Article Figures & Data
Figures
- Fig 1
Differential expression of four efflux genes in selected mutant strains in four different lineages. The data were generated by real-time RT-PCR in comparison with the parent strain (NCTC 11168 or 81-176).
- Fig 2
Immunoblotting analysis of CmeABC expression in selected strains in four different lineages. In panel A, numbers 1, 2, 3, 4, 5, and 6 represent C. jejuni NCTC 11168, 68E1-3, 68E4-3, 68E8-3, 68E32-3, and 68E64-3, respectively. In panel B, numbers 1, 2, 3, 4, 5, and 6 correspond to C. jejuni NCTC 11168, 68E1-1, 68E4-1, 68E8-1, 68E32-1, and 68E64-1. In panel C, numbers 1, 2, 3, and 4 correspond to C. jejuni 81-176, 76E2-6, 76E8-6, and 76E64-6. In panel D, numbers 1, 2, 3, and 4 correspond to C. jejuni 81-176, 76T8-7, 76T16-7, and 76T32-7. Cell envelopes prepared from each strain were blotted with polyclonal antibodies to CmeA, CmeB, and CmeC. The same amount of total proteins was loaded in each lane. Prestained molecular mass markers (lane M; Bio-Rad) were used to estimate the sizes of the proteins (shown in kilodaltons). The positions of CmeA, CmeB, and CmeC are indicated.
- Fig 3
HCL analysis of C. jejuni genes differentially expressed in the three strains (68E1-3, 68E8-3, and 68E64-3) in lineage 68Ex-3. For each of the four clusters, hierarchical clustering was performed both on the genes and on the strains to visualize the relationships among the genes and strains, respectively. Four clusters are presented in four different panels. For each cluster, the color scale shows the n-fold change range of up- and down-regulated genes. The three mutant strains are indicated at the top of each panel.
Tables
- Table 1
Primers used for PCR amplification and real-time RT-PCR
Primer Sequence (5′–3′) Reference 23S rRNA gene amplification FI CCCTAAGTCAAGCCTTTCAATCC 20 FII CGTTATAGATACGCTTAGCGGTTATG 20 FIII CATCGAGCAAGAGTTTATGCAAGC 20 CJ-copy-R CTACCCACCAGACATTGTCCCAC 20 Fragment of 23S rRNA gene 23S-domV-F GTAAACGGCGGCCGTAACTA This study 23S-domV-R GACCGAACTGTCTCACGACG This study 23S-2611F ATGTCGGCTCATCGCATCCTGG 20 23S-2611R CATCCATTACACACCCAGCCTATC 20 Ribosomal protein L4 L4-F GTAGTTAAAGGTGCAGTACCA This study L4-R GCGAAGTTTGAATAACTACG This study Ribosomal protein L22 L22-F GAATTTGCTCCAACACGC This study L22-R ACCATCTTGATTCCCAGTTTC This study CmeR and cmeABC promoter cmeR-F TAGAAAAGTATATTTGTATACCCT 16 cmeR-R CGCCACTAACTTGAGGCTTTA 16 Real-time RT-PCRa 16S-F TACCTGGGCTTGATATCCTA 21 16S-R GGACTTAACCCAACATCTCA 21 CmeB-F ACGATTCAACCTTTTCCCAGC This study CmeB-R TTTGCTACTTGAGCAATCGCTTC This study Cj1687-F TGGTGGTTTGGACTCTTTCTGGGA This study Cj1687-F TCTCTGCGATTAAAGCCACCACGA This study Cj1375-F GCTTTACCACGATTTTCTTCTGTGA This study Cj1375-R TACACCATGCTTTTAGGAAGAATGC This study Cj1257c-F ATCGCCGTGATAGCGCCTAAAGAA This study Cj1257c-R GCCACCAAACAAAGGCCCAAGTAA This study ↵a Sequences and gene names are based on C. jejuni NCTC 11168.
- Table 2
Characteristics of C. jejuni mutants selected by Ery
Lineagea and strainb MIC (μg/ml) Mutation in 23S rRNA genec Change in protein L4 Change(s) in protein L22 Mutation in cmeR Ery Tyl Parent, NCTC 11168 1 4 —d — — 68Ex-1 68E1-1 4 8 — — A88E — 68E4-1 32 256 — — A88E Del490A 68E8-1 64 128 — — A88E Del490A 68E32-1 256 128 — G67V A88E Del490A 68E64-1 >512 256 C2627A (3) G67V A88E Del490A 68Ex-2 68E1-2 4 8 — 59-G-60 — 68E4-2 32 128 — 59-G-60 G86E — 68E8-2 64 128 — 59-G-60 A84D, G86E — 68E32-2 128 128 — 59-G-60 A84D, G86V — 68E64-2 512 256 — 59-G-60 A84D, G86V — 68Ex-268Ex-3 68E1-3 4 16 — R72I — — 68E4-3 64 64 — R72I V100L H174N 68E8-3 128 256 — R72I 91-S-92 H174N 68E32-3 >512 >512 — R72I — H174N 68E64-3 >512 >512 A2074C (3) R72I — H174N Parent, 81176 1 4 — — — — 68Ex-268Ex-376Ex-6 76E2-6 32 64 — — — — 76E8-6 256 >512 A2074C (2) — — — 76E64-6 >512 >512 A2074C (3) — — — ↵a The lineages are named according to the parent strain and the antibiotic used for selection. 68 and 76 indicate that the parent strains are 11168 and 81-176, respectively, E represents selection by Ery, x depicts the various concentrations used for selection, and the last number indicates the lineage number.
↵b The strains in the lineages are named similarly to the lineages, except that x is replaced by the actual antibiotic concentration from which the mutant was selected.
↵c Each number in parentheses is the number of mutated copies of the 23S rRNA gene.
↵d —, no mutations detected.
- Table 3
Characteristics of C. jejuni mutants selected by Tyl
Lineagea and strainb MIC (μg/ml) Mutation in 23S rRNA gene Change in protein L4 Change(s) in protein L22 Mutation in cmeR Ery Tyl Parent, NCTC 11168 1 4 —c — — 68Tx-1 68T4-1 4 8 — — A88E 68T16-1 32 256 — R72I A88E H174N 68T32-1 64 256 — R72I A88E H174N 68T128-1 128 256 — R72I A88E H174N 68Tx-2 68T4-2 4 8 — — — 68T16-2 128 256 — G57V — — 68T32-2 256 512 — G57V G86E — 68T128-2 128 256 — G57V G86E — 68 Tx-3 68T4-3 2 8 — — — — 68T16-3 32 256 — A71D — S117Y 68T32-3 64 512 — A71D A88E S117Y 68T64-3 16 256 — A71D A88E S117Y 68Tx-4 68T4-4 2 8 — — — — 68T8-4 16 64 — A71D — — 68T16-4 32 128 — A71D A88E G204L 68T32-4 64 256 — A71D A88E G204L 68T64-4 128 512 — A71D A88E, G86V G204L Parent, 81176 1 4 — — — — 76Tx-7 76T8-7 8 32 — — — — 76T16-7 32 128 — G57D G86E — 76T32-7 64 256 — G57D G86E —
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Figure S1, Venn diagram showing the numbers of genes differentially expressed in three selected mutant strains (68E1-3, 68E8-3, and 68E64-3) of lineage 68Ex-3 in comparison with wild-type NCTC 11168.
PDF, 34K
- Supplemental file 2
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Supplemental Table S1
XLSX, 35K
- Supplemental file 1 -
