Random transposon mutagenesis in pathogenic Leptospira spp.
The genetics of the pathogenic Leptospira
spp. is in its infancy. Transposon mutagenesis is a powerful, broadly applicable 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 saprophyte L. biflexa
) for use with pathogenic Leptospira
spp. Initially, the plasmid vector pSC189 (4
), containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, was used to deliver Himar1
into the L. interrogans
). The only origin of replication present in the plasmid construct was that from the Escherichia coli
plasmid vectors, which is nonfunctional 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 promoter increased the yield of transformants in L. interrogans
). 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 replicative plasmid vector available for pathogenic Leptospira
, reintroduction of an intact copy of disrupted genes can be achieved via a transposon with alternative selection (26
) or by homologous 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 approximately 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 μg of DNA did not significantly improve the yield of transformants, although there was no reduction of transformation efficiency observed when using up to 50 μg of DNA. The inserted transposons remained stable after 100 generations 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 pathogenic and saprophytic species (12
). To identify a strain with improved transformation efficiency, we examined the transformability of laboratory and clinical isolates of pathogenic Leptospira
spp., including pathogenic strains from L. noguchii
and L. weilii
(Table ), 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 10−7
to 9 × 10−6
; some strains were completely resistant to transformation (Table ). The plating efficiency (the ratio of number of CFU to number of bacteria enumerated in a Petroff-Hauser counting chamber) of pathogenic 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 differences in the transformation efficiency between high and low in vitro-passaged variants of the same strain.
The poor transformability of leptospires may reflect the involvement 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 produced from a dam dcm
double mutant of E. coli
. In addition, treatment of plasmid DNA with crude protein extracts from Leptospira
) 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 individual strains could be due to variations in leptospiral cell surface properties, as previously suggested for the poorly transformable mycobacteria and Borrelia
). 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 uncharacterized 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 μg 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 transposon. 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 sequenced 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
serovar Lai strain 56601 chromosomes (Fig. ).
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 (more ...)
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 transcription 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. Although the profile indicates a random distribution throughout 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. ). For the LPS locus (position, kilobases 1570 to 1688 of the large chromosome), 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 proteins.
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. ). The proportion of Himar1
insertions in coding sequences was 78% (721/929), a frequency that closely approximates the proportion 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 exactly the same genomic location, further suggesting that Himar1
inserts randomly into chromosomal DNA. Surprisingly, 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 Leptospira
spp., rRNA genes are not linked, and L. interrogans
contains 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 transcribed regions that favors transposon integration remains to be determined. Excluding insertions in 16S and 23S rRNA genes and transposases, 551 individual genes have been interrupted in L. interrogans
. Of these, 266 (48%) encode hypothetical 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 pathogenic strains (Table ) (see the table in the supplemental material).
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 (more ...)
These observations, together with the high A+T content of the L. interrogans
genome, suggest that the mariner
transposition system is suitable for the generation of libraries of random mutants. The L. interrogans
genome contains approximately 3,400 predicted protein coding regions (excluding transposases and pseudogenes), of which half have been assigned no biological 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 reasonable 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 flagella, essential for motility, that are inserted at each end of the cell and extend toward the middle of the cell body. Approximately 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 paralogs), 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 colonies of the uvrB
mutant at the lowest UV dose tested, compared 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 involved in heme acquisition (LB191, encoding a TonB-dependent transporter) and utilization (LB186, encoding a heme oxygenase) (1
). The iron chelator dipyridyl was used to produce iron-limited conditions that inhibited the growth of Leptospira
). Addition of 10 μM 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
), 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 rRNAs.
To establish a system for the identification of virulence-associated genes, 29 mutants were selected for virulence testing using the hamster model of acute infection (Table ). 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 stability.
Virulence of mutants in hamster model of acute infection
The majority of mutants retained full virulence (Table ), indicating that the mutagenesis process and the necessary associated in vitro passage do not per se lead to attenuation. Two mutants, with mutations in LA1641 and LA0615, were identified 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 function 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 (unpublished data) and was selected for the virulence assay because mutations affecting LPS can lead to attenuation in other bacterial pathogens (10
). LA0615 is located downstream of the gene encoding LipL41 and was selected for the virulence assay because the gene is unique to pathogenic species of Leptospira
. The system outlined here demonstrates the feasibility of using random transposon mutagenesis in conjunction 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 observation 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 important 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 chemotaxis 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 mutations in lenB
(with six paralogs in the genome), which encode proteins binding host extracellular matrix components in vitro (28
), did not show an attenuated phenotype (Table ). Redundancy in the genome may make the identification of virulence factors in L. interrogans
more difficult; only one attenuated transposon mutant has been described to date, with a mutation in the gene encoding LA0222, an OmpA family protein (26
). Although the majority of mutants do not demonstrate 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, this work represents a major advance. Clearly, additional work 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 apparently attenuated mutants demonstrates the value of this work in identifying novel virulence mechanisms of L. interrogans. 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 pathology. Further increases in transformation efficiency, through the identification of more transformable strains or the development of new genetic tools, will provide opportunities to generate extensive mutant libraries that may subsequently be used to screen for phenotypes affecting diverse aspects of the physiology of Leptospira.