Transposon Tn21, Flagship of the Floating Genome

Tn 3-Like Transposable Elements—Subtype Tn21 Tn3-like transposable elements are characterized by flanking inverted repeats of about 38 bp and have two genes,tnpA and tnpR, which encode a transposase and resolvase, respectively (29, 95). The tnpR gene and the res site (the site at which the resolvase acts) are located just upstream of tnpA. In the Tn21transposon subgroup, the tnpA and tnpR genes are transcribed in the same direction. The TnpA proteins of this transposon family are at least 70% homologous to each other, as are their TnpR proteins (29). Transposases from this family share, as do many other transposases, a DD(35)E motif, which may form part of the catalytic center (125).

Tn 21, a Class II Replicative Transposon Transposition of transposon Tn21 is carried out by the transposase, TnpA (29). The sites of insertion, defined by a 5-bp duplication of target DNA, are usually AT rich but have no other obvious consensus sequence (77). Replicative transposition of Tn21 involves specific recognition of and binding to the terminal inverted-repeat (IR) elements of Tn21 by TnpA, which then mediates joining of the donor and recipient replicons (77). This cointegrate intermediate contains a copy of Tn21 and is resolved by the action of the tnpRgene product, a resolvase which acts at a specific site (res) adjacent to the tnpR gene (85).

The resolvase catalyzes site-specific recombination between supercoiled DNA and two directly repeated copies of the transposon DNA (102). Resolvase subunits bind to the three subsites (I, II, and III) of res within both recombination loci (100). These protein-DNA assemblies at each ressite then interact with each other to form a synaptic complex (101) in which the resolvase separates the cointegrate to give a new recombinant replicon with one copy of the transposon and the original donor replicon, which retains its copy. In the Tn21subgroup of transposons, the transposase-IR interactions and the resolvases are interchangeable; e.g., Tn21 tnpA can act on the Tn501 IRs and Tn21 tnpR can act on the Tn501 res site (29).

Putative tnpM. An adjacent putative gene of undefined function, tnpM (351 bp), was originally defined as starting at the junction of the tnp region and the integron IR_i (leftward inverted repeat) and terminating withinresI (42). The ribosomal binding site and the promoter −10 and −35 regions for this putative tnpM gene are located within the integron, in IR_i and in the noncoding region downstream of the intI1 gene, respectively.

Although the roles of tnpA and tnpR have been defined, regulation of the tnp genes in Tn21 is less well understood. Studies by de la Cruz and Grinsted with Tn21 (19) and by Tanaka et al. with Tn2603 (112) showed that transposition and resolution functions were intact in deletion derivatives which left thetnpA, tnpR, and tnpM reading frames intact but disrupted the putative −35 promoter region oftnpM. These authors concluded that tnpM was not necessary for transposition. However, Hyde and Tu (42) proposed that the tnpM gene produces a protein which enhances Tn21 transposition and suppresses cointegrate resolution. The basis for their proposal was the finding that a strain carrying a nonsense mutation in the tnpM reading frame was less effective than its parent in effecting Tn21transposition and resolution. Furthermore, they observed that when a multicopy derivative carrying only Tn21 tnpM was present, transposition of Tn21 increased by 2.5-fold and resolution decreased 22-fold. They also noted an open reading frame (ORF) in Tn501 (orf2) at the same relative location and orientation, whose product was 84.6% similar to the putative Tn21 TnpM protein (Fig. 3). These similarities led Hyde and Tu to examine whether transposition and resolution of Tn501 was susceptible to modulation by Tn21 tnpM. They found that with the Tn21 tnpM gene in trans, transposition of Tn501also increased by 2.5-fold and resolution decreased by 1.5-fold. However, no TnpM protein has been demonstrated. Thus, at present, neither the exact nature of the control for TnpA and TnpR nor the role of the putative TnpM is understood. We offer here the possible alternative that the tnpM reading frame is actually derived from a longer ORF which was interrupted by the insertion of In2 (see below). These alternatives are not necessarily mutually exclusive.

