JOURNAL OF BACTERIOLOGY, Nov. 2006, p. 7893–7904
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 22
Comparative and Functional Genomic Analyses of Iron Transport and
Regulation in Leptospira spp.?†
H. Louvel,1S. Bommezzadri,1,2N. Zidane,3C. Boursaux-Eude,4S. Creno,3A. Magnier,3Z. Rouy,5
C. Me ´digue,5I. Saint Girons,1C. Bouchier,3and M. Picardeau1*
Laboratoire des Spiroche `tes, Institut Pasteur, Paris, France1; Department of Pathology and Laboratory Medicine, University of Parma,
Parma, Italy2; Plate-forme Ge ´nomique3and Plate-forme Inte ´gration et Analyse Ge ´nomiques,4Institut Pasteur, Paris, France; and
Pasteur Genopole Ile de France, Genoscope, and CNRS-UMR8030, Atelier de Ge ´nomique Comparative, Evry, France5
Received 18 May 2006/Accepted 5 September 2006
The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being
responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the
slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work
with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa
enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that
Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis
of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is
presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12
genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent
receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron.
In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron
regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant
functional information on iron homeostasis in Leptospira spp.
Leptospira belongs to the bacterial phylum of spirochetes,
which has a deep branching lineage in Bacteria, as indicated by
16S rRNA analysis (42). The genus Leptospira was initially
divided into two groups: the pathogenic Leptospira referred to
as Leptospira interrogans sensu lato and the saprophytic Lep-
tospira referred to as L. biflexa sensu lato (9). Saprophytic and
pathogenic Leptospira spp. were first classified into serovars,
with more than 220 serovars defining the pathogens. More
recently, DNA-DNA hybridization studies separated Lepto-
spira species into 17 genomospecies, including 7 pathogenic
In the past decade, leptospirosis has emerged as a wide-
spread zoonosis, and its incidence is high in tropical countries.
Leptospirosis is acquired by direct or indirect contact with the
urine of infected animals such as rodents (9). Virulence mech-
anisms and more generally the fundamental understanding of
the biology of the causative agent of leptospirosis remain
largely unknown. Recently, the genome sequencing of two
serovars of L. interrogans sensu stricto, the main species asso-
ciated with human leptospirosis, has been achieved (38, 48).
However, the lack of genetic tools in pathogenic Leptospira
does not allow the full characterization of genes of interest.
Only recently the first evidence of gene transfer has been
demonstrated in L. interrogans by transposition of Himar1, a
transposon of eukaryotic origin (13). In contrast, numerous
tools for genetic manipulation of saprophytic Leptospira spe-
cies have been developed in recent years (7, 27, 35, 43, 44, 51,
56). These studies enable the use of the saprophyte L. biflexa as
a model spirochete. The availability of the genome sequence of
the saprophyte L. biflexa (unpublished data) and its compari-
son with the genomes of pathogenic species give us functional
information on the lifestyles of Leptospira spp. in the environ-
ment and the infected host.
Iron plays a central role in many major biological processes,
such as the electron transport chains for most living cells,
including Leptospira spp. However, a few organisms, such as
the spirochete Borrelia burgdorferi, do not require iron for
growth (46). Spirochetes possess a double-membrane structure
composed of a cytoplasmic membrane that differs substantially
from that of gram-negative bacteria, the periplasm, and the
outer membrane (9), which may constitute a barrier for mol-
ecules that could be used as an iron source. In gram-negative
bacteria, iron sources can be recognized by specific outer mem-
brane receptors, called TonB-dependent receptors, and then
transported across the inner membrane by periplasmic binding
protein-dependent ABC permeases (38, 48). We recently char-
acterized the fecA- and feoB-like genes by random transposon
mutagenesis in L. biflexa (35). Genes involved in iron acquisi-
tion are usually transcriptionally regulated by the availability of
iron through regulators such as the ferric uptake regulator
protein Fur. Cullen et al. have shown that the expressions of
some genes from L. interrogans were regulated by iron (19),
and fur-like genes are present in the L. interrogans genomes
In this study, the analysis of the genome sequence of L.
biflexa allowed us to identify putative genes involved in iron
transport and regulation. We showed that some of these pu-
* 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:
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 15 September 2006.
tative genes modulate their expression in response to iron. To
study the function of these genes, we generated several mu-
tants in L. biflexa, and their phenotypes were characterized.
Since pathogenic Leptospira spp. as well as saprophytic species
need to obtain iron to grow in vitro, and probably in vivo in the
host, better knowledge of the iron uptake systems and their
regulation is essential to understand the pathogenesis of this
intriguing group of organisms.
MATERIALS AND METHODS
Bacterial strains and growth conditions. L. biflexa serovar Patoc strain Patoc1
and L. interrogans serovar Lai strain Lai (National Reference Center for Lepto-
spira, Paris, France) were grown at 30°C in EMJH (22, 31) medium. When
necessary, kanamycin and spectinomycin were added at 40 ?g/ml. Minimal iron
EMJH medium was prepared by either omitting iron sulfate (normally 330 ?M)
or, prior to inoculation, treating normal EMJH medium with 50 ?M 2,2?-dipyri-
dyl (Sigma-Aldrich, St. Louis, MO) overnight. Media for testing the ability of
Leptospira to use specific sources of iron was prepared by supplementing the
dipyridyl-treated EMJH medium. The siderophores ferrichrome (final concen-
tration, 20 ?M), enterobactin (final concentrations, 10 to 100 ?M), and aerobac-
tin (final concentration, 50 ?M) were purchased from EMC Microcollections
GmbH (Germany). Desferrioxamine (final concentration, 10 ?M), also called
ferrioxamine B, refers to deferoxamine mesylate (Sigma-Aldrich, St. Louis, MO).
Bovine hemin (final concentration, 10 ?M) and lactoferrin (final concentration,
10 ?M) were obtained from the Sigma-Aldrich Company (St. Louis, Mo). Iron
citrate and iron chloride were used at 100 ?M.
DNA and RNA manipulations. Genomic DNA of Leptospira was isolated as
previously described (44). Plasmid DNA was purified using the Plasmid Mini-
prep kit (QIAGEN GmbH, Hilden, Germany). Total RNA was isolated by using
RNAwiz (Ambion Inc.) and treated with DNaseI. For transcription studies,
RNA was isolated from exponential-phase cultures of L. biflexa grown in EMJH
or in dipyridyl-treated EMJH. The absence of DNA contamination was con-
firmed by PCR. RNA concentration was measured by spectrophotometry at 260
nm. Reverse transcription-PCR (RT-PCR) of RNA was performed using con-
ditions recommended by the manufacturer (SuperScript One Step RT-PCR with
Platinum Taq; Invitrogen) with primer pairs (primer nucleotide sequences are
available on request) corresponding to selected open reading frames as previ-
ously described (12). The semiquantitative determination of transcript levels by
RT-PCR was performed with 1, 10, and 100 ng of total RNA from L. biflexa. The
amplified products were analyzed by agarose gel electrophoresis. The assays were
performed in triplicate. For real-time quantitative reverse transcription-PCR
(qRT-PCR), RNA (800 to 1,500 ng) was reverse transcribed using random
primers and reverse transcriptase as described in the manufacturer’s instructions
(Roche Diagnostics, GmbH, Mannheim, Germany). The cDNA was used as
template for gene-specific primer pairs with a LightCycler FastStart DNA
MasterPLUSSYBR Green (Roche Diagnostics) in a LightCycler apparatus
(Roche Diagnostics). The thermal cycling conditions were as follows: 10 min at
95°C, followed by 45 cycles of 10 s at 95°C, 5 s at 60°C, and 13 s at 72°C, then 1
cycle of 5 s at 95°C, 15 s at 65°C, and 30 s at 40°C. Data were analyzed using
RelQuant (Roche Diagnostics). In all cases, transcript levels were determined in
duplicate and at least two independent RNA samples were used for each con-
dition tested. The relative expression of target genes was normalized to the level
of 23S rRNA as an endogenous control.
