The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

Summary The genetic environment of plasmid-borne bla _TEM mutant genes, encoding nine distinct TEM-type extended-spectrum -lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine bla _TEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes bla _TEM-1 or _–2. A 6.6 kb DNA fragment of pCFF04 containing bla _TEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that bla _TEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was, in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with bla _TEM-3.     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

Summary A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3 ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3 ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Applications of transposition mutagenesis in antibiotic producing streptomycetes

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the no rmally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

IS231 and otherBacillus thuringiensis transposable elements: A review

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of -endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 fromClostridium perfringens and distantly related to IS4 and IS186 fromEscherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231 A inEscherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained inBacillus subtilis andB. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.     

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758 bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.     

On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette

Summary The aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that theaacC gene is coded by a precise insertion of 833 bp between theaadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 β-lactamase gene insertion in Tn2603. Upstream of theaacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression casssettes.     

Evolutionary relationships between catabolic pathways for aromatics: Conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids

Summary TOL plasmid pWW0 and plasmid NAH7 encode catabolic enzymes required for oxidative degradation of toluene and naphthalene, respectively. The gene order of the catabolic operon of NAH7 for salicylate oxidation was determined to be: promoter-nahG (the structural gene for salicylate hydroxylase)-nahH (catechol 2,3-dioxygenase)-nahI (hydroxymuconic semialdehyde dehydrogenase)-nahN (hydroxymuconic semialdehyde hydrolase)-nahL (2-oxopent-4-enoate hydratase). This order is identical to that of the isofunctional genes of TOL plasmid pWW0. The complete nucleotide sequence of nahH was determined and compared with that of xylE, the isofunctional gene of TOL plasmid pWW0. There were 20% and 16% differences in their nucleotide and amino acid sequences, respectively. The homology between the NAH7 and TOL pWW0 plasmids ends upstream of the Shine-Dalgarno sequences of nahH and xylE, but the homology continues downstream of these genes. This observation suggested that genes for the catechol oxidative enzymes of NAH7 and TOL pWW0 were derived from a common ancestral sequence which was transferred as a discrete segment of DNA between plasmids.     

Conjugal transfer of enterococcal transposons in Bacillus megaterium

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10^-6 to 6.6×10^-7. The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.     

Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^–5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing

Summary The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.