonstrated gene
transfer in a pathogenic Leptospira strain, involving the trans-
position of Himar1, a transposon of eukaryotic origin (2). We
* Mailing address for Mathieu Picardeau: Unite ´ de Biologie des Spi-
roche `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:
email@example.com. Mailing address for Ben Adler: Department of
Microbiology, Monash University, Wellington Road, Victoria 3800,
Australia. Phone: 61 3 9905 4815. Fax: 61 3 9905 4811. E-mail: ben.adler
† Present address: CSIRO Livestock Industries, Australian Animal
Health Laboratory (AAHL), Geelong, VIC 3220, Australia.
‡ Supplemental material for this article may be found at http://iai
?Published ahead of print on 1 December 2008.
identified genes interrupted by Himar1 insertion in 35 mutants
of L. interrogans serovar Lai. Since that study, transposon mu-
tagenesis in L. interrogans has allowed the identification of a
mutant, lacking expression of Loa22, exhibiting attenuated vir-
ulence in animal models (26) and a mutant obtained by inser-
tion of the transposon Himar1 into a gene encoding heme
Low electroporation efficiency and a low growth rate have
limited the generation of L. interrogans random mutants and
mean that the generation of high-coverage libraries, common-
place in most mutagenesis studies, is not feasible for L. inter-
rogans. Under these circumstances, each mutant isolated is
worth characterizing. Notably, there have been only three stud-
ies published on the topic in the last three years. Over this
period, we have generated libraries of random mutants for
different pathogenic strains. In this study we present a library
of approximately 1,000 defined mutants with characterized
transposon insertion points. This collection of insertional mu-
tants constitutes an extremely valuable resource for functional
studies of pathogenic Leptospira. The library will be particu-
larly useful for identifying new genes, validating the functions
of predicted proteins, and discovering novel virulence factors.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The strains used for this study are
listed in Table 1. All strains were obtained from the collection of the Centro de
Pesquisas Gonc ¸alo Moniz, Fundac ¸a ˜o Oswaldo Cruz, Salvador, Brazil, except the
L. interrogans serovars Lai and Manilae. L. interrogans serovar Manilae was
provided by N. Koizumi, National Institute of Infectious Diseases, Tokyo, Japan,
while L. interrogans serovar Lai was obtained from the National Institute for
Communicable Disease Control and Prevention, Beijing, China (25). High-pas-
sage strains refer to strains that were subcultured in EMJH liquid medium more
than 10 times. All strains were cultured at 30°C in liquid EMJH medium (7, 11)
or on EMJH plates containing 1.5% agar. Kanamycin or spectinomycin was
added at 40 ?g/ml when required.
For UV irradiation, cells were spread at appropriate dilutions on EMJH agar
plates and irradiated under UV light (254 nm, 10 ?W/cm2) for various time
periods (from 2 to 10 s). UV sensitivity was evaluated by colony counting, with
untreated cells serving as a control. Medium for testing the ability of Leptospira
strains to use hemin was prepared by supplementing EMJH medium with 50 ?M
2,2?-dipyridyl (Sigma-Aldrich, St. Louis, MO). Bovine hemin was then added at
a final concentration of 10 ?M.
Transposon mutagenesis. The plasmid pSC189ColE1 was constructed by am-
plifying the ColE1 origin of replication from pBluescript II (using primers 5?-A
AAATACGTAAGCAAAAGGCCAGGAAC-3? and 5?-AAAACTGCAGGAT
CAAAGGATCTTCTTG-3?), and the product was digested with SnaBI and PstI
then ligated into similarly digested pSC189, replacing OriR6k. The plasmids
pSHT, pKMars (14), and pSC189ColE1 were used to perform random transpo-
son mutagenesis in L. interrogans strains as described previously (2). Briefly, L.
interrogans was grown to exponential phase and then washed and concentrated in
water. For electroporation, approximately 1010cells in ?100 ?l were mixed with
1 ?g of plasmid DNA in 2-mm chilled cuvettes. The electroporator was set to 1.8
kV, 25 ?F, and 200 ?. One milliliter of EMJH medium was immediately added
to the cuvette, and the cells were incubated overnight at 30°C. Finally, transfor-
mants were plated on EMJH agar plates containing antibiotic. Plates were
incubated for 4 weeks at 30°C in sealed plastic bags or wrapped in foil to avoid
desiccation. Transformants were then picked and subcultured in 5 ml of EMJH
liquid medium. Genomic DNA was extracted, and the Himar1 insertion site was
identified by ligation-mediated PCR (LM-PCR) (15, 24) or direct sequencing
(19). Confirmation of genotypes was performed by PCR with primers located in
the flanking sequences of the predicted transposon insertion site. We did not
observe any kanamycin-resistant colonies that did not contain the transposon.
