Relationships among the streptothricin resistance transposons Tn1825 and Tn1826 and the trimethoprim resistance transposon Tn7

The streptothricin resistance transposons Tn1825 and Tn1826 are closely related, based on physical and genetic characteristics, to the trimethoprim resistance transposon Tn7. These transposons may be considered to be members of a transposon family sharing in common the transposition functions and a basic streptomycin/spectinomycin resistance determinant but differing from one another with respect to particular additional resistance genes inserted to the left of the aadA gene.     

Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related.     

Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons

Tn21- and Tn3-related transposons are widespread and carry various resistance determinants. The insertion points of different resistance genes were precisely defined in Tn2424, Tn1696, Tn2410, Tn4000 and its derivatives and compared to the corresponding sites in Tn7, pSA, R388, R46, Tn2603, Tn1331 and in Tn3-related elements. Insertional 'hot spots' located at the 3' end of different genes comprised 55 nucleotides and yielded more than 90% homology to the corresponding consensus sequence, termed hs1. Elements of this class were found to direct recA-independent generation of deletions. Flanking the 5' ends, hs2 (CTAAAACAAAGTTA) comprised the terminal nucleotides of hs1. Functional properties of hot spots as recognition sites for site-specific recombination and regulation of gene expression indicate that they might be involved in transfer, stable inheritance and expression of prokaryotic genes.     

IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.     

The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene

The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.     

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.     

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.     

Characterization of transposon Tn5086, carrying the site-specifically inserted gene dhfrVII mediating trimethoprim resistance.

Two different enteric plasmids of widely separate origins were observed to carry a new 15.3-kb trimethoprim resistance transposon, Tn5086, also mediating resistance to mercuric ions and to a low level of sulfonamide. The trimethoprim resistance gene characterized from Tn5086 was found to be distinct from those found earlier and was designated type VII. Molecular analysis demonstrated that Tn5086 is closely related to Tn21. The internal part of Tn21 and Tn5086, the element referred to as the integron, was found to be different. First, the integron of Tn5086 contains a 0.62-kb cassette formed by the trimethoprim resistance gene dhfrVII and its immediate surroundings instead of the 0.86-kb aadA1 cassette of Tn21. Second, the integron of Tn5086 lacks a 4.2-kb segment 3' of sulI in Tn21. The dhfrVII gene commences with a UUG codon but was otherwise seen to be markedly related to the cassette genes dhfrI, dhfrV, and dhfrVI. The four related dihydrofolate reductases of 157 amino acids encoded by these genes contain a glutamate instead of the aspartic acid residue found at position 27 of the active center of the chromosomal enzyme from Escherichia coli.     

Tn5 carries a streptomycin resistance determinant downstream from the kanamycin resistance gene

In Rhizobium meliloti, Tn5 conferred resistance not only to kanamycin but to streptomycin, as well, in Escherichia coli, however only to kanamycin. Using in vitro recombinant DNA techniques, it was shown that the streptomycin resistance determinant was located downstream from the kanamycin resistance gene in the unique central region of Tn5. Expression of various cloned fragments of Tn5 suggested that both kanamycin and streptomycin resistance genes were transcribed from the same promoter. E. coli mutants allowing the expression of streptomycin resistance from Tn5 were isolated. The differential expression of the streptomycin resistance gene provides a simple selection/counterselection criterion, using only streptomycin in transfer experiments of Tn5 between E. coli and R. meliloti.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat.

We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.     

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.     

Transductional mapping of ksgB and a new Tn5-induced kasugamycin resistance gene, ksgD, in Escherichia coli K-12

We have mapped the Escherichia coli ksgB gene to min 36.5, 0.8 min from man and 0.7 min from aroD. A new kasugamycin resistance (Ksgr) gene, ksgD, has been isolated, using a transposon, Tn5. ksgD::TN5 is 44% cotransducible with sbcA, unlinked to trp, and unlinked to man (by P1 transduction). The ksgD::Tn5 has a late time of entry from HfrB7 (PO43). These data place ksgD clockwise from sbcA (which enters early from HfrB7) at min 30.4. The reistance of ksgB ksgD single and double mutant strains has been quantitated. Single mutations, ksgB or ksgD, gave resistance to 600 micrograms of kasugamycin per ml, whereas a ksgB ksgD strain was able to grow in the presence of kasugamycin levels in excess of 3,000 micrograms/ml. This indicates that the mechanisms of resistance coded for by the two genes are independent and synergistic.     

Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance

The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains.     

Characteristics of transposition of Tn7-like elements determining resistance to trimethoprim and streptomycin

The transposing elements Tn7, Tn1824, controlling the resistance to trimethoprim and Tn1925, Tn1826, carrying the streptothricin resistance genes were classified as a new transposon family on the basis of their physical structure. The comparative genetic analysis of the frequency, specificity and insertion orientation in different replicons, obtained in independent research systems in this study, demonstrated the identity of transposition characteristics of the transposons. The latter makes it possible to classify them as an independent transposon family. The peculiar feature of the Tn7-like elements family is their RecA-dependent transposition into the chromosome of Escherichia coli stimulated by bacteriophage Plkc transduction of the transposons.     

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.