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Fig. 3.

Defined or hypothetical proteins of Tn21, the closely related Tn501 and Tn5036, the distantly related transposons Tn5041 and Tn5053, and othermer 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 tnigenes, 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 meroperon has not been sequenced.

Mercury ResistanceDivalent mercuric ion, Hg(II), has a high affinity for thiol groups of proteins and for this reason is extremely toxic in biological systems. The current model of the mercury resistance (Hg^r) mechanism of gram-negative bacteria posits that Hg(II) enters the periplasm and is bound to a pair of cysteine residues in MerP which transfer Hg(II) to cysteine residues in MerT or MerC. The latter move the ion across the cytoplasmic membrane in a series of ligand exchange reactions, resulting in the delivery of Hg(II) to the active site of the cytoplasmic flavin disulfide oxidoreductase, mercuric reductase (MerA) (98). Mercuric reductase catalyzes the reduction of Hg(II) to the highly volatile and less reactive monoatomic gas, Hg⁰, which diffuses from the cell and from the medium.

mer GenesThe mer region (Table 4) (1, 65, 66), flanked on its right by Tn21 IR_merand on its left by the rightward IR (designated IR_t) of the integron, is sufficient to confer resistance to Hg(II). The mercury resistance operon of Tn21 contains two regulatory genes,merR and merD, and four structural genes,merT, merP, merC, and merA, encoding the NADH-dependent flavin oxidoreductase, mercuric reductase (MerA); two inner membrane proteins (MerT and MerC); and a periplasmic protein (MerP). Further genes may also play a role in mercury resistance.

urf2 and the Hypothetical urf2MOverlapping the 3′ end of merE by 4 bp is the second reading frame of unknown function, urf2 (708 bp), whose stop codon lies 67 bp inside the right end of the integron rightward boundary at IR_t. However, if the integron and one of its flanking 5-bp direct repeats were removed, urf2 would fuse in frame with the putativetnpM gene to produce a 987-bp hypothetical gene which we designate Tn21 urf2M (Fig. 3). Its 328-aa predicted product, Urf2M, is 76.9% identical and 79.3% similar to the predicted 329-aa product of orf2 found in transposon Tn501. Transposon Tn501 is a Tn21 subgroup transposon with a gene organization similar to that of Tn21 but lacking bothmerC and the integron (29). Also, the predicted product of urf2 in the mer operon of plasmid pPB is 78.7% similar to Tn21 Urf2M. The predicted product of orf2 (an incomplete sequence) in the mer operon of plasmid pDU1358 is 80.0% similar to Tn21 Urf2M.

Further, part of Tn21 Urf2M (226 of 328 aa) (Fig. 3) is also about 62% similar to a group of hypothetical proteins which belong to the YEGE/YHDA/YHJK/YJCC family of proteins (4, 121). These putative proteins (referred to here as Y proteins) are characterized by transmembrane regions and tyrosine kinase domains as found in two-component signal regulators (41). The carboxyl termini of the Y proteins are 62.3% ± 1.0% similar (40.3% ± 3.5% identical) to Tn21 Urf2M beginning at aa 22 and ending 35 aa into the beginning of putative TnpM (a total of 226 contiguous residues). This similarity to the carboxyl termini of the Y proteins also occurs in Tn501 Orf2 and pPB Urf2.

These similarities suggest that before In2 insertion, a distinct insertion or deletion occurred, forming an ORF (encoding a 328-aa product) between the mer and tnp genes or within a gene at the 3′ end of an ancestral mer operon. Possible remnants of this event are the last 66 aa of Tn21 TnpM, the last 67 aa of Tn501 Orf2 and pPB Urf2, and the last 70 aa of Tn5041 Urf2, pKLH2 Urf2y, and Tn5036 Urf2y, which are conserved (including three conserved cysteine pairs) (Fig. 3). Ribosomal binding sites in front of possible translation start sites occur in the genes encoding each of the aforementioned 66-, 67-, and 70-aa predicted products, suggesting that a smaller gene may be present in the larger ORFs.