Hamster model of infection. Four-week-old hamsters were injected intraperi-
toneally with leptospires at the stated inoculum in 100 ?l of EMJH. The 50%
lethal dose for L. interrogans serovar Manilae was approximately 10 leptospires.
Hamsters were monitored for 14 days postinfection and euthanized if moribund
in accordance with animal ethics requirements. Lungs were inspected for hem-
orrhage to confirm infection with Leptospira. Culture isolation was performed
with kidney tissues from hamsters for approximately half of the strains tested.
The genotype of the recovered leptospires was confirmed by PCR amplifying the
region across the transposon insertion.
Sequence analysis. The Himar1 insertion site sequences were compared with
the complete genome sequence of L. interrogans serovar Lai strain 56601 by using
the SpiroScope (http://www.genoscope.cns.fr/agc/mage) (30) and Wasabi (3) da-
tabases. Multiple sequence alignments of a conserved ?15-bp region surround-
ing each insertion point were generated with scripts coded in Perl. Consensus
sequences were visualized with Sequence Logo analysis using WebLogo (http:
RESULTS AND DISCUSSION
Random transposon mutagenesis in pathogenic Leptospira
spp. The genetics of the pathogenic Leptospira spp. is in its
infancy. Transposon mutagenesis is a powerful, broadly appli-
cable tool for the generation of libraries of random mutants.
Himar1, of the mariner family, is one of the most widely used
transposons for random mutagenesis in bacteria and other
organisms (23). In this article, we describe methods for the use
of Himar1 for transposon mutagenesis in L. interrogans.
We have applied the method previously used with the sa-
prophyte L. biflexa (14, 15) for use with pathogenic Leptospira
spp. Initially, the plasmid vector pSC189 (4), containing both
the hyperactive transposase C9 and transposon terminal in-
verted repeats flanking a kanamycin resistance gene, was used
TABLE 1. Bacterial strains used for random transposon mutagenesis
L1 130 LP
L1 130 HP
L1 133 LP
2 ? 10?7
2 ? 10?7
7 ? 10?6
9 ? 10?6
1 ? 10?6
8 ? 10?6
?1 ? 10?8
9 ? 10?6
9 ? 10?6
8 ? 10?6
5 ? 10?6
aWhen an entry is underlined, only the serogroup of the studied strain is indicated (the serovar was not identified).
bLP, low-passage strain; HP, high-passage strain.
cTransformation frequency is defined as the number of transposon mutants divided by the number of cells which survived electroporation (approximately 10%). A
transformation frequency of ?1 ? 10?8represents the limit of detection in these transformations.
VOL. 77, 2009TRANSPOSON MUTAGENESIS IN PATHOGENIC LEPTOSPIRA SPP.811
to deliver Himar1 into the L. interrogans genome (2). The only
origin of replication present in the plasmid construct was that
from the Escherichia coli plasmid vectors, which is nonfunc-
tional in Leptospira spp. Thus, any resistant colonies arising
after electroporation of this plasmid into L. interrogans are the
result of random insertion into the host genome.
We made a number of modifications of the original vector to
potentially improve its use in transforming L. interrogans. The
ColE1 replication origin was introduced to replace OriR6K
from the original pSC189 to simplify preparation of vector
DNA. Increased expression of the hyperactive transposase C9
gene by substituting a spirochetal promoter for the native pro-
moter increased the yield of transformants in L. interrogans
10-fold (2). In addition, a transposon carrying a spectinomycin
resistance gene has been constructed; electroporation of this
plasmid construct into L. interrogans resulted in spectinomycin-
resistant colonies at a frequency similar to that generated by
the kanamycin-resistant transposon. Since there is no replica-
tive plasmid vector available for pathogenic Leptospira, rein-
troduction of an intact copy of disrupted genes can be achieved
via a transposon with alternative selection (26) or by homolo-
gous recombination (5).
Transformation of L. interrogans was optimal at 9 kV cm?1
for a pulse time of 5 ms. This field strength resulted in approx-
imately 10% viability for all pathogenic strains tested. The L.
interrogans strains exhibited maximal electrocompetence when
harvested in mid- to late exponential growth phase. Use of
more than 2 ?g of DNA did not significantly improve the yield
of transformants, although there was no reduction of transfor-
mation efficiency observed when using up to 50 ?g of DNA.
