PLASMID 12,218-221(1984)

The Role of Tn A Transposase in Transposition Immunity

JOHNHERITAGE ANDPETER M. BENNETT

University of Bristol, Department of Microbiology. The Medical School, University WaIk, Bristol BS8 1 TD, United Kingdom

Received June 26, 1984

The strength of expression of transposition immunity depends on the level of transposase available in the system, and on the location of the element to be transposed. 0 1984 Academic

Transposition immunity has been defined as the inability of a replicon carrying a copy of a transposon to acquire a second copy of that transposon by normal transposition (I- 3). In an attempt to define the sequence(s) within Tn4 which are responsible for the expression of this phenomenon, Wallace et al. (3) proposed that there existed an “Im- munity Site” located within the first 1000 bp on the left of the element. Furthermore, since plasmids carrying the terminal 450 bp, in- cluding the inverted repeat sequence, from the right of the element displayed no im- munity, it was concluded that the inverted repeat sequences played no part in transpo- sition immunity. In contrast, Lee et al. (I) demonstrated that a single copy of the Tn4 38-bp inverted-repeat sequence was sufficient to confer transposition immunity. In an at- tempt to resolve the apparent contradic- tion between these two reports, we have re- peated, in part, the work of Wallace et al. (3) by generating deletion derivatives of the R388: :Tn802 recombinant plasmid pUBSO 1 in vitro using the restriction endonuclease PstI. Plasmids R388, pUBSO 1, pUB305 1, pUB3052, pUB3053, and pUB3054 (Fig. 1) were then tested for their ability to acquire a copy of Tn4 from the chromosome of Esch- erichiu cofi UB1780 (Fig. 1). Retention of both ends of Tn4 conferred transposition immunity (Fig. 1 ), as was reported by Wallace

et al. (3). Transposition of Tn4 was observed onto derivatives which retain only the right- hand end of TnA; however, the frequency of transposition was about 20-fold lower than onto R388. Thus removal of the left-hand end of Tn4 relieves immunity, in part, but does show that the right-hand end of Tn4 is indeed capable of expressing transposition immunity (Fig. 1). In that the only sequences common to both ends of the element are the 38-bp inverted repeat sequences (4), then these data agree with those of Lee et al. (I), namely, that the inverted repeat sequence is necessary for the expression of transposition immunity. We would add, however, that some other sequence(s) within the element probably potentiates the effect with respect to the left-hand inverted repeat, in that im- munity conferred by the left-hand appears to be absolute (3).

Accepting that the immunity site is the terminal inverted repeat sequence (I), it seems likely that transposase is somehow implicated in the expression of transposition immunity, since this enzyme must recognise the extrem- ities of the transposon in order accurately to transpose the element. Wallace et al. (5) reported that transposition of tnpR mutants of T&4 onto an immune plasmid is not inhibited. In agreement with this, we have demonstrated transposition of the tnpR mu- tant Tn3.E.5 (4) from pUB1290 (6) to

0147-619X/84 $3.00 Copyright Q 1984 by Academic Press. Inc. All ri&ts of reproduction in any form reserved.

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Frequency of

transDOdtion products

(x 100000) RP B PP L BBIIP BP B

not detected pUB50 1

3- pUB305l

not detected - pUB3062

not detected - - pUB3063

5- pUB3054

100 - R388

FIG. 1. Restriction maps of the relevant sections of plasmids R388, pUBSOl, and its derivatives, and the frequency of transposition of Tn802 from UBl780 to these plasmids. The Tn802amp of pUBSO is ~npA+, mpR+, and rest. The bold lines denote Tn802 sequences. L and R denote the left- and right-hand inverted repeat sequences of Tn802 (4). Restriction sites are designated BumHI, B;Eg!lI, BII; and &I, P. The limit of detection of Tn802 transposition in these experiments was about IO-‘.

pUB501 (data not shown). Wallace et al. (5) suggested that the tnpR gene product itself was necessary for the expression of transpo- sition immunity, but the transposition of Tn3.E.5 from pUB1290 onto pUBSOl, which is itself tnpR+, is not consistent with this explanation, since tnpR gene product is pres- ent in trans. An alternative explanation for these results is that mutations in the tnpR gene derepress the tnpA gene (7), resulting in an increased transposase level, which, in turn, overcomes immunity. Consequently, one might expect that, although Tn4 will not transpose from the bacterial chromosome onto an immune plasmid, such as pUB50 1, it will transpose from a multicopy plasmid such as pACYC184. Plasmid pUB2583 is a pACYCl84: :Tn3 recombinant. The Tn4 element on this plasmid transposed to R388 at a frequency of 2.6 X 10p2, and onto pUBSOl, an immune derivative, at a fre- quency of 2 X lo-‘. Molecular analysis of a number of pUBSOl::Tn3 recombinants re- vealed two classes of molecule: in one the plasmids carry two copies of Tn4, as inverted repeats; in the other the plasmids carry a single copy of Tn4, but have, in addition, undergone deletion of pUB50 1 sequences. All deletions extend in the same direction from the site of insertion of Tn4 in pUB50 1.