To assess the possible origin of Tn21 urf2M and Tn501 orf2, we compared codon usage patterns of Tn21 urf2M with those of the Tn21 and Tn501 merA and tnpA genes (Tables7 and 8). The GCG program Correspond (24) compares two codon frequency tables (20) and yields the residuals statistic D² (Table 7, footnote b), which, when greater than 3, indicates different codon usage patterns. D² values of <3.0 are consistent with a common origin for the genes (24). We largely limited our analysis to sequences of >1,000 bp since we observed that D² increased when the sequence was small.

The D² values, 0.98 to 1.31, indicated that Tn21 merA, Tn501 merA, and Tn501 orf2 have similar codon usage patterns to Tn21 urf2M (Table 7). Higher D² values (3.21 and 3.61) indicated that Tn21 tnpA and Tn501 tnpAhave dissimilar codon usage patterns compared to Tn21 urf2M. Codon usage in each of the two ORFs which comprise the putative Tn21 urf2M (tnpM and urf2) shows that each is as distant from tnpA and as close to merA as is the putative composite gene; thus, by the D²statistic, neither component gene is related to tnpA or tomerA any differently than the putative fused gene. We also noted a surprisingly low D² value of 1.13 between one of the genes which encodes the Y protein CY02B10.18C (Mycobacterium tuberculosis) and Tn21 urf2M. On the basis of these comparisons, Tn21 urf2M is more likely to have been associated longer with the mer genes than with thetnp genes, although its function in mercury resistance and its relationship to the Y proteins is unknown. An alternative hypothesis is that the origins of these ORFs cannot be discerned by codon usage comparisons because they are nonfunctional and therefore are freer to drift than is a functional gene.

5′-Conserved SegmentThe 5′-CS or intI module of class 1 integrons extends from the left IR, designated IR_i, to the boundary of the first integrated cassette (Fig. 2). The 5′-CS includes three repeats at its left end that are predicted to bind the tnitransposase (79) (described in more detail below): theintI1 gene, encoding the class 1 integrase; the promoter regions for the intI1 and aadA1 genes; and the bulk of the attI1 site, the IntI1-specific recombination site into which cassettes are inserted. IntI1 is a member of the integrase family of site-specific recombinases (14, 26, 74) and catalyzes recombination between various pairs of recombination sites (59-be [see below] × 59-be, 59-be × attI1, and attI1 × attI1) in experimental conduction assays (32, 38, 60, 61, 82, 105). IntI1 also catalyzes the integration of additional gene cassettes atattI1 (10) and both the excision and rearrangement of gene cassettes (12, 13). Experimentally, the attI1 site consists of at least 34 bp but no more than 64 bp of the 5′-CS and includes 6 to 10 bp at the first adjacent cassette (Fig. 4) (82), although a shorter length has recently been claimed (38). There are four IntI1 binding sites in attI1: two sites appear to consist of a simple integrase recombination site made up of a pair of inversely oriented IntI1-binding sites and two additional IntI1-binding sites (DR1 and DR2) that enhance the efficiency of the simple recombination site (Fig. 4) (14, 25).

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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 andintI1 are indicated by the dashed arrows. TheattI1 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 theattI1-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 sequenceAF071413.

