Transposon Tn21, flagship of the floating genome

Abstract

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.

FIG. 1

NR1, an IncFII self-transmissible, multiple-antibiotic resistance R plasmid. Inserts indicate mobile genetic elements.

FIG. 2

Symbols for the transposition (tnp) region, the integron, and the mer operon of transposon Tn21 (GenBank accession no. AF071413). Vertical bars indicate flanking inverted repeats (IR) of transposons and insertion sequences (IS). The tnp region consists of genes for the transposase (tnpA), the resolvase (tnpR), the putative transposition regulator (tnpM), and the resolution site (res) for Tn21. The 5′-CS of the integron includes the integrase gene (intI1) and the attI1 insertion site. The aadA1 gene cassette contains the aminoglycoside adenylyltransferase gene and the 59-be. The arrow indicates the direction of transcription. The 3′-CS includes genes encoding resistance to quaternary ammonium compound disinfectants (qacEΔ1) and sulfonamide resistance (sul1), an ORF (orf5) of unknown function, and two insertion sequences (IS1353 is inserted into IS1326). The tni (transposition of the integron) gene region has suffered deletion in Tn21, and only tniA and a portion of tniB remain. The mercury resistance (mer) operon consists of the regulatory genes merR and merD and the structural genes merT, merP, merC, and merA. There are two unknown reading frames, urf1 (also called merE) and urf2, downstream of merD. urf2M is a hypothetical gene defined in this work, which may have existed before integron insertion.

FIG. 3

Defined or hypothetical proteins of Tn21, the closely related Tn501 and Tn5036, the distantly related transposons Tn5041 and Tn5053, and other mer operons in plasmids pDU1358, pKLH2, and pPB (narrow- and broad-spectrum mercury resistance operons). References are given in Table 6. Numbers in boxes and triangles representing mer or transposition genes are percent amino acid similarities to corresponding proteins of Tn21. Tall shaded boxes represent regions similar to putative Tn21 Urf2 (light gray), to the last 66 aa of putative TnpM (medium gray), or to hypothetical Tn21 Urf2M (dark gray). Black boxes represent small regions (5 to 7 aa) of similarity to the first 7 aa in Urf2M. Numbers below the tall shaded boxes are percent similarities to corresponding proteins in Tn21. Horizontal striped boxes represent tni genes, and spotted boxes represent tnp genes. The slashed box represents a region similar to Tn21 Urf2M and Y proteins. merA of the pPB broad-spectrum mer operon has not been sequenced.

FIG. 4

In2 promoter and attI1 regions. The direction and position of transcription from the promoters P_c and P2 for the aadA1 gene and transcription from the promoter P_int for the intI1 gene are indicated by arrows with parentheses. Translation start sites of aadA1 and intI1 are indicated by the dashed arrows. The attI1 site includes a 64-bp region (sufficient for full recombination site activity for attI1). The numbering scheme relates to the number of base pairs to the left (negative) and to the right (positive) of the recombination crossover point (designated position 0), which is marked with a vertical arrow. A box surrounds the attI1-aadA1 recombination site. Integrase binding sites DR1 and DR2 (closely related to the 7-bp consensus sequence, GTTRRRY) and the “simple site” recognized by IntI1 are underscored with arrows. The numbers 5100 to 5500 indicate the location in the GenBank sequence AF071413.

FIG. 5

The aadA1 cassette containing the aadA1 (aminoglycoside adenylyltransferase) gene and 59-be. The base pairs comprising the 59-be in its free circular form are indicated by the heavy line below them, and those comprising its integrated form are indicated by the heavy line above them. Small boxes surround the composite attI/aadA1 (left) and aadA1/qacE 59-be (right) recombination sites. The putative initiation codon of the aadA1 gene and its stop codon are in bold (large box). The putative ribosomal binding site (rbs) is marked in italics. Arrows indicate imperfect inverted repeat sequences. The numbering scheme is as described in the legend to Fig. 4.

FIG. 6

Evolution of Tn21. Dark gray represents the 5′-CS, and light gray represents the 3′-CS. Cassette insertion is indicated by plus (+), and cassette excision is indicated by minus (−).

FIG. 7

PCR primers used to detect integron components and physical linkage between mer operons and class 1 integrons in gram-negative bacteria. Primers I1 and I2 are located in the intI1 gene, and intergenic primers, 5′-CS and 3′-CS, lie between the intI1 gene and the antibiotic resistance (AbiR) gene cassette and between the AbiR gene cassette and the qacEΔ1 gene, respectively (56). S1 and N1 primers are located in the sul1 and tniA genes of the integron, and primer A9 is located in the merA gene of the mer operon (57). Numbers under bars indicate the expected size (in kilobases) of the PCR products for Tn21.