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.     

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.     

DNA damage differentially activates regional chromosomal loci for Tn7 transposition in Escherichia coli

The bacterial transposon Tn7 recognizes replicating DNA as a target with a preference for the region where DNA replication terminates in the Escherichia coli chromosome. It was previously shown that DNA double-strand breaks in the chromosome stimulate Tn7 transposition where transposition events occur broadly around the point of the DNA break. We show that individual DNA breaks actually activate a series of small regional hotspots in the chromosome for Tn7 insertion. These hotspots are fixed and become active only when a DNA break occurs in the same region of the chromosome. We find that the distribution of insertions around the break is not explained by the exonuclease activity of RecBCD moving the position of the DNA break, and stimulation of Tn7 transposition is not dependent on RecBCD. We show that other forms of DNA damage, like exposure to UV light, mitomycin C, or phleomycin, also stimulate Tn7 transposition. However, inducing the SOS response does not stimulate transposition. Tn7 transposition is not dependent on any known specific pathway of replication fork reactivation as a means of recognizing DNA break repair. Our results are consistent with the idea that Tn7 recognizes DNA replication involved in DNA repair and reveals discrete regions of the chromosome that are differentially activated as transposition targets.     

Independence of the processes of transposition and genome rearrangements induced by transposons

The data on the influence of the tnm mutations affecting transposition process on the deletion formation promoted by Tn and IS elements are presented. It was shown that the tnm mutations did not affect the frequency of deletion formation. The results of genetic analysis of the tnm mutant deficient in both transposition and genomic rearrangements induced by Tn9 inserted into lambda prophage, indicated that the mutant phenotype was caused by two different but linked mutations. A mutation affecting the process of genomic rearrangements was designated gerA2. The gerA2 mutation decreased sharply the frequency of rearrangements promoted by Tn9, Tn10 or Tn601 inserted into lambda prophage. However, this mutation had no influence upon transposition of the same Tn elements. The data obtained could be interpreted as indicating the independence of the processes of transposition and genomic rearrangements or as indication of the existence of specific steps of these processes.     

Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.     

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.     

Integration host factor plays a role in IS50 and Tn5 transposition.

In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.     

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.     

Temperature sensitivity of transposition of class II transposons

It has been reported that transposition of Tn3 is temperature-sensitive. The effect of temperature on the transposition of other class II bacterial transposable elements is reported here: Tn21, Tn501, Tn1721, Tn2501 and Tn3926 all also display temperature-sensitivity of transposition. The temperature at which the highest transposition frequency was observed varied between room temperature and 30 degrees C.     

The global bacterial regulator H-NS promotes transpososome formation and transposition in the Tn5 system

The histone-like nucleoid structuring protein (H-NS) is an important regulator of stress response and virulence genes in gram-negative bacteria. In addition to binding regulatory regions of genes in a structure-specific manner, H-NS also binds in a structure-specific manner to sites in the Tn10 transpososome, allowing it to act as a positive regulator of Tn10 transposition. This is the only example to date of H-NS regulating a transposition system by interacting directly with the transposition machinery. In general, transposition complexes tend to include segments of deformed DNA and given the capacity of H-NS to bind such structures, and the results from the Tn10 system, we asked if H-NS might regulate another transposition system (Tn5) by directly binding the transposition machinery. We show in the current work that H-NS does bind Tn5 transposition complexes and use hydroxyl radical footprinting to characterize the H-NS interaction with the Tn5 transpososome. We also show that H-NS can promote Tn5 transpososome formation in vitro, which correlates with the Tn5 system showing a dependence on H-NS for transposition in vivo. Taken together the results suggest that H-NS might play an important role in the regulation of many different bacterial transposition systems and thereby contribute directly to lateral gene transfer.     

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.     

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.     

Genetic organization of transposon Tn10

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.     

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.     

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.     

Copy Number Control of Tn5 Transposition

Transposition of Tn5 in Escherichia coli strains containing one or multiple copies of the transposable element was investigated. It was found that the overall frequency of transposition within a cell remained constant regardless of the number of copies of Tn5 present in that cell. Experiments measuring the transposition frequency of differentially marked Tn5s confirmed that the frequency of transposition of an individual Tn5 decreased proportionally with the total number of copies of the element present in a cell. The IS50R -encoded function, protein 2, which has previously been shown to be an inhibitor of transposition, is sufficient to mediate this inhibitory effect. The concentration of protein 2 in a cell appears to modulate the transposition of individual Tn5 elements in such a way that the overall transposition of Tn5 in a cell remains constant.     

Transpososome dynamics and regulation in Tn10 transposition

Tn10 is a bacterial transposon that transposes through a non-replicative mechanism. This mode of DNA transposition is widely used in bacteria and is also used by "DNA-based" transposons in eukaryotes. Tn10 has served as a paradigm for this mode of transposition and continues to provide novel insights into how steps in transposition reactions occur and how these steps are regulated. A common feature of transposition reactions is that they require the formation of a higher order protein-DNA complex called a transpososome. A major objective in the last few years has been to better understand the dynamics of transpososome assembly and progression through the course of transposition reactions. This problem is particularly interesting in the Tn10 system because two important host proteins, IHF and H-NS, have been implicated in regulating transpososome assembly and/or function. Interestingly, H-NS is an integral part of stress response pathways in bacteria, and its function is known to be sensitive to changes in environmental conditions. Consequently, H-NS may provide a means of allowing Tn10 to responed to changing environmental conditions. The current review focuses on the roles of both IHF and H-NS on Tn10 transposition.     

Effect of mutations in recB, recC, sbcB and recF genes controlling the homologous recombination in Escherichia coli cells on transposition of Tn1

The mutations in the genes controlling the homologous DNA recombination in Escherichia coli cells effect the efficiency of Tn1 transposition. Mutations in recB and recC genes decrease 50-fold the frequencies of Tn1 transposition. Introduction of an additional mutation in sbcB gene increase transposition frequency for three orders as compared with the one registered in wild type cells. Inactivation of sbcB gene in the wild type cells does not affect transposition significantly. Mutation in recF gene results in the great decrease of transposition when it is introduced into multiple recBC sbcB mutant, but not into the wild type bacteria. The possibility of two pathways for Tn1 transposition existing in Escherichia coli cells is discussed, as well as possibility of existence of similar stages in transposition and recombination controlled by the same genes.     

Transposition of Plasmid-Borne Tn10 Elements Does Not Exhibit Simple Length-Dependence

The transposition frequencies of Tn10 elements from the bacterial chromosome to an F epitome decrease 40% for every kilobase increase in transposon length. The basis for this relationship is not known. We have now examined complemented transposition of defective Tn10 elements off small multicopy plasmids. We find that length dependence in this situation is either reduced or absent, depending on the specific class of transposition events involved. These observations can be interpreted as evidence against the model that chromosomal length dependence occurs because of decay of a transposition-associated replicative complex. This interpretation is consistent with unrelated experiments suggesting that Tn10 transposition is normally nonreplicative. Alternative explanations of length dependence phenomena are discussed.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.