Mutagenesis in L. biflexa. To mediate allelic exchange, a pGEM7Z-f?(Pro-
mega) derivative plasmid was used for the construction of plasmids containing
insertional inactivated genes. The process was as follows: PCR primers for the
amplification of the kanamycin or spectinomycin resistance cassette and the left
and right arms of the target gene were designed, and in each instance a restric-
tion endonuclease site was introduced at each end of each PCR product. The
resulting three PCR products were digested with the appropriate restriction
endonucleases and ligated into the pGEM7Z-f?derivative plasmid. The plasmid
constructs delivering the inactivated allele were formed by insertion of a resis-
tance cassette between the right and left arms (?0.5 kb in length) of the target
gene; this introduces a partial gene deletion. The plasmids, which are not rep-
licative in Leptospira spp., were then subjected to UV irradiation and used to
deliver the inactivated alleles in L. biflexa as previously described (44). Kanamy-
cin- or spectinomycin-resistant colonies were picked and tested for the insertion
of the resistance cassette in the target gene by PCR as previously described (44).
Random insertion mutagenesis using Himar1 was carried out in medium with or
without hemin as previously described (35). We used RT-PCR assays to check
that the mutations did not prevent transcription of genes downstream of the
Sequencing and annotation of the genome of L. biflexa. The sequenced strain,
L. biflexa serovar Patoc strain Patoc1, was initially isolated from stream water (3)
and maintained in the collection of the National Reference Center of Leptospira
(Institut Pasteur, Paris, France). L. biflexa genomic DNA was randomly sheared
by nebulization (hydroShear; GeneMachines) to short (1.5 to 2.5 kb) and long
(35 to 45 kb) DNA fragments, and the insert DNAs were end repaired and
ligated into a derivative of plasmid pGEM7-Zf?(Promega) and fosmid
pCC1FOS (Epicenter, Madison, WI), respectively. Sequencing reactions were
performed, from both ends of DNA template, using an ABI PRISM BigDye
Terminator cycle sequencing ready reaction kit and run on a 3700 or a 3730 xl
Genetic Analyzer (Applied Biosystems) at the Genomics Platform (Pasteur
Genopole Iˆle-de-France). Phred (23), Phrap (26), and in-house software (cover
and coverparse; unpublished data) were used for genome assembly. The com-
plete genome sequence was obtained from 58,663 end sequences (giving ?8?
coverage). Annotation was done using MaGe (http://www.genoscope.cns.fr/agc
/mage/) (59), which allows graphic visualization of the L. biflexa annotations
enhanced by a synchronized representation of synteny groups in other genomes
chosen for comparisons. Coding sequences (CDSs) likely to encode proteins
were predicted with the AMIGene (10) and MICheck (18) software. Putative
orthologs showed at least 30% identity and a minimum ratio of 0.8 to the length
of the smallest protein. Each predicted gene was assigned a unique identifier
prefixed with “LEPBIa” for the large chromosome, “LEPBIb” for the small
chromosome, and “pLEPBI” for the 74-kb plasmid. TMHMM, version 2.0 (34), and
PRED-TMBB (4) were used to identify putative transmembrane domains and
?-barrel domains, respectively. Deduced amino acid sequences were also analyzed
using the databases of Pfam protein families (6), membrane transport systems
(http://www.membranetransport.org/) (47), and ABC systems (ABSCISSE v3.0 da-
tabase; http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml) (11). The
complete genomic sequence of L. biflexa serovar Patoc strain Patoc1 analyzed in this
work will be published in another study.
Nucleotide sequence accession numbers. Nucleotide sequences of predicted
CDSs involved in iron uptake and regulation (Table 1) were deposited into
EMBL under accession numbers AM162599 to AM162646 (see the supplemen-
RESULTS AND DISCUSSION
Iron is an essential nutrient for Leptospira spp. Iron is es-
sential for the growth of both saprophytic and pathogenic
Leptospira spp. (24). In biological systems, iron is typically
complexed to other molecules, and free iron is virtually absent.
Leptospira spp. are usually cultivated in a standard albumin
Tween 80 medium, the EMJH medium, containing iron sulfate
as an iron source. To test for other iron sources, iron sulfate
was omitted during the preparation of the EMJH medium. We
find that whereas L. biflexa was not able to grow in iron sulfate-
free EMJH, L. interrogans retained a wild-type growth. It is
postulated that iron traces in iron sulfate-free EMJH were
sufficient for the observed growth of L. interrogans. Further
experiments were therefore done in iron-depleted EMJH that
had been preincubated with 2,2?-dipyridyl. In these conditions,
growth tests demonstrated that both L. biflexa and L. interro-
gans were able to use iron chloride, iron sulfate, and iron
citrate. The most abundant source of iron in the host is heme
and heme-containing proteins (62). Leptospira spp. were able
to use exogenous hemin/hemoglobin as an iron source. To-
gether with our previous study on an L. biflexa hemH mutant
(27), this suggests that L. biflexa uses heme/hemoglobin as an
iron as well as a heme source. Lactoferrin is another host
protein that binds iron with high affinity, but neither L. biflexa
nor L. interrogans was able to use lactoferrin as an iron source.
Siderophores are high-affinity ferric chelators which are gen-
erally low-molecular-weight compounds synthesized and se-
creted by microorganisms in response to iron restriction. The
7894LOUVEL ET AL.J. BACTERIOL.
secretion of siderophores by L. interrogans could explain its
ability to grow in iron-depleted medium. However, analysis of
the Leptospira genomes did not allow the identification of any
genes that encoded proteins related to proteins involved in
siderophore synthesis or siderophore secretion. In addition, L.
biflexa failed to grow in the supernatant derived from an iron-
depleted culture of L. interrogans. This result suggests that L.
interrogans does not produce siderophore or that this sid-
TABLE 1. Putative genes discussed in this study
1. Iron transport
?-barrel OMP/plug domain;
?-barrel OMP/plug domain
?-barrel OMP/plug domain
?-barrel OMP/plug domain
?-barrel OMP/plug domain;
?-barrel OMP/plug domain
?-barrel OMP/plug domain;
?-barrel OMP/plug domain;
?-barrel OMP/plug domain
NH LA0572LIC12998e-09 (22); E. coli
e-41 (26); Salmonella
e-17 (23); Yersinia
e-22 (20); Colwellia
NH LA2641LIC11345 TB-DR
?-barrel OMP/plug domain
?-barrel OMP/plug domain
?-barrel OMP/plug domain;
?-barrel OMP/plug domain;
1.2. Divalent metal
transporters of the
LEPBIa0902 NH NHe-72 (37); Vibrio
Mn(II) and Fe(II) transporters
of the NRAMP family
1.3. Ferrous iron
of the Feo type
LEPBIa0879LA2579LIC11402 e-203 (53)Ferrous iron transport protein
Ferrous iron transport protein
1.4. ABC transport
systems of the
pLEPBIa0012NH NHe-66 (42); Yersinia
HmuS hemin transport proteinSimilar to Bordetella sp.
BhuS, Shigella dysenteriae
ShuS, and Y. enterocolitica
Similar to Bordetella sp.
BhuV, Shigella dysenteriae
Y. enterocolitica HemV
Similar to Bordetella sp.
BhuU, Shigella dysenteriae
Y. enterocolitica HemU
Similar to Bordetella sp.
