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

Victoria B, Ahmed A, Zuerner RL, Ahmed N, Bulach DM, Quinteiro J, Hartskeerl RA - PLoS ONE (2008)

Bottom Line: In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela, Galicia, Spain.

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.

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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 [2].
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pone-0002752-g003: 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 [2].

Mentions: The 20-mer primers G1 and G2 amplify a 285 bp fragment of secY, and these primers were developed previously as a diagnostic PCR for the detection of Leptospira DNA [5]. A 245 bp fragment flanked by the G1–G2 primers has been shown previously to be a useful tool for discriminating between species [18]–[21]. This study provides an opportunity to broaden the evaluation of the G1–G2 domain by comparing the discriminative value of this domain with the majority of the secY sequence. Sequences for secY were obtained from 131 Leptospira strains (GenBank accession numbers EU357938–EU358070). The phylogenetic tree produced from secY sequence data was compared to a tree derived from the extracted sequences of the 245 bp fragment flanked by primers G1 and G2 (Fig. 3). These two trees are similar, resolve Leptospira species, and discriminate between strains. With few exceptions, all strains clustered with other members of the same species as determined by DNA-DNA hybridization analysis [4]. Because of a limitation presented by the original G2/G2 primer pair, it does not amplify DNA from L. kirschneri; two new primers were designed (SecYII and SecYIV) that flank the G1 and G2 annealing sites. These primers amplify secY sequences from all pathogenic strains (data not shown).


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

Victoria B, Ahmed A, Zuerner RL, Ahmed N, Bulach DM, Quinteiro J, Hartskeerl RA - PLoS ONE (2008)

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 [2].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481283&req=5

pone-0002752-g003: 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 [2].
Mentions: The 20-mer primers G1 and G2 amplify a 285 bp fragment of secY, and these primers were developed previously as a diagnostic PCR for the detection of Leptospira DNA [5]. A 245 bp fragment flanked by the G1–G2 primers has been shown previously to be a useful tool for discriminating between species [18]–[21]. This study provides an opportunity to broaden the evaluation of the G1–G2 domain by comparing the discriminative value of this domain with the majority of the secY sequence. Sequences for secY were obtained from 131 Leptospira strains (GenBank accession numbers EU357938–EU358070). The phylogenetic tree produced from secY sequence data was compared to a tree derived from the extracted sequences of the 245 bp fragment flanked by primers G1 and G2 (Fig. 3). These two trees are similar, resolve Leptospira species, and discriminate between strains. With few exceptions, all strains clustered with other members of the same species as determined by DNA-DNA hybridization analysis [4]. Because of a limitation presented by the original G2/G2 primer pair, it does not amplify DNA from L. kirschneri; two new primers were designed (SecYII and SecYIV) that flank the G1 and G2 annealing sites. These primers amplify secY sequences from all pathogenic strains (data not shown).

Bottom Line: In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela, Galicia, Spain.

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.

Show MeSH
Related in: MedlinePlus