These molecules were probably generated by insertion of the second copy of Tn4 into pUBSO as a direct repeat of the first copy. “Resolution” of the cointegrate-like structure by resolvase (8) would allow the generation of a hybrid transposon carrying the blu+ gene at the original site of insertion of Tn4 in pUB50 1, and the deletion of all the interven- ing sequences.

The above results suggested to us that the inverted repeat sequences present on an im- mune molecule might interact with the small amount of transposase normally present in a cell, thus sequestering it. If this is so, then transposition onto a nonimmune plasmid should be less efficient in the presence of an immune plasmid. We have tested a num- ber of R388derived recombinant plasmids known to express transposition immunity, and have found that they all depress trans- position of T&02 onto pUB307 by a factor of about 10 (data not shown). Interestingly, those plasmids carrying the left-hand end of Tn4 were more inhibitory than were recom- binants carrying just the right-hand end. R388 itself was not inhibitory. The simplest expla- nation for these results is that the inverted repeat sequence on the immune plasmid competes with those carried on the chromo- somal elements for transposase. This effec-

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tively lowers the concentration of available transposase, and, as a consequence, depresses the frequency of transposition.

One plasmid tested for its effect on trans- position of Tn,4 was pUB3065, a multicopy plasmid specifying chloramphenicol and tet- racycline resistance, the former carried on the Tn802-derived transposon Tn825. Tran- sposon Tn825 has the two internal PstI frag- ments from Tn802 replaced with a single PstI fragment carrying the cat gene of plasmid Sa (9). Thus Tn825 is able to confer resistance to chloramphenicol but it is tnpA, tnpR, res, and bla. Transposon Tn825 will transpose efficiently from pUB3065 to R388 when the missing transposition functions are supplied in tram from an intact copy of Tn4 carried on the chromosome or on a plasmid. In either case transposition of Tn 825 is more efficient than transposition of the comple- menting element. Hence, although plasmid R388 transferred readily from E. coli UB 1780 (R388, pUB3065) (frequency 10-l) transfer of carbenicillin resistance was not detected. In contrast in control experiments in the absence of pUB3065, carbenicillin resistance was routinely transferred at a frequency of about IO-’ relative to trimethoprim resistance (R388) (2). The limit of detection of carben- icillin resistance transfer in these experiments would have been lo-‘. In contrast to the lack of transfer of carbenicillin resistance, chloramphenicol resistance transferred at a frequency of about lop4 ( lop3 relative to that of trimethoprim resistance). Molecular anal- ysis of transconjugants revealed that these contained cointegrate molecules comprising R388 and pUB3065. These were not resolved by Tn4 resolvase, being res, but were resolved upon introduction into a recA+ host cell, as demonstrated by marker segregation, e.g., loss of tetracycline resistance in a PolA back- ground. Trimethoprim resistance and chlor- amphenicol resistance remained linked.

A control experiment using UB5201 which has no Tn4 inserted in the chromosome, instead of UB1780, as the host strain failed

to demonstrate transfer of chloramphenicol resistance. Therefore the transfer of chlor- amphenicol resistance from UB 1780 (R388, pUB3065) was as a consequence of Ttut- mediated transposition of Tn825, despite the fact that transposition of Tn4 itself was severely inhibited. In a further experiment, in which R388::TnA recombinants were se- lected by plating UB1780 (R388, pUB3065) on cephaloridine (3), it was possible to dem- onstrate that Tn4 does transpose in the pres- ence of Tn825, but the frequency of trans- position could not be quantified. Hence the Tn4 element does retain its ability to trans- pose from the chromosome in the presence of pUB3065 but the frequency of this event is greatly decreased. In another experiment, transposition of Tn4 onto R388, in the pres- ence of pUB3065, from the temperature sen- sitive plasmid pMR5, was demonstrated. The transposition frequency was about 10e5 com- pared with a frequency of 10e3 for Tn825. Again Tn825 transposed rather more effi- ciently than the complementing Tn4.

These data are in agreement with an old observation, namely, that Tn4 transposes from one plasmid to another more readily than from the chromosome to a plasmid (10). This preferential transposition of plas- mid-carried elements may indicate a prefer- ence for transposase to associate with inverted repeat sequences on plasmid rather than on chromosomal elements. In conclusion, this work indicates that the expression of trans- position immunity depends upon the pres- ence of an inverted repeat sequence on the potential recipient replicon. In addition, the observed degree of immunity will be dictated not only by the level of transposase present, but also by the location of the transposing element.

ACKNOWLEDGMENTS

This work was supported by a grant from the Medical Research Council (UK) to P.M.B. We thank Dr. Helen Dodd for the gift of plasmid pUB3003, the immediate precursor of pUB3065. We also thank Dr. John Grinsted

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for the gift of plasmid pUB2583, and for his helpful discussions.

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