Integrons also include a promoter, P_c (formerly known as P_ant or P1), that directs transcription of the cassette-borne gene(s) (Fig. 4). The sequences of the 5′-CS of class 1 integrons found in several different locations are >99% identical. The most frequent differences between the sequences of the 5′-CS from different integrons occur within the −10 or −35 regions of the P_c promoter (104), giving rise to variants with different strengths (9, 11, 55); the variant of P_c in In2 is the weakest. However, the 5′-CS of In2 contains a unique insertion of three G residues (bases 5278 to 5280 [Table 5]; bold G’s in Fig. 4) that increases the spacing between a distinct set of potential −10 and −35 hexamers from 14 to 17 bp, thereby creating a second promoter, P2, which is more efficient than P_c (11, 55). P2 is a secondary promoter found only in In2 and close relatives. Constitutive transcripts from both P_c and P2 can be detected (11); there is no information on whether either is subject to regulation by other factors.

aadA1 CassetteIn2 includes a single gene cassette containing theaadA1 gene, which determines resistance to streptomycin and spectinomycin. The AAD(3") protein is an aminoglycoside adenylytransferase that inactivates these antibiotics by adding an adenyl or other nucleotidyl group. The cassette also contains an IntI1-specific recombination site, the aadA1 59-base element (59-be; also called attC) located downstream of the gene (Fig. 5). The aadA1 cassette thus has the features typical of gene cassettes, i.e., a gene and a 59-be. The aadA1 cassette was presumably incorporated into In2 by IntI1-mediated recombination between the attI1 site and the 59-be in a free circular aadA1 cassette. Experimental evidence for the free circular intermediate and for cassette integration has been reviewed extensively elsewhere (34,35, 80).

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Fig. 5.

The aadA1 cassette containing theaadA1 (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) andaadA1/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.

The aadA1 cassette is organized compactly (Fig. 5). The first possible in-frame ATG codon for aadA1 is only 8 bp from the cassette boundary but is not preceded by a consensus ribosome binding site within the cassette. Alternatively, translation ofaadA1 may initiate at a GTG codon, 12 bp downstream, which is preceded by a ribosome binding site. Translation of theaadA2 gene in a related cassette is initiated in this manner (3). The 59-be of the aadA1 cassette lies only 1 bp from the termination codon of the gene.

In the aadA1 cassette, the 59-be is actually 60 bp long and has features common to this family of recombination sites (13, 32,105). Since the 59-be, of the free circular cassette form, is separated into two parts by the recombination crossover, the 59-be immediately downstream from aadA1 in In2 actually includes 54 bp of the aadA1 59-be (from the circular cassette form) and at least 6 bp (lowercase in Fig. 5) of the adjacent module, which is the 3′-CS of In2. This composite 59-be is called theaadA1/qacE 59-be. The terminal 6 bp of the aadA159-be in the circular cassette form lie at the beginning of the integrated aadA1 cassette and constitute part of the composite attI1/aadA1 recombination site of In2. The outer 25 bp of all 59-be are relatively highly conserved (13,105). Within each 25-bp consensus region is a simple site configuration (1L-2L and 2R-1R [Fig. 5]), as found with other integrase recognition sites, and each simple site includes a pair of inversely oriented 7-bp core sites. Only 8 base of the composite core sites are completely conserved: three each in 1L and 1R (ACC and GTT) and one each in 2L and 2R (A and T) in all 59-be (105).

3′-Conserved SegmentIn early studies of class 1 integrons, the 3′-CS was defined as a conserved region containing three ORFs (qacEΔ1,sul1, and orf5) located to the right of the different cassette arrays found in various integrons (Fig. 2) (104). More recent studies (5, 33) have revealed that the length of the 3′-CS region is variable and that some integrons have additional sequence (orf6 or portions of orf6) downstream of orf5. However, not all class 1 integrons have this region (Fig.6); e.g., Tn402, also recently called Tn5090 (79), does not have a 3′-CS region.

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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 (−).

