Article Figures & Data
Figures
- FIG. 1.
Southern blotting reveals the presence of two copies of dosR in recombinant W/Beijing strains. Genomic DNA from wild-type and two independent dosR::hyg recombinants generated for H37Rv (clones 19 and 24) and the W/Beijing isolates HN878 (clones 25 and 28) and G4B1.2 (clones 30 and 32) was digested with PstI and transferred by Southern blotting. (A) The membrane was hybridized with a 2.4-kb PCR fragment (generated using dosR-1 and dosS-1) that includes Rv3134c, dosR, and 739 bp of dosS. Note the presence of an intact copy of dosR retained by the HN878 and G4B1.2 dosR::hyg strains. (B) Based on the published H37Rv sequence, a genetic map of the dosR region in the recombinant strains is shown. By replacing 486 bp of dosR with the hyg resistance cassette, an additional PstI site has been introduced.
- FIG. 2.
Characterization of the W/Beijing duplication through whole-genome microarray. Comparative array hybridizations were carried out for genomic DNA prepared from wild-type W/Beijing isolates G4B1.2 (A) and HN878 (B) versus H37Rv. For each, the average Z scores from three arrays for each of the 3,924 genes present in H37Rv are shown. The RD105 and RD207 deletions unique to the W/Beijing lineage are indicated, as are the RD150 and RD142 deletions that are characteristic of group 4 and 5 W/Beijing isolates, respectively. The RD3 and RD152 deletions were also identified. The majority of the ∼300 genes in the contiguous stretch from Rv3128c to Rv3427c showed a Z score of ≥2.0, consistent with the presence of a 350-kb duplicated region in G4B1.2 and HN878 relative to H37Rv.
- FIG. 3.
The two copies of the 350-kb duplicated region are arranged in tandem. (A) A schematic representation of the duplicated region is shown. Genes Rv3128c and Rv3427c mark the approximate beginning and end of each duplicated segment, respectively. Genes Rv3127c and gadB flank the duplication externally. The length of the duplication is indicated in parentheses, and the two copies (Copy 1 and Copy 2) are arranged in a direct, tandem duplication. The arrangement of the genes indicated (boxed) was confirmed by Southern blotting of genomic DNA comparing wild-type H37Rv and the W/Beijing isolates HN878 and G4B1.2. The approximate locations of the restriction enzyme sites are based upon the published H37Rv sequence. (B and C) Southern blots confirming that the beginning of the duplication is located between Rv3127c (single copy) and tgs1 (duplicated). DNAs were digested with XbaI and hybridized with a 620-bp probe derived from Rv3127c (B; primers Rv3127c-A and -B) or with a 920-bp tgs1 probe (C; primers Rv3130-1 and Rv3130c-F). (D and E) Southern blots confirming the location of the junction of the duplication and that the two copies are arranged in a direct, tandem duplication. DNAs digested with HindIII and SspI were hybridized with a 540-bp alr probe (D; primers alr-C and alr-R) or with a 1-kb dosR probe [E; primers dosR-C and dosRrev(HindIII)]. (F and G) Southern blots confirming that the end of the duplication is located between Rv3427c (duplicated) and gadB (single copy). DNAs were digested with BamHI and NheI and hybridized with the 540-bp alr probe (F) or a 1.2-kb gadB probe (G; primers gadB-C and gadB-D).
- FIG. 4.
