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.     

The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology

The inverted repeats of Tn5 are functionally different. One repeat codes for larger polypeptides, which are required for transposition. The other repeat has a better promoter for the neomycin resistance gene in the region of the repeat near the unique sequences. These dissimilarities are now shown to be caused by a single base pair difference. This change both creates a better promoter sequence and codes for part of a new UAA nonsense codon. Mutants in which the DNA sequence of a repeat is altered only at this base pair are shown to function like the opposite repeat. Furthermore, it is possible to suppress the UAA nonsense codon with an ochre suppressor, making the previously abbreviated polypeptides functional in transposition.     

The tetracycline resistance transposons Tn1721 and Tn1771 have three 38-base-pair repeats and generate five-base-pair direct repeats

Summary The 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a minor transposon and a tet region. Identical sequences of at least 87 base pairs providing recombination hot spots for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.To Professor Wolfram Heumann on the occasion of his 65th birthday     

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.     

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

A pathway for the evolution of the plasmid NTP16 involving the novel kanamycin resistance transposon Tn4352

The kanamycin resistance determinant of the drug resistance plasmid NTP16 has been characterized by DNA sequencing and has been shown to possess all of the structural features of a transposable element. It is made up of a 1040-bp central region encoding a protein identical to the aminoglycoside 3'-phosphotransferase of Tn903, flanked by direct repeats of an element identical to IS26. This novel transposon has been designated Tn4352. Analysis of the host sequences flanking the transposon reveal that they are derived from a Tn3-like element, and contain no 8 base pair target size duplications which are normally created by the insertion of IS26-like elements. Comparison to the Tn3 sequence shows that the flanking sequences are noncontiguous within Tn3, with the clear implication that NTP16 has evolved from a similar plasmid encoding only ampicillin resistance (presumably NTP1) by the insertion of Tn4352 into the Tn3-like element, followed by a substantial deletion. The sequence analysis suggests that the initial insertion was into the tnpR gene of the ampicillin transposon, followed by a deletion extending to a specific site within tnpA.     

Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10.

We describe an unusual DNA alteration, "nearly precise excision," which has been identified among tetracycline-sensitive deletion derivatives of lambda phages carrying the translocatable tetracycline-resistance element Tn10. DNA sequence analysis of two such derivatives demonstrates that each retains exactly 50 bp of Tn10 material. The original junctions between lambda and Tn10 sequences remain intact; however, an internal deletion has occurred within Tn10 which eliminates all but the last few base pairs at each end of the element. This deletion occurs within a short A + T-rich inverted repeat which is present near each end of Tn10. Nearly precise excisions occur at frequencies comparable to Tn10-promoted deletions, inversions and translocations, and, like these other events, are independent of phage and bacterial functions for homologous recombination (recA, recB, red). It is not yet clear, however, whether nearly precise excisions are specifically promoted by Tn10 or whether they arise during the course of normal DNA replication processes as a consequence of unusual symmetries present in the DNA sequence at the ends of Tn10.     

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5′GTATT3′ at the target site which included the first base at the 5′ end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5′GACAC3′ also involved the same direct repeat also at the 5′ end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5′ to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.     

The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR.

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.     

Transposon Tn951 (TnLac) is defective and related to Tn3

Summary Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene.In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition.Transposition of Tn951 duplicates 5 bp of target DNA sequence.     

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.     

The transposon Tn9 generates a 9 bp repeated sequence during integration.

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.     

Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3.

Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements. Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides. Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis. A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site. Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J. Bacteriol. 136: 742--756, 1978; Heffron et al., Proc. Natl. Acad. Sci U.S.A. 72:3632--3627, 1975). No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.     

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.