The inserted transposons remained stable after 100 genera-
tions in the absence of antibiotic selection. In addition, all
random mutants that were recovered from animals maintained
the antibiotic resistance cassette (data not shown), indicating
that transposon insertions are extremely stable.
The genus Leptospira is composed of more than 16 patho-
genic and saprophytic species (12). To identify a strain with
improved transformation efficiency, we examined the trans-
formability of laboratory and clinical isolates of pathogenic
Leptospira spp., including pathogenic strains from L. noguchii
and L. weilii (Table 1), with plasmids delivering Himar1. For all
the tested strains, transformation of Himar1 in pathogenic
leptospires occurred at a low frequency. There was significant
strain-dependent variation in transformation competence, with
frequencies varying from 10?7to 9 ? 10?6; some strains were
completely resistant to transformation (Table 1). The plating
efficiency (the ratio of number of CFU to number of bacteria
enumerated in a Petroff-Hauser counting chamber) of patho-
genic strains ranged between 70 and 90%, suggesting that the
low-transformation efficiency was not due to poor viability of
pathogenic strains in solid medium. We did not observe dif-
ferences in the transformation efficiency between high and low
in vitro-passaged variants of the same strain.
The poor transformability of leptospires may reflect the in-
volvement of DNA restriction and modification mechanisms.
The genome sequences of L. interrogans showed one complete
putative type I restriction and modification system (LA3197 to
LA3200), which is not found in the saprophyte L. biflexa, and
a total of 12 putative DNA methyltransferase genes. However,
transformation efficiency did not increase in any of the strains
when transformation was carried out with plasmid DNA pro-
duced from a dam dcm double mutant of E. coli. In addition,
treatment of plasmid DNA with crude protein extracts from
Leptospira strains (6) prior to electroporation had no effect on
the transformation efficiency (data not shown). These results
suggest the absence of a strong restriction-modification system
in pathogenic leptospires. The transformable character of in-
dividual strains could be due to variations in leptospiral cell
surface properties, as previously suggested for the poorly trans-
formable mycobacteria and Borrelia (8, 29). For example, the
low-level-transformable Fiocruz strain was found to aggregate
more than did the Lai strain in liquid cultures, reflecting as yet
undefined differences in surface properties.
Transposon integration sites were identified by either LM-
PCR (24) (304 mutants) or direct genome sequencing of the
genomic DNA (19) (624 mutants). LM-PCR is a commonly
used technique for amplifying the DNA flanking sequences of
transposon insertion sites. However, we have found that this
method is laborious and time-consuming. In addition, using
this amplification method, we could not amplify insertion sites
in 60% of the mutants. Several mutants remained uncharac-
terized by LM-PCR, despite repeated efforts and modifications
to the procedure. Sequencing directly from the chromosome
using a primer within the transposon was successful in more
than 75% of reactions. Typically, 200 to 1,000 bp of quality
sequence was obtained, though only 30 bp or so were required
to locate the transposon on the chromosomes. Since signal
strength was usually low, reactions were improved with a larger
amount of template (up to 2 ?g total DNA).
Library of transposon mutants. Sequences were compared
with the complete genome sequence of L. interrogans serovar
Lai strain 56601 to identify the genomic location of the trans-
poson. A total of 929 different genomic sites for transposon
insertion were identified in L. interrogans strains (see the table
in the supplemental material): 617 in L. interrogans serovar
Manilae strain L495, 250 in L. interrogans serovar Lai strain
56601, 32 in L. interrogans serogroup Canicola strain Kito, 17
in L. interrogans serovar Pomona strain PO-06-047, 9 in L.
interrogans serovar Copenhageni strain Fiocruz L1-130, and 4
in L. interrogans serovar Canicola strain L1-133. The insertion
sites of two random mutants were also identified in L. weilii
serogroup Hebdomadis strain EcoChallenge. All of the se-
quenced insertion sites could be mapped using the available L.
interrogans serovar Lai genome sequence. This is consistent
with the fact that gene content is highly conserved between L.
interrogans serovars, with sequences of L. interrogans serovars
Lai and Copenhageni having 95% identity at the nucleotide
level (20). The position of the transposon in every mutant was
plotted on a circular map representing the L. interrogans sero-
var Lai strain 56601 chromosomes (Fig. 1).
