A genomic island of the pathogen Leptospira interrogans serovar Lai can excise from its chromosome

Abstract

An examination of the two Leptospira interrogans genomes sequenced so far reveals few genetic differences, including an extra DNA region, 54 kb in length, in L. interrogans serovar Lai. This locus contains 103 predicted coding sequences that are absent from the genome of L. interrogans serovar Copenhageni, of which only 20% had significant BLASTP hits in GenBank. By analyzing the L. interrogans serovar Lai genome by pulsed-field gel electrophoresis, we also found that this 54-kb DNA fragment exists as a circular plasmid. This was confirmed by amplification of a DNA fragment corresponding to that of the predicted fragment if this region excised from the chromosome and its left and right ends joined together. In addition, cloning of the putative rep gene of this DNA region was responsible for autonomous replication in Leptospira spp., therefore generating a new Escherichia coli-Leptospira sp. shuttle vector. Taken together, our results show that this genomic island can excise from the chromosome and form a replicative plasmid. Analysis of the distribution of this genomic island revealed that highly related sequences exist in other L. interrogans virulent strains. This genomic island, containing a high proportion of novel genes, may have an important role in spreading genes, including virulence factors, among bacterial populations.

FIG. 1.

Genomic island of L. interrogans serovar Lai in comparison with the corresponding region in L. interrogans serovar Copenhageni. (A) A chromosomal segment, extending between bp positions 1755000 and 1825000, is represented on this graphical map of the MaGe interface (24). Annotated CDSs are represented in the six reading frames of the sequence by red rectangles, and coding prediction curves are superimposed on the predicted CDSs (blue curves). The synteny maps, calculated on a set of selected genomes, are displayed below. A line contains the similarity results between L. interrogans serovar Lai and one given genome. A rectangle represents a putative ortholog between one CDS of the compared genome and one CDS of the L. interrogans serovar Lai genome. If, for several colocalized CDSs on the L. interrogans serovar Lai genome, there are several colocalized orthologs in the compared genome, the rectangles are all of the same color; otherwise, the rectangle is white. A group of rectangles of the same color thus indicates synteny between L. interrogans serovar Lai and the compared genome. L. interrogans serovar Copenhageni LIC12047 and LIC12044 proteins are similar to LA1765 (the LA1766 protein is a duplication of a 3′ region of the LA1765 protein) and LA1848 proteins, respectively, in L. interrogans serovar Lai. Homologs of the two IS1500 open reading frames have been found in synteny with Shewanella oneidensis (second line of the synteny map). The third line shows Bacillus halodurans synteny results in which similar integrases are found but not colocalized (white rectangles). (B) A line plot has been obtained comparing synteny results between L. interrogans serovar Lai and L. interrogans serovar Copenhageni. Putative orthologous relations between the two genomes are defined as gene couples satisfying the bidirectional best hit criterion or a BLASTP alignment threshold, a minimum of 35% sequence identity on 80% of the length of the smallest protein. These relations are subsequently used to search for conserved gene clusters, e.g., synteny groups (syntons). All possible kinds of chromosomal rearrangements are allowed (inversion, insertion, and deletion). A gap parameter, representing the maximum number of consecutive genes which are not involved in a synteny group, is set to five genes. Green, synteny groups are organized on the same strand; red, synteny groups are organized on two opposite strands. chr., chromosome.

FIG. 2.

Organization of the genes located in the genomic island locus of L. interrogans serovar Lai. Comparison of the overall organization of the genomic island of L. interrogans serovar Lai (including the published genome sequence and the amplified products that were sequenced) with that of the corresponding region in L. interrogans serovar Copenhageni. Empty boxes indicate the 812- and 170-bp direct repeats at the boundaries of the 54-kb DNA fragment. DNA fragments belonging to LaiGI I are shadowed in gray. CDSs with the same motif are homologs. The locations of primers (Table 1) used for PCR amplification are indicated. The figure is drawn to scale.

FIG. 3.

PCR results showing DNA excision and integration of the 54-kb DNA fragment. PCR amplification for detection of the left end of LaiGI I into the chromosome (P1-P2), the right end of LaiGI I into the chromosome (P3-P4), the circular intermediate with junction of left and right ends of the chromosomal LaiGI I region (P2-P3), and the excision event (P1-P4) in L. interrogans serovar Lai.

FIG. 4.

Detection of the circular intermediate of LaiGI I by PFGE. (A) Whole genomic analysis of L. interrogans serovar Lai DNA by PFGE. ND, undigested DNA; NotI, digestion with NotI. The arrowhead indicates the additional restriction fragment when the NotI macrorestriction profile was compared with predictions made from the whole-genome sequence of L. interrogans serovar Lai. Asterisks indicate DNA fragments reported as absent (22-kb fragment) or possibly different in length (720-kb fragment) by PFGE compared with predictions made from the whole-genome sequence of L. interrogans serovar Lai (20). For Southern blot analysis, the genomic DNA of L. interrogans serovar Lai was blotted to a nylon membrane and then hybridized at 60°C with the radiolabeled probe B (corresponding to the rep gene). The size marker, on the left, is the bacteriophage λ DNA multimer (monomer = 48.5 kb). (B) Schematic representation of the macrorestriction patterns from L. interrogans serovar Lai. For Southern blot analysis, the genomic DNA of L. interrogans serovar Lai was blotted to a nylon membrane and then hybridized at 60°C with radiolabeled probes. Probes A, B, C, and D were generated by PCR of total genomic DNA with primer pairs L9a-L9b, 1809a-1809b, 1839a-1839c, and 1850a-1851b, respectively (Table 1). The probes B and C hybridized to the same fragment (located between the 48- and 97-kb fragments of the molecular size standard), indicating the linkage between these two probes. Similarly, probes A and D hybridized to a single macrorestriction fragment (located between the 679- and 727-kb fragments of the molecular size standard). The 22-kb fragment predicted from the published genome sequence (20) is reported as absent. This suggests that the majority of LaiGI I (black box) is not inserted into the chromosome but as a circular plasmid, which is linearized after NotI restriction. Arrows indicate the NotI restriction sites on the large chromosome of L. interrogans serovar Lai (the sizes of restriction fragments are indicated in kilobases).