BhuT, Shigella dysenteriae
ShuT, and Y. enterocolitica
pLEPBI0013NH NH e-39 (37); Y. pestis
HmuV hemin transport system
pLEPBI0014NH NHe-63 (39); Y. pestis
HmuU hemin transport system
pLEPBI0015NHNHe-47 (38); Y. pestis
Continued on following page
VOL. 188, 2006IRON TRANSPORT AND REGULATION IN LEPTOSPIRA SPP.7895
erophore is not utilized by L. biflexa. Leptospira spp. do not
appear to synthesize siderophores; however, they could use
exogenous siderophores of other microorganisms as an iron
source. Among the hydroxamate-type siderophores, aerobactin
and ferrichrome were used by both L. biflexa and L. interro-
gans, while desferrioxamine was only used by L. biflexa. Des-
ferrioxamine is produced by the gram-positive Streptomyces
pilosus (37) and is utilized by some gram-negative bacteria,
1.5. ABC transport
systems of the
LEPBIa1264NH NHe-40 (34); Aquifex
ABC-type metal ion transport
NHNH e-25 (36); Bacillus
ABC transport system (8
2. Hemolysins and
Hemolytic protein-like protein
Hemolytic activity (68)
Hemolytic activity (68)
Hemolytic activity (68)LEPBIa2015LA1650LIC12134e-86 (50) Hemolysin hemolytic protein
HlyX hemolysin LEPBIa2375LA0378LIC10325 e-104 (48)Hemolytic activity (68)
6 TMS LEPBIa2477NHNHe-53 (47); Bacillus
S. aureus P09978
e-49 (51); LA3540/
S. aureus P09978
e-58 (42); Bacillus
Predicted membrane protein,
Phospholipase C precursorNH LA1029 LIC12631 Sphingomyelinase activity
NH LA3050 LIC11040 Hemolytic protein-like protein
2.2. ABC transporter
LEPBIa0357NH NHe-87 (34); Xyllela
Toxin secretion ABC
Hemolysin secretion protein D
RTX toxin transporter(6
LEPBIa0358 NH NHe-18 (22); E. coli
e-77 (28); E. coli
e-13 (23); Bacteroides
e-15 (25); Vibrio
RTX toxin transporter(1
RTX toxin transporter
LEPBIa0359 LA0150LIC10136Hemolysin secretion protein B
LEPBIa0360 NHNHOuter membrane efflux
Multidrug efflux transporter
NH LA3926 LIC13134
NH LA3927LIC13135 Outer membrane efflux
3. Fur-like proteinsLEPBIa2152
e-07 (25); Borrelia
e-11 (37); Rhizobium
PerR “CXXC” motifs
PerR “CXXC” motifs
4. Iron storage LEPBIa1791
DNA-binding stress protein
aEMBL accession numbers are indicated in the supplemental material.
bNH, no homolog.
cUnderlined gene names indicate that a mutant is available (see Table 2).
dShown is the E value (% identity) between L. biflexa and L. interrogans orthologs, except where otherwise stated.
eTB-DR, TonB-dependent receptor.
fProtein sequences were analyzed for the prediction of ?-barrel/plug domains (PRED-TMBB) and transmembrane helices (TMHMM). OMP, outer membrane
protein; TMS, transmembrane segments (the number of putative TMS is indicated).
7896LOUVEL ET AL. J. BACTERIOL.
such as Yersinia enterocolitica (8) or Vibrio vulnificus (65), and
aerobactin is synthesized by Shigella spp. as well as some Esch-
erichia coli clinical isolates, while the siderophore ferrichrome
is synthesized by various fungal species. The catechol sid-
erophore enterobactin is also produced by enterobacteria but
was not utilized as an iron source by Leptospira spp.
The in vitro utilization of exogenous siderophores suggests
that Leptospira spp. encounter the corresponding siderophores
in their environment. While it is not surprising that Leptospira
would use exogenous siderophores as an expedient way to
acquire iron, it is not clear why saprophytes have the ability to
use hemin and hemoglobin as an iron source.
Genomic sequences of Leptospira spp. Recently, the com-
plete genome sequences of L. interrogans serovar Lai (48) and
L. interrogans serovar Copenhageni (38) have been deter-
mined. The two genomes exhibit 95% identity at the nucleotide
level and a further 99% identity for predicted protein-coding
genes that are orthologs. The original annotations found that
L. interrogans serovar Lai has nearly 1,000 more genes (4,727
open reading frames versus 3,658 CDSs) than L. interrogans
serovar Copenhageni (38). However, this discrepancy may not
reflect the reality and rather may be due to the annotation
criteria used by the two genome projects (58). Using the
MICheck software (18) on the two genomes, we predicted a
total number of 3,798 and 3,651 CDSs for L. interrogans sero-
var Lai and L. interrogans serovar Copenhageni, respectively.
Since all the homologous gene products of serovars Lai and
Copenhageni share more than 95% identity, the term L. inter-
rogans will therefore refer to both of the serovars. The com-
plete genome sequence of the saprophyte L. biflexa serovar
Patoc strain Patoc1 is also available and consists of approxi-
mately 3,790 predicted coding genes (M. Picardeau et al., un-
Iron participates as a metabolic cofactor in a variety of
biochemical processes involving electron transfer. Genes en-
coding proteins that typically require iron as a cofactor, such as
cytochromes, catalase, and tricarboxylic acid enzymes, were
detected in both L. biflexa and L. interrogans. In contrast, a
putative superoxide dismutase gene (LEPBIa0027) was only
detected in L. biflexa. As previously shown, Leptospira spp.
possess a complete heme biosynthesis pathway, including a
ferrochelatase (27). A putative heme oxygenase was also found
in the genomes of L. biflexa (LEPBIa0669) and L. interrogans
(LB186/LIC20148). We have used comparative genomics to
identify putative genes involved in iron acquisition systems and
iron regulation in the genomes of L. biflexa and L. interrogans.
We used the BLAST program and queried all L. biflexa protein
sequences exhibiting significant similarities with iron transport-
ers and iron regulators against the L. interrogans databases and
vice versa. Genes encoding putative hemolysins, hemolysin ex-
cretion apparatus, and iron storage proteins were also included
in this study (Table 1).
Transport across the outer membrane via TonB uptake sys-
tems. We have shown that Leptospira spp. were able to utilize
various iron sources such as siderophores, but the uptake sys-
tem mechanisms are not known. The acquisition of molecules
by bacteria often relies on the active transport through dedi-
cated outer membrane receptors. In gram-negative bacteria,
transport of iron sources is mediated by outer membrane re-
ceptors, also called TonB-dependent receptors, which utilize
the energy produced by the inner membrane complex of TonB,
ExbB, and ExbD.
Bacteria often possess multiple TonB-dependent receptors,
each providing the bacterium with specificity for different iron
sources. For example, E. coli has one set of TonB, ExbB, and
ExbD as well as eight TonB-dependent receptors (29). Other
gram-negative organisms may have two or more TonB-ExbB-
ExbD-like systems with distinct specificities for the TonB-de-
pendent receptors (36). Analysis of the predicted proteins en-
coded by the L. biflexa and L. interrogans genomes revealed five
putative ExbB and ExbD proteins and three putative TonB
proteins. These genes are grouped in five distinct loci, and
their arrangements are similar in both L. biflexa and L. inter-
rogans. Consistent with an inner membrane localization, the
putative TonB and ExbD proteins possess a single transmem-
brane segment (TMS) near the N-terminal end, and the puta-
tive ExbB proteins have three TMS (Table 1). Further analysis
of the genomes reveals a total of 8 L. biflexa and 12 L. inter-
rogans genes encoding proteins related to TonB-dependent
receptors (Table 1), and it is notable that only one gene en-
coding a protein related to a TonB-dependent receptor (LEP
BIa3017/LB279/LIC20214) is genetically linked to an ExbB-
ExbD-TonB system. Despite low primary sequence similarity,
the structure of TonB-dependent receptors is usually well-
conserved in bacteria; all share a 22-stranded transmembrane
?-barrel that forms a pore, which is plugged from the
periplasm by a globular N-terminal domain (67). The 19
putative TonB-dependent receptors of L. biflexa and L. in-
terrogans are 684 to 991 amino acids in length. Protein
analysis using the Pfam database showed that they all exhibit
a putative plug domain (E value from e-03 for LA3102 to
e-24 for LA3021) and a TonB-dependent receptor domain
(E value from e-03 for LEPBIa3354 to e-31 for LEP-
BIa3432). Further analysis predicts that these proteins are
hydrophobic and have a putative large ?-barrel domain typ-
ical of a TonB-dependent receptor. None of the putative
TonB-dependent receptors of Leptospira possesses an N-
terminal extension that could interact with anti-sigma fac-
tor, as in the E. coli fecA-fecIR system (15).
