Properties of the Translocatable Tetracycline-Resistance Element Tn10 in ESCHERICHIA COLI and Bacteriophage Lambda

A number of independent insertions into bacteriophage lambda of the translocatable tetracycline-resistance element Tn10 have been isolated and characterized. The physical positions and relative orientations of several such insertions were determined. Two independent insertions appear to lie in the same orientation at or very near the same site in the cI gene, and two more lie in opposite orientations at or near the same position in or near the rex gene. Insertions in or near genes cI, rex, and cIII have been characterized genetically for their effects on expression of nearby genes. Tn10 appears to exert a polar effect on expression of distal genes when it is inserted within an operon, even when expression of that operon is under the influence of lambda N-function. In addition, Tn10 insertions in rex appear to influence in some way expression of an "upstream" gene, cI. Lambda derivatives carrying Tn10 give rise to spontaneously occurring, tetracycline-sensitive deletions at high frequencies. It is likely that formation of these deletions is promoted in some way by the Tn10 element. Lambda::Tn10 phages carrying a Tn10 element that has undergone several successive cycles of translocation since its first isolation and characterization have been analyzed. The results confirm that Tn10 often retains its physical and functional integrity during many cycles of translocation. Lambda derivatives carrying Tn10 have been used to generate insertions of Tn10 in the chromosome of Escherichia coli. This process is independent of recA function, and seems to be quite analogous to the translocation of Tn10 in Salmonella typhimurium as studied previously.     

Regional Specificity of Illegitimate Recombination by the Translocatable Amplicillin-Resistance Element Tn1 in the Genome of Phage P22

Insertions of the translocatable ampicillin-resistance element Tn1 were selected in the genome of the temperate Salmonella phage P22 by growing the phage on hosts carrying the resistance plasmid RP4. Insertions of Tn1 into phage P22 are rare (10(-10) per phage) and nonrandomly distributed in the P22 genome. They are found mainly in the vicinity of the P22 ant gene. Insertions within the ant gene are found at many (at least 15) genetically separable sites, are found equally frequently in both orientations and cause irreversible loss of gene function. Some insertions in ant appear to be associated with an adjecent deletion. Prophage deletions were derived from P22::Tn1 phages by two methods. Low multiplicity transductants have nonrandomly distributed endpoints. One end is at or very near the site of the Tn1 insertion, and the other is in the vicinity of gene 12; however, there are many genetically distinguishable endpoints within gene 12. Prophage deletions selected as survivors of induction of a P22Ap mnt-ts lysogen have similarly nonrandom endpoints, with the Tn1-distal end frequently near the ant gene, as well as gene 12. Physical analysis of several prophage deletions suggests that the Tn1 is intact to the resolution of DNA electron microscopy and that the deletions begin at the end of the Tn1 insertion. These results suggest that illegitimate recombination associated with Tn1 shows regional specificity (i.e., preference for some large areas of the P22 genome over other areas), but that within these regions is quite nonspecific.     

Specificity of Transposon Tn5 Insertion

Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.     

ESCHERICHIA COLI K-12 Auxotrophs Induced by Insertion of the Transposable Element Tn5

The sites of insertion of the transposable kanamycin-neomycin resistance-determining element, Tn5, in the E. coli K-12 chromosome were assessed in a collection of over 300 auxotrophs. Although mutations in at least 45 different cistrons were obtained, the distribution of insertion sites was not completely random: proA or proB; cysG; and cysH, cysD or cysC mutants were found in excess.     

Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra⁺) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10^?8 indicating adequate stability of Tn10 mutations.     

Transposon Tn5 Target Specificity: Preference for Insertion at G/C Pairs

The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly. Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion. In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter. We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling. We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold. The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.     

Intramolecular transposition by Tn10

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.     

Insertion site specificity of the transposon Tn3.

The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer. Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (approximately 37% of sites are potential targets). In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently. The 935 bp cold spot showed no unusual features when analysed with the consensus sequence. The consensus can be used to accurately predict likely insertion sites in a new sequence. Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised. These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor, such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.     

The tetracycline-resistance transposon Tn10 inhibits translocation of Tn10

Summary Using a set of overlapping deletion mutants in the tetracycline-resistance transposon Tn10, it has been established that certain regions of the Tn10 genome exert a powerful inhibition on translocation of an intact Tn10 element into the bacterial genome. Such inhibition is strongly temperature dependent: at 37° C translocation is inhibited by at least a factor of 100; no inhibition of translocation is detected at 30° C.     

Analysis of the Complete Nucleotide Sequence of the Tetracycline-Resistance Transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10^R100, demonstrating the conservation that exists between these transposons. A previously uncharacterized 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

Specificity of transposition of Tn7 in Vibrio parahaemolyticus

Tn7 was found to transpose at a high frequency from the plasmid, RP4::Tn7, to the chromosome of Vibrio parahaemolyticus. Seven isolates carrying Tn7 insertions were derived from three wild-type strains isolated from geographically distinct areas, and HindIII and BstEII DNA digests of these strains were probed with a ColE1::Tn7 biotinylated probe. The results indicated that V. parahaemolyticus is similar to several other species which have been studied in having a highly preferred site of insertion of Tn7 in the chromosome.     

Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance

Summary We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.     

A comparison of intramolecular rearrangements promoted by transposons Tn5 and Tn10.

The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target. Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert. The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative. In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements. Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates. This behaviour is fully consistent with a conservative mechanism. In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates. These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition. Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition. These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition.     

recA-dependent genetic switch generated by transposon Tn10

We describe a new type of regulatory switch generated in bacteriophage lambda by transposon Tn10. By this switch, phage genes alternate reversibly between expressed and non-expressed states as the direct consequence of a reversible DNA rearrangement. The switch itself has arisen via Tn10-promoted recombination. The subsequent “flip-flop” in gene expression occurs by general recombination between two IS10 elements serving as “portable regions of homology”.     

Transposable Element ISHp608 of Helicobacter pylori: Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity

A new member of the IS605 transposable element family, designated ISHp608, was found by subtractive hybridization in Helicobacter pylori. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (orfA and orfB), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes; orfB is also related to the Salmonella virulence gene gipA. PCR and hybridization tests showed that ISHp608 is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. ISHp608 also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively; P = 0.04). Two ISHp608 types differing by approximately 11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by < or = 2% in DNA sequence, but several recombinant elements were also found. ISHp608 marked with a resistance gene was found to (i) transpose in Escherichia coli; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require orfA but not orfB for its transposition. ISHp608 represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.     

Two Classes of Tn10 Transposase Mutants That Suppress Mutations in the Tn10 Terminal Inverted Repeat

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named ``SEM'''' for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.     

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.