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Mapping of genotype-phenotype diversity among clinical isolates of mycobacterium tuberculosis by sequence-based transcriptional profiling.

Rose G, Cortes T, Comas I, Coscolla M, Gagneux S, Young DB - Genome Biol Evol (2013)

Bottom Line: First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression.Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences.Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.

ABSTRACT
Genome sequencing has identified an extensive repertoire of single nucleotide polymorphisms among clinical isolates of Mycobacterium tuberculosis, but the extent to which these differences influence phenotypic properties of the bacteria remains to be elucidated. To determine whether these polymorphisms give rise to phenotypic diversity, we have integrated genome data sets with RNA sequencing to assess their impact on the comparative transcriptome profiles of strains belonging to M. tuberculosis Lineages 1 and 2. We observed clear correlations between genotype and transcriptional phenotype. These arose by three mechanisms. First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression. Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences. We show that in some cases this mechanism is expected to generate functionally active truncated proteins involved in innate immune recognition. Finally, genes showing lineage-specific patterns of differential expression not linked directly to primary mutations were characterized by a striking overrepresentation of toxin-antitoxin pairs. Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

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DosR regulon and SNP-associated TSS. (A) Heatmap of regulon (comprising of 48 genes and 1 sRNA) in the six strains. Unsupervised hierarchical clustering of strains by normalized gene expression of the regulon separates Lineage 2 Beijing subgroup strains (N0145 and N0052). Scale bar indicates fold change, from 10-fold downregulation (blue) to 10-fold upregulation (red). Genes not expressed are colored black. (B) Exponential phase mapped RNA-seq reads in Lineage 2 strains for dosR. Plot shows reads mapping to the forward (blue) and reverse (red) DNA strand. Plots are shown at an identical scale with scale bar indicating maximum read depth included in the bottom panel (this convention is kept for all plots). The C to T SNP in Beijing strains is indicated with an asterisk (*) and the new TSS 7 nucleotides from the created -10 box highlighted. (C) RNA-seq TSS mapping for strain N0145 (Lineage 2) and N0153 (Lineage 1) grown at exponential and stationary phases; TSS shown with arrows. The Beijing-specific TSS within Rv3134c is expressed in both phases.
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evt138-F2: DosR regulon and SNP-associated TSS. (A) Heatmap of regulon (comprising of 48 genes and 1 sRNA) in the six strains. Unsupervised hierarchical clustering of strains by normalized gene expression of the regulon separates Lineage 2 Beijing subgroup strains (N0145 and N0052). Scale bar indicates fold change, from 10-fold downregulation (blue) to 10-fold upregulation (red). Genes not expressed are colored black. (B) Exponential phase mapped RNA-seq reads in Lineage 2 strains for dosR. Plot shows reads mapping to the forward (blue) and reverse (red) DNA strand. Plots are shown at an identical scale with scale bar indicating maximum read depth included in the bottom panel (this convention is kept for all plots). The C to T SNP in Beijing strains is indicated with an asterisk (*) and the new TSS 7 nucleotides from the created -10 box highlighted. (C) RNA-seq TSS mapping for strain N0145 (Lineage 2) and N0153 (Lineage 1) grown at exponential and stationary phases; TSS shown with arrows. The Beijing-specific TSS within Rv3134c is expressed in both phases.

Mentions: It has previously been reported that genes belonging to the DosR regulon are overexpressed during exponential growth in strains belonging to the Beijing family (Reed et al. 2007; Homolka et al. 2010). Among these genes, only Rv1733c met statistical criteria for upregulation in Lineage 2 in the lineage comparison (supplementary table S6A, Supplementary Material online), but an enhanced DosR response was clearly seen in the individual strains N0145 and N0052 (fig. 2A). The outlying strain, N0031, belongs to a basal branch of Lineage 2 that diversified prior to expansion of the major Beijing branches represented by N0145 and N0052 (fig. 1A). A 350 kb genomic duplication that includes the DosR operon has been suggested to contribute to increased constitutive expression of the regulon in Beijing strains (Domenech et al. 2010). This duplication is present in N0145 and N0031, but absent from N0052, and therefore cannot account for the observed differential pattern of DosR expression in our study (supplementary fig. S4, Supplementary Material online). A previously identified indel in dosT linked to the Beijing strains was also not found to be responsible for the observed DosR regulon phenotype (Fallow et al. 2010).Fig. 2.—


Mapping of genotype-phenotype diversity among clinical isolates of mycobacterium tuberculosis by sequence-based transcriptional profiling.

