Article Figures & Data
Figures
- FIG 1
Relative expression levels of pbp5 and vanB ligase in the naive TC-A transconjugant (TCA P0) or in the resulting selected ones (TCA-A P9, which was continuously passaged in the presence of ampicillin for ∼400 generations, and TCA-V P13, which was continuously passaged in the presence of vancomycin for ∼400 generations). The parental strains (D344RRF and C68) were included for comparison. The parents and the three versions of TC-A were grown with either ampicillin or vancomycin, and relative expression was calculated using the levels of expression for the corresponding group without antibiotic treatment. Expression levels of 16S rRNA were used to normalize the data. Error bars indicate the standard errors of the means for biological triplicates. (A) pbp5 expression is not modified with respect to that for the control (untreated cells) in the presence of half the MIC of ampicillin. pbp5 expression was induced in C68 and the three different TC-A groups in the presence of vancomycin. (B) vanB ligase expression was very low in the absence of vancomycin. In the presence of the antibiotic, there was a significant induction of vanB ligase expression. Unpassaged TC-A (P0) had lower vanB ligase expression levels than C68 and the passaged groups, which correlates with a lower vancomycin MIC. White bars, BHI-grown cells (calibrator); gray bars, ampicillin-grown cells; black bars, vancomycin-grown cells. ****, P < 0.0001; ***, P < 0.0002; *, P < 0.05.
- FIG 2
pbp5 and TC-D insertion site. (A) pbp5 operon cartoon showing the differences between strains D344RRF and C68. In C68, an insertion (C) 153 bp downstream of the ribosomal binding site causes a frameshift introducing a premature stop codon 309 bp downstream of the start of the gene, possibly generating a nonfunctional gene. (B) The sequence of the pbp5 gene in C68 codes for an extra serine and has two amino acid substitutions compared with the sequence of D344RRF (red). (C) Alignment of D344RRF, TC-D, and C68 in the region of integration of Tn5382 carrying vanB. Green, the vancomycin resistance-carrying transposon; light blue, the transposon (Tn) ends flanking the vancomycin resistance-carrying transposon; light gray, the segments in which the C68 genome replaced that of D344RRF in the transconjugant; purple, the crossover regions (COR) flanked by the SNV used to identify the region. Note the additional C68 integration in TC-D upstream of the pbp5 operon. pbp5 from D344RRF was not replaced by that of C68 in this transconjugant. SNV, single nucleotide variant.
- FIG 3
Crossover regions. (A) Cartoon representation of crossover regions in all individual transconjugants. The area of the crossover is measured from the last SNV corresponding to the donor strain, C68, to the first SNV corresponding to the recipient strain, D344RRF. Transconjugants are organized by groups with shared left crossover regions. Notice the additional crossover regions in TC-D and TC-M (light gray). The amount of chromosomal DNA integrated in addition to Tn5382 is measured from the left or right end of the element up to the last SNV between D344RRF and each transconjugant. The amount of integrated DNA is shown in kilobases. (B) Plot of GC and AT contents of C68 (donor) and D344RRF (recipient) in the chromosomal region where crossovers occurred. Blue, GC content; green, AT content. The high-GC-content area in C68 corresponding to Tn5382 carrying vanB is highlighted between red lines.
Tables
- TABLE 1
Antibiotic resistance profile of the parental strains and selected transconjugants
Strain Resistance traitsa Origin (reference) C68 Ampr, Vanr, Eryr, Tetr, Strr, Genr Clinical isolate (13) D344RRF Ampr,b Rifr, Fusr, Eryr, Tetr, Strr, Kanr Derived from clinical isolate D344R (22) Transconjugants Ampr, Vanr, Rifr, Fusr, Eryr, Tetr, Strr, Kanr Transconjugants obtained from mating C68 and D344RRF during this work - TABLE 2
MICs for parental strains and transconjugants
Straina MICb (μg/ml) Van Amp Fus Rif C68 >400 >400 1.6 <0.2 D344RRF 1.6 12.5 >400 >400 TC-A P0 100 50 >400 >400 TC-A BHI P13 400 200 >400 >400 TC-A Van P13 400 >400 >400 >400 TC-A Amp P9 400 400 >400 >400 TC-B P0 200 50 >400 >400 TC-B BHI P13 400 >400 >400 >400 TC-B Van P13 400 200 >400 >400 TC-B Amp P9 400 >400 >400 >400 TC-C 25 25 200 >400 TC-D 50 25 400 >400 TC-E 50 50 200 >400 TC-F 50 100 400 >400 TC-G 25 12.5 200 400 TC-H 50 25 200 >400 TC-I 200 25 >400 >400 TC-J 200 25 >400 >400 TC-K 100 100 >400 >400 TC-L 50 6.3 >400 >400 TC-M 100 25 >400 >400 TC-N 12.5 6.3 200 200 - TABLE 3
Integration site and size of integrated DNA in the studied transconjugants
TC Position of integration sitea Size (kb) of integrated DNA Additional integration site First SNV of CORb left of vanB First SNV of COR right of vanB (integrase site) Minimum amt transferredc COR between the two delimiting SNVs Left Right TC-A −49.87 +100.8 184.6 574 307 TC-B −10.6 +49.9 94.5 3,112 4,315 TC-C −16.7 +27.7 78.4 6,229 1,159 TC-D 0 +2.9 36.9 654 2,466 9,098d TC-E −14.9 +15.1 64 2,005 2,171 TC-F −10.9 +47.9 92.8 3,112 2,049 TC-G −61.8 +27.6 123.4 2,878 1,157 TC-H −60.2 +81.3 175.5 593 126 TC-I −16.9 +124 174.9 6,182 11 TC-J −16.9 +79.9 130.8 6,182 1,240 TC-K −10.9 +78.7 123.6 3,112 185 TC-L −16.9 +48.4 99.3 6,182 947 TC-M −10.9 +36 80.9 3,113 7,373 11,719e TC-N −14.9 +5.9 54.8 2,005 371
Supplemental material
- Supplemental file 1 -
Supplemental Tables S1 to S4
PDF, 159K
- Supplemental file 2 -
Supplemental Figures S1 and S2
PDF, 3.6M
- Supplemental file 3 -
Supplemental Table S5: (A) pLRM23 gene content, (B) putative pRIH77 gene content, and (C) pLRM23 coverage and pRIH77 coverage in parents and transconjugants.
XLSX, 52K
- Supplemental file 1 -
