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Conceived and designed the experiments: BA CB MP RZ DB PW EK AM CB-E. Performed the experiments: CM ZR NZ SC SB JD MJ AL. Analyzed the data: RC BA JR JD MP RZ DB AM TS. Wrote the paper: BA JR MP RZ DB.
Leptospira biflexa is a free-living saprophytic spirochete present in aquatic environments. We determined the genome sequence of L. biflexa, making it the first saprophytic Leptospira to be sequenced. The L. biflexa genome has 3,590 protein-coding genes distributed across three circular replicons: the major 3,604 chromosome, a smaller 278-kb replicon that also carries essential genes, and a third 74-kb replicon. Comparative sequence analysis provides evidence that L. biflexa is an excellent model for the study of Leptospira evolution; we conclude that 2052 genes (61%) represent a progenitor genome that existed before divergence of pathogenic and saprophytic Leptospira species. Comparisons of the L. biflexa genome with two pathogenic Leptospira species reveal several major findings. Nearly one-third of the L. biflexa genes are absent in pathogenic Leptospira. We suggest that once incorporated into the L. biflexa genome, laterally transferred DNA undergoes minimal rearrangement due to physical restrictions imposed by high gene density and limited presence of transposable elements. In contrast, the genomes of pathogenic Leptospira species undergo frequent rearrangements, often involving recombination between insertion sequences. Identification of genes common to the two pathogenic species, L. borgpetersenii and L. interrogans, but absent in L. biflexa, is consistent with a role for these genes in pathogenesis. Differences in environmental sensing capacities of L. biflexa, L. borgpetersenii, and L. interrogans suggest a model which postulates that loss of signal transduction functions in L. borgpetersenii has impaired its survival outside a mammalian host, whereas L. interrogans has retained environmental sensory functions that facilitate disease transmission through water.
The genus Leptospira contains pathogenic and saprophytic species that differ in their capacity for survival in a vast array of environments that range from soil and water  to the tissues of mammalian hosts during acute and chronic infection . Typically, long term colonization by pathogenic Leptospira of the proximal renal tubules of mammalian maintenance host species provides a persistent source of infection; thus, pathogenic Leptospira is shed in the urine of chronically infected animals, facilitating transmission to naïve hosts . Leptospirosis is of considerable importance to international public health, with more than half a million cases reported annually due largely to environmental exposure to pathogenic Leptospira species, with mortality rates of up to 25% in some outbreaks. In addition, leptospirosis in production animals results in a significant economic burden worldwide .
Recent applications of molecular taxonomy techniques to this genus reveal extensive genetic diversity within Leptospira, with more than 16 pathogenic and saprophytic species recognized , . A significant challenge in the future will be to more precisely correlate these genetic differences with biological differences. The relationship between leptospiral genome content, pathogenesis and the ability to survive in diverse environmental niches is a particularly important area of investigation, highlighted by our recent findings. A process of genome erosion and loss of gene function in L. borgpetersenii serovar Hardjo  limits its viability outside a mammalian host and likely impairs disease transmission through water, a route commonly used by L. interrogans to infect new hosts.
To gain insight into the genetic potential of Leptospira and to help identify genes that contribute to long-term survival in surface water, we determined the genome sequences of two L. biflexa strains, the first saprophytic Leptospira species to be characterized by genome analysis. Comparison of these data to genomes of the pathogenic species L. borgpetersenii , and L. interrogans ,  provides an opportunity to identify features that are unique to pathogenic and saprophytic species, thereby providing new experimental directions and novel perspectives on leptospiral evolution, environmental persistence and the causation of disease.
The genome of Leptospira biflexa serovar Patoc strain Patoc1 (Ames strain) consists of three replicons with a total of 3,956,086 base pairs (bp) (Figure 1). The two larger replicons share extensive similarity to the two chromosomes that comprise the genomes of L. borgpetersenii and L. interrogans and are therefore referred to as chromosome I (CI; 3,603,977 bp; GC% 38.89) and chromosome II (CII; 277,995 bp; GC% 39.27). L. biflexa possesses a third circular replicon (74,114 bp; GC% 37.47), that we designate p74, not found in the previously sequenced pathogenic Leptospira species. (The complete genomic sequences of L. biflexa serovar Patoc strain Patoc1, strains Paris and Ames, have been deposited in GenBank under the Accession Numbers: CP000777, CP000778, CP000779, CP000786, CP000787 and CP000788).
