target specificity of insertion element is30

Target specificity of insertion element IS 30

Ferenc Olasz, 1,2* Janos Kiss, 2 Peter Ko nig, 1 ZsuzsaBuzas,3 Rolf Stalder 1 and Werner Arber 1

1Biozentrum der Universitat Basel, AbteilungMikrobiologie, Klingelbergstrasse 70, CH-4056 Basle,Switzerland.Institutes for 2Molecular Genetics and 3Biochemistry,Agricultural Biotechnology Centre, Szent-Gyorgyi Albertu. 4, H-2101 Godollo, Hungary.

Summary

The Escherichia coli resident mobile element IS 30 haspronounced target specificity. Upon transposition, theelement frequently inserts exactly into the same posi-tion of a preferred target sequence. Insertion sites inphages, plasmids and in the genome of E. coli arecharacterized by an exceptionally long palindromicconsensus sequence that provides strong specificityfor IS 30 insertions, despite a relatively high level ofdegeneracy. This 24-bp-long region alone determinesthe attractiveness of the target DNA and the exactposition of IS 30 insertion. The divergence of a targetsite from the consensus and the occurrence of ‘non-permitted’ bases in certain positions influence thetarget activity. Differences in attractiveness are empha-sized if two targets are present in the same replicon, aswas demonstrated by quantitative analysis. In a systemof competitive targets, the oligonucleotide sequencerepresenting the consensus of genomic IS 30 inser-tion sites proved to be the most efficient target. Havingcompared the known insertion sites, we suppose thatIS30-like target specificity, which may represent analternative strategy in target selection among mobileelements, is characteristic of the insertion sequencesIS3, IS6 and IS21, too.

Introduction

Mobile genetic elements in bacteria exhibit different pat-terns of target specificity (for a recent review, see Craig,1997). Earlier classifications suggested by Iida et al.(1983) divided the elements into three major groups. Thefirst group of elements (such as bacteriophage Mu) insertsnearly randomly into target sequences (for a review, see

Pato, 1989). The ISs belonging to the second group havepreferences for larger regions (in the order of 100 bp) andcan be inserted into several sites within these regions(IS1: Galas et al., 1980; Zerbib et al., 1985; IS2: Sengstaget al., 1983; Sengstag and Arber, 1987; Szeverenyi et al.,1996a,b; and IS50: Berg et al., 1983; Lupski et al., 1984).The target specificity of members in the third group can becharacterized by a consensus sequence of various lengths(e.g. IS4: Mayaux et al., 1984; IS5: Engler and van Bree,1981; IS10: Foster, 1977; Halling and Kleckner, 1982), inwhich the frequency of transposition may depend on thedivergence of a given target sequence from the consen-sus. Transposon Tn7 shows particular target site selectiv-ity. It transposes frequently into a unique sequence calledatt Tn7, but other sites can also be used (for a review, seeCraig, 1996; 1997).

The Escherichia coli mobile element IS30 mediatessimple insertion, inversion, deletion and transpositionalfusion and, upon these processes, 2 bp of the targetsequence are duplicated (Caspers et al., 1984). The1221 bp element contains one large open reading frame(ORFA) with a coding capacity for a 44.3 kDa protein (Dal-rymple et al., 1984). The 17 kDa N-terminal part of thetransposase protein interacts specifically with the terminalinverted repeats (IRs) of IS30 (Stalder et al., 1990).

The transposition of IS30 occurs via a very active andunstable (IS30)2 intermediate (Dalrymple, 1987; Olaszet al., 1993; 1997; Farkas et al., 1996; Fig. 1B), which isformed by site-specific dimerization and composed oftwo directly repeated elements that are generally sepa-rated by 2 bp, occasionally 1 or 3 bp (T. Naas, A. Ariniand W. Arber, unpublished). The dimer structure is fre-quently produced from composite transposons and, ingeneral, from replicons carrying two IS30 elements (Fig.1A). Inverse transposition, the most preferred reaction ofthe IS30 composite transposon Tn2706, and transposi-tional fusion mediated by a single copy of IS30 are pre-sented schematically in Fig. 1B. (IS)2-like transpositionalintermediates have also been detected with IS21 (Reim-mann et al., 1989) and IS2 (Szeverenyi et al., 1996a).(IS30)2 generates transpositional fusion, deletion andinversion during interaction with different types of targets.Further IR–IR junctions, resulting from the formation of cir-cular elements, were referred for Tc1 (Rose and Snutch,1984), Tn10 (Morisato and Kleckner, 1987), IS1 (Turlanand Chandler, 1995), IS911 (Polard and Chandler, 1995;Polard et al., 1996; Ton-Hoang et al., 1997) IS2 (Lewisand Grindley, 1997) and IS30 (J. Kiss, unpublished), but

