Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira

Abstract

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.

Figure 1. Consensus tree based on PCR amplification data.

Majority-rule consensus tree elaborated under the parsimony criterion and based on binary data (absence/presence) coded from amplification patterns in the S10-spc-α locus for different Leptospira species. Numbers on nodes are bootstrap support after 100 replicates. Only bootstrap values above or equal to 50% are shown. Species included in the sequence analysis are coded in color. L. biflexa was used as the outgroup. CI = 0.346.

Figure 2. Phylogenetic trees based on Tamura-Nei distances and elaborated using the Neighbor-Joining method.

Distances were calculated from the 300–301 (A), 621–625 (B), 624–650 (C) and G1–G2 (D) sequence fragments within the S10-spc-α locus of pathogenic species of Leptospira. The total evidence was combined and analyzed under identical conditions (E). In addition, data available from 16S rDNA (rrs) sequences were used to obtain an alternative hypothesis for the relationships of diverse Leptospira strains (F). Dotted lines show alternative branching patterns, with bootstrapping values ≥50%, obtained in the consensus majority rule tree obtained by parsimony criterion. Numbers above branches represent the percentage of bootstrapping results (2000 replicates). Trees are drawn to scale as indicated by the bar depicted below each tree; bars represent the estimated distance in units of the number of base substitutions per site. The scale the 16S rRNA-based tree is expanded relative to other loci. L. biflexa was used as the outgroup.

Figure 3. Circular phylogenetic trees based in Tamura-Nei distances and elaborated using Neighbor-Joining method.

Distances were calculated from G1–G2 (A) restricted sequences or the secY sequences (B), and are based on analysis of 131 strains of pathogenic species of Leptospira. Numbers above branches represent the percentage of bootstrapping results (2000 replicates). Only bootstrap values above or equal to 50% are shown. L. biflexa was used as the outgroup. Dots indicate strains with divergent positions compared to those from DNA-DNA reassociation analysis .