The qacE gene encodes an inner membrane protein with four transmembrane segments and determines resistance to quaternary ammonium compounds (antiseptics and disinfectants) by an efflux mechanism (71, 88). The last 66 bp of qacEΔ1 (encoding 115 aa) differs from the complete qacE gene (encoding 110 aa); the qacEΔ1 gene confers only marginal resistance to quaternary ammonium compounds (75). The sul1 gene (formerly sulI) encodes a sulfonamide-resistant dihydropteroate synthase (279 aa), which can replace the normal bacterial enzyme targeted by this group of drugs (111). The function of orf5 is not known; however, the predicted protein is 38% identical and 47% similar to a puromycinN-acetyltransferase (gi 1346911) of Streptomyces alboniger and is 36% identical and 50% similar to a putative acetyltransferase (gi 3449263) of Streptomyces coelicolor(121). Although in In2, transcription originating from P_c or P2 in the 5′-CS is likely to extend into the 3′-CS, affording expression at these three 3′-CS genes, a weak promoter that is sufficient for expression of sulfonamide resistance has also been identified upstream of the qacEΔ1 gene (30).

In Tn402, the qacE gene is associated with a 59-be and hence is part of a mobile cassette (81), whereasqacEΔ1 is not mobile. The sul1 and adjacent sequence may also have originally been part of cassettes which integrated at attI1 and subsequently became immobilized by loss of their adjacent 59-be recombination sites (Fig. 6) (5,104). Thus, the 3′-CS appears to contain vestiges of the earliest insertion events. The presence of the sul1 gene would explain the wide distribution of integrons containing this region, since sulfonamides were the first widely used antimicrobial agents.

tni Transposition ModuleOf the class 1 integrons, where sufficient sequence is available, only Tn402 (Fig. 6) carries the full complement of fourtni genes (5, 79), believed to encode transposition functions on the basis of their similarity to thetni genes of Tn5053 (Fig. 3) (48). ThetniA, tniB, and tniQ genes of Tn5053 are required for transposition, and the fourth gene,tniR, encodes a resolvase which resolves the cointegrates as does TnpR of Tn21 (48). In In2, only one complete gene, tniA, and a truncated version of the tniBgene are present. All class 1 integrons, including Tn402, have complex ends (79). Both ends have three copies of a 19-bp sequence, the outermost of which overlaps the terminal 25-bp IRs. By analogy to Tn7, these three 19-bp repeats are believed to function as transposase binding sites, although the identity of the Tni protein involved is not known.

IS 1326 Insertion Sequence The insertion sequence IS1326 has terminal 26-bp IRs and includes two ORFs that overlap by 14 bp (5). These ORFs have been designated istA (1,524 bp) and istB(786 bp) due to the similarity of their predicted products to IstA and IstB, required for transposition in IS21 (83). IstA has a DD(45)E transposase motif and a helix-turn-helix motif, and IstB has a distinctive set of nucleoside triphosphate binding domains (5). These two genes have share a common promoter upstream of istA. However, in In2, IS1353 has inserted between the −10 and −35 promoter regions of IS1326, and thus istAB may not be expressed. IS1326 also occurs in other integrons, e.g., integrons In0 and In5 in plasmids pVS1 and pSCH884, respectively (5).

IS 1353 Insertion Sequence The acquisition of IS1353 by In2 appears to be a recent event that occurred after the movement of the In2 to its present location in Tn21 (5). IS1353 is a member of the IS3 family (5) and has the characteristic 5′-TG and CA-3′ at the outer ends of its terminal 12-bp IRs. IS1353 is flanked by a direct duplication of 2 bp of the target sequence. It contains two ORFs, orfA andorfB, transcribed in the opposite direction fromistA and istB of IS1326. A potential promoter for orfA and orfB lies 24 bp upstream of the putative OrfA translational start. OrfB has a DD(35)E transposase motif similar to other transposases of the IS3 family (23). A potential frameshift motif, A₆G, and a predicted mRNA stem-loop are present where the orfA andorfB reading frames overlap, suggesting that (like other IS3 relatives, IS911 and IS150), three polypeptides, OrfA, OrfB, and a fusion protein (OrfAB) that is the actual transposase, may be produced (78). The initiation codon of OrfB has yet to be determined; it may be at the ATT codon 3 bp upstream of the A₆G motif (as seen in IS911) or at the ATG codon 6 bp downstream of the A₆G motif.

