Kanamycin Resistance Transposon Research Articles

Julian Davies and the discovery of kanamycin resistance transposon Tn5.

This paper recounts some of my fond memories of a collaboration between Julian Davies and myself that started in 1974 in Geneva and that led to our serendipitous discovery of the bacterial kanamycin resistance transposon Tn5, and aspects of the lasting positive impact of our interaction and discovery on me and the community. Tn5 was one of the first antibiotic resistance transposons to be found. Its analysis over the ensuing decades provided valuable insights into mechanisms and control of transposition, and led to its use as a much-valued tool in diverse areas of molecular genetics, as also will be discussed here.

IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit.

ABSTRACTWe recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end.

Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test.The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.

Molecular cloning of cellobiose and other β-glucosidase determinants from Klebsiella oxytoca

A gene bank of the β-glucoside-utilizing Klebsiella oxytoca DNA has been constructed using cosmid pHC79 in Escherichia coli K12. The DNA encoding cellobiose, salicin and arbutin utilization as well as esculin hydrolysis have been isolated from this gene bank by direct selection on indicator plates. Subsequent subcloning into the kanamycin resistance transposon Tn 5 has created a transposable β-glucosidase element (Tn 5 bgl) with an insert of approximately 5 kb. Insertion of Tn 5 bgl into the broad host range plasmid pR751 allowed transfer to and expression of β-glucosidase activity in a range of Gram negative bacteria. Two polypeptides were found to be encoded by the cloned DNA fragment.

A small nonconjugative plasmid encoded for streptomycin-resistant character in Shigella dysenteriae.

Conjugative transfers of drug-resistant plasmids in Shigella dysenteriae type 1 isolated from the epidemic in West Bengal were unsuccessful. The plasmids were non-transmissible in spite of having the fertility factor F, genetically labelled with kanamycin-resistant transposon Tn903 (pWS 7). A small 2.5 kb nonconjugative plasmid encoded for the streptomycin-resistant character. Cured plasmid-less strains of S. dysenteriae 1 showed resistance to sulfonamides.

Construction of transposons encoding genes for β-glucosidase, amylase and polygalacturonatetrans-eliminase fromKlebsiella oxytoca and their expression in a range of gram-negative bacteria

DNA fragments containing theKlebsiella oxytoca genes encoding β-glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.

Conjugal mobility of the multicopy plasmids NTP1 and NTP16

Mobilization of the multicopy plasmid NTP16, like that of ColE1, is promoted by a range of conjugal plasmids. However, the mechanisms employed for NTP16 mobilization differ between groups. Mobilization by the IncI1 plasmid R64 requires trans-acting products from NTP16 plus a cis-acting region of the small plasmid. In contrast, this system is used inefficiently by the F plasmid and instead, a high-frequency conduction process occurs. Analysis of exconjugant cells reveals that F-mediated mobilization of NTP16 frequently involves rearrangements of NTP16 DNA, promoted by the Tn1000 transposon of F and/or by the kanamycin resistance transposon (Tn4352) of NTP16. Possible mechanisms for the high-frequency F-mediated mobilization of NTP16 are discussed. The plasmid NTP1, which is closely related to NTP16, is also mobilized efficiently by R64. It is not however efficiently mobilized by F, demonstrating the requirement for the Tn4352 element, which is not present in this plasmid, for effective F-mediated transfer.

Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001.

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.

Pseudomonas mutants able to accumulate phenolic and catecholic 9,10-secosteroids from bile acids

An NTG induced mutant of a bile acid utilizing strain of Pseudomonas putida was isolated which was blocked in the steroid catabolic pathway. This mutant (PS5-7) was able to accumulate both phenolic and catecholic secosteroid products from cholic acid. Using a transposing system containing the kanamycin resistance transposon Tn5, mutants of PS5-7 were isolated which produced only the phenolic secosteroid. One of these mutants (PS5-25) was able to produce close to theoretical yields of various phenolic secosteroids from corresponding bile acids.

Expression ofRhodopseudomonas sphaeroides carotenoid photopigment genes in phylogenetically related nonphotosynthetic bacteria

The carotenoid photopigment genes of the purple nonsulfur photosynthetic bacteriumRhodospeudomonas sphaeroides have been cloned into the kanamycin resistance transposon Tn5 to create a carotenoid transposon-Tn5-Crt+. Transposition of Tn5-Crt+ onto the broad host range plasmid pR751 produced a broad host range carotenoid plasmid-pJP115. Transfer of pJP115 to the phylogenetically related nonphotosynthetic bacteriaParacoccus denitrificans, Agrobacterium tumefaciens, Agrobacterium radiobacter, andAzotomonas insolita resulted in carotenoid biosynthesis by these strains. Expression of carotenoid photopigment genes in nonphotosynthetic bacteria lends credence to the evolutionary relationships between photosynthetic and non-photosynthetic bacteria.

Characterization of the drug resistance plasmid NTP16

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.

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 transposn 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.

Construction of a transposon containing a gene for polygalacturonate trans-eliminase from Klebsiella oxytoca.

A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5. This new transposon, designated Tn5-Pga+, had a transposition frequency of 1 X 10(-6). The broad host range plasmid pR751::Tn5-Pga+ was conjugally transferred to a variety of genetic backgrounds. The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.

Contributions oftraTandissgenes to the serum resistance phenotype of plasmid ColV2-K94

A genetic determinant for serum resistance, designated iss, has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT, on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT+ Iss+) contained the kanamycin (Km) resistance transposon Tn903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT+), pWS16 (TraT+) and pWS18 (TraT+ Iss+) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.

Evidence for clustered pathogenicity genes in Xanthomonas campestris pv. campestris

Plasmid pIJ3000 contains DNA from the plant pathogen Xanthomonas campestris pv. campestris which restores symptom production to a non-pathogenic mutant. pIJ3000 was mutagenised with the kanamycin resistance transposon Tn5 in Escherichia coli, and the sites of insertion were mapped physically. Derivatives of pIJ3000 containing Tn5 inserted in X. c. campestris DNA were introduced into a wild-type X. c. campestris strain, and the Tn5 insertions recombined into the genome by a marker exchange process. The resulting strains were tested for pathogenicity to turnip seedlings.

A vehicle for the introduction of transposons into plant-associated pseudomonads

A recombinant plasmid with wide-host-range transfer functions, narrow-host-range replication functions, and carrying a kanamycin-resistant transposon transferred kanamycin resistance to a number of plant-associated pseudomonads. Southern hybridization studies suggest that only a small portion of the plasmid, coinciding with the location of the transposon, is present in the kanamycin-resistant Pseudomonas derivatives. The plasmid sequences appear to be inserted at a number of different sites in the recipient genome. This plasmid can thus be used as a vehicle for the introduction of transposons into some plant-associated pseudomonads and should be useful in both genetic and ecological studies of these bacteria.

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production.

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.

IS50-mediated inverse transposition: specificity and precision

The IS 50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn 5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a λ :: Tn 5 phage genome are composed of two overlapping transposons — the kan segment bracketed by IS 50 elements (Tn 5), and λ bracketed by IS 50 elements. During direct transposition, mediated by IS 50 “O” (outside) ends, the kan gene is moved and the λ vector is left behind. During inverse transposition, mediated by the “I” (inside) ends of the IS 50 elements, the λ vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a λ :: Tn 5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn 5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS 50 junctions, and in eleven others by sequencing one junction. The sequence of the “I” end of IS 50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of “I” ends is similar in its specificity and precision to the more frequent transposition mediated by IS 50 “O” ends.

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (Km R) transposon Tn5 was constructed (Tn 5-751) that carried the trimethoprim-resistance (Tp R) determinant from plasmid R 751 as an additional marker. Double selection for Km R and Tp R avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasmid RP1, proved effective; pME305 is temperature-sensitive at 43°C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS 21 and the Km R and primase genes. In matings with an E. coli donor carrying pME9 (= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 × 10 −7/donor at 43°C. Among Tn 5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn 5-751 has a single EcoRI site between the Tp R and Km R genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.