Rose G, Cortes T, Comas I, Coscolla M, Gagneux S, Young DB - Genome Biol Evol (2013)

DosR regulon and SNP-associated TSS. (A) Heatmap of regulon (comprising of 48 genes and 1 sRNA) in the six strains. Unsupervised hierarchical clustering of strains by normalized gene expression of the regulon separates Lineage 2 Beijing subgroup strains (N0145 and N0052). Scale bar indicates fold change, from 10-fold downregulation (blue) to 10-fold upregulation (red). Genes not expressed are colored black. (B) Exponential phase mapped RNA-seq reads in Lineage 2 strains for dosR. Plot shows reads mapping to the forward (blue) and reverse (red) DNA strand. Plots are shown at an identical scale with scale bar indicating maximum read depth included in the bottom panel (this convention is kept for all plots). The C to T SNP in Beijing strains is indicated with an asterisk (*) and the new TSS 7 nucleotides from the created -10 box highlighted. (C) RNA-seq TSS mapping for strain N0145 (Lineage 2) and N0153 (Lineage 1) grown at exponential and stationary phases; TSS shown with arrows. The Beijing-specific TSS within Rv3134c is expressed in both phases.
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Related In: Results  -  Collection

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evt138-F2: DosR regulon and SNP-associated TSS. (A) Heatmap of regulon (comprising of 48 genes and 1 sRNA) in the six strains. Unsupervised hierarchical clustering of strains by normalized gene expression of the regulon separates Lineage 2 Beijing subgroup strains (N0145 and N0052). Scale bar indicates fold change, from 10-fold downregulation (blue) to 10-fold upregulation (red). Genes not expressed are colored black. (B) Exponential phase mapped RNA-seq reads in Lineage 2 strains for dosR. Plot shows reads mapping to the forward (blue) and reverse (red) DNA strand. Plots are shown at an identical scale with scale bar indicating maximum read depth included in the bottom panel (this convention is kept for all plots). The C to T SNP in Beijing strains is indicated with an asterisk (*) and the new TSS 7 nucleotides from the created -10 box highlighted. (C) RNA-seq TSS mapping for strain N0145 (Lineage 2) and N0153 (Lineage 1) grown at exponential and stationary phases; TSS shown with arrows. The Beijing-specific TSS within Rv3134c is expressed in both phases.
Mentions: It has previously been reported that genes belonging to the DosR regulon are overexpressed during exponential growth in strains belonging to the Beijing family (Reed et al. 2007; Homolka et al. 2010). Among these genes, only Rv1733c met statistical criteria for upregulation in Lineage 2 in the lineage comparison (supplementary table S6A, Supplementary Material online), but an enhanced DosR response was clearly seen in the individual strains N0145 and N0052 (fig. 2A). The outlying strain, N0031, belongs to a basal branch of Lineage 2 that diversified prior to expansion of the major Beijing branches represented by N0145 and N0052 (fig. 1A). A 350 kb genomic duplication that includes the DosR operon has been suggested to contribute to increased constitutive expression of the regulon in Beijing strains (Domenech et al. 2010). This duplication is present in N0145 and N0031, but absent from N0052, and therefore cannot account for the observed differential pattern of DosR expression in our study (supplementary fig. S4, Supplementary Material online). A previously identified indel in dosT linked to the Beijing strains was also not found to be responsible for the observed DosR regulon phenotype (Fallow et al. 2010).Fig. 2.—

Bottom Line: First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression.Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences.Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.

ABSTRACT
Genome sequencing has identified an extensive repertoire of single nucleotide polymorphisms among clinical isolates of Mycobacterium tuberculosis, but the extent to which these differences influence phenotypic properties of the bacteria remains to be elucidated. To determine whether these polymorphisms give rise to phenotypic diversity, we have integrated genome data sets with RNA sequencing to assess their impact on the comparative transcriptome profiles of strains belonging to M. tuberculosis Lineages 1 and 2. We observed clear correlations between genotype and transcriptional phenotype. These arose by three mechanisms. First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression. Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences. We show that in some cases this mechanism is expected to generate functionally active truncated proteins involved in innate immune recognition. Finally, genes showing lineage-specific patterns of differential expression not linked directly to primary mutations were characterized by a striking overrepresentation of toxin-antitoxin pairs. Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

Show MeSH
Related in: MedlinePlus