A total of 3,590 protein-coding genes (CDSs) was identified in L. biflexa (Table 1). Most of these genes are located on CI, including two rrf genes, two rrl genes and two rrs genes, coding for 5S, 23S and 16S rRNA molecules respectively. These rRNA genes are not linked to each other, a feature unusual among most bacteria but common among Leptospira. Similar to the slower-growing pathogenic species L. interrogans and L. borgpetersenii, each of which has a relatively low number (37) of transfer RNA (tRNA) genes, the faster-growing L. biflexa surprisingly has only 35 tRNA genes. This finding indicates that the growth rate of Leptospira is not restricted by the low number of tRNA and rRNA genes as previously suggested , but rather may be due to other differing metabolic capacities amongst Leptospira spp. As reported for other Leptospira spp. , essential genes such as gltB (glutamate synthase) and asd (aspartate semialdehyde dehydrogenase) are carried on CII in L. biflexa.
It is unclear if replicon III (p74) functions as a chromosome, i.e. carries genes essential for survival, or is an extrachromosomal element or plasmid. Thirteen genes on p74 have orthologs located on CI in pathogenic Leptospira (Table 2). For example, recBCD are located on CI in L. interrogans and L. borgpetersenii, but are located on p74 in L. biflexa. Mutation of these housekeeping genes in other bacterial species can affect transformation competence and viability , suggesting that p74 is essential for the survival of L. biflexa.
GC skew analysis (Figure 1) suggests that CI, CII and p74 are theta-type replicons that replicate bi-directionally from a unique origin. Each L. biflexa replicon encodes its own partition proteins from origin-proximal genes that may recognize and interact with a replication-specific binding site. The replication origin of CI resembles typical circular chromosomes from other bacteria, i.e. it is adjacent to dnaA, which encodes the initiator protein, and other genes such as dnaN, recF and gyrAB. In contrast, the replication origins of CII, replicon p74 and the previously reported leptophage LE1  resemble phage and plasmid replicons; they contain both a partition operon and a downstream putative rep gene. For p74, the predicted replication origin based on GC skew analysis includes a partition operon (pLEPBI0001/LBF_5000 and pLEPBI0002/LBF_5001) and an adjacent gene, pLEPBI0003/LBF_5003, the product of which shares 63% similarity with the leptophage LE1 Rep protein. Regardless of the similarity between the LE1 Rep protein and the product of pLEPBI003, p74 and the LE1 prophage can co-exist (data not shown). Furthermore, we demonstrated that this region of p74 directs autonomous replication in L. biflexa (Figure S1). This will facilitate the construction of new shuttle vectors for the genetics of Leptospira and their use in co-transformation experiments with the L. biflexa-E. coli shuttle vector derived from LE1 .
We suggest that the CII and p74 replicons evolved from a stabilized circular intermediate of a progenitor phage related to LE1 . Intermediates in this process may be related to the LaiGI-1 Genomic Island , which exists both as an element integrated into CI of L. interrogans serovar Lai or as an autonomously replicating plasmid. Stabilization of these intermediates could have occurred through illegitimate recombination resulting in incorporation of essential genes from CI into the smaller replicons. Evidence for this model is provided by the presence of genes located on the CI replicon in pathogenic Leptospira on either CII or p74 in L. biflexa, as noted above.