TonB-dependent receptors usually have high affinity and
specificity for their substrates. In E. coli, the well-characterized
TonB-dependent receptors FecA, FhuA, FepA, and BtuB are
required for the active transport of ferric citrate, ferrichrome,
enterobactin, and vitamin B12, respectively. However, sub-
strate specificity is difficult to identify by sequence analysis of
The construction or identification of mutants in each of the
genes encoding TonB-dependent receptors and subsequent
characterization of substrate specificity of the mutants is the
preferred approach to the characterization of these receptors.
Such an approach would not have been possible until the
recent development of some key genetic manipulation tech-
niques for Leptospira (35). By random transposon mutagenesis
in L. biflexa, we recently identified mutants with insertions in a
gene (LEPBIa1883) encoding a protein that shares homology
with FecA (35). By screening a total of 6,000 L. biflexa trans-
poson mutants for hemin auxotrophy, 11 out of 14 auxotrophic
mutants exhibited the transposon inserted in LEPBIa1883 at
distinct locations (data not shown). These strains were im-
paired in their ability to use iron citrate, iron chloride, iron
VOL. 188, 2006IRON TRANSPORT AND REGULATION IN LEPTOSPIRA SPP. 7897
sulfate, and aerobactin as an iron source (Table 2). Interest-
ingly, aerobactin-like siderophores are derived from citrate
(39), and therefore aerobactin and iron citrate share a similar
structure that is evidently recognized by the same receptor.
The gene product of LEPBIa1883 (642 amino acids in length)
has 48% identity (E value, e-229) with the L. interrogans
LA3468/LIC10714 product (650 amino acids in length). The
reciprocal best BLAST hit test indicates that these proteins are
orthologous and strongly suggests that they share the same
Besides fecA, we have attempted to disrupt the other puta-
tive genes encoding TonB-dependent receptors by allelic
exchange in L. biflexa. Gene inactivation of pLEPBI0018, LEP
BIa3432, LEPBIa3362, and LEPBIa3354 resulted in a wild-
type phenotype in iron-depleted medium supplemented with
different iron sources (Table 2). The lack of a phenotype in
these mutants could be due to functional redundancy with
another iron uptake system.
Disruption of LEPBIa2760 resulted in a mutant that was
impaired in its ability to use desferrioxamine as an iron source
(Table 2). Introduction of a recombinant plasmid harboring
the LEPBIa2760 locus restored the ability of the mutant to use
desferrioxamine (data not shown). Desferrioxamine is also uti-
lized via a TonB-dependent receptor, called FoxA, in Yersinia
enterocolitica (8). These results are evidence that LEPBIa2760
encodes the receptor protein for ferrioxamines in L. biflexa.
Finally, we failed to obtain double-crossover events in LEP
BIa0500 and LEPBIa3017. This may indicate that these genes
are essential for the survival of L. biflexa. Leptospira spp. have
an absolute requirement for vitamin B12, which is usually trans-
ported via TonB-like systems in other gram-negative bacteria.
Since vitamin B12is a cofactor for enzymes of major biological
processes, inactivation of its receptor should result in nonvia-
ble mutants. Amino acid comparisons of TonB-dependent re-
ceptors of heme, hemoglobin, siderophores, and vitamin B12
revealed a highly conserved domain containing the FRAP and
NPNL amino acid box (14, 53). This conserved domain was
found in some TonB-dependent receptors from Leptospira,
including LEPBIa0500 (Table 1; Fig. 1).
It is important to note that the current understanding about
the mechanism of TonB-dependent transport across the outer
membrane comes from studies in E. coli. Further studies are
required to understand the precise physiological role of the
TonB-dependent receptors of Leptospira spp. in iron uptake.
Transport across the cytoplasmic membrane. The TonB
periplasm, and these complexes are transported across the
inner membrane via ATP-binding cassette (ABC) transport
systems. In bacteria, the passive diffusion of Fe(II) through the
complexes into the
FIG. 1. Alignment of the FRAP/NPNL motif from several pre-
dicted TonB-dependent receptors. Similar residues are shaded in
black. The His461 marked by an “H” has been shown to be essential
for the Y. enterocolitica HemR function (14). The TonB-dependent
receptors from L. biflexa (pLEPBI0018, LEPBIa0500, LEPBIa3354,
and LEPBIa3432), L. interrogans (LA0706, LA1356, LA3149, LA3258,
and LB191), E. coli (BTUB_ECOLI), and Yersinia pestis (HmuR_
YERPE) are shown.
TABLE 2. Effect of iron sources on growth of L. biflexa mutant strainsd
Interrupted gene in
Hemin DfrxAerobact Ferrichr
aFor growth tests, iron-depleted EMJH medium was supplemented with iron sulfate (100 ?M), iron chloride (100 ?M), iron citrate (100 ?M), hemin (10 ?M),
desferrioxamine (Dfrx; 10 ?M), aerobactin (Aerobact; 50 ?M) and ferrichrome (Ferrichr; 20 ?M). “?” indicates wild-type growth in liquid media (see legend of Table
2), and “0” indicates no growth or poor growth after 1 week in liquid media. The growth curve of L. biflexa in iron-depleted EMJH liquid medium supplemented with
distinct iron sources (at the concentration indicated above) was similar to the growth curve obtained in EMJH liquid medium. ND, not determined.
bTonB-DR, TonB-dependent receptor; ATP-ABC, ATP-binding protein of an ABC transport system.
cSee reference 35.
dLate-exponential-phase cultures (optical density at 420 nm ?OD420?, 0.5) were diluted 1:2,000 in fresh iron-depleted culture medium containing distinct iron sources
and incubated at 30°C with shaking for 1 week. At each time point (48, 72, 96, 120, and 146 h), aliquots were removed from the liquid medium, and growth was
determined by measuring the OD420. For each mutant, growth was compared to the L. biflexa wild-type strain in similar conditions.
7898LOUVEL ET AL.J. BACTERIOL.
outer membrane can represent a second source of iron; active
transport of ferrous irons across the cytoplasmic membrane is
normally distinct from the transport of ferric iron. In E. coli,
uptake of Fe(II) across the cytoplasmic membrane is per-
formed by the energy-driven high-affinity transporter FeoAB
(32) and by the proton-dependent MntH transporter from the
NRAMP (natural resistance-associated macrophage proteins)
The NRAMP family of membrane metal transporters was
originally identified in eukaryotes and then in prokaryotes.
Phylogenetic analyses of the NRAMP family of proteins sug-
gested horizontal transfer from eukaryotes to bacteria (17).
Characterized eukaryotic NRAMP proteins transport divalent
cations such as iron, manganese, and zinc through the cyto-
plasmic membrane. In bacteria, these proteins have been most
extensively characterized in enterobacteria where they act as
manganese transporters, hence the designation MntH pro-
teins. The Mycobacterium tuberculosis MntH also transports
significant amounts of both iron and zinc (1). L. biflexa LEP
BIa0902 displays a small but significant similarity to MntH
proteins (Table 1). This protein contains 11 putative TMS,
which is consistent with the predicted topology model of MntH
proteins (28). However, most of the conserved residues found
essential for the function of E. coli MntH (28), i.e., Asp-34,
Glu-102, Asp-109, Glu-112, and Asp-238, were not found in
LEPBIa0902. Disruption of LEPBIa0902 resulted in a wild-
type phenotype in iron-depleted medium supplemented with
different iron sources (Table 2), manganese, or zinc (data not
shown). In conclusion, we do not have evidence on the role of
LEPBIa0902 as a member of the NRAMP family.