Molecular Microbiology (1998) 28(4), 691–704

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Received 9 December, 1997; revised 4 February, 1998; accepted 12February, 1998. *For correspondence at the Institute for MolecularGenetics. E-mail [email protected]; Tel. (28) 430600; Fax (28)430416.

in these cases the possibility of dimerization was notinvestigated.

The ends of IS30 serve as functional target sites for thetransposition of the element. The 26-bp-long IRs alone aresufficient in these reactions, but their attractiveness can beinfluenced by bordering sequences. The functional part ofIRs corresponds to the binding site of IS30 transposase(Olasz et al., 1997).

IS30 was isolated independently several times in thegenome of P1 prophage exactly in the same position (Cas-pers et al., 1984), and this finding suggested a pronouncedtarget specificity. Non-random distribution of insertionswas also found during transpositional fusion or simpleinsertion of an IS30 composite transposon (Caspers etal., 1984; Stalder and Arber, 1989), which also supportedthe primary observation.

We report in this study that IS30 frequently transposesinto certain DNA sequences. The analysis of these targetsites resulted in the deduction of a nearly perfect palin-drome consensus sequence, in which the central two posi-tions are occupied by the bases that are duplicated upon

insertion. The unusually long (24 bp) consensus sequenceexhibits a high degree of degeneracy. The sequencessynthesized on the basis of the consensus showed allthe characteristics of a hot-spot. Quantitative analysis ofthe activities of different target sequences allowed theirranking and revealed some features of their interactions.Furthermore, it turned out that target sequence GOHSsynthesized on the basis of genomic insertion sites wasby far the most preferred hot-spot investigated so far.

Results

Detection of IS30 hot-spots

In preliminary studies of IS30 transposition, non-randomutilization of target sites was observed (Caspers et al.,1984; Stalder and Arber, 1989). In order to investigatethe target specificity of IS30, the P1HS segment (P1hot-spot), in which the first IS30 insertions were foundin phage P1 (Caspers et al., 1984), was cloned in the l

phage derivative lgt11 (for relevant properties of phagesand plasmids, see Table 1). The resulting phagelgt11::P1HS was offered as target in transpositionalexperiments (see Experimental procedures), in which theKmR donor plasmid pAW332 contained the CmR compositetransposon Tn2706. The products of transposition weredetected as CmR transductant colonies at a frequency ofabout 10¹8 (Table 2). The CmR colonies representedtwo different types of rearrangements: insertion (CmRKmS

phenotype) and co-integration (CmRKmR phenotype) ofTn2706. Phage production of the transductant colonieswas also investigated: phage-producing (Phþ) and non-producing (Ph¹) phenotypes indicated insertions intonon-essential and essential genes of bacteriophage l res-pectively. In the majority of transducing phages, Tn2706targeted non-essential genes (CmRKmSPhþ phenotype).The relatively small number of CmRKmRPhþ andCmRKmRPh¹ colonies can be explained by the low effi-ciency of IS30-mediated co-integration in a recA¹ geneticbackground (Olasz et al., 1993).