To evaluate the distribution of Himar1 in the L. interrogans
genome and determine any site specificity, we analyzed the
insertion site sequences. The two possible orientations of the
transposon with respect to the direction of replication or tran-
scription were present in nearly equal proportions, indicating
that neither orientation is favored (data not shown). We found
that transposon insertion was uniformly distributed across the
two chromosomes (4,333 and 358 kb in size). The mapping of
826 insertion sites over a 4,690-kb target genome yields a
density of approximately one transposon integration per 5 kb.
812MURRAY ET AL.INFECT. IMMUN.
Although the profile indicates a random distribution through-
out the genome, some regions of the genome showed few
insertion sites. These regions generally contained genes that
are notionally essential, such as the lipopolysaccharide (LPS)
biosynthetic locus in the large chromosome and the heme
biosynthetic genes in the small chromosome (Fig. 1). For the
LPS locus (position, kilobases 1570 to 1688 of the large chro-
mosome), the few insertion sites (9 insertions, in comparison to
21 predicted, if random insertion was normally distributed)
map to an intergenic region or genes encoding hypothetical
We examined the occurrence of bases in 15-bp sequences
upstream and downstream of the target site. Consistent with
mariner-based mutagenesis systems used for other bacterial
species (23), all Himar1 insertions in L. interrogans occurred at
a TA dinucleotide. Statistical target site analyses revealed an
absence of any additional target site preference (Fig. 2). The
proportion of Himar1 insertions in coding sequences was 78%
(721/929), a frequency that closely approximates the propor-
tion of the genome that is protein coding (75% of the genome).
With only one exception (mutants FLaiS270 and AMan990),
no two transformants contained a transposon insertion at ex-
actly the same genomic location, further suggesting that
Himar1 inserts randomly into chromosomal DNA. Surpris-
ingly, the transposon insertion sites of several mutants were
within the 16S (18 mutants) or 23S (7 mutants) rRNA gene,
with each mutant showing a different insertion site. In Lepto-
spira spp., rRNA genes are not linked, and L. interrogans con-
tains one rrf gene, two rrl genes, and two rrs genes, encoding 5S,
23S, and 16S rRNA molecules, respectively. Whether there is
something unusual about the architecture of these highly tran-
scribed regions that favors transposon integration remains to
be determined. Excluding insertions in 16S and 23S rRNA
genes and transposases, 551 individual genes have been inter-
rupted in L. interrogans. Of these, 266 (48%) encode hypothet-
ical proteins. Among the disrupted genes, 437 have orthologs
in the pathogen L. borgpetersenii, 312 have orthologs in the
saprophyte L. biflexa, and notably, 139 are unique to patho-
genic strains (Table 2) (see the table in the supplemental
These observations, together with the high A?T content of
the L. interrogans genome, suggest that the mariner transposi-
tion system is suitable for the generation of libraries of random
mutants. The L. interrogans genome contains approximately
FIG. 1. Mapping of transposon insertions on the genome of L. interrogans. Insertion sites of Himar1 in 826 transposon mutants (excluding
insertions into 16S and 23S rRNA and transposases) of L. interrogans were mapped onto circular representations. From the outside in: first,
coordinates of the circular chromosome; next, insertion sites of the random mutants in (i) L. interrogans serovar Manilae strain L495; (ii) L.
interrogans serovar Lai strain 56601; (iii) L. interrogans serovar Copenhageni strain Fiocruz L1-130; (iv) L. interrogans serogroup Canicola strain
Kito; (v) L. interrogans serovar Pomona strain PO-06-047; and (vi) L. interrogans serovar Canicola strain L1-133 (no random mutants in the small
chromosome for this strain). Positions of the LPS and hem loci are indicated on the large (CI) and small (CII) chromosomes, respectively.
FIG. 2. Himar1 target site consensus sequence. Sequence logo is drawn from 100 distinct Himar1 insertion sites in L. interrogans serovar Lai
strain 56601. The degree of sequence conservation at each position is indicated by the height of letters (maximum of 2 bits for a nucleotide
VOL. 77, 2009TRANSPOSON MUTAGENESIS IN PATHOGENIC LEPTOSPIRA SPP.813
3,400 predicted protein coding regions (excluding transposases
and pseudogenes), of which half have been assigned no bio-
logical role whereas the remainder have been assigned roles
that await experimental validation. Based on recent whole-
genome analyses of essential genes in bacteria (9), it is reason-
able to assume that approximately 3,000 out of a total of 3,400
are nonessential and can therefore be mutated. Therefore, at
this stage the transposon insertion library for L. interrogans is
clearly not saturated.