In an earlier study, random transposon mutagenesis allowed
us to identify an feoB-like gene in L. biflexa (35). By using the
same methodology, we also identified an L. biflexa feoA mutant
(Table 2). FeoA, a protein of approximately 75 amino acids in
length, and FeoB were shown to be the only ferrous ion trans-
port systems in both E. coli and Legionella pneumophila (32,
49). FeoB proteins are cytoplasmic membrane proteins that
have two main regions: a hydrophilic N-terminal domain with
GTPase activity and a hydrophobic C-terminal region with 7 to
12 transmembrane-spanning ?-helices (FeoB in L. biflexa has 8
predicted TMS). L. biflexa feoA and feoB mutants were im-
paired in the uptake of ferrous/ferric iron, iron citrate, and the
siderophores desferrioxamine, aerobactin, and ferrichrome
(Table 2). This suggests that iron is released from the sid-
erophores in the periplasm and then transported into the cy-
toplasm via FeoAB. Previous studies have demonstrated that
FeoB was important for the infectivity of pathogenic bacteria
such as Legionella pneumophila, Helicobacter pylori, and Shi-
gella flexneri (49, 50, 60). The L. interrogans feoA and feoB
genes (Table 1) may also have a major role in the acquisition
of ferrous iron while in the host.
Besides the NRAMP-type and the FeoAB-type iron trans-
porters, four distinct families of ABC transporters related to
iron uptake are known (33). Their components can mediate
the transfer of ferric iron, siderophores, heme, and vitamin B12
into the cytosol of prokaryotes. The ABC transporters are
typically composed of periplasmic binding proteins, one or two
identical or homologous permeases, and one or two ATPases
located on the inner surface of the cytoplasmic membrane and
supplying the system with energy. The ATPases are the most
conserved modules among the ABC transporters, and the per-
meases are characterized by their overall hydrophobicity. Only
a few ABC transport systems of the iron/metals type has
been described in spirochetes (21, 30). By similarity search-
ing against a database of characterized ABC transporters
(ABSCISSE v3.0 database), the L. biflexa locus containing
genes LEPBIa1264 to LEPBIa1266 is predicted to encode a
MET (metallic cation uptake) family ABC transporter (11).
No orthologous locus of this putative ABC transporter was
detected in L. interrogans (Table 1). Inactivation of the gene
encoding the ATP-binding protein, LEPBIa1265, had pleiotro-
pic effects on growth in the presence of an Fe(III) source but
not with Fe(II), i.e., iron sulfate (Table 2).
The characterization of an L. biflexa hemH mutant suggested
that Leptospira can transport the entire heme molecule into the
cell (27). Transport of heme across the cytoplasmic membrane
is usually mediated via ABC transporters (62). In L. biflexa, but
not in L. interrogans, four genes encoding a putative ABC
transport system (genes pLEPBI0012 to pLEPBI0015; Fig. 2)
showed significant similarity to the corresponding proteins
encoded by a well-defined hemin uptake system operon,
hmuSTUV. Similar operons have been described in numer-
ous pathogenic bacteria (57). Interestingly, pLEPBI0018,
encoding a putative TonB-dependent receptor, is located
immediately downstream of the putative hmuSTUV operon
in the L. biflexa circular plasmid p74 (Fig. 2). In Yersinia
pestis, the gene encoding the hemin receptor HmuR is also
linked to the operon hmuSTUV. This genetic organization
suggests that L. biflexa pLEPBI0018 encodes the hemin re-
ceptor. However, hemin uptake was not affected in the
pLEPBI0018 mutant (Table 2). In addition, the alignment of
putative leptospiral TonB-dependent receptor sequences
(Fig. 1) reveals that a His residue, thought to be essential for
heme binding in the HemR receptor from Y. enterocolitica
(14), was not found in pLEPBI0018 and was only in the
protein encoded by LA3149 (Fig. 1).
The ABC transporter proteins of the L. biflexa hmu locus
exhibit similarities to Y. pestis HmuS, HmuT, HmuU, and
HmuV proteins (Table 1). It was previously proposed that
HmuS was a heme oxygenase (55), hence the designation of
heme-degrading protein. However, the inactivation of either Y.
pestis hmuS or Shigella dysenteriae shuS showed that these
mutants were still able to use hemin and all hemoproteins as an
iron source (57, 66). In addition, no heme oxygenase activity
was detected for the HmuS homolog protein, ShuS, of S. dys-
enteriae (63). Similarly, an L. biflexa mutant of the hmuS-like
gene grew like the wild-type strain when hemin was provided as
the sole source of iron (Table 2). It has to be noted that the L.
biflexa genome also contains another putative gene, LEP
BIa0669, encoding a protein with 62% and 55% similarity to
the heme oxygenase from Synechocystis sp. strain PCC6803 and
human, respectively. Another hypothesis is that HmuS is in-
volved in heme storage and/or the oxidative stress. Two types
of iron storage proteins are also found in the genomes of
Leptospira spp.: the heme-containing bacterioferritin and the
Dps protein (Table 1). In E. coli, Dps does not have a strict
function in iron storage, and it also protects DNA against the
combined action of ferrous iron and hydrogen peroxide in the
production of a hydroxy radical. In Y. pestis, HmuT is proposed
to be a periplasmic binding protein which specifically binds
VOL. 188, 2006IRON TRANSPORT AND REGULATION IN LEPTOSPIRA SPP. 7899
heme and acts as a receptor for the active uptake of heme into
the cytoplasm. The HmuU and HmuV proteins have been
proposed to comprise the cytoplasmic membrane permease
and ATPase, respectively. Transcriptional analysis by RT-PCR
with a set of primers for the genes pLEPBI0012, pLEPBI0013,
pLEPBI0014, and pLEPBI0015 revealed that these genes form
an operon (data not shown). Inactivation of the putative hmuV
(pLEPBI0013) by allelic exchange had no effect on hemin
utilization (Table 2). This hemin uptake system was also found
not to be essential for hemin utilization in Vibrio cholerae and
Bradyrhizobium japonicum (40, 41). This might be explained by
the presence of alternative mechanisms or low-affinity systems
for transporting hemin across the cytoplasmic membrane.
Secretion of putative hemolysins. Extracellular bacteria such
as L. interrogans could release heme and hemoglobin from host
red blood cells by the secretion of hemolysins, constituting a
mechanism by which bacteria can gain access to ready sources
of iron in the host. Once released, hemin may be rapidly bound
to host proteins but may also be directly transported by bac-
teria or by binding of hemin or hemin complexes to TonB-
dependent receptors (62). The genomes of Leptospira spp.
contain several genes encoding putative hemolysins, even in
the saprophyte L. biflexa (Table 1). There are at least five L.
biflexa and eight L. interrogans genes that encode products that
exhibit similarities to hemolysins (Table 1). In a recent study,
Zhang et al. (68) demonstrated that the recombinant proteins
encoded by the hemolysin genes of L. interrogans have hemo-
lytic activities in E. coli (Table 1). In addition, a putative
hemolysin secretion system similar to the E. coli-hemolysin
(HlyA) secretion system (16) was identified in L. biflexa (LEP
BIa0357-LEPBIa0360), but no orthologous system was found
in L. interrogans (Table 1). The putative L. biflexa hemolysin
secretion system comprises HlyB-, HlyD-, and TolC-related
proteins. Feasibly, L. interrogans could use an alternative
TolC-based secretion system, given the presence of a gene
encoding a TolC-related protein (LA3927/LIC13135). Other
genes encoding putative proteases may also be involved in
degrading heme-containing compounds like in Porphyromo-
nas gingivalis (54).