The genus Leptospira is renowned for the stability of its agglutinating antigens during in vitro culture, with examples of strains maintaining serovar identity during more than 80 years of propagation , implying considerable genomic stability in the absence of selective pressure for antigenic change. Furthermore, more than 2% of the L. interrogans serovar Copenhageni genome is dedicated to LPS biosynthesis. In L. biflexa about 1.4% of the genome encodes LPS biosynthesis functions with essentially all relevant CDS encoded on the same strand, and these genes are identical in the Ames and Paris strains. In contrast, non-LPS encoding regions in the two L. biflexa strains maintained separately for 17 years show some evidence of minor accumulation of changes. Comparison of the Paris and Ames strains of L. biflexa reveals four indels, three of which are insertions of ISLbi1 elements into the coding regions of genes in the Ames strain (LBF_0259, LBF_2295 and LBF_2512). The first two genes encode proteins of unknown function, and the third encodes a protein with a predicted role in lipid metabolism; the significance of these insertions is not known. In the Paris strain, the insertion element is restricted to one copy each on CII and p74; these copies are maintained in the Ames strain. The fourth difference occurs in CII in which an additional 250 bp are found in an intergenic region in the Ames strain. Aside from these differences, the genomes of the Paris and Ames strains are virtually identical; in this article the term L. biflexa will refer to the Ames strain unless stated otherwise.
Approximately two thirds of the genes in L. biflexa have orthologous genes in the pathogens L. interrogans and L. borgpetersenii (Figure 2), consistent with a common origin for leptospiral saprophytes and pathogens. The genes conserved across the three species are distributed in the two chromosomes of all Leptospira species and strains (Table 3). The high sequence identity of the small chromosome parA gene product (>87%) between all Leptospira spp. suggests a shared ancestry for this replicon. Moreover, there is no difference in inter-chromosomal DNA parameters such as GC%, codon preferences and gene density within the three L. biflexa replicons, despite the relatively wide range of these parameters in pathogenic Leptospira. By contrast, codon preferences of genes of the leptospiral bacteriophage LE1 and its host, L. biflexa  are different (data not shown). These data suggest a long-standing relationship between the three L. biflexa replicons and perhaps a more recent acquisition of LE1.
Phylogenetic analysis based on comparison of 16S rRNA sequences indicates that members of the family Leptospiraceae form the deepest branch in spirochete evolution, with divergence of saprophytic and pathogenic Leptospira likely being the result of a single event . Further diversification of species within each of these two evolutionary branches of Leptospira is supported by multilocus sequence analysis and DNA renaturation kinetics , , .
We used comparative genome analysis to help identify key features showing patterns of variation consistent with the action of selective evolutionary pressure within Leptospira. The L. biflexa and L. borgpetersenii genomes are similar in size (Table 1), but the gene density in L. biflexa is much higher, probably as a result of IS-mediated genome erosion in L. borgpetersenii. In contrast, the L. interrogans genome is larger, probably reflecting the added genetic information required for survival both within mammalian hosts and aquatic environments, whereas L. biflexa and L. borgpetersenii are restricted to aquatic and mammalian host environments, respectively. The dearth of IS-elements in the L. biflexa genome (five IS-elements) is in stark contrast to their abundance in the L. interrogans (36 and 69 IS-elements in the Fiocruz LI-130 and Lai 56601 strains, respectively) and L. borgpetersenii (167 IS elements) genomes. The presence of large numbers of IS elements is an indicator of genome plasticity in Leptospira species. Taken together, our results suggest that the L. biflexa gene order is more likely to have a closer relationship to the progenitor genome for the genus Leptospira. There is low synteny between the sequenced pathogenic Leptospira (Figure 3) , despite the short evolutionary distance separating them.
Gene and functional redundancy is also more common in the pathogens in comparison to the saprophytes (Table 4). The pathogens have more paralogs (excluding transposases), with 203 (5.01%) and 438 (10.52%) paralogs for L. interrogans serovar Copenhageni Fiocruz L1-130 and L. borgpetersenii serovar Hardjo L550, respectively, compared to 62 paralogs (1.65%) in the saprophyte L. biflexa. We identified 43, 67, and 53 loci with gene duplication in the genomes of L. biflexa, L. interrogans, and L. borgpetersenii, respectively. The genome of L. biflexa therefore appears to be more stable than those of L. interrogans and L. borgpetersenii. Moreover, the L. biflexa genome (92%) has a greater gene density than the L. borgpetersenii (80%) and L. interrogans genomes (75%) (Table 1). Greater gene density can contribute to a relatively stable gene order. For example, the spirochete Borrelia has a gene density of more than 92% and the B. garinii and B. burgdorferi genomes are essentially collinear .