Phenotypic analysis of a subset of mutants. Some mutants
were further characterized by comparing their phenotypes to
that of the parental strain. L. interrogans has periplasmic fla-
gella, essential for motility, that are inserted at each end of the
cell and extend toward the middle of the cell body. Approxi-
mately 80 genes encode proteins involved in motility (20).
Mutants were identified with transposon insertions in putative
motility genes, including LA0025 (encoding FliG, one of the
four paralogs, associated with the flagellar motor switch in E.
coli), LA2417 (encoding the flagellar hook protein FlgL-1, one
of four paralogs), LA2069 (encoding FliN, a putative flagellar
motor switch protein, one of two paralogs), LA2215 (encoding
a putative flagellar motor protein, one of three or more para-
logs), and LA2592 (encoding FliI, a putative flagellum-specific
ATP synthase). Unexpectedly, these mutants were motile in
liquid culture and did not exhibit any in vitro growth defects
compared to the parental strain (data not shown). This may be
due to functional redundancy; as indicated above, these genes
of L. interrogans have multiple paralogs that may compensate
for the motility-associated mutations.
Leptospires have a full nucleotide excision repair system
(UvrA, UvrB, UvrC, and UvrD). A mutant with transposon
disruption in uvrB was assayed for its ability to recover from
DNA damage produced by exposure to UV irradiation. In
three independent experiments, there were no detectable col-
onies of the uvrB mutant at the lowest UV dose tested, com-
pared to 10% survival for the wild-type strain. This treatment
therefore had a significantly greater effect on mortality of the
uvrB mutant than on that of the wild-type strain. We also
identified transposon mutants in a locus containing genes in-
volved in heme acquisition (LB191, encoding a TonB-depen-
dent transporter) and utilization (LB186, encoding a heme
oxygenase) (1, 19). The iron chelator dipyridyl was used to
produce iron-limited conditions that inhibited the growth of
Leptospira strains (13). Addition of 10 ?M hemin restored the
ability of the L. interrogans wild-type strain to grow under iron
starvation conditions, but not in the mutant strains. These
results suggest that disruption of LB186 and LB191, which
encode the heme oxygenase and a TonB-dependent receptor
(1, 19), resulted in mutants that were impaired in their ability
to use hemin as an iron source.
We obtained several mutants exhibiting insertions in the 16S
and 23S rRNA genes. The growth rates of all mutants were
comparable to that of the parental strain, with no mutants
showing altered motility or morphology, consistent with the
notion that the mutants are functionally able to overcome
inactivation of one of the two copies of the 16S and 23S
To establish a system for the identification of virulence-
associated genes, 29 mutants were selected for virulence test-
ing using the hamster model of acute infection (Table 3).
Analysis of the L. interrogans genome identified few obvious
virulence factors, most likely due to the evolutionary distance
between L. interrogans and prototypic bacterial pathogens.
This is consistent with the notion that Leptospira has unique
virulence mechanisms. Therefore, mutants were selected based
on the following criteria for the disrupted gene: the absence of
an orthologous gene in L. biflexa, a predicted outer membrane
location, indicating likelihood of interaction with the host, and
a potential role in signaling, motility, or chemotaxis, all of
which may be required in the in vivo dissemination of L.
interrogans. Mutants recovered from host animals were tested
for stability of the transposon by PCR. In each mutant tested,
the transposon remained in situ, indicating a high degree of
The majority of mutants retained full virulence (Table 3),
indicating that the mutagenesis process and the necessary as-
sociated in vitro passage do not per se lead to attenuation. Two
mutants, with mutations in LA1641 and LA0615, were identi-
fied to have lost virulence, with all hamsters surviving infection
and exhibiting no lung pathology or signs of disease. Kidneys
from these hamsters were also culture negative for Leptospira.
In both instances, the interrupted gene had no predicted func-
tion and showed normal in vitro growth. LA1641 is located in
the LPS biosynthesis locus and is found only in L. interrogans.
The mutant expressed a lower-molecular-weight LPS structure
TABLE 2. L. interrogans mutant libraries
No. of mutants with defined insertion locations
No. of mutants:
For L. interrogans serovar Manilae strain L495
For L. interrogans serovar Lai strain 56601
For L. interrogans serogroup Canicola strain Kito
For L. interrogans serovar Pomona strain EUA
For L. interrogans serovar Copenhageni strain
For L. interrogans serovar Canicola strain L1-133
No. (%) of mutations:
In chromosome I (92.4% of genome)a
In chromosome II (7.6% of genome)a
In coding region
In rRNA gene
In intergenic region
No. of ORFs disrupted:
Encoding hypothetical proteins
Encoding proteins with predicted L. biflexa
Encoding proteins with predicted L.
