A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

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.     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

Expression of Kanamycin resistance introduced byAgrobacteriutn binary vector intoNicotiana tabacum andAtropa belladonna

Kanamycin resistance gene was introduced into tobacco and Atropa belladonna cells by binary vectors, based on Agrobacterium, by means of inoculation of seedlings. The plasmid pGA472, which carries chimaeric kanamycin resistance gene expressed in plants was introduced by transformation into A. tumefaciens Bo542, harbouring pTiBo542 plasmid and A. rhizogenes 8196, carrying pRi8196 plasmids and the resulting two strains were used as binary vectors. Tobacco tumors induced by A. tumefaciens Bo542(pGA472) grew as undifferentiated, kanamycin resistant tissues. Those induced by A. rhizogenes 8196(pGA472) differentiated into transformed plants. When cultivated in vitro on 200 μg ml^-1 kanamycin medium, they showed yellow green sectoring, which was not selected out during vegetative propagation. Atropa belladonna tissues transformed by both A. tumefaciens Bo542(pGA472) and A. rhizogenes 8196(pGA472) differentiated plants which grew well on 200 μg ml^-1 kanamycin as green, non-sectoring plants; sensitive cells obviously did not divide at all. Selection of Atropa belladonna transformed tissues on kanamycin medium is much more efficient than selection of transformed tobacco tissues with introduced kanamycin resistance gene.     

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.     

Molecular properties of transmissible R factors of Haemophilus influenzae determing tetracycline resistance.

The tetracycline-resistant Haemophilus influenzae strains LU121 and FR16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU12U) has a mol. wt of 31.5 X 10(6) and that of the latter (pFR16017) has a mol. wt of 33 X 10(6). Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain LU121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR15017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR15017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.     

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.     

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

A pathway for the evolution of the plasmid NTP16 involving the novel kanamycin resistance transposon Tn4352

The kanamycin resistance determinant of the drug resistance plasmid NTP16 has been characterized by DNA sequencing and has been shown to possess all of the structural features of a transposable element. It is made up of a 1040-bp central region encoding a protein identical to the aminoglycoside 3'-phosphotransferase of Tn903, flanked by direct repeats of an element identical to IS26. This novel transposon has been designated Tn4352. Analysis of the host sequences flanking the transposon reveal that they are derived from a Tn3-like element, and contain no 8 base pair target size duplications which are normally created by the insertion of IS26-like elements. Comparison to the Tn3 sequence shows that the flanking sequences are noncontiguous within Tn3, with the clear implication that NTP16 has evolved from a similar plasmid encoding only ampicillin resistance (presumably NTP1) by the insertion of Tn4352 into the Tn3-like element, followed by a substantial deletion. The sequence analysis suggests that the initial insertion was into the tnpR gene of the ampicillin transposon, followed by a deletion extending to a specific site within tnpA.     

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.     

A novel large plasmid carrying multiple beta-lactam resistance genes isolated from a Klebsiella pneumoniae strain

Klebsiella pneumoniae clinical isolates were selected according to the results of antibiotic susceptibility tests. Most of them were resistant to multiple antibiotics, including ampicillin, ceftazidime, cefotaxime and aminoglycosides. Large plasmids were observed in these Kl. pneumoniae strains by pulsed-field gel electrophoresis with S1 nuclease digestion. The Kl. pneumoniae strains investigated produced one to two extrachromosomal bands with a mobility corresponding to 97 approximately 145 kbp linear DNA molecules. A 100 kbp plasmid, designated pK1, was observed in the multiply resistant strain K250. pK1 had sequences homologous to both the TEM-1 and the aphD probe which were associated with beta-lactam and aminoglycoside resistance. pK1 was transformed into Escherichia coli strain DH5alpha and was found to confer resistance to ampicillin, ceftazidime, cefotaxime and kanamycin. A 8 kbp BamHI DNA fragment of pK1 that carried the ampicillin resistance gene (minimum inhibitory concentration > 1000 microgram ml-1) was cloned into the BamHI site of pACYC184. Sequence determination showed that this cloned fragment carried a TEM-1 gene. These findings suggest that pK1 is novel in that it appears to carry genes for resistance to ampicillin, cefotaxime and ceftazidime, as well as kanamycin.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Molecular Genetics and Genomics - The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the...     

Robust multi-type plasmid modifications based on isothermal in vitro recombination

A robust strategy for multi-type plasmid modifications is developed based on the isothermal in vitro recombination technology, by which any combination of the sequence modifications can be efficiently achieved in plasmids at any desired position in a seamless manner. As an example, we showed that a plasmid modification with insertion of a GFP gene, deletion of a 623-bp fragment, and substitution of an ampicillin resistance gene by a kanamycin resistance gene was accomplished simultaneously by this method. Therefore, the isothermal in vitro recombination-based multi-type plasmid modification strategy is a useful approach for broad application prospects in molecular biology studies.     

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.