Transposition of the carbenicillin-hydrolyzing beta-lactamase gene

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.     

Denaturation map of the ColE1-Km plasmid pCR11.

The denaturation map of EcoRI-digested pCR11, a ColE1-Km plasmid, is described. The 2.0 kilobase ColE1-derived segment contains an adenine+thymine rich site in the colicin immunity gene region. In the 7.2 kilobase kanamycin resistance region, the transposon Tn903 consists of an adenine+thymine rich 0.98 kilobase kan gene region flanked by a guanine+cytosine rich 1.09 kilobase inverted duplication.     

Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21

Two transposable elements, Tn2410 and Tn2411, were isolated from Salmonella typhimurium R-factor R1767. They have sizes of 18.5 and 18.0 kilobases, respectively. Tn2411 mediates resistance to streptomycin, sulfonamides, and mercury. In Tn2410, the streptomycin resistance gene was replaced by a gene coding for the production of the beta-lactamase OXA-2, which is responsible for ampicillin resistance. Physical and functional maps of both transposons were compared with those of Tn21, Tn4, and Tn2603. From these data it appeared that Tn21 could be an ancestral transposon from which Tn2411, Tn2410, Tn2603, and Tn4 were evolved by the addition or deletion of small DNA segments.     

Transposition of the oxacillin-hydrolyzing penicillinase gene.

We have found that the oxacillin-hydrolyzing penicillinase gene (oxa) encoded by plasmid RGN238 transposes to various plasmids in a recA background. We call this transposable element Tn2603. Tn2603 encodes the genes for streptomycin, sulfonamide, and mercury resistance in addition to the oxa gene. Tn2603 has a molecular size of 19.6 kilobase pairs and appears to be flanked by small inverted repeat sequences of about 200 base pairs long.     

A gene and its product required for transposition of resistance transposon Tn2603.

Tn2603 is a multiple-resistance transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury and having a molecular size of 20 kilobase pairs, with 200-base-pair inverted repeats at both ends. The essential sites and functions of Tn2603 which are required for its transposition were determined through construction and characterization of various deletion mutants affecting the efficiency of transposition. Deletions were introduced in plasmid pMK1::Tn2603 by partial digestion with restriction endonuclease EcoRI in vitro. Analysis of deletion mutants showed that the inverted repeat segments at both ends of the trans-acting diffusible product(s) encoded in the right-hand side of the central portion were required for the transposition of Tn2603. An essential gene product was revealed as a protein having a molecular weight of 110,000 by analysis of polypeptides synthesized in Escherichia coli minicells. This protein was assumed to be the so-called transposase.     

Pseudomonas streptomycin resistance transposon associated with R-plasmid mobilization.

Plasmid pMG1 encodes resistance to gentamicin, streptomycin, sulfonamides, and mercuric ions and also mobilizes pRO161, a transfer-deficient plasmid derived from RP1. Upon mobilization, pRO161 acquires streptomycin resistance (Smr) and can subsequently be remobilized by pMG1 at significantly higher frequencies than pRO161 itself. Both the initial acquisition of Smr and the subsequent mobilization of the transfer-deficient plasmid are recA independent: thus, the Smr determinant appears to be located on a transposon, disignated Tn904. Tn904 transposes to a variety of other plasmids, including RP1, FP2, R388, K, pRO1600, and pBR322, and in some cases the acquisition of this transposon accompanied deletions in the target plasmid. When no deletion occurred, target plasmids gained 5.2 kilobase pairs of DNA and new restriction endonuclease cleavage sites for AvaI, BglII, PstI, SmaI, and SstI. Physical analysis of such plasmids showed that the Tn904 termini are inverted repeat DNA sequences of approximately 124 base pairs. After cloning into vector pRO1723, a single site for restriction endonuclease AvaI was identified within the Smr determinant of Tn904. In Escherichia coli, but not in Pseudomonas aeruginosa. Tn904 shows a gene dosage-dependent expression of streptomycin resistance.     