The spectrum of ecological niches occupied by diverse Leptospira species raises questions as to how the capacity to survive in these diverse environments has evolved within different species in the genus Leptospira. We propose that their common progenitor had a genome more like L. biflexa. Subsequently, acquisition of genes that enabled Leptospira to infect mammals would have expanded the range of environments that it could successfully occupy. Loss of environmental survival genes then would lead to dependence on a mammalian host and eventually return the genome to a smaller size (i.e. L. borgpetersenii), consistent with our model for IS-mediated genome degradation .
The extensive repertoire of genes (Table 5) encoding proteins involved in signal transduction in L. biflexa (287 CDS) compared with L. interrogans (214 CDS) and L. borgpetersenii (167 CDS) is consistent with an enhanced metabolic capability in L. biflexa reflected by its environmental habitat and likely contributes to its enhanced growth rate relative to the pathogens. An analogous situation is seen in Mycobacterium, where M. marinum which occupies both animal and aquatic environments has many more genes encoding environmental sensing and metabolic proteins than the closely related, obligate human pathogen M. tuberculosis , .
The genomes of Leptospira spp. contain a number of genes involved in the production of exopolysaccharides. These genes, such as those encoding glycosyltransferases, alginate biosynthesis, and lipopolysaccharide transport systems, may contribute to colonization of both biotic and abiotic surfaces. When investigating the formation of biofilms on solid surfaces, we have observed the production of a strong biofilm by L. biflexa and L. interrogans (Unpublished results). The formation of such a biofilm is consistent with the life of saprophytic species in water; it may facilitate L. biflexa occupying particular environmental niches. The presence of biofilms may also play an important role in chronic carriage of the pathogen L. interrogans in animal reservoirs by facilitating colonization of the renal tubules. Interestingly, genes involved in alginate biosynthesis are present in both L. biflexa (11 genes) and L. interrogans (8 genes), but are absent in L. borgpetersenii, a finding consistent with the reduced environmental survival of L. borgpetersenii .
The molecular mechanisms of pathogenesis in leptospirosis remain almost entirely unknown. The set of leptospiral, pathogen-specific genes, defined as those with no orthologous gene in L. biflexa, is likely to be enriched for genes that play a role in pathogenesis. Significantly, the majority (893) of these 1,431 genes has no known function (Table S1abc), suggesting the presence of pathogenic mechanisms unique to Leptospira. Among the remaining 538 genes, there are several genes encoding response regulators and environment sensing proteins; these genes are likely to represent adaptations that enable survival in environments not encountered by the saprophytic strain. There is an expansion of genes encoding leucine–rich repeat (LLR) proteins from one gene in L. biflexa to eight and 18 genes in L. borgpetersenii and L. interrogans, respectively. Although these LLR proteins have no obvious function, in Treponema denticola the LRR protein LrrA appears to have roles in attachment and to, and penetration of, host tissues ; the diversity of LRR proteins in pathogenic Leptospira may be important for succesful infection of a wide variety of mammalian host species.
The regulation of transcription differs significantly between the saprophytic and pathogenic strains as indicated by the presence of more than 50 saprophyte-specific, and more than 20-pathogen specific, transcription regulators, whereas there are only 27 transcription regulators that are common to all of the sequenced Leptospira species.
Because the bacterial surface is an interface between the pathogen and the host, any differences in cell-surface proteins might reflect variation in pathogenesis mechanisms among Leptospira spp., which contain a relatively low repertoire of trans-membrane proteins in their outer membranes . However, the outer membranes contain a predominance of lipoproteins, that may be either surface-exposed or located in the periplasm. Predicted lipoproteins are prominent in both saprophytic (164 predicted lipoproteins) and pathogenic Leptospira (184 and 130 predicted lipoproteins in L. interrogans and L. borgpetersenii respectively). Despite these similarities, there are significant differences, most notably the absence in L. biflexa of an ortholog of the major outer membrane lipoprotein, LipL32 . Moreover, 89 L. biflexa lipoprotein genes have no orthologs in other Leptospira species, and more than 90 lipoproteins from the pathogenic species have no orthologs in the L. biflexa genome. In addition to LipL32, other characterized lipoproteins that have no orthologs in L. biflexa include LipL41, LipL36 and several LipL45 related proteins . However, LipL21, which was reported as a pathogen-specific lipoprotein  based on antibody reactivity, has an ortholog in L. biflexa with 50% similarity. This level of similarity is consistent with a different function for the LipL21 orthologs in the two Leptospira species.