Fur and other regulatory proteins. Bacteria typically regu-
late their metabolism in response to iron availability. In E. coli,
most of the genes involved in iron acquisition are transcrip-
tionally regulated by the ferric uptake regulator protein Fur
(29). The expression of seven L. biflexa genes of interest, in-
cluding four fur-related genes, the ferrous iron transporter
gene (feoB), and two TonB-dependent receptors involved in
iron transport (fecA and tbr3), were analyzed by quantitative
reverse transcription-PCR (qRT-PCR). Our results were sup-
ported by the semiquantitative RT-PCR method (data not
shown). No significant change in the relative expression of fur1
(LEPBIa2461) was determined in response to iron availability.
Iron depletion led to a threefold increase in transcript levels of
tbr3 (LEPBIa2760) encoding the receptor for desferrioxamine,
whereas the other genes (fecA, feoB, fur2, fur3, and fur4)
showed more than a 10-fold decrease in expression (Fig. 3).
Interestingly, fecA and tbr3 have different transcription pro-
files. Under iron limitation, the expression of tbr3 is induced to
utilize siderophores, which are high-affinity iron-chelating mol-
ecules. Conversely, the expression of fecA, which encodes a
receptor for a relatively large number of iron sources (i.e., iron
citrate, iron sulfate, iron chloride, and aerobactin), is more
important in the presence of higher iron concentrations (Fig.
3). The expression of these genes involved in iron uptake is
therefore sensitive to iron level, but expression is likely to be
regulated by a complex regulatory mechanism in which Fur
may not be the exclusive regulatory protein.
In the genomes of both L. biflexa and L. interrogans, four
fur-like genes were identified (Table 1). On chelation of iron
from the growth medium, the level of transcripts from the L.
biflexa fur-like genes LEPBIa2152 (fur4), LEPBIa2330 (fur2),
and LEPBIa2849 (fur3) decreased at least 10-fold, while the
expression of LEPBIa2461 (fur1) was independent of iron con-
centration (Fig. 3). Among the Fur family of proteins, there
are three types of proteins (Fur, Zur, and PerR) sharing high
sequence identity. In E. coli, Fur regulates iron uptake and
siderophore biosynthesis, Zur regulates zinc uptake systems,
and PerR regulates oxidative stress response genes (29). We
should find Fur, Zur, and PerR proteins in the genomes of
Leptospira spp. Indeed, zinc and iron are important nutrients
for Leptospira, and high concentrations of these metals are
usually toxic for bacteria. A leptospiral PerR protein may also
play an important role when Leptospira spp. encounter envi-
FIG. 2. Schematic representation of the L. biflexa hmuSTUV locus and the corresponding locus in Y. pestis. Orthologous proteins are shaded
in gray and joined by a line, and the percentage of identity is indicated below.
7900LOUVEL ET AL.J. BACTERIOL.
ronmental oxidative stresses. All Fur family proteins share an
N-terminal DNA-binding domain with a helix-turn-helix motif
and conserved metal-binding sites (45). PerR regulators typi-
cally have two separate CXXC motifs in the C terminus (29).
Such motifs were found in the leptospiral LB183, LEPBIa2152,
and LEPBIa2849 Fur-like proteins. LEPBIa2849 also presents
significant similarities with an oxidative stress regulator from
Borrelia burgdorferi (52). In addition to iron-binding sites, E.
coli Fur possesses one zinc-binding site composed of Cys93 and
Cys96, which are also perfectly conserved in Zur proteins (25).
Analysis of the Fur-like proteins from Leptospira spp. indicates
that LEPBIa2461/LA1857 on one hand and LA2887 on the
other hand do not contain this motif and therefore may not
function as Fur or Zur proteins (Fig. 4). Since it is not possible
to distinguish between Zur and Fur regulators on the basis of
sequence, we have attempted to disrupt each gene by allelic
exchange in L. biflexa to determine their biological role. Inac-
tivation of LEPBIa2152 and LEPBIa2461 resulted in a wild-
type phenotype in EMJH liquid medium (Table 2) as well as a
wild-type peroxide sensitivity (data not shown). The lack of
obvious phenotype in the LEPBIa2461 and LEPBIa2152 mu-
tants could be due to functional redundancy with another
member of the Fur family. The fur-like genes LEPBIa2330 and
LEPBIa2849 showed different expression under iron-replete
and -deplete conditions, and in the absence of allelic exchange
mutants they appeared essential, as is the case in some other
fur genes (20).
Fur-binding sites, known as the Fur boxes, were originally
identified as a 19-bp inverted repeat sequence in the promoter
region of iron-regulated genes. A 150-bp region preceding the
start codon of putative iron-regulated genes from L. biflexa and
L. interrogans such as fecA, feoAB, and fur-like genes were
analyzed for the presence of putative Fur-binding sites (5).
Although these genes are likely members of the Fur regulon,
we were not able to identify a leptospiral Fur box. The DtxR
protein family is another family of iron regulators that were
first found in gram-positive organisms with a high-GC content
and also in the spirochete Treponema pallidum (30). No DtxR-
like proteins were detected in the genomes of Leptospira spp.
Despite the large number of putative extracytoplasmic func-
tion sigma factors in both L. biflexa and L. interrogans, no
FecI-related proteins, which are referred to as iron starvation
sigma factors (15, 61), were identified. This suggests the ab-
sence of this type of signal transduction cascade in Leptospira.
A high concentration of extracellular iron, which can be toxic
for bacteria, can be detected by two-component systems (64).
At least 47 potential response regulator genes were identified
in the L. interrogans genome (2). This indicates that Leptospira
spp. have developed a vast array of detection systems that
enable them to respond to environmental signals, one of which
could be iron.
Comparative genomics between saprophytes and pathogens.
The possession of specialized iron transport systems for the
saprophyte L. biflexa and the pathogen L. interrogans may
reflect the various iron sources they may encounter in their
diverse habitats. A more detailed comparative genomic anal-
ysis should provide clues to the lifestyle of Leptospira in the
environment and in the infected host, increasing our under-
standing of the transition from environmental bacterium to
major human and animal pathogen (unpublished data).
FIG. 3. Effects of iron depletion on mRNA levels of feoB (LEPBIa0880), tbr3 (LEPBIa2760), fecA (LEPBIa1883), and fur-like homologs (fur1,
LEPBIa2461; fur2, LEPBIa2330; fur3, LEPBIa2849; and fur4, LEPBIa2152) in L. biflexa. For qRT-PCR, RNA was isolated from L. biflexa cells
grown in EMJH (high-iron conditions) and iron-depleted EMJH media (low-iron conditions). The amount of a specific mRNA transcript in
low-iron conditions is relative to the quantity of that particular mRNA transcript in high-iron conditions. As an endogenous control, the 23S rRNA
was used for normalization of transcript levels (i.e., 23S rRNA ? 1.0).
VOL. 188, 2006 IRON TRANSPORT AND REGULATION IN LEPTOSPIRA SPP.7901
Based on our findings, a model for iron uptake in Leptospira
can reasonably be proposed (Fig. 5). The analysis of the ge-
nome of the pathogen L. interrogans has allowed the identifi-
cation of 12 putative TonB-dependent receptors, while L. bi-
flexa possesses 8 putative TonB-dependent receptors. This
difference suggests that pathogenic species are able to use a
wider range of iron sources than saprophytes. Alternatively,
the pathogens may also present redundancy in their genome
content. As in gram-negative bacteria, periplasmic binding
proteins may shuttle iron-containing complexes from TonB-
FIG. 4. Alignment of Fur-like proteins from L. interrogans and L. biflexa. Similar residues are shaded in black. Metal-binding sites defined by Pohl
et al. (45) are indicated by asterisks. Cysteine residues corresponding to Cys93 and Cys96 of the E. coli Fur were shown to be the zinc-binding site by
Gonzales de Peredo et al. (25). The putative DNA-binding ?-helix is indicated by arrows. LB183, LA2887, LA3094, and LA1857 are L. interrogans Fur
homologs; LEPBIa2152, LEPBIa2330, LEPBIa2461, and LEPBIa2849 are L. biflexa Fur-like proteins; Fur_Ecol indicates E. coli Fur.