Phage DNA was purified from independent CmRKmSPhþ

colonies (representing simple insertions of Tn2706), and thelocation of insertions was determined (Fig. 2). The clonedP1HS segment preserved its hot-spot function: out of 13insertions, six were found in this region (Fig. 2A). All thesequenced insertions in P1HS (K6, K9 and K10) were inthe same position as the first three IS30 isolates (positions1027–1028; Sengstag et al., 1983). A new hot-spot calledLHS (lambda hot-spot) was also discovered in lgt11::P1HS(five out of 13 insertion events). It was proved that LHSwas used as target by Tn2706 in phage lgt11, too (fiveout of eight insertions; Fig. 2B), and all the insertionswere in the same position (33291–33292 bp of wild-typel). Other insertion sites in l DNA (K8, K11, K17, K25and K27) were targeted only once.

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Fig. 1. The schematic presentation of the site-specific dimerization(A) and transpositional fusion (B) mediated by IS30. The IS elementis represented by a box carrying open and filled triangles indicatingthe left and right IR ends respectively. Small arrows indicate theactive IR ends and target site in the given reaction. Grey circle,target region for transposition; thin line, IS30 donor replicon; thickline, target replicon; KmR, ApR and CmR, selectable reporter genes.

692 F. Olasz et al.


Table 1. Description of plasmids and bacteriophages used in transposition experiments.

Name Relevant properties Reference

Donor plasmidspAW332 KmR CmR pACYC177 derivative containing the IS30 composite transposon Tn2706 Olasz et al. (1993)pAW1039 KmR pACYC177 derivative containing (IS30)2, a tandem dimer of IS30 Olasz et al. (1993)

TargetsBacteriophages

lgt11 l bacteriophage derivative containing lacZ gene Young and Davis(1983)

lgt11::P1HS lgt11 containing P1HS (Sengstag et al., 1983) from bacteriophage P1 This work

Plasmids withone target site

Target region

pEMBL19 none ApR cloning vector Dente et al.(1983)

pAW782 LHS The 260 bp Sal I–XhoI fragment containing a hot spot from phage l (33 244–33 498 bp) was cloned into the Sal I site of pEMBL19

Olasz et al. (1993)

pAW1016 P1HS The 96 bp Taq I fragment containing a hot spot from phage P1 (989–1086 bp;Sengstag et al., 1983) was cloned into Acc I site of pUC7 (Vieira and Messing,1982) and its 160 bp EcoRI fragment was inserted into the EcoRI site of pEMBL19

This work

pAW780 K8HS The 598 bp ClaI–Bgl I fragment containing a hot-spot from l phage (30 290–30 888 bp) was blunt ended and ligated into the SmaI site of pEMBL19

This work

pAW495 POHS The synthesized oligonucleotide 58-TCGACAAAAATTGCAGCTGCAATTTTTG-38was annealed and ligated into the Sal I site of pEMBL19

This work

pFOL201 LSHS The oligonucleotide 58-CTAAATTCACTATCGCCACTTTTA-38 and its complementarystrand were synthesized, annealed and ligated into the SmaI site of pEMBL19

This work

pFOL682 1/2POHS682 The Pvu II–EcoRI fragment of pAW495 containing the half POHS sequence wascloned into the SmaI–EcoRI site of pEMBL18 (Dente et al., 1983)

This work

pFOL820 1/2POHS820 The Pvu II–EcoRI fragment of pAW495 containing the half POHS sequence wascloned into the HincII–EcoRI site of pEMBL18

This work

pFOL821 1/2POHS821 The Pvu II–HindIII fragment of pAW495 containing the half POHS sequence wascloned into the SmaI–HindIII site of pEMBL18

This work

pFOL822 1/2POHS822 The Pvu II–HindIII fragment of pAW495 containing the half POHS sequence wascloned into the HincII–HindIII site of pEMBL18

This work

pFOL546 GOHS The palindromic oligonucleotide 58-TAAAAATGGCGATCGCCATTTTTA-38 wassynthesized, annealed and ligated into the SmaI site of pEMBL19

