Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the environments ofaac(6′)-Iy in BM4361 and BM4362 and of the 97.6-min chromosomal region of E. coli K12. Arrows indicate the direction of transcription. The ORFs upstream fromaac(6′)-Iy in BM4361 (open arrows) had ca. 75% identity with the 97.6-min chromosomal region of E. coli K12. The nucleotides adjacent to the deletion are indicated. The inserts of recombinant plasmids are represented by lines between vertical lines, and the sequenced portions are indicated by thick lines. The oligodeoxynucleotides used for PCR amplification are indicated (O1 from 3034 to 3053, O2 from 3451 to 3470, and O3 from 2177 to 2196; the numbering is in accordance with that for the sequence with GenBank accession no. AF144881 ). Probes B1 and B2 used for screening recombinant plasmids and probes A to F used for Southern and Northern analyses are indicated. The ca. 60-kb deletion is indicated by a double-headed arrow.
- Fig. 2.
PFGE (A) and Southern hybridization (B and C) of total DNA from BM4361 (lanes 1) and BM4362 (lanes 2) restricted withXbaI and bacteriophage lambda concatamers (lanes M). (B) Hybridization with a probe for aac(6′)-Iy. (C) Hybridization with a probe for the nmpC 5′ end.
- Fig. 3.
Analysis of aac(6′)-Iy transcription by Northern hybridization. Total RNA from BM4361 (lanes 1) and BM4362 (lanes 2) was hybridized with aac(6′)-Iy probe A (A),nmpC 5′ end probe C (B), probe D (C), and probe E (D) (Table2). The sizes of the transcripts relative to the RNA molecular weight marker I were determined (Boehringer).
- Fig. 4.
Identification of the transcriptional start site foraac(6′)-Iy in BM4362 by primer extension analysis. (Left panel) Lane 1, control without RNA; lane 2, primer elongation product obtained with oligodeoxynucleotide O4 and 50 μg of total RNA from BM4362 (arrowhead); lanes T, G, C, and A, results of sequencing reactions performed with pAT711 DNA as the template and O4 as the primer. (Right panel) Sequence from nucleotide positions 721 to 960 (numbering in accordance with that for sequence with GenBank accession no. AF144881 ). +1, transcriptional start site for aac(6′)-IymRNA in BM4362. The −35 and −10 promoter sequences upstream from the transcriptional start site are underlined with thick lines. The ATG start codon of nmpC is boxed, and the RBS is underlined with a thin line.
Tables
- Table 1.
Strains and plasmids
Strain or plasmid Relevant characteristicsa Source or reference E. coli JM83 F−ara Δ(lac-proAB) rpsL[F80dlacΔ(lacZ)M15] 37 MC1061 F−araD139Δ(ara-leu)7696 galE15 galK16Δ(lac)X79 rpsL hsdR2(rK−mK+)mcrA mcrB1 36 C600 F− e14− (mcrA)thr-1 leuB6 thi-1 lacY1 supE44 rfbD1 fhuA21 3 Cla F−restriction defective 31 C. freundii ATCC 8090 Reference strain 8 Salmonella BM4361 S. enterica subsp. I serotype Enteridis; clinical isolate This study BM4362 S. entericasubsp. I serotype Enteridis; clinical isolate; Tm This study LT2 S. enterica subsp. I serotype Typhimurium WHOCCSb BM4410 S. enterica subsp. II; 6.8:m.t:1.5 WHOCCS BM4411 S. enterica subsp. IIIa; 21:f.z51 WHOCCS BM4412 S. enterica subsp. IIIb; 11:1.u:z WHOCCS BM4413 S. enterica subsp. IV; 6.7:z4.z24 WHOCCS BM4414 S. enterica subsp. VI; 1.6.14.25:Q:e.u.x WHOCCS BM4415 S. bongori; 44:r WHOCCS Plasmids pAT713 2.2-kb SspI fragment from BM4361 cloned into pUC18 This study pAT714 1.6-kbSalI fragment from BM4361 cloned into pUC18 This study pAT715 1.3-kb PCR fragment from BM4361 cloned into pCR-Blunt vector; Tm This study pAT716 438-bp PCR fragment ofaac(6′)-Iy from BM4361 cloned into pCR-Blunt vector This study pAT703 2.8-kb Sau3AI fragment from BM4362 cloned into pUC18; Tm This study pAT711 438-bp PCR fragment of aac(6′)-Iy from BM4362 cloned into pUC19; Tm This study pAT712 6-kb SalI-BamHI fragment from BM4362 cloned into pUC18 This study pAT718 1.17-kbHindIII PCR fragment of nmpC from BM4361 cloned into pSU19 This study - Table 2.
Probes
Probe Positiona Size (bp) Location A 3034–3471 438 aac(6′)-Iy B1 1674–2294 621 sgcEs 3′ end B2 1552–2176 625 Upstream fromsgcAs to sgcEs 5′ end C 840–1534 695 nmpC 3′ end D 112–688 577 140 bp upstream from the nmpCstart codon E 3664–4190 527 Downstream fromaac(6′)-Iy F 731–1154 424 Internal tosgcQs - Table 3.
Susceptibilities of strains to selected aminoglycosides
Strain MIC (μg/ml) of: Amikacin Gentamicin Netilmicin Tobramycin S. enterica BM4361 1.5 0.5 0.5 1 S. entericaBM4362 8 1 2 16 S. entericaBM4362/pAT718 8 1 2 16 E. coliJM83 1 ≤0.25 ≤0.25 ≤0.25 E. coliJM83/pAT711 32 ≤0.25 4 16