Encoding pathogen-specific proteins
aExcluding insertion locations corresponding to multiple locations (trans-
b473 in L. interrogans serovar Manilae strain L495, 200 in L. interrogans
serovar Lai strain 56601, 24 in L. interrogans serogroup Canicola strain Kito, 13
in L. interrogans serovar Pomona strain PO-06–047, 7 in L. interrogans serovar
Copenhageni strain Fiocruz L1–130, and 4 in L. interrogans serovar Canicola
cSix in L. interrogans serovar Lai strain 56601, four in L. interrogans serovar
Manilae strain L495, and one in L. interrogans serogroup Canicola strain Kito.
dNineteen in L. interrogans serovar Manilae strain L495, five in L. interrogans
serovar Lai strain 56601, and one in L. interrogans serogroup Canicola strain
814MURRAY ET AL.INFECT. IMMUN.
(unpublished data) and was selected for the virulence assay
because mutations affecting LPS can lead to attenuation in
other bacterial pathogens (10, 18). LA0615 is located down-
stream of the gene encoding LipL41 and was selected for the
virulence assay because the gene is unique to pathogenic spe-
cies of Leptospira. The system outlined here demonstrates the
feasibility of using random transposon mutagenesis in conjunc-
tion with the hamster animal model to identify novel virulence
factors in L. interrogans.
A number of mutants of particular interest were examined.
These include the ligC mutant (LA3075, an intact gene in L.
interrogans serovar Manilae). Members of the lig family of
genes in L. interrogans encode outer membrane proteins with
immunoglobulin-like repeats (16). The lack of attenuation in
the ligC mutant is consistent with ligC being a pseudogene in
the pathogenic serovar Copenhageni and the recent observa-
tion that mutation of ligB does not impair virulence in the
hamster model of infection (5). An unexpected finding was that
inactivation of a number of chemotaxis-related genes did not
result in attenuation. It is possible that chemotaxis is not im-
portant in the hamster model of infection, but a more likely
explanation is that the mutations may be compensated for by
other genes; the L. interrogans genome has a high degree of
apparent gene duplication and redundancy, with at least 24
chemotaxis genes, including 12 encoding methyl-accepting che-
motaxis proteins. Likewise, mutation of the putative OmpA
family protein LB328 (with 7 paralogs in the genome), the
TonB-dependent receptor LA3258 (with 10 paralogs), or the
fur gene LA1857 (4 paralogs) may have been compensated for
through functional redundancy. Finally, strains carrying muta-
tions in lenB and lenE (with six paralogs in the genome), which
encode proteins binding host extracellular matrix components
in vitro (28), did not show an attenuated phenotype (Table 3).
Redundancy in the genome may make the identification of
virulence factors in L. interrogans more difficult; only one at-
tenuated transposon mutant has been described to date, with a
mutation in the gene encoding LA0222, an OmpA family pro-
tein (26). Although the majority of mutants do not demon-
strate an impairment in growth in vivo, further studies may find
that these genes play a role under different conditions, such as
at the mucosal surface.
This study presents the results of an extensive mutagenesis
project generating 929 transposon insertion mutants. Given
the low growth rate and genetic intractability of L. interrogans,
TABLE 3. Virulence of mutants in hamster model of acute infection
Location of insertion Predicted function of mutated gene or descriptionb
OmpA family protein
Intergenic mutant control
CheX, inhibitor of MCP methylation
HP, contains leucine-rich repeats
HP, ankyrin repeat protein
CheB, chemotaxis response regulator
HP, LipL45-related protein
Flagellar motor protein/OmpA family protein
RNA polymerase sigma subunit
aMutants were constructed in L. interrogans serovar Manilae L495. ?, the genotype of reisolates was confirmed by PCR; #, bacteria could not be recovered from
bPredicted function of protein encoded by disrupted gene. HP, hypothetical protein; MCP, methyl-accepting chemotaxis protein.
cPredicted subcellular location of disrupted gene product using the psortb software program (www.psort.org/psortb/); C, cytoplasmic; OM, outer membrane; IM,
dIndicates the presence of a deduced protein in L. biflexa with ?50% similarity by BLASTP. Y, yes; N, no.
eMutants were injected intraperitoneally in two doses (103and 105leptospires) into groups of two or four hamsters, or a single dose was injected into groups of five
fNo. surviving/total. Survival data are pooled for both doses if appropriate.