This work

Plasmids withtwo target sitesa

Targetregion 1

Targetregion 2

pFOL387 LSHS LHS Derivative of pFOL201 and pAW782 This workpAW432 POHS K8HS Derivative of AW1063 and pAW780 This workpAW1193 P1HS K8HS Derivative of pAW1016 and pAW780 This workpFOL17 P1HS POHS Derivative of pAW1016 and pAW495 This workpAW1109 LHS K8HS Derivative of pAW782 and pAW780 This workpAW1153 LHS POHS Derivative of pAW782 and pAW495 This workpFOL388 LSHS POHS Derivative of pFOL201 and pAW495 This workpAW1028 LHS P1HS Derivative of pAW782 and pAW1016 This workpFOL519 GOHS POHS Derivative of pFOL546 and pAW495 This workpFOL518 GOHS LSHS Derivative of pFOL546 and pFOL201 This work

a. Plasmids containing two target sites were constructed from plasmids with single target sites by multiple cloning steps. The cloning vector wasidentical (pEMBL) in all constructs.

Table 2. Analysis of transposition products mediated by the composite transposon Tn2706 using lgt11 and lgt11::P1HS bacteriophages as targetreplicons.

Phenotypes of transductantsTargetphages

Frequency of CmR

transductants ×10¹8Totaltested CmRKmSPhþ CmRKmSPh¹ CmRKmRPhþ CmRKmRPh¹

lgt11::P1HS 2.8 337 247 66 23 1lgt11 2.7 235 145 36 47 7

The data reflect the results of 9 (lgt11) and 13 (lgt11::P1HS) independent experiments.

Target specificity of IS30 693

Analysis of cloned segments containing targetsequences

For further analysis of target sites (TS), LHS, P1HS andK8 were cloned into the ApR vector pEMBL19 (Table 1)and examined in transpositional fusion experiments, inwhich the (IS30)2-containing KmR plasmid pAW1039served as donor (see Experimental procedures). All threetarget regions behaved as a real hot-spot, and the fre-quency of fusion between the donor and target replicons(presented schematically in Fig. 1B) was 0.8–10.0 ×10¹6 KmRApR/ApR transductants (Table 3A). Structural

analysis of the fusion products revealed that the majorityof insertions occurred in the offered target sites, in bothpossible orientations. With respect to the orientation ofinsertions, an interesting observation was made. The twoorientations did not occur with equal frequency, and thisbias was not changed when the direction of the targetsite itself was changed to the opposite orientation (datanot shown). This fact suggests that the orientation effectis not the inherent feature of the target sequence.

Moreover, sequencing 10 junctions of IS30 in LHS (fiveisolates) and P1HS (five isolates) revealed that the inser-tions were exactly in the same positions as previously. In


Fig. 2. Insertion sites of the IS30 compositetransposon Tn2706 in phages lgt11::P1HS(A) and lgt11 (B). lgt11::P1HS phage alsocontains an additional insertion of the mobileelement IS2. Phage DNAs are represented byboxes. The black arrowheads refer to theinsertions of Tn2706. The lacZ, P1HS andIS2 sequences are indicated by variouspatterns. Abbreviations for restriction sites:BamHI (B); EcoRI (E); HindIII (H); Sal I (S);XbaI (X); XhoI (Xh).

Table 3. Detection of IS30 insertions inplasmids containing hot-spots.

Targetplasmids Hot-spot

Frequency ofKmRApR

colonies ×10¹6Totaltested

Transpositionalevents into theoffered hot-spot

ApEMBL19 None 0.1 48 0pAW782 LHS 9.0 94 76pAW1016 P1HS 8.0 160 134pAW780 K8HS 0.8 31 22

BpAW495 POHS 2.1 86 63

CpFOL201 LSHS 5.0 19 16

DpFOL682 1/2POHS682 0.2 30 0pFOL820 1/2POHS820 0.1 30 0pFOL821 1/2POHS821 0.2 30 0pFOL822 1/2POHS822 0.3 30 0

EpFOL546 GOHS 11.0 19 19

Each tested transpositional product originated from independent experiments. Transpositionalways occurred in the offered hot-spots; other tested KmRApR colonies contained productsof multiple rearrangements or illegitimate recombination. The frequency of rearrangementswas calculated as described in Experimental procedures.