Several putative virulence factors previously identified in pathogenic Leptospira spp. are not present in L. biflexa, including the Lig surface proteins containing immunoglobulin-like repeats predicted to play a role in the adhesion to host tissues . Similarly, LfhA, a putative factor H binding protein  that has also been shown to bind the extracellular matrix protein laminin , is shared among pathogenic Leptospira, but is lacking in L. biflexa. Although the genome of L. biflexa contains putative hemolysins , its genome is devoid of genes encoding enzymes capable of degrading tissues, such as the range of sphingomyelinases found in pathogenic species , , . The role of sphingomyelinases in the pathogenesis of leptospirosis has been controversial; are they key virulence factors or do they merely play a role in nutrient acquisition? Their absence in L. biflexa strongly supports their involvement in survival within mammalian hosts. Interestingly, the membrane protein, Loa22, the only protein to date that has been shown genetically to be required for virulence in L. interrogans , has a L. biflexa ortholog with 73% similarity. Its role in either pathogenic or saprophytic species is unknown, but its presence in the saprophytic species suggests that it is involved in survival rather than being a direct virulence factor and is consistent with the common progenitor hypothesis.
L. interrogans is the most frequently reported agent of human leptospirosis. The disease is also generally more severe with L. interrogans than with L. borgpetersenii . On this basis we propose that the subset of L. interrogans genes that have no orthologs in either L. biflexa or L. borgpetersenii may contain virulence factors that are responsible for the more severe form of leptospirosis. Other subsets that may be enriched for genes involved in particular biological functions include those genes that have orthologs in L. biflexa and L. interrogans and not L. borgpetersenii which may contain genes involved in survival outside the animal host. While the loss of many genes from the L. borgpetersenii genome has occurred through genome reduction , the presence of 265 unique genes in L. borgpetersenii (Figure 2; Table S1abc) indicates that these genomes have also gained some additional genes during the course of their evolution. The lateral acquisition of genetic material is often associated with IS-elements. While there is an association between IS-elements and genes that are isolate- or species-specific, in particular in L. interrogans, no mechanism has been determined. Other possible mechanisms for horizontal gene acquisition may include the involvement of bacteriophage, as integrated prophages or bacteriophage remnants are present.
Saprophytic and pathogenic Leptospira species belong to two distinct phylogenetic groups, leading us to conclude that the 2,052 genes shared by both groups constitute the core genome of this genus (Figure 2). As expected, many of the functional categories that are involved in essential housekeeping functions, such as DNA and RNA metabolism, protein processing and secretion, cell structure, cellular processes, and energetic and intermediary metabolism, are represented in the core gene set.
The presence of orthologous genes in all the sequenced leptospiral species is an indicator that these genes were acquired prior to the radiation of the genus, and as such, is a strong indicator that these genes have not been laterally acquired. The substantial proportion of the total leptospiral genes that are in this category is perhaps an indicator that lateral transfer of genes into the genus Leptospira is a minor contributor to the overall genetic composition of the genus and an indicator that the genus has undergone an extended period of ‘genetic isolation’. This notion is supported by the fact that approximately 20% of the core leptospiral genes are unique to the genus. However, this does not preclude horizontal acquisition of some genes or gene clusters.
Despite the development of basic tools such as transposon mutagenesis for the pathogenic species of Leptospira, targeted gene inactivation is not yet possible. Therefore, there is likely to be continued interest in the use of L. biflexa as a model bacterium for genetic analysis . Knowledge of the distribution of orthologous genes in L. biflexa will be an important resource for the elucidation of function for genes common to pathogenic and saprophytic species.