Model for Tn 21 Evolution Several transposons, considered to be very close relatives of Tn21, have nearly identical DNA sequences or restriction maps in the tnp and mer regions but differ in that they carry no or different antibiotic resistance genes. Tanaka et al. (112) first proposed that those Tn21-like transposons which confer multiple antibiotic resistance had descended from an ancestral mercury resistance transposon like Tn501by successive insertions of antibiotic resistances and/or insertion sequences. Their restriction endonuclease analysis indicated similarity between the mer and the tnp regions of Tn2613, which lacks antibiotic resistance genes, and the corresponding regions of Tn2608, which has a 6.3-kb insertion between the mer and tnp regions and has also gained streptomycin and sulfonamide resistances. These observations suggested that Tn2613 might reasonably be considered the predecessor of Tn2608. However, Tn21 itself has a slightly different restriction map in the Tn21 IR_mer and in the tnpM regions from those of both Tn2613 and Tn2608, suggesting that neither of these transposons was the immediate predecessor of Tn21.

A more recent model for the evolution of Tn21 and other family members (5) is based on restriction maps, heteroduplex analyses, and sequence data for a larger group of these transposons (19, 53, 63, 94, 110, 113, 117, 119, 122, 123). In this analysis, those considered members of the family are similar to Tn21 in carrying the same tnpRA transposition genes and an integron in the same position as In2. They may or may not carry the complete mer locus, and they also differ from Tn21 in either the identity or number of gene cassettes in the integron or by the acquisition or loss of other IS elements or transposons. For example, the original Tn21, isolated in Japan, includes a 1.6-kb region corresponding to IS1353 that is not present in related isolates from Germany (29), which suggests that Tn21 acquired IS1353 after the global spread of an ancestor such as Tn2411 that does not contain this IS (5).

Brown et al. (5) proposed that Tn2411 arose from a hypothetical ancestor called Tn21Δ, which lacks an integron but contains the mer operon (Fig. 6). In Tn21Δ, urf2M would be present (as seen in orf2 and urf2 adjacent to mer operons in Tn501[6] and pPB [84], respectively [Fig.3]). The ancestor of In2 (without IS1353) inserted into Tn21Δ, perhaps via a transposition event catalyzed intrans by another transposon with a complete set oftni transposition genes. The alternative, i.e., that the In2 ancestor might have been complete and later lost its tnigenes by a deletion event occurring on or after insertion of IS1353, seems unlikely since integrons with very similar organization to In2 are found in other locations (5).

Subsequently, transposon Tn2411 became widely distributed, experiencing integration, excision, or exchange of gene cassettes, insertion of transposons or insertion sequences, and IS-mediated deletions or excisions (Fig. 6), which have given rise to the examples of this group described so far (19, 53, 63, 94, 110, 119, 122,123) and probably many others.

Model for In2 EvolutionThe In2 integron is believed to have been a highly evolved element (Fig. 6) before it integrated into Tn21Δ. Brown et al. (5) have proposed that the central components of In2 evolved from a progenitor, similar to Tn402, which had experienced several genetic events prior to insertion into Tn21Δ. These genetic events would include the creation of the 3′-CS via the integration of qacE, as well as sul1, and other putative cassettes and subsequent events to immobilize them. After the insertion of IS1326, this IS caused the partial deletion of the adjacent 3′-CS and tni module. The insertion of IS1353 occurred after the integron moved into Tn21Δ and is likely to be the latest event in the creation of both In2 and Tn21.

Codon Usage ComparisonsTn21 can be viewed as an assemblage of genes captured by transposition genes: the tnp genes acquired themer operon, and the tni genes acquired the integrase system. After these two independent events, the integron progenitor, itself an assemblage of genes captured by either thetni module or the integrase module, inserted into Tn21Δ, followed by insertion of IS1353 (Fig.6). Some hints about the variety of sources of these many components is given by their codon usage patterns.