694 F. Olasz et al.

the control experiment, in which the pEMBL19 vectorserved as target without cloned hot-spots, IS30 insertionwas never detected. In this case, the frequency of KmRApR

colonies was at least one order of magnitude lower, andthe few KmRApR colonies were products of illegitimaterecombination. The results indicated that the hot-spotregions carried by the cloned fragments preserved theirtarget activity, despite the fact that they were deprived oftheir original DNA surroundings.

The consensus sequence of IS30 target sites

The fact that IS30 insertions always occurred exactly inthe same position in a certain hot-spot incited us to deducea consensus sequence from the 11 known insertion sites(Fig. 3A). The consensus sequence was created accord-ing to the following criteria: (i) a single base was acceptedat a given position if it occurred there with at least 40% fre-quency; (ii) alternative bases were used if two bases werefound in a certain position with about equal frequency, andthey represented at least 70% of the cases.

The deduced consensus CIP (consensus of insertionsin phages/plasmids) had 21 conserved bases out of 24(Fig. 3B), although there was no position at which thesame base always occurred. The sequence showed nearlypalindromic symmetry to the central two bases. The 61positions of the consensus corresponded to the basesduplicated in the target sequences and were characterizedby RY (purine–pyrimidine bases). The most frequentlyused target duplication was AT (four cases out of 11).The consensus was rich in A and T stretches that wereinterrupted by conserved GC bases at positions 63 and64. The homology of individual target sequences to theconsensus varied between 50% (pMAD33TS) and 86%(pAW340TS). The sequences LHS, P1HS and K8HS(chosen for detailed analysis) matched to the consensusin 17, 16 and 15 positions (71%, 67% and 63% homology)respectively.

The consensus sequence as target DNA

In order to verify the prediction that the consensus behavesas a hot-spot, an oligonucleotide sequence POHS (plasmid/phage derived oligonucleotide hot-spot; Fig. 3C) wassynthesized on the basis of the following criteria: (i) thelength of the sequence had to be 24 bp; (ii) the unambigu-ous bases in the consensus sequence should be pre-served (positions ¹2, 63, 64, 67, 68, 69, 610 andþ11; Fig. 3B). The ambiguous positions (61, þ2, 65,66, ¹11 and 612) were determined so that (iii) the palin-dromic structure of the sequence should be conserved;therefore, at position ¹11, base A and, at position þ2,base T were chosen, because their symmetric counterparts

were T and A respectively; (iv) the positions 61, 66 and612, where the consensus predicted R, Y or W, weredetermined considering the palindromic symmetry. Inthe 61 positions of the sequence, GC bases were usedto create a PvuII restriction site that was used in furtherexperiments (see The fidelity of target duplication). Atthe remaining positions 65, where consensus was notfound, A and T bases were used respectively.

The synthesized POHS sequence was cloned intopEMBL19, and the resulting plasmid pAW495 was offeredas target in transpositional fusion experiments. Out of 86independent transpositional fusion products, in 63 theIS30 insertions occurred exactly in the predicted positionof POHS (Table 3B). The target activity of POHS indicatesthat the consensus CIP really reflects the characteristics ofIS30 target sites. Remarkable bias was observed betweenthe frequencies of the two orientations of insertions,despite the perfect symmetry of POHS, which finding sup-ported our earlier assumption that this effect was indepen-dent of the hot-spot sequence.

At this stage in the experiments, the possibility thatthe bordering sequences of the 24-bp-long hot-spot coreregion play a role in the target specificity cannot beexcluded. In an attempt to clarify this question, the oligo-nucleotide sequence LSHS (lambda synthetic hot-spot;Fig. 3C) was synthesized, which contained just the 24-bp-long core region of LHS. LSHS carried by pEMBL19 wasas efficient a target for IS30 (Table 3C) as LHS, whichconfirmed the hypothesis that the 24 bp region alone issufficient to determine a functional hot-spot.