A chromosomally located transposon in Pseudomonas aeruginosa

A new transposon, Tn2521, coding for carbenicillin, streptomycin, spectinomycin, and sulfanilamide resistance, has been identified in Pseudomonas aeruginosa. The transposon occurs naturally in the chromosome of clinical strains of P. aeruginosa isolated in geographically separated hospitals. This has been demonstrated by its transductional linkage to the pur-136 marker and also by Southern hybridization. Tn2521 is 6.8 kilobases, can transpose from the chromosome to both IncP-1 and IncP-2 plasmid genomes, and has a pattern of restriction endonuclease sites unlike that of any previously described transposon. The carbenicillin resistance carried by Tn2521 is due to the PSE-4 type of beta-lactamase.     

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.     

Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp.

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.     

Identification of a Staphylococcus aureus transposon (Tn4291) that carries the methicillin resistance gene(s).

We isolated a transposon (Tn4291) that carries the resistance gene(s) for methicillin in a secondary insertion site on the penicillinase plasmid pI524. Transposition of Tn4291 into pI524 occurred during the transduction of the tetracycline resistance plasmid pSN1 from a methicillin-resistant donor into a recipient that carried the mec allele in the primary site on the chromosome. Insertion of Tn4291 caused extensive rearrangement of pI524 and resulted in the formation of a 27.9-kilobase-pair plasmid (pIT103) which coded for resistance to methicillin and cadmium, but not penicillin. Although resistance to methicillin and cadmium were always linked, Tn4291 was stably maintained only in the presence of a chromosomal mec allele, while in its absence the plasmid was unstable and transposition to the primary site occurred. Subsequently, a 20.1-kilobase-pair plasmid, pIT203, was formed which retained cadmium resistance and regained the ability to express beta-lactamase activity.     

Primary structure of an aminoglycoside 6''-N-acetyltransferase AAC(6'')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase.

The gene aacA4 encoding an aminoglycoside 6'-N-acetyltransferase, AAC(6')-4, was cloned from a natural multiresistance plasmid, and its nucleotide sequence was determined. The gene was 600 base pairs (bp) long, and the AAC(6')-4 had a calculated molecular size of 22.4 kilodaltons and an isoelectric point of 5.35. The sequence of the 17 N-terminal amino acids was determined from the purified enzyme. The AAC(6')-4 gene was part of a resistance gene cluster, and its expression was under the control of the regulatory sequences of the beta-lactamase encoded by Tn3. The five N-terminal amino acids were identical to those of the signal peptide of the Tn3-encoded beta-lactamase, and the entire 5' region of aacA4, as far as it was sequenced (354 bp, including the promoter and the ribosome-binding site sequences), was identical to that of the beta-lactamase gene. This led us to presume an in vivo fusion between the beta-lactamase and the acetyltransferase genes. The latter was followed, in a polycistronic arrangement, by an aminoglycoside 3",9-adenylyltransferase gene, aadA, with an intergenic region of 68 bp. At a distance of ca. 1.3 kilobases in the 3' direction, we found remnants of a second Tn3-like element specifying an active beta-lactamase. At their 5' extremities, the two incomplete copies of Tn3, which were in tandem orientation, were interrupted within the resolvase gene. We speculate that Tn3-related sequences have played a role in the process of selection and dissemination of the AAC(6')-4 gene, which specifies resistance to amikacin and related aminoglycosides.     

Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.     

Beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae have retained an intact right part of a Tn3-like transposon.

In three beta-lactamase-producing strains of Neisseria gonorrhoeae, ampicillin resistance is due to the presence of a 7.4-kilobase plasmid. Heteroduplex analysis has shown that the R plasmid contains a 1.6-kilobase segment homologous to the right part (the region coding for the beta-lactamase) of the Tn3-like transposon Tn2301 the 1.6-kilobase DNA segment is not transposable, but it can give rise to a functional transposon, when linked to the left part of TN2301. This provides strong evidence that the R plasmids of N. gonorrhoeae are deletion derivatives of a plasmid that contained an entire TN3-like transposon.     

Molecular structure and interrelationships of multiresistance beta-lactamase transposons

Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes. Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin. Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components. Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation. Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative. Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes.     

Transposon Tn21, Flagship of the Floating Genome

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.     

Transposon Tn21, flagship of the floating genome.

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.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.     

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.