FIG. 5. Diagram showing an overview of iron acquisition systems in L. biflexa. Eight putative TonB-dependent receptors were identified in the L.
biflexa genome. The five TonB loci identified could be involved in the formation of the ExbB-ExbD-TonB complex (for simplicity, only one ExbB-
ExbD-TonB system is indicated). By mutagenesis in L. biflexa, we characterized the function of two TonB-dependent receptors (LEPBIa1883 and
LEPBIa2760), the FeoAB system, and the metal-type ABC transporter (LEPBIa1265). The absence of knockout mutants, suggesting that the product
is essential, indicates that pLEPBI0018 or LEPBIa0500 is the TonB-dependent receptor for either vitamin B12or hemin. The HmuSTUV transport
proteins may be involved in the periplasmic transport of hemin. An unidentified system may also exist for the periplasmic transport of siderophores
(indicated in italics). Leptospira spp. could also release heme and hemoglobin from host red blood cells by the secretion of hemolysins. There is no
evidence of siderophore synthesis in Leptospira spp. All the molecules participating in each step of the transport process have not been identified; other
proteins such as reductases and other periplasmic proteins may also be involved. mb, membrane.
7902LOUVEL ET AL.J. BACTERIOL.
dependent receptors to cytoplasmic membrane ABC trans-
porters that in turn deliver them in the cytoplasm. The patho-
gen L. interrogans may obtain iron from heme by secreting
hemolysins to lyse red blood cells and thereby making the iron
available for uptake. Surprisingly, L. biflexa, a nonpathogenic
species, has putative hemin uptake and hemolysin secretion
systems. Notably, the L. biflexa genome does not contain or-
thologs of the L. interrogans sphingomyelinases, which may be
involved in the typical vascular damage seen in acute leptospi-
rosis. Leptospira spp. also possess uptake systems that use sid-
erophores produced by other bacteria or fungi. This ability to
utilize xenosiderophores broadens the available sources of iron
and allows the bacterium to occupy extended ecological niches.
Bacterial iron homeostasis is best understood in E. coli, a
bacterium phylogenetically distant from Leptospira. This study
is a first step towards understanding iron acquisition systems in
Leptospira. This study highlights the importance of mutagene-
sis tools, such as random transposon mutagenesis systems, to
the molecular analysis of Leptospira.
This work was supported by Pasteur Genopole Iˆle-de-France
(Genomic Platform of the Institut Pasteur) and the French Ministry of
Research ACI IMPBio (Genoscope). S.B. was supported by a grant
from the Scientific Research FIL (Parma, Italy).
We thank Dieter Bulach for critical reading of the manuscript.
1. Agranoff, D., I. M. Monahan, J. A. Mangan, P. D. Butcher, and S. Krishna.
1999. Mycobacterium tuberculosis expresses a novel pH-dependent divalent
cation transporter belonging to the Nramp family. J. Exp. Med. 190:717–724.
2. Ashby, M. K. 2004. Survey of the number of two-component response reg-
ulator genes in the complete and annotated genome sequences of pro-
karyotes. FEMS Microbiol. Lett. 231:277–278.
3. Babudieri, B. 1961. Presented at the XI Congresso Societa Italiana di Mi-
crobiologia, Cagliari-Sassari, Italy.
4. Bagos, P. G., T. D. Liakopoulos, I. C. Spyropoulos, and S. J. Hamodrakas.
2004. PRED-TMBB: a web server for predicting the topology of beta-barrel
outer membrane proteins. Nucleic Acids Res. 32:W400–W404.
5. Baichoo, N., and J. D. Helmann. 2002. Recognition of DNA by Fur: a
reinterpretation of the Fur box consensus sequence. J. Bacteriol. 1841:5826–
6. Bateman, A., E. Birney, R. Durbin, S. R. Eddy, K. L. Howe, and E. L. L.
Sonnhammer. 2000. The Pfam protein families database. Nucleic Acids Res.
7. 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.
8. Ba ¨umler, A. J., and K. Hantke. 1992. Ferrioxamine uptake in Yersinia en-
terocolitica: characterization of the receptor protein FoxA. Mol. Microbiol.
9. Bharti, A. R., J. E. Nally, J. N. Ricaldi, M. A. Matthias, M. M. Diaz, M. A.
Lovett, P. N. Levett, R. H. Gilman, M. R. Willig, E. Gotuzzo, J. M. Vinetz, et
al. 2003. Leptospirosis: a zoonotic disease of global importance. Lancet
Infect. Dis. 3:757–771.
10. Bocs, S., S. Cruveiller, D. Vallenet, G. Nuel, and C. Medigue. 2003. AMIGene:
annotation of microbial genes. Nucleic Acids Res. 31:3723–3726.
11. Bouige, P., D. Laurent, L. Piloyan, and E. Dassa. 2002. Phylogenetic and
functional classification of ATP-binding cassette (ABC) systems. Curr. Pro-
tein Pept. Sci. 3:541–559.
12. Bourhy, P., L. Frangeul, E. Couve, P. Glaser, I. Saint Girons, and M.
Picardeau. 2005. Complete nucleotide sequence of the LE1 prophage from
the spirochete Leptospira biflexa and characterization of its replication and
partition functions. J. Bacteriol. 187:3931–3940.
13. 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.
14. Bracken, C. S., M. T. Baer, A. Abdur-Rashid, W. Helms, and I. Stojiljkovic.
1999. Use of heme-protein complexes by the Yersinia enterocolitica HemR
receptor: histidine residues are essential for receptor function. J. Bacteriol.
15. Braun, V., S. Mahren, and M. Ogierman. 2003. Regulation of the FecI-type
ECF sigma factor by transmembrane signalling. Curr. Opin. Microbiol.
16. Braun, V., R. Schonherr, and S. Hobbie. 1993. Enterobacterial hemolysins:
activation, secretion and pore formation. Trends Microbiol. 1:211–216.
17. Cellier, M. F., I. Bergevin, E. Boyer, and E. Richer. 2001. Polyphyletic origins
of bacterial Nramp transporters. Trends Genet. 17:365–370.
18. Cruveiller, S., J. Le Saux, D. Vallenet, A. Lajus, S. Bocs, and C. Medigue.
2005. MICheck: a web tool for fast checking of syntactic annotations of
bacterial genomes. Nucleic Acids Res. 33:W471–W479.
19. Cullen, P. A., S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler. 2002.
Global analysis of outer membrane proteins from Leptospira interrogans
serovar Lai. Infect. Immun. 70:2311–2318.
20. de Luca, N. G., M. Wexler, M. J. Pereira, K. H. Yeoman, and A. W. B.
Johnston. 1998. Is the fur gene of Rhizobium leguminosarum essential?
FEMS Microbiol. Lett. 168:289–295.
21. Dugourd, D., C. Martin, C. R. Rioux, M. Jacques, and J. Harel. 1999.
Characterization of a periplasmic ATP-binding cassette iron import system
of Brachyspira (Serpulina) hyodysenteriae. J. Bacteriol. 181:6948–6957.
22. 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.
23. Ewing, B., L. Hillier, M. C. Wendl, and P. Green. 1998. Base-calling of
automated sequencer traces using phred. I. Accuracy assessment. Genome
24. Faine, S. 1959. Iron as a growth requirement for pathogenic Leptospira.
J. Gen. Microbiol. 20:246–251.
25. Gonzalez de Peredo, A., C. Saint-Pierre, A. Adrait, L. Jacquamet, J. M.
Latour, I. Michaud-Soret, and E. Forest. 1999. Identification of the two
zinc-bound cysteines in the ferric uptake regulation protein from Escherichia
coli: chemical modification and mass spectrometry analysis. Biochemistry
26. Gordon, D., C. Abajian, and P. Green. 1998. Consed: a graphical tool for
sequence finishing. Genome Res. 8:195–202.