Loss of function of half target sites

Our earlier studies revealed that the IRs of IS30 could alsoserve as target for the element (Olasz et al., 1997). Inthese cases, not only was the symmetric (IS30)2 structurecomposed of the two joined IRs of IS30 a very efficient tar-get, but so were the IRs alone, which could be consideredas the half of (IS30)2. In an attempt to examine the role ofthe symmetry of non-IR hot-spots, the 24-bp-long coreregion was investigated further. The half of POHS (repre-senting positions from ¹1 to ¹12) was inserted into differ-ent sites of the cloning vector pEMBL19 (1/2POHS682,1/2POHS820, 1/2POHS821 and 1/2POHS822) and testedas target. It turned out that the truncated hot-spots com-pletely lost their target function in transpositional fusionexperiments (Table 3D). In these constructs, the missinghalf of the core region was replaced by different parts ofthe multicloning site matching the consensus only bychance (in 1–5 positions out of the 11; Fig. 3D). Consider-ing these flanking sequences and the half POHS as 24 bpunits, they were identical to the consensus in 11–15 posi-tions. In spite of this agreement, which was similar to thatof the real target sequences K11TS and pAW83TS (12




Fig. 3. The consensus sequence of IS30 insertions in phages and plasmids.A. The sequences of different IS30 insertion sites. The two bases in the middle correspond to the target duplication (they are separated fromthe flanking sequences by a space). P1HS, pAW33TS, pAW340TS and pAW341TS originated from phage P1; LHS, K8HS and K11TS derivedfrom l phage; pAW83TS and R124TS were insertion sites in plasmids NR-1 and R124 respectively; d1822TS and pMAD33TS representend-points of IS30 internal deletions generated by the element. In the latter two cases, only the sequenced half of the insertion sites isshown, and it is not completed according to the IS30 sequence.B. Consensus sequence of IS30 insertion sites. The positions and symmetry of the CIP sequence are also indicated.C. Sequence of POHS and LSHS hot-spots and their homology to the CIP consensus.D. The sequence of ‘half ’ POHS sites.n, consensus cannot be determined; R, A or G; Y, T or C; W, A or T. Capital letters correspond to bases matching the consensus in A and D.The fractions give the proportion of matching bases to the number of conserved positions in CIP.

696 F. Olasz et al.

and 13 matches respectively), the half of POHS wasinactive in all four constructs.

Study of genomic insertion sites of IS30

In previous experiments, we used plasmids or phages toinvestigate target sites. The probability that relatively smalltarget DNAs contain optimal target sites is quite low (e.g.we could not find any hot-spot in the 4 kb vectorpEMBL19; Table 3A); therefore, the deduced CIP sequencemay not represent the optimal consensus. In order to over-come this problem, the largest available target DNA, thegenome of E. coli, was offered for IS30 transposition(see Experimental procedures).

Consensus was made from eight newly isolated inser-tion sites and the flanking sequences of the four IS30copies resident in the genome of E. coli (Fig. 4A and B).The deduced consensus CIG (consensus of insertions ingenome of E. coli ; Fig. 4C) was also a 24-bp-long palin-dromic sequence and was very similar to CIP (Fig. 4D),although they originated from independent assays. CIGcontained only two non-conserved positions (¹2 andþ5), and the occurrence of alternative residues alsodecreased (positions 61 and 66). The homology of indivi-dual target sites to the CIG sequence varied from 50%(IGEN221) to 92% (ECAE499, ECIS1B and IGEN1319).

The construction of the synthetic GOHS (E. coli geno-mic oligonucleotide hot-spot; Fig. 4D) was based on CIGusing the same criteria that were applied in the case ofPOHS (see The consensus sequence as target DNA).The oligonucleotide GOHS was cloned in vector pEMBL19to test its target activity. In fusion experiments, the syntheticsequence behaved as a very effective target (Table 3E),indicating that GOHS is a real hot-spot and CIG properlyreflects the attributes of the genomic insertion sites.

Direct comparison of target sequences

In the first transpositional experiments, when bacterio-phages lgt11 and lgt11::P1HS were applied as targets,the attractiveness of the hot-spots seemed to be different,e.g. LHS and P1HS were used several times, while othertarget sites only once (Fig. 2A and B). To examine thisphenomenon, a direct comparative system was developed,in which the donor was the same as previously (pAW1039),but the target plasmids contained two target regions insteadof one. The same system was used to study the target activ-ities of IS