Genetic map and PCR-based screening for identifying isolates bearing the 350-kb duplication. The genetic arrangement of the cloned fragments representing the beginning (A), end (B), and junction (C) regions of the duplication is shown. Genes present in “Copy 1” of the duplication are indicated in black, while those in “Copy 2” are in dark gray. The three sets of primers used to screen for the presence of the duplication in clinical isolates are highlighted (boxed). The screening method is based on detecting the IS6110 transposase insertions (white) located in the beginning (A and D), end (B and E), and junction (C and F) regions. (D to F) Lanes: 1, M. tuberculosis West African-I; 2, M. bovis BCG; 3, M. bovis; 4, M. canetti; 5 and 6, M. tuberculosis Indo-Oceanic (I-O) lineage; 7 and 8, East African-Indian (E-A-I); 9 and 10, Euro-American (E-A); 11 and 12, W/Beijing group 1 (G-1); 13 and 14, W/Beijing group 2 (G-2); 15 to 20, W/Beijing group 3 (G-3); 21 to 24, W/Beijing group 4 (G-4); 25 to 28, W/Beijing group 5 (G-5); 29, negative control. In the example shown, the W/Beijing isolates in lanes 23 and 24 (group 4) and in lanes 27 and 28 (group 5) are positive in all three reactions, suggesting that they contain the full duplication.
- FIG. 5.
Impact of the W/Beijing duplication on dosR expression. (A) qRT-PCR analysis of dosR expression in the wild type (wt) and the single or double dosR mutant strains indicated in panel B. The expression levels of dosR are normalized to the sigA housekeeping gene and are plotted relative to H37Rv. Each sample was assayed in quadruplicate, and at least two independent biological replicates were analyzed for each strain. Data from a single representative experiment are presented. Error bars represent standard deviation. (B) Schematic representation depicting the duplication and dosR genotypes for each of the strains analyzed in panel A. Further details for each strain can be found in the text.
Tables
- TABLE 1.
Functional categories of the 313 genes present in the 350-kb duplication
Functional category No. of genes in duplication % of total genesa Virulence, detoxification, adaptation 15 0.38 Lipid metabolism 17 0.43 Information pathways 20 0.51 Cell wall and cell wall processes 35 0.89 Insertion sequences and phages 17 0.43 PE/PPE proteins 14 0.36 Intermediary metabolism and respiration 89 2.27 Regulatory proteins 21 0.54 Conserved hypothetical proteins 85 2.17 All genes in the duplication 313 7.98 ↵ a Relative to the complete H37Rv genome.
- TABLE 2.
Distribution of the 350-kb duplication in M. tuberculosis clinical isolates
Isolate group na No. of isolates with IS6110 transposase atb: No. of isolates with results confirmed byd: Southern blotting DNA microarray Beginning Junction End Dupl+ Dupl− Dupl+ Dupl− Non-W/Beijing 33 0 0 0c ND ND ND ND Group 1 6 0 0 0 ND ND ND ND Group 2 2 0 0 0 ND ND ND ND Group 3 61 42 3 54 ND ND ND ND Group 4 14 14 6 13 6 (6) 8 (8) 3 (3) 1 (1) Group 5 10 10 4 10 4 (4) 4 (4) 2 (2) 3 (3) Total 126 66 13 77 10 12 5 4 ↵ a Number of isolates screened in each group.
↵ b Isolates were screened by PCR for the presence of the IS6110 transposase at each of the regions indicated. See Fig. 4 for further details.
↵ c No “end” product was detected for 6 strains. This is due to the RD6 deletion that occurs in M. bovis, M. canetti, and some M. tuberculosis isolates (5).
↵ d PCR screening results were confirmed by Southern blotting for 22 isolates and by DNA microarray for 9 isolates. The number of isolates in each group that were tested by the methods indicated is given in parentheses. Dupl+, duplication positive; Dupl−, duplication negative. ND, not done.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1, primers; Table S2, genes included in W/Beijing duplication; Table S3, MICs
Zipped MS Word document, 38K. - Supplemental file 2
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Fig. S1, confirmation of the presence of two dosR copies in wild-type W/Beijing isolates; Fig. S2, IS6110 RFLP and cluster analyses; Fig. S3, whole-genome microarray analysis; Fig. S4, generation of a double dosR knockout
Zipped PDF file, 16.6MB.
- Supplemental file 1
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Table S1, primers; Table S2, genes included in W/Beijing duplication; Table S3, MICs