27. 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.
28. Haemig, H. A., and R. J. Brooker. 2004. Importance of conserved acidic
residues in MntH, the Nramp homolog of Escherichia coli. J. Membr. Biol.
29. Hantke, K. 2001. Iron and metal regulation in bacteria. Curr. Opin. Micro-
30. Hazlett, K. R., F. Rusnak, D. G. Kehres, S. W. Bearden, C. J. La Vake, M. E.
La Vake, M. E. Maguire, R. D. Perry, and J. D. Radolf. 2003. The Treponema
pallidum tro operon encodes a multiple metal transporter, a zinc-dependent
transcriptional repressor, and a semi-autonomously expressed phosphoglyc-
erate mutase. J. Biol. Chem. 278:20687–20694.
31. Johnson, R. C., and V. G. Harris. 1967. Differentiation of pathogenic and
saprophytic leptospires. J. Bacteriol. 94:27–31.
32. Kammler, M., C. Schon, and K. Hantke. 1993. Characterization of the
ferrous iron uptake system of Escherichia coli. J. Bacteriol. 175:6212–6219.
33. Koster, W. 2005. Cytoplasmic membrane iron permease systems in the bac-
terial cell envelope. Front. Biosci. 10:462–477.
34. Krogh, A., B. Larsson, G. von Heijne, and E. L. Sonnhammer. 2001.
Predicting transmembrane protein topology with a hidden Markov model:
application to complete genomes. J. Mol. Biol. 305:567–580.
35. Louvel, H., I. Saint Girons, and M. Picardeau. 2005. Isolation and character-
ization of FecA- and FeoB-mediated iron acquisition systems of the spirochete
Leptospira biflexa by random insertional mutagenesis. J. Bacteriol. 187:3249–
36. Mey, A. R., and S. M. Payne. 2003. Analysis of residues determining speci-
ficity of Vibrio cholerae TonB1 for its receptors. J. Bacteriol. 185:1195–1207.
37. Muller, G., and K. N. Raymond. 1984. Specificity and mechanism of fer-
rioxamine-mediated iron transport in Streptomyces pilosus. J. Bacteriol. 160:
38. 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.
39. Neilands, J. B. 1995. Siderophores: structure and function of microbial iron
transport compounds. J. Biol. Chem. 270:26273–26276.
40. Nienaber, A., H. Hennecke, and H. M. Fischer. 2001. Discovery of a haem
uptake system in the soil bacterium Bradyrhizobium japonicum. Mol. Micro-
VOL. 188, 2006IRON TRANSPORT AND REGULATION IN LEPTOSPIRA SPP.7903
41. Occhino, D. A., E. E. Wyckoff, D. P. Henderson, T. J. Wrona, and S. M. Download full-text
Payne. 1998. Vibrio cholerae iron transport: haem transport genes are linked
to one of two sets of tonB, exbB, exbD genes. Mol. Microbiol. 29:1493–1507.
42. Paster, B. J., F. E. Dewhirst, W. G. Weisburg, L. A. Tordoff, G. J. Fraser,
R. B. Hespell, T. B. Stanton, L. Zablen, L. Mandelco, and C. R. Woese. 1991.
Phylogenetic analysis of the spirochetes. J. Bacteriol. 173:6101–6109.
43. 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.
44. 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.
45. Pohl, E., J. C. Haller, A. Mijovilovich, W. Meyer-Klaucke, E. Garman, and
M. L. Vasil. 2003. Architecture of a protein central to iron homeostasis:
crystal structure and spectroscopic analysis of the ferric uptake regulator.
Mol. Microbiol. 47:903–915.
46. Posey, E., and F. C. Gherardini. 2000. Lack of a role for iron in the Lyme
disease pathogen. Science 288:1651–1653.
47. Ren, Q., K. H. Kang, and I. T. Paulsen. 2004. TransportDB: a relational
database of cellular membrane transport systems. Nucleic Acids Res. 32:
48. 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.
49. Robey, M., and N. P. Cianciotto. 2002. Legionella pneumophila feoAB pro-
motes ferrous iron uptake and intracellular infection. Infect. Immun. 70:
50. Runyen-Janecky, L. J., S. A. Reeves, E. G. Gonzales, and S. M. Payne. 2003.
Contribution of the Shigella flexneri Sit, Iuc, and Feo iron acquisition systems
to iron acquisition in vitro and in cultured cells. Infect. Immun. 71:1919–
51. 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.
52. Seshu, J., J. A. Boylan, J. A. Hyde, K. L. Swingle, F. C. Gherardini, and J. T.
Skare. 2004. Conservative amino acid change alters the function of BosR,
the redox regulator of Borrelia burgdorferi. Mol. Microbiol. 54:1352–11363.
53. Simpson, W., T. Olczak, and C. A. Genco. 2000. Characterization and ex-
pression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromo-
nas gingivalis. J. Bacteriol. 182:5737–5748.
54. Sroka, A., M. Sztukowska, J. Potempa, J. Travis, and C. A. Genco. 2001.
Degradation of host heme proteins by lysine- and arginine-specific cysteine
proteinases (gingipains) of Porphyromonas gingivalis. J. Bacteriol. 183:5609–
55. Stojiljkovic, I., and K. Hantke. 1994. Transport of haemin across the cyto-
plasmic membrane through a haemin-specific periplasmic binding-protein-
dependent transport system in Yersinia enterocolitica. Mol. Microbiol. 13:
56. 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–
57. Thompson, J. M., H. A. Jones, and R. D. Perry. 1999. Molecular character-
ization of the hemin uptake locus (hmu) from Yersinia pestis and analysis of
hmu mutants for hemin and hemoprotein utilization. Infect. Immun. 67:
58. Ussery, D. W., and P. F. Hallin. 2004. Genome update: annotation quality in
sequenced microbial genomes. Microbiology 150:2015–2017.
59. 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.
60. Velayudhan, J., N. J. Hughes, A. A. McColm, J. Bagshaw, C. L. Clayton, S. C.
Andrews, and D. J. Kelly. 2000. Iron acquisition and virulence in Helicobacter
pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol.
61. Visca, P., L. Leoni, M. J. Wilson, and I. L. Lamont. 2002. Iron transport and
regulation, cell signalling and genomics: lessons from Escherichia coli and
Pseudomonas. Mol. Microbiol. 45:1177–1190.
62. Wandersman, C., and I. Stojiljkovic. 2000. Bacterial heme sources: the role
of heme, hemoprotein receptors and hemophores. Curr. Opin. Microbiol.
63. Wilks, A. 2001. The ShuS protein of Shigella dysenteriae is a heme-seques-
tering protein that also binds DNA. Arch. Biochem. Biophys. 387:137–142.
64. Wo ¨sten, M. M. S. M., and L. Fox. 2000. A signal transduction system that
responds to extracellular iron. Cell 103:113–125.
65. Wright, A. C., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the
pathogenesis of Vibrio vulnificus infections. Infect. Immun. 34:503–507.
66. Wyckoff, E. E., D. Duncan, A. G. Torres, M. Mills, K. Maase, and S. M.
Payne. 1998. Structure of the Shigella dysenteriae haem transport locus and its
phylogenetic distribution in enteric bacteria. Mol. Microbiol. 28:1139–1152.
67. Yue, W. W., S. Grizot, and S. K. Buchanan. 2003. Structural evidence for
iron-free citrate and ferric citrate binding to the TonB-dependent outer
membrane transporter FecA. J. Mol. Biol. 332:353–368.
68. Zhang, Y. X., Y. Geng, B. Bi, J. Y. He, C. F. Wu, X. K. Guo, and G. P. Zhao.
2005. Identification and classification of all potential hemolysin encoding
genes and their products from Leptospira interrogans serogroup Icterohaem-
orrhagiae serovar Lai. Acta Pharmacol. Sin. 26:453–461.
7904 LOUVEL ET AL. J. BACTERIOL.