Absence of direct repeats flanking transposons resulting from intramolecular transposition

Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3. The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660. In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation. In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.     

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.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

A system for in vivo selection of genomic rearrangements with predetermined endpoints in Escherichia coli using modified Tn10 transposons

Using recombinant DNA techniques, the Tn10-specific tetA gene (coding for tetracycline resistance) has been mutagenized by insertion of a streptomycin-resistance or a kanamycin-resistance gene. The insertions occurred at loci separated by 920 bp. The mutated tetA fragments, respectively designated as Tes (for tetracycline-streptomycin) and Tek (for tetracycline-kanamycin), were subsequently cloned into a phage lambda cIII+cIts857cII+ in replacement of the att lambda region. The two recombinant phages are convenient delivery vehicles which permit the in vivo substitution of the tetA locus of any Tn10 insertion with the Tes or the Tek fragment. The procedure involves two selectable steps: (i) integration of a lambda-Tes (or lambda-Tek) prophage into the Tn10 of interest; (ii) excision of the prophage by a second exchange which leaves the extra resistance gene installed within the Tn10. A major interest of the system is that, once a bacterium carries both Tn10-Tes and Tn10-Tek insertions, a recombination event between the two Tn10 sequences can reconstitute an active tetA gene. This selectable event may be associated with the rearrangement of the sequences surrounding the transposons. This unique property of the "Tes and Tek" system makes it very useful for selection of genomic rearrangements using the Tn10-Tes and Tn10-Tek as predetermined endpoints. The successful isolation of a chromosomal inversion is reported.     

Single-strand conformational polymorphism and direct sequencing applied to carrier testing in families with ornithine transcarbamylase deficiency

Single-strand conformational polymorphism (SSCP) and direct sequencing were used to confirm or deny carrier status in three families with ornithine transcarbamylase (OTC) enzyme deficiency. Two male probands with late onset OTC deficiency, whose private mutations were previously characterized, inherited the mutations form their heterozygous mothers. One of the heterozygous mothers had a false negative allopurinol test. Three female siblings of the two male probands were tested, one proved to be a carrier of the respective mutation while the other two were found to have normal alleles. In the third family, the proband was a female with late onset presentation of OTC deficiency. We found a new point mutation in this girl consisting of a guanine-tocytosine transversion at nucleotide 520 resulting in a substitution of proline for alanine at amino acid 142 of the mature OTC protein. We confirmed that this mutation occurred spontaneously and that neither of the two parents carries this mutation. We conclude that SSCP, in conjunction with direct sequencing, is a useful technique that can be practically applied for carrier testing in families with OTC deficiency.     

Nucleotide sequence of the kanamycin resistance transposon Tn903

The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.     

Analysis of Tn3 sequences required for transposition and immunity

Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity.     

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.     

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.     

Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E. coli

To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.     

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.     

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn 5053 ) has been determined. Tn 5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD , and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn 21 and Tn 501 . The transposition module of Tn 5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ , and tniR . Transposition of Tn 5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR . The same pathway of transposition is used by Tn 402 (Tn 5090 ) which carries the integron of R751. Transposition genes of Tn 5053 and Tn 402 are interchangeable. Sequence analysis suggests that Tn 5053 and Tn 402 are representatives of a new family of transposable elements, which fall into a recently recognized superfamily of transposons including retroviruses, insertion sequences of the IS 3 family, and transposons Tn 552 and Tn 7 . We suggest that the tni genes were involved in the dissemination of integrons.     

tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution

We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein. The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo. A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences. Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments. Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene. We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

Factors that affect transposition mediated by the Tn21 transposase

The frequencies of one-ended transposition mediated by the Tn21 transposase acting on plasmids containing 38-bp inverted repeat sequences (IRs) of both Tn21 and of Tn501/Tn1721 and Tn2501 were measured. The enzyme acted on all these IRs, but more efficiently on the homologous sequences. These differences were magnified when the enzyme acted on plasmids containing two copies of the IRs, inverted with respect to each other. The Tn21 enzyme did not recognize the IR of Tn3. The Tn501 transposase did not mediate measurable one-ended transposition of any of the plasmids used, including those containing an IR of Tn501.     

Tn5253, the pneumococcal omega (cat tet) BM6001 element, is a composite structure of two conjugative transposons, Tn5251 and Tn5252.

Tn5253, carrying tetracycline and chloramphenicol resistance determinants, is a 65.5-kb conjugative transposon originally detected in the chromosome of Streptococcus pneumoniae BM6001. We have identified an 18-kb segment of DNA carrying the tet determinant within Tn5253 to be an independent conjugative transposon when removed from the context of the larger element. In vivo deletion of this DNA segment, now termed Tn5251, from within Tn5253 did not affect the conjugative transposition properties of the remaining sequences. Thus, Tn5253 is a composite element of two conjugative structures: Tn5252, constituting the sequences beyond Tn5251 within Tn5253, and Tn5251. The transfer properties of Tn5252 and Tn5251 suggest that these may belong to two different classes of mobile elements even though they were initially found associated. The notion that a tet-carrying transposon like Tn5251 may have been the ancestral element in the evolution of the larger streptococcal conjugative transposons must be reevaluated in the light of present observations.     

Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40-54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all beta-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the beta-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.     

Transposition of the carbenicillin-hydrolyzing beta-lactamase gene

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.