VOL. 77, 2009TRANSPOSON MUTAGENESIS IN PATHOGENIC LEPTOSPIRA SPP.815
this work represents a major advance. Clearly, additional work Download full-text
is required to fully understand the phenotypes of randomly
constructed mutants. Complementation of the disrupted genes
and/or independent generation of further mutants in the same
gene will need to be performed to provide confirmation for the
phenotypes observed. However, the identification of two ap-
parently attenuated mutants demonstrates the value of this
work in identifying novel virulence mechanisms of L. interro-
gans. The use of different routes of inoculation, quantitative
PCR, and histopathological analyses may further reveal the
role of different genes in spirochete burden and tissue pathol-
ogy. Further increases in transformation efficiency, through the
identification of more transformable strains or the develop-
ment of new genetic tools, will provide opportunities to gen-
erate extensive mutant libraries that may subsequently be used
to screen for phenotypes affecting diverse aspects of the phys-
iology of Leptospira.
We thank David Haake for kindly providing Leptospira interrogans
strains EcoChallenge and PO-06-047. We also thank Caroline Bour-
saux-Eude for graphic support.
This work was supported by the National Health and Medical Re-
search Council, Australia; the Australian Research Council; Institut
Pasteur, Paris, France; the French Ministry of Research ANR Jeunes
Chercheurs (no. 05-JCJC-0105-01); the Fiocruz-Pasteur Scientific Co-
operation Agreement; the Brazilian National Research Council (Insti-
tuto Mile ˆnio 420067/2005); and the National Institutes of Health (5
R01 AI052473, 2 R01 AI034431, and 2 D43 TW-00919). G.L.M. is
supported by a National Health and Medical Research Council
(NHMRC) Peter Doherty Fellowship.
1. Asuthkar, S., S. Velineni, J. Stadlmann, F. Altmann, and M. Sritharan.
2007. Expression and characterization of an iron-regulated hemin-binding
protein, HbpA, from Leptospira interrogans serovar Lai. Infect. Immun. 75:
2. Bourhy, P., H. Louvel, I. Saint Girons, and M. Picardeau. 2005. Random
insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis,
using a mariner transposon. J. Bacteriol. 187:3255–3258.
3. Bulach, D. M., R. L. Zuerner, P. Wilson, T. Seemann, A. McGrath, P. A.
Cullen, J. Davis, M. Johnson, E. Kuczek, D. P. Alt, B. Peterson-Burch, R. L.
Coppel, J. I. Rood, J. K. Davies, and B. Adler. 2006. Genome reduction in
Leptospira borgpetersenii reflects limited transmission potential. Proc. Natl.
Acad. Sci. USA 103:14560–14565.
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.
5. Croda, J., C. P. Figueira, E. A. J. Wunder, C. S. Santos, M. G. Reis, A. I. Ko,
and M. Picardeau. 2008. Targeted mutagenesis in pathogenic Leptospira:
disruption of the ligB gene does not affect virulence in animal models of
leptospirosis. Infect. Immun. 76:5826–5833.
6. Donahue, J. P., D. A. Israel, R. M. Peek, M. J. Blaser, and G. G. Miller. 2000.
Overcoming the restriction barrier to plasmid transformation of Helicobacter
pylori. Mol. Microbiol. 37:1066–1074.
7. 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.
8. Etienne, G., F. Laval, C. Villeneuve, P. Dinadayala, A. Abouwarda, D. Zer-
bib, A. Galamba, and M. Daffe ´. 2005. The cell envelope structure and
properties of Mycobacterium smegmatis mc(2)155: is there a clue for the
unique transformability of the strain? Microbiology 151:2075–2086.
9. Gil, R., F. J. Silva, J. Pereto ´, and A. Moya. 2004. Determination of the core
of a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68:518–537.
10. Harper, M., A. Cox, F. St Michael, I. W. Wilkie, J. D. Boyce, and B. Adler.
2004. A heptosyltransferase mutant of Pasteurella multocida produces a trun-
cated lipopolysaccharide structure and is attenuated in virulence. Infect.
11. Johnson, R. C., and V. G. Harris. 1967. Differentiation of pathogenic and
saprophytic leptospires. J. Bacteriol. 94:27–31.