Codon usage comparisons of Tn21 genes point to significant diversity in their origins (Table 8). Codon usage comparisons ofmerA and urf2M reveal low D ² values with respect to each other but not with respect to other integron ortnp genes, with the exception of orfAB of IS1353. Codon usage in aadA1, as in the majority of genes found in integrated cassettes (2), is quite dissimilar to that in other genes within the integron, presumably reflecting diverse origins of the cassette-associated genes. In Tn21, the exception to this appears to be the low D² values betweenaadA1 and istAB (IS1326) and orfAB (IS1353). High D² values are also observed between qacEΔ1 (a former cassette) and all other Tn21 genes compared with it. Codon usage comparisons of genes within the integron revealed low D² values between intI1, sul1, and tniA, suggesting that these genes originated from closely related microorganisms which are different from E. coli orPseudomonas aeruginosa (2, 79). This observation supports the view that sul1 was integrated early in the evolution of class 1 integrons (104). Comparison of codon usage in Tn402 genes revealed similar low D² values (0.81) between int andtniA as for those genes in Tn21 and high D² (9.27 to 6.98) values between qacEand other genes within this integron (79). The low D² values between most of the Tn21genes and the orfAB genes in IS1353 suggest that perhaps IS1353 was acquired while Tn21 was in a host with codon utilization similar to that of the ancestral host of Tn21.

Environmental OccurrencesPearson et al. observed the widespread distribution of Tn21- and Tn501-related tnpA andtnpR sequences in soil bacteria from three sites (two mercury-polluted sites and one pristine site); 20 of 30 isolates hybridized to transposase and resolvase gene probes (76). They observed extensive recombination between different transposition genes and reassortment of the mer and the transposition gene regions. Further evidence for widespread distribution includes a recent report of the occurrence of Tn21-, Tn501-, and Tn3-related tnpA sequences in total bacterial DNA from the marine environment (15). The Tn21 tnpA sequence occurred once per 10³ or 10⁴ bacteria.

Clinical OccurrencesApart from the above environmental isolates, Tn21carriage of integrons and integrated cassette-associated resistance genes in gram-negative pathogens is also well documented (32, 53,80). The Tn21 family encodes resistances against both older and newer antibiotics. Strains of bacteria resistant to streptomycin, an antibiotic rarely used medically in the past 30 years but still extensively used in animal husbandry, are frequently found (68). In a 1995 study, over half (53%) of 49 clinical isolates with transferable resistance to aminoglycosides carried integrons with aadA1 (25%) or aacA4 (75%) gene cassettes (92). In an earlier (1992) study, 807 unselected gram-negative clinical isolates were screened to determine the distribution of the integron and Tn21 transposition genes (128). A total of 19% of the isolates carried some part of the tnpA tnpR, or intI1 gene and 8% of the isolates had all of the genes, although linkage of tnp genes and integrons was not demonstrated. Besides complete Tn21-like transposons, a great variety of defective transposons were observed in many genera of bacteria (128).

Tn21 and Tn21-like transposons with integrons have also been observed in primate intestinal bacteria that were challenged with Hg(II) released from dental amalgams (108). There was a strong association of multiple-antibiotic resistance with two of the most abundant phylogenetic types of Hg resistance locus (locus 1 and locus 4) in these bacteria (58, 118). Using PCR, we have established (Fig. 7) thatmer locus 1 carries a In2-like integron and that some examples of mer locus 4 carry a class 1 integron similar to In4 in Tn1696 (119). In both mer locus types, the adjacent integrons carry from one to four antibiotic resistance genes. Hg exposure experienced by the fecal flora of these animals led to enrichment for strains carrying Hg resistance loci closely linked to integron multiple-antibiotic resistant transposons.

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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 theintI1 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 theqacEΔ1 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.

The causes of the dissemination of the Tn21 family of transposons are presently unknown but may be due to the chance association of the integron and mer at a time when the rich ecosystem of the human intestinal microflora was experiencing increasing antibiotic usage coinciding with exposure to Hg due to its use in the treatment of syphilis and in amalgam dental restorations.