Some properties of the chloramphenicol resistance transposon Tn9

Summary We have isolated variants of the plasmid RTF which have received the transposon Tn9 from bacteriophage P1Cm. We have shown by the formation of heteroduplex molecules between one RTF: Tn9 derivative and R100.1 that Tn9 is homologous to the r-determinant region of R100.1 which carries the determinants for chloramphenicol resistance. This suggests that Tn9 was derived from an r-det like structure by deletion, possibly mediated by one of the flanking IS1 elements. In spite of the similarity in structure between Tn9 and r-det however, we have demonstrated two distinct differences in the behavior of these two elements: 1) Tn9 but nor r-det, is able to amplify, by a recA dependent mechanism, when cells harboring RTF::Tn9 are grown in the presence of chloramphenicol, and 2) Tn9, unlike r-det, does not form extrachromosomal circular molecules when RTF::Tn9 is tegrated into the bacterial chromosome.     

Specificity of introducing the transposon that determines chloramphenicol resistance (Tn9) into the chromosome of Escherichia coli K-12]

Translocation of Tn9 coding for chloramphenicol-resistance from lambdaatt80 genome into bacterial chromosome was studied. Three preferential sites of Tn9 integration, attTn9A, ATTTn9B and attTn9C, were found on the chromosome of Escherichia coli K-12; attTn9A was mapped between purD and rpoB loci, attTn9B was cotransducible with argG, attTn9C was located between 61 and 66 minutes of the standard E. coli K-12 genetic map. The integration of Tn9 in these att sites occurs with almost equal probability. Tn9 integrated in attTn9A shown significantly higher frequency of excision and P1-transduction frequency than Tn9 integrated in either of two other attTn9 sites.     

Insertion of a transposon for chloramphenicol resistance into bacteriophage Mu.

We have isolated mutants of bacteriophage Mu carrying the X mutations caused by the insertion of cam (Tn9), a transposon for chloramphenicol resistance. The Mu X cam mutants were obtained by selecting for heat-resistant survivors of a Mucts62, P1cam dilysogen. Like the previously described X mutants, Mu X cam mutants are defective prophages which can be excised from the host DNA at a frequency of 10(-5) to 10(-7) per cell. Tn9 insertions in Mu X cam mutants are located within 5000 base pairs of the left end of Mu DNA in a region that controls early replication functions of Mu. There is one EcoRI cleavage site in Tn9. The Tn9 transposon itself can be excised precisely from the Mu X cam mutants to generate wild type Mu. In most Mu X cam mutants, precise excision of Tn9 occurs at a low frequency (10(-6) per cell), whereas in some, the frequency is higher (10(-4) per cell). Mu X cam prophages can replicate after induction with the help of wild type Mu. The lysates containing Mu X cam particles, however, fail to transduce chloramphenicol resistance at a high frequency; Mu X cam mutants apparently have a cis dominant defect in integration.     

The Clostridium perfringens chloramphenicol resistance transposon Tn4451 excises precisely in Escherichia coli

Nucleotide sequence analysis of the Tn4451-deletion derivatives, pJIR47 and pJIR86, which were derived from Escherichia coli and Clostridium perfringens, respectively, showed that the deletion events that led to the formation of these plasmids were identical and precise. The results also showed that the termini of this C. perfringens-derived transposon contained imperfect 12-bp inverted repeat sequences which had some sequence similarity with the termini of Tn3-like transposons.     

Nature of the plasmids determining the unstable inheritance of transposon Tn9 in Escherichia coli K-12

Unstable inheritance of transposon Tn9 in the Escherichia coli strain KS7201 had been connected with its integration into a certain bacterial chromosome site (attTn9A). However, the present work shows that the transposon is situated within an unstable plasmid in this strain. A possibility of such plasmid's formation, as a result of a deletion of a part of bacteriophage lambda DNA, is shown.     