12. Levett, P. N. 2001. Leptospirosis. Clin. Microbiol. Rev. 14:296–326.
13. Louvel, H., S. Bommezzadri, N. Zidane, C. Boursaux-Eude, S. Creno, A.
Magnier, Z. Rouy, C. Medigue, I. S. Girons, C. Bouchier, and M. Picardeau.
2006. Comparative and functional genomic analyses of iron transport and
regulation in Leptospira spp. J. Bacteriol. 188:7893–7904.
14. Louvel, H., and M. Picardeau. 2007. Genetic manipulation of Leptospira
biflexa. J. Wiley and Sons, Hoboken, NJ.
15. Louvel, H., I. Saint Girons, and M. Picardeau. 2005. Isolation and charac-
terization of FecA- and FeoB-mediated iron acquisition systems of the spi-
rochete Leptospira biflexa by random insertional mutagenesis. J. Bacteriol.
16. Matsunaga, J., M. A. Barocchi, J. Croda, T. A. Young, Y. Sanchez, I.
Siqueira, C. A. Bolin, M. G. Reis, L. W. Riley, D. A. Haake, and A. I. Ko.
2003. Pathogenic Leptospira species express surface-exposed proteins be-
longing to the bacterial immunoglobulin superfamily. Mol. Microbiol. 49:
17. McBride, A. J., D. A. Athanazio, M. G. Reis, and A. I. Ko. 2005. Leptospi-
rosis. Curr. Opin. Infect. Dis. 18:376–386.
18. Murray, G. L., S. R. Attridge, and R. Morona. 2003. Regulation of Salmo-
nella typhimurium lipopolysaccharide O antigen chain length is required for
virulence; identification of FepE as a second Wzz. Mol. Microbiol. 47:1395–
19. Murray, G. L., K. M. Ellis, M. Lo, and B. Adler. 2008. Leptospira interrogans
requires a functional heme oxygenase to scavenge iron from hemoglobin.
Microbes Infect. 10:791–797.
20. 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.
21. 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.
22. Picardeau, M., D. M. Bulach, C. Bouchier, R. L. Zuerner, N. Zidane, P. J.
Wilson, S. Creno, E. S. Kuczek, S. Bommezzadri, J. C. Davis, A. McGrath,
M. J. Johnson, C. Boursaux-Eude, T. Seemann, Z. Rouy, R. L. Coppel, J. I.
Rood, A. Lajus, J. K. Davies, C. Me ´digue, and B. Adler. 2008. Genome
sequence of the saprophyte Leptospira biflexa provides insights into the
evolution of Leptospira and the pathogenesis of leptospirosis. PLoS ONE
23. Plasterk, R. H., Z. Izsva ´k, and Z. Ivics. 1999. Resident aliens: the Tc1/
mariner superfamily of transposable elements. Trends Genet. 15:326–332.
24. 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:
25. 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. Q., A. Danchin, I. Saint
Girons, R. L. Somerville, Y. M. Weng, M. Shi, Z. Chen, J. G. Xu, and G. P.
Zhao. 2003. Unique and physiological and pathogenic features of Leptospira
interrogans revealed by whole genome sequencing. Nature 422:888–893.
26. Ristow, P., P. Bourhy, F. W. da Cruz McBride, C. P. Figueira, M. Huerre, P.
Ave, I. S. Girons, A. I. Ko, and M. Picardeau. 2007. The OmpA-like protein
Loa22 is essential for leptospiral virulence. PLoS Pathog. 3:e97.
27. 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.
28. Stevenson, B., H. A. Choy, M. Pinne, M. L. Rotondi, M. C. Miller, E. Demoll,
P. Kraiczy, A. E. Cooley, T. P. Creamer, M. A. Suchard, C. A. Brissette, A.
Verma, and D. A. Haake. 2007. Leptospira interrogans endostatin-like outer
membrane proteins bind host fibronectin, laminin and regulators of comple-
ment. PLoS ONE 2:e1188.
29. Tilly, K., A. F. Elias, J. L. Bono, P. Stewart, and P. Rosa. 2000. DNA
exchange and insertional inactivation in spirochetes. J. Mol. Microbiol. Bio-
30. Vallenet, D., L. Labarre, Z. Rouy, V. Barbe, S. Bocs, S. Cruveiller, A. Lajus,
G. Pascal, C. Scarpelli, and C. Me ´digue. 2006. MaGe—a microbial genome
annotation system supported by synteny result. Nucleic Acids Res. 34:53–65.
Editor: A. J. Ba ¨umler
816MURRAY ET AL.INFECT. IMMUN.