Activation of the cryptic aac(6')-Iy aminoglycoside resistance gene of Salmonella by a chromosomal deletion generating a transcriptional fusion

Abstract

Salmonella enterica subsp. enterica serotype Enteritidis BM4361 and BM4362 were isolated from the same patient. BM4361 was susceptible to aminoglycosides, whereas BM4362 was resistant to tobramycin owing to synthesis of a 6'-N-acetyltransferase type I [AAC(6')-I]. Comparative analysis of nucleotide sequences, pulsed-field gel electrophoresis patterns, and Southern hybridizations indicated that the chromosomal aac(6')-Iy genes for the enzyme in both strains were identical and that BM4362 derived from BM4361 following a ca. 60-kb deletion that occurred 1.5 kb upstream from the resistance gene. Northern hybridizations showed that aac(6')-Iy was silent in BM4361 and highly expressed in BM4362 due to a transcriptional fusion. Primer extension mapping identified the transcriptional start site for aac(6')-Iy in BM4362: 5 bp downstream from the promoter of the nmpC gene. Study of the distribution of aac(6')-Iy by PCR and Southern hybridization with a specific probe indicated that the gene, although not found in S. enterica subsp. arizonae, was specific for Salmonella. In this bacterial genus, aac(6')-Iy was located downstream from a cluster of seven open reading frames analogous to an Escherichia coli locus that encodes enzymes putatively involved in carbohydrate transport or metabolism. This genomic environment suggests a role in the catabolism of a specific sugar for AAC(6')-Iy in Salmonella.

FIG. 1

Schematic representation of the environments of aac(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 from aac(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 with XbaI 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) (Table 2). The sizes of the transcripts relative to the RNA molecular weight marker I were determined (Boehringer).

FIG. 4

Identification of the transcriptional start site for aac(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′)-Iy mRNA 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.