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PubMed2003

Transposon mutagenesis of the mouse germline.

Sleeping Beauty is a synthetic "cut-and-paste" transposon of the Tc1/mariner class. The Sleeping Beauty transposase (SB) was constructed on the basis of a consensus sequence obtained from an alignment of 12 remnant elements cloned from the genomes of eight different fish species. Transposition of Sleeping Beauty elements has been observed in cultured cells, hepatocytes of adult mice, one-cell mouse embryos, and the germline of mice. SB has potential as a random germline insertional mutagen useful for in vivo gene trapping in mice. Previous work in our lab has demonstrated transposition in the male germline of mice and transmission of novel inserted transposons in offspring. To determine sequence preferences and mutagenicity of SB-mediated transposition, we cloned and analyzed 44 gene-trap transposon insertion sites from a panel of 30 mice. The distribution and sequence content flanking these cloned insertion sites was compared to 44 mock insertion sites randomly selected from the genome. We find that germline SB transposon insertion sites are AT-rich and the sequence ANNTANNT is favored compared to other TA dinucleotides. Local transposition occurs with insertions closely linked to the donor site roughly one-third of the time. We find that approximately 27% of the transposon insertions are in transcription units. Finally, we characterize an embryonic lethal mutation caused by endogenous splicing disruption in mice carrying a particular intron-inserted gene-trap transposon.

Animals↗
PubMed1980

Chi mutation in a transposon and the orientation-dependence of Chi phenotype.

Chi, an element that stimulates recombination via the E. coli RecBC pathway, can arise by spontaneous mutation in the transposon Tn5. When in phage lambda in one orientation, the mutant transposon confers Chi+ phenotype (large plaque and a high rate of exchange near the transposon). In the other orientation, however, the transposon does not confer Chi+ phenotype. The mobility of the transposon allows us to show that the Chi+ orientation of the mutant Tn5 is the same at different locations in lambda. These include a site near gene J, one in gam at 69, one to the right of gam at 73 and several to the right of R between 95.7 and 99.5. To the right of R, the mutant transposon could be found in only one orientation, that which confers Chi+ phenotype. We speculate that the other orientation of Tn5 in that locale is lethal to lambda. The orientation-dependence of Chi+ phenotype also revealed that Tn5 flip-flops in lambda.

Bacteriophage lambda↗
PubMed2000

Nature of transposon-mediated high-level gentamicin resistance in Enterococcus faecalis isolated in the United Kingdom.

Forty-two high-level gentamicin-resistant (MIC > 1000 mg/L) strains of Enterococcus faecalis, isolated from diverse geographical locations throughout the UK between 1993 and 1995, were studied to identify the nature of the high-level gentamicin-resistant determinants and the possibility of these determinants being associated with a transposon. High-level gentamicin resistance was attributed to the synthesis of the bifunctional (AAC6'-APH2") aminoglycoside-modifying enzyme. The aac6'-aph2" gene, which was present on a 70 kb plasmid in all 42 isolates, could be transferred by conjugation in association with the 70 kb plasmid in 39 of the isolates studied. In three E. faecalis isolates, however, the high-level gentamicin resistance was transferable independent of the 70 kb plasmid, suggesting the presence of a conjugative transposon. Long-PCR studies showed that all 42 clinical isolates harboured a transposon similar to Tn5281, originally identified in E. faecalis strain HH22 isolated in the USA. Restriction endonuclease and Southern hybridization analysis of the UK transposon showed that it is closely related to the high-level gentamicin resistance-conferring transposon Tn5281. However, the UK transposon lacks the HaeIII site identified in Tn5281. Pulsed-field gel electrophoresis analysis identified seven different patterns. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with nine different plasmid types in E. faecalis.

Anti-Bacterial Agents↗
PubMed2003

Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme.

OBJECTIVE: In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated. METHODS: The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach. RESULTS: Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland. CONCLUSIONS: The resistance determinant of P. aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

Amino Acid Sequence↗
PubMed2003

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms.

The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.

Binding Sites↗
PubMed1995

beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Base Sequence↗
PubMed2005

Gambol and Tc1 are two distinct families of DD34E transposons: analysis of the Anopheles gambiae genome expands the diversity of the IS630-Tc1-mariner superfamily.

Tc1 is a family of DNA transposons found in diverse organisms including vertebrates, invertebrates and fungi. Tc1 belongs to the IS630-Tc1-mariner superfamily, which is characterized by common 'TA' target site and conserved D(Asp)DE(Glu) or DDD catalytic triad. All functional Tc1-like transposons contain a transposase with a DD34E catalytic triad. We conducted a systematic analysis of DD34E transposons in the African malaria mosquito, Anopheles gambiae, using a reiterative and exhaustive search program. In addition to previously described Tc1-like elements, we uncovered 26 new DD34E transposons including a novel family that we named gambol. Designation of family status to gambol is based on phylogenetic analyses of transposase sequences that showed gambol and Tc1 transposons as distinct clades that were separated by mariner and other families of the IS630-Tc1-mariner superfamily. The distinction between Tc1 and gambol is also consistent with the unique TIRs in gambol elements and the presence of a 'W[I/L/V]DEDC' signature near their N-termini. This signature is predicted as part of the 'RED' domain, a component of the 'PAI' and 'RED' DNA binding domains in Tc1 and possibly mariner. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol has been reported in any other insects or animals thus far. Several gambol and Tc1 elements have intact ORFs and different genomic copies with high sequence identity, which suggests that they may have been recently active.