The strain, L. biflexa serovar Patoc strain Patoc1, was initially isolated from stream water , maintained in the collection of the National Reference Center of Leptospira (Institut Pasteur, Paris, France) and designated the Paris strain. A second strain was derived from the same source but kept in the culture collection at the National Animal Disease Center (NADC), Ames, IA since 1990 and is designated the Ames strain. Each strain was colony purified before growth for genomic DNA isolation. For each isolate, genomic DNA was randomly sheared by nebulization (HydroShear, GeneMachines) and the ends repaired enzymatically. Small fragments (~1.5–4 kb) were ligated either to a derivative of plasmid pGEM7Zf+ (Promega) (Paris strain) or pSMART-HC and pSMART-LC (Lucigen Corp.) (Ames strain). Large (35–45 kb) DNA fragments were ligated to fosmid pCC1FOS (Epicentre, Madison, WI) (Paris strain). Intermediate sized DNA fragments (7–12 kb) were prepared from partially BamHI-digested Ames strain DNA and ligated into pZERO-1 (Invitrogen). Plasmid DNA preparation was performed with the TempliPhi DNA sequencing template amplification kit (GE Healthcare -Bio-Sciences) or SprintPrep plasmid preparation kits (Agencourt Bioscience). Fosmid DNA purification was performed with the Montage BAC Miniprep96 Kit (Millipore) . Sequencing reactions were performed from both ends of DNA templates using ABI PRISM BigDye Terminator cycle sequencing ready reactions kits and run on a 3700 or a 3730 xl Genetic Analyzer (Applied Biosystems) at the Genomics platform (Pasteur Genopole Île-de-France), the Australian Genome Research Facility, Brisbane, Australia or at the Genomics Facility at the NADC. Base-calls from sequence data were made using Phred . Sequences not meeting our production quality criteria (at least 100 bases called with a quality over 20) were discarded. The traces were assembled using Phrap and Consed .
Whole genome shotgun sequencing was performed until approximately 6× genome coverage was achieved. Autofinish  was used to design primers for improving regions of low quality sequence and for primer walking along templates that spanned the gaps between contigs. Several strategies were used to orientate contigs to enable directed PCR-based approaches to span gaps between contigs. These strategies included Blast-based approaches that identified contig ends with hits to the same gene or to genes within the same locus, provided that there was conservation of gene order in both the L. interrogans and the L. borgpertersenii genomes. Repeated sequences longer than 600 bases, such as IS-elements and the rrl and rrn genes, were identified and curated manually to ensure that at least two templates spanned these regions, thus confirming the assembly of these regions. At this stage, single, circularised contigs representing CII and and p74 were identified. Combinational PCR was used to close the gaps between the final contigs making up CI. Outward-directed primers were designed for each of the contig ends; the primer sequences were subsequently checked and confirmed to be unique on the genome. The combinational PCR process required approximately 600 PCR reactions pairing each of the primers. In each instance where a gap was spanned, the size of the PCR product was less than 2 kb. The sequence of these PCR products was determined and added to the assembly to enable the closing of CI. PCR with independent primers was used to confirm the joins determined by the combinational PCR. In addition, for the Paris strain five fosmid clones were completely sequenced by transposon-assisted sequencing (Finnzymes, TGS II kit). Seven fosmid clones were also selected by Southern hybridization and sequenced in order to verify the 23S RNA and 16S RNA regions and 4 repeat regions. Validation of the final assembly was achieved by comparison of in silico digestion patterns with macrorestriction patterns obtained by PFGE with NotI and AscI. For each genome, the error rate was less than 1 error per 10,000 bp.
The complete genome sequence was obtained from 58,663 and 40,260 sequences for the Paris and Ames strains respectively (giving >8× and 7× coverage). For the Paris strain, coding sequences (CDSs) likely to encode proteins were predicted with the AMIGene software . Annotation was performed as described previously  using the MaGe annotation platform . All the data were stored in SpiroScope, a relational database which is publicly available (http://www.genoscope.cns.fr/agc/mage). The Ames strain was annotated as described previously for the L. borgpetersenii serovar Hardjo genome using the Wasabi interactive platform .
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We thank David Alt, Richard Hornsby, A. Magnier and A. Blanchard for technical support.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the following: National Health and Medical Research Council, Australia; Australian Research Council; U.S. Department of Agriculture; Institut Pasteur, Paris, France; French Ministry of Research ANR Jeunes Chercheurs (no. 05-JCJC-0105-01); Consortium National de Recherche en Génomique (no. RNG-20040057); ACI IMPBio 2004 (MicroScope project). Funders had no role in the planning, performance or analysis of the work.