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Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.

Dos Vultos T, Mestre O, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Gicquel B - PLoS ONE (2008)

Bottom Line: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study.Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious.This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique mycobactérienne, Institut Pasteur, Paris, France.

ABSTRACT

Background: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task.

Principal findings: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains.

Conclusions/significance: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

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Geographic origin of the haplotypes identified.This phylogenetic network constructed using the median-joining algorithm with a final set of 252 SNPs characterized in 92 clinical strains of the Mycobacterium tuberculosis complex (MTC). Deletions were excluded from the analysis. Geographical origin is classified with a color code. Node sizes indicate the number of strains belonging to the same haplotype.
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pone-0001538-g003: Geographic origin of the haplotypes identified.This phylogenetic network constructed using the median-joining algorithm with a final set of 252 SNPs characterized in 92 clinical strains of the Mycobacterium tuberculosis complex (MTC). Deletions were excluded from the analysis. Geographical origin is classified with a color code. Node sizes indicate the number of strains belonging to the same haplotype.

Mentions: A comprehensive set of 56 genes encoding 3R system components was analyzed by sequencing (Tables 1 and 2) in a spoligotype-based set of 92 clinical strains; 45 of these strains are representative of global MTC diversity [3] and were included to ascertain the global diversity of 3R genes in M. tuberculosis. The other 47 strains were chosen to allow evaluation of the resolution power of 3R-SNP-based variations in strains from very precise geographical locations. One group of strains was from Bangui, CAR, where the predominance of two major families of strains has been described: they were used to determine whether this approach could discriminate between these strains. The second group was from Madagascar, a country where both human and bacterial diversity is high. Analysis of 6.7 Mbp of MTC nucleotide sequence, corresponding to roughly 1.5 times the genome of M. tuberculosis H37Rv, showed an unexpectedly large set of highly polymorphic genes, implicating 3R systems in MTC evolution. We identified 259 polymorphisms, in 52 variable genes from 92 clinical isolates. These polymorphisms comprised 161 non synonymous (ns) SNPs, including three encoding stop codons, 91 synonymous (s) SNPs, and 7 deletions (Tables 1, 3, 4, 5 and 6, see Supplementary Information Text S1, Table S1). As previously reported, nsSNPs were much more abundant than sSNPs (Figure 1, Table 1) [7]. SOS repair, Holliday junction-resolving genes and NER were the classes of genes that showed nucleotide diversity lower, although similar, than that of the housekeeping genes. This surely reflects the importance of these genes for mycobacteria. It seems logical that an obligate intracellular pathogen such as M. tuberculosis maintains a stable SOS repair machinery and consequently stable Holliday junction-resolving genes, which are induced as part of the SOS response. NER stabillity might indicate the importance of UV radiation resistance for M.tuberculosis. Nevertheless, these results reveal a wealth of polymorphisms, very different from the restricted allelic variation generally observed for M. tuberculosis housekeeping genes (Tables 3, 4 and 5). We identified 74 haplotypes, with a nucleotide diversity per site of 0.00024, approximately twice that reported for the control group of housekeeping genes (Mann-Whitney p< = 0.01082). Due to technical limitations, the analysis of housekeeping genes was restricted to a control group of strains whose genomic sequences were available online (see Materials and Methods). In the control group, 3R and housekeeping genes showed a nucleotide diversity of 0,00042 and 0,00012, respectively, which represents a 3.5-fold difference. In total, 115 informative sites marking the evolutionary history of M. tuberculosis and 137 non-informative sites specific to single strains were identified. No recombination events were detected. Figures 2 and 3 show the phylogenetic networks constructed using the data obtained [15]. Polymorphic site parsimony was perfectly correlated with spoligotype signature and could therefore be used to trace the evolutionary history of MTC (Figure 4). Principal genetic group 1 (PGG1) strains, such as the W-Beijing, CAS, EAI and M. bovis families of strains, appear to be only very distantly related to the strains of PGG2 and PGG3, providing a strong argument for the use of ecotype categorization for MTC members rather than the traditional subspecies classification. Our results suggest a high degree of functional redundancy among 3R genes. The occurrence of an ns variation in one gene in a particular 3R system is generally accompanied by neutral mutations or wild-type copies of other genes from the same system. This may reflect the existence of an equilibrium, demonstrating the importance of these 3R systems for the genomic integrity in mycobacteria and the significance that even individual nsSNPs may have. This is demonstrated by the analysis of average nucleotide diversity per classes of genes (Figure 1), where only the groups involved in direct repair and alkylation damage, ligases and AP endonucleases show a bigger than 2-fold average non-synonymous diversity in comparison with synonymous diversity.


Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.

Dos Vultos T, Mestre O, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Gicquel B - PLoS ONE (2008)

Geographic origin of the haplotypes identified.This phylogenetic network constructed using the median-joining algorithm with a final set of 252 SNPs characterized in 92 clinical strains of the Mycobacterium tuberculosis complex (MTC). Deletions were excluded from the analysis. Geographical origin is classified with a color code. Node sizes indicate the number of strains belonging to the same haplotype.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211405&req=5

pone-0001538-g003: Geographic origin of the haplotypes identified.This phylogenetic network constructed using the median-joining algorithm with a final set of 252 SNPs characterized in 92 clinical strains of the Mycobacterium tuberculosis complex (MTC). Deletions were excluded from the analysis. Geographical origin is classified with a color code. Node sizes indicate the number of strains belonging to the same haplotype.
Mentions: A comprehensive set of 56 genes encoding 3R system components was analyzed by sequencing (Tables 1 and 2) in a spoligotype-based set of 92 clinical strains; 45 of these strains are representative of global MTC diversity [3] and were included to ascertain the global diversity of 3R genes in M. tuberculosis. The other 47 strains were chosen to allow evaluation of the resolution power of 3R-SNP-based variations in strains from very precise geographical locations. One group of strains was from Bangui, CAR, where the predominance of two major families of strains has been described: they were used to determine whether this approach could discriminate between these strains. The second group was from Madagascar, a country where both human and bacterial diversity is high. Analysis of 6.7 Mbp of MTC nucleotide sequence, corresponding to roughly 1.5 times the genome of M. tuberculosis H37Rv, showed an unexpectedly large set of highly polymorphic genes, implicating 3R systems in MTC evolution. We identified 259 polymorphisms, in 52 variable genes from 92 clinical isolates. These polymorphisms comprised 161 non synonymous (ns) SNPs, including three encoding stop codons, 91 synonymous (s) SNPs, and 7 deletions (Tables 1, 3, 4, 5 and 6, see Supplementary Information Text S1, Table S1). As previously reported, nsSNPs were much more abundant than sSNPs (Figure 1, Table 1) [7]. SOS repair, Holliday junction-resolving genes and NER were the classes of genes that showed nucleotide diversity lower, although similar, than that of the housekeeping genes. This surely reflects the importance of these genes for mycobacteria. It seems logical that an obligate intracellular pathogen such as M. tuberculosis maintains a stable SOS repair machinery and consequently stable Holliday junction-resolving genes, which are induced as part of the SOS response. NER stabillity might indicate the importance of UV radiation resistance for M.tuberculosis. Nevertheless, these results reveal a wealth of polymorphisms, very different from the restricted allelic variation generally observed for M. tuberculosis housekeeping genes (Tables 3, 4 and 5). We identified 74 haplotypes, with a nucleotide diversity per site of 0.00024, approximately twice that reported for the control group of housekeeping genes (Mann-Whitney p< = 0.01082). Due to technical limitations, the analysis of housekeeping genes was restricted to a control group of strains whose genomic sequences were available online (see Materials and Methods). In the control group, 3R and housekeeping genes showed a nucleotide diversity of 0,00042 and 0,00012, respectively, which represents a 3.5-fold difference. In total, 115 informative sites marking the evolutionary history of M. tuberculosis and 137 non-informative sites specific to single strains were identified. No recombination events were detected. Figures 2 and 3 show the phylogenetic networks constructed using the data obtained [15]. Polymorphic site parsimony was perfectly correlated with spoligotype signature and could therefore be used to trace the evolutionary history of MTC (Figure 4). Principal genetic group 1 (PGG1) strains, such as the W-Beijing, CAS, EAI and M. bovis families of strains, appear to be only very distantly related to the strains of PGG2 and PGG3, providing a strong argument for the use of ecotype categorization for MTC members rather than the traditional subspecies classification. Our results suggest a high degree of functional redundancy among 3R genes. The occurrence of an ns variation in one gene in a particular 3R system is generally accompanied by neutral mutations or wild-type copies of other genes from the same system. This may reflect the existence of an equilibrium, demonstrating the importance of these 3R systems for the genomic integrity in mycobacteria and the significance that even individual nsSNPs may have. This is demonstrated by the analysis of average nucleotide diversity per classes of genes (Figure 1), where only the groups involved in direct repair and alkylation damage, ligases and AP endonucleases show a bigger than 2-fold average non-synonymous diversity in comparison with synonymous diversity.

Bottom Line: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study.Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious.This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique mycobactérienne, Institut Pasteur, Paris, France.

ABSTRACT

Background: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task.

Principal findings: We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains.

Conclusions/significance: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

Show MeSH
Related in: MedlinePlus