Amino Acid Sequence↗
PubMed1993

Deletions of Tn916-like transposons are implicated in tetM-mediated resistance in pathogenic Neisseria.

Using the tetM-containing conjugative transposon Tn916 as a mutagenesis tool, we identified two distinct classes of transposon insertions in the meningococcal chromosome. Class I insertions have an intact copy of Tn916 that appears to have transposed by a novel recombinational mechanism, similar to the transposition of conjugative transposons in Gram-positive bacteria. Class II insertions were characterized by deletions of Tn916 but preservation of the tetM determinant. In addition, we identified Class II Tn916-like insertions in the naturally occurring 25.2 MDa tetM-containing plasmids of both Neisseria meningitidis and Neisseria gonorrhoeae. The turncated Tn916-like insertions appeared to be present in the same site in these two plasmids; however, the deletions of the transposon were different. Plasmid sequence adjacent to the truncated transposon in the 25.2 MDa plasmids was found in a tetracycline-sensitive N. gonorrhoeae 24.5 MDa conjugative plasmid. These data suggest that the 25.2 MDa plasmids are the result of one or a series of Class II Tn916-like insertions into 24.5 MDa conjugative plasmids. Class II insertions of Tn916-like transposons are implicated in the dissemination of tetM resistance in pathogenic Neisseria.

Base Sequence↗
PubMed2001

Characterization of the 13-kilobase ermF region of the Bacteroides conjugative transposon CTnDOT.

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351 element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2, an open reading frame located immediately downstream of the integrase (int) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the Bacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverse Bacteroides population.

Anti-Bacterial Agents↗
PubMed2003

Selection on the genes of Euplotes crassus Tec1 and Tec2 transposons: evolutionary appearance of a programmed frameshift in a Tec2 gene encoding a tyrosine family site-specific recombinase.

The Tec1 and Tec2 transposons of the ciliate Euplotes crassus carry a gene for a tyrosine-type site-specific recombinase. The expression of the Tec2 gene apparently uses a programmed +1 frameshift. To test this hypothesis, we first examined whether this gene has evolved under purifying selection in Tec1 and Tec2. Each element carries three genes, and each has evolved under purifying selection for the function of its encoded protein, as evidenced by a dearth of nonsynonymous changes. This distortion of divergence is apparent in codons both 5' and 3' of the frameshift site. Thus, Tec2 transposons have diverged from each other while using a programmed +1 frameshift to produce recombinase, the function of which is under purifying selection. What might this function be? Tyrosine-type site-specific recombinases are extremely rare in eukaryotes, and Tec elements are the first known eukaryotic type II transposons to encode a site-specific recombinase. Tec elements also encode a widespread transposase. The Tec recombinase might function in transposition, resolve products of transposition (bacterial replicative transposons use recombinase or resolvase to separate joined replicons), or provide a function that benefits the ciliate host. Transposons in ciliated protozoa are removed from the macronucleus, and it has been proposed that the transposons provide this "excisase" activity.

Animals↗
PubMed1995

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

Animals↗
PubMed1984

Transposon mutagenesis of marine Vibrio spp.

Coliphage P1 was used to transduce derivatives of transposons Tn5 and mini-Mu into marine Vibrio spp. Transposon Tn5 encoding tetracycline resistance (Tn5-132) was used to isolate mutants of Vibrio harveyi defective in genes for bioluminescence (lux). Insertion of transposon Tn5-132 into the lux gene region was demonstrated by intraspecific transduction with phage hv-1 and by Southern blot hybridization. Transposon mini-Mu, modified to specify tetracycline resistance, was employed to mutagenize genes for lateral flagella synthesis of Vibrio parahaemolyticus. Mini-Mu contains the lacZ structural gene, and transposition results in transcriptional fusion of Vibrio genes with the transposon lacZ gene. Thus, in these fusions, lacZ expression was proportional to the level of transcription of the target gene. Regulation of lateral flagella gene expression was studied in vivo by measuring beta-galactosidase activity, and conditions which activate transcription of these genes were identified. A method for gene cloning with transposon-induced mutations is discussed.

Coliphages↗
PubMed1993

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.

Actinobacillus pleuropneumoniae↗
PubMed1993

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.

Bacteroides↗
PubMed1994

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853.

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.

Bacterial Proteins↗
PubMed1997

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.

Bacteroides fragilis↗
PubMed1999

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans.

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53.

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.

Bacterial Proteins↗