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.     

Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2.

A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated. The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively. Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found. The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E. coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine. In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA). A region containing res-like sites was located immediately upstream of the partial tnpR gene. Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans. Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.     

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.     

Overproduction of the Tn3 transposition protein and its role in DNA transposition

Five single base pair mutations that increase expression of the tnpA (transposase) gene of the Tn3 transposon approximately 30-fold, but which still allow the gene to be regulated, have been isolated by using a generally applicable procedure that involves distally linked lac gene fusions. The mutations, which are all located in a region controlling initiation of translation of the tnpA gene, do not affect normal repression of tnpA by the tnpR gene product, and yield up to a 9000-fold increase in tnpA protein production when combined with a tnpR mutation and placed on a high copy number plasmid. The mutation yielding the highest expression level was separated from the fused lac gene segment by homologous recombination and was found to increase the rate of transposition without altering the nature of the transposition product; in cells defective in both the E. coli recA gene and the tnpR gene of tn3, cointegrate transposition-intermediate structures occur with the overproducing--as well as with the wild-type--tnpA gene. In the presence of a functional Tn3 tnpR gene or the related transposon delta gamma, such cointegrate structures are resolved into the final products of transposition.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.     

Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein.

The complete 11,139-nucleotide sequence of transposon Tn1721 has been determined. It contains three 38-bp inverted repeats, and (in this order) a new orfI, a resolution site (res), genes encoding resolvase (tnpR), transposase (tnpA), tetracycline-resistance (TcR) repressor (tetR), TcR (tetA) and a truncated transposase gene (tnpA'). The modulator origin of Tn1721 from at least three separate sources is supported by the distinctive codon usages of orfI, tnpR/tnpA and tetR/tetA, and by sequence similarities with Tn501 (tnpR/tnpA) and RP1 (tetR/tetA). The ORFI-encoded 56-kDa polypeptide exhibits features of a methyl-accepting chemotaxis protein (MCP) with a conserved signal domain and a potential transmembrane domain; this polypeptide cross-reacts with anti-MCP antiserum. Like chemotaxis genes, orfI is transcribed from a sigma 28-like promoter. The overexpressed orfI gene product interferes with MCP-dependent chemotaxis suggesting that it completes for soluble transducer protein(s) in the cell. The potential selective advantage of this novel transposon-borne gene is discussed.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

Purification of the Tn3 transposase and analysis of its binding to DNA

The transposase encoded by the tnpA gene of Tn3 is a protein specifically required for Tn3 transposition. We have purified it to homogeneity from an Escherichia coli strain containing a mutant Tn3 that overproduces transposase. About a 10-fold additional increase in transposase resulted from growth into stationary phase. The initial purification was guided by the presence of a protein band with the electrophoretic mobility of the tnpA gene product. The identity of the purified protein was proven by the agreement of five NH2-terminal amino acids with the nucleotide sequence of the A gene; this, in turn, fixed the initiation codon. Transposase formed large aggregates in the absence of Mg2+ at salt concentrations of 0.1 M or less. In nonaggregating conditions, it had 1 or 2 copies of 113,000-dalton protomers. Subsequent purifications exploited the rapid and simple assay of transposase-mediated retention of labeled DNA to a nitrocellulose filter. Transposase bound tightly to single-stranded DNA but weakly to intact duplex DNA. DNA binding did not require Mg2+ and was highly salt-resistant. Binding did not require specific sequences, because poly(dT) was as good a substrate as phi X174 viral DNA. The high DNA binding constant of 4 X 10(9) M-1 is about the same as for some single-stranded DNA binding proteins.     

Fine structure of transposition genes on Tn2603 and complementation of its tnpA and tnpR mutations by related transposons

The fine structure of the genes tnpA, tnpR and res of Tn2603 required for its own transposition, was determined. The order of the genes was tnpA-tnpR-res from the right end of the right hand side region in Tn2603, the tnpA and tnpR encoded gene products having molecular weights of 110,000 and 21,000, respectively. The 110,000 molecular weight polypeptides was absolutely required for replicon fusion as the first stage of transposition, and named transposase. On the other hand, the 21,000 molecular weight polypeptide was necessary for resolution of the cointegrate as the second stage of transposition, and named resolvase. We also examined the ability of various transposons, assumed to be closely related, to complement the tnpA and tnpR mutations of Tn2603. The results indicated that the mercury resistance transposon, Tn2613, and Tn501, can complement both genes, but TnAs and gamma delta cannot at all. Tn501 had much less efficiency of complementation for tnpA than Tn2613. We have also discovered that the transposition frequency of transposons in the tn2613 family systematically depend on their size of transposon.     

The transposon Tn1 as a probe for studying ColE1 structure and function.

Summary Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be -lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.     

The nucleotide sequence of the tnpA gene of Tn21.

The nucleotide sequence of the tnpA gene of Tn21 is presented. The transposase encoded by this gene is exactly the same length (988 amino acids) as the Tn501 transposase (4), and shows 72% homology overall with this protein, with greater homology towards the C-terminus. The sequence of the transposase is discussed in the context of the evolution of Class II transposable elements and of the characteristics of the enzyme's action.     

Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon.

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.     

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells. All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein. Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids. The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells. A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin. Two mutants with insertions within the structural gene for colicin E3 were also characterized. They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene. These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein. Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3.