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae)

A gene (ORFB) from Streptomyces antibioticus (an oleandomycin producer) encoding a large, multifunctional polyketide synthase (PKS) was cloned and sequenced. Its product shows an internal duplication and a close similarity to the third subunit of the PKS involved in erythromycin biosynthesis by Saccharopolyspora erythraea, showing the equivalent nine active site domains in the same order along the polypeptide. An unusual feature of this ORF is the GC content of most of the sequence, which is surprisingly low, for a Streptomyces gene; the large number of codons with T in the third position is particularly striking. The last 800 by of the gene stand out as being normal in their GC content, this region corresponding almost exactly to the thioesterase domain of the gene and suggesting that this domain was a late addition to the PKS. Based on the high degree of similarity between the ORFB product and the third subunit of the erythromycin PKS and the occurrence nearby of a gene conferring oleandomycin resistance, it is possible that this gene might be involved in the biosynthesis of the oleandomycin lactone ring.     

Transfer and expression of the tetracyclin resistance transposon Tn925 in Acetobacterium woodii

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.     

Mutagenesis of dimeric plasmids by the transposon gamma delta (Tn1000).

The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon gamma delta (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single gamma delta insertion, the occasional recovery of transposon-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization. We show here that gamma delta-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec+.     

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae

Summary Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.     

The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.     

Detection of free cytoplasmic circles of transposon Tn9 multimers in Escherichia coli

Extrachromosomal circular DNA molecules consisting of IS1-cat repeats, (IS1-cat)_n, were isolated from an E. coli strain harboring nearly 30 copies of tandemly amplified transposon Tn9 located on the chromosome. The DNA circles were characterized by restriction analysis followed by Southern blotting and electron microscopic examination. Their size varied from approximately 5.5 kb to 53 kb.     

Participation of the uvrD gene in the precise excision of the Tn9 transposon

HfeTn5-(04,06) and IfeTn9 mutations increase efficiency of precise excision of Tn5, Tn10 and decrease that of Tn9. These mutations have been mapped in uvrD gene. In LFETn9 and UVRE502 mutants, the multicopy plasmid pEM61 carrying the cloned uvrD gene complements LFETn9- phenotype (Low Frequency Excision of Tn9). These results indicate that the uvrD gene product plays different role in excision of transposons with long and short inverted repeats. The mechanism of this effect is discussed.     

Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9'

In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region. The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1. The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied. The three types of cointegrates were found. Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene. These cointegrates contain three copies of IS1 and are of high stability. The cointegrates of the type II contain two entire copies of delta Tn9' (i.e. four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1. Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells. It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1.     

G418 resistance in the yeast Saccharomyces cerevisiae: comparison of the neomycin resistance genes from Tn5 and Tn903

Summary The neo genes of Tn5 and Tn903 (Tn601) coding for amigoglycoside phosphotransferase type II and type I, respectively, were joined to the yeast adc 1 promoter and trp1 terminator and introduced into yeast (Saccharomyces cerevisiae) cells. Transformants were obtained by direct selection for G418 resistance. Plasmids containing the Tn5 neo gene induced antibiotic resistance only at low frequency, whereas colonies transformed with the Tn903 neo gene could be selected at high frequency (300–400 transformants/g plasmid DNA). The resistance threshold of transformed strains was increased to 30 mg G418/ml by both genes and high level expression of the bacterial genes in yeast could be shown using an in vitro phosphotransferase assay. The results indicate that this system can be used for high frequency transformation of wild-type strains and might in addition be used for the identification and isolation of promoter-active sequences.Abbreviations adc 1 alcoholdehydrogenase I gene - APH aminoglycoside-3-phosphotransferase - leu2 -isopropylmalate dehydrogenase gene - neo aminoglycoside-3-phosphotransferase gene - trp1 N-(5-phosphoribosyl)-anthranilate isomerase gene Dedicated to Prof. Dr. Dr. h. c. Karl Esser on the occasion of his 65th birthday     

Reduced expression of Tn 10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together (Shales et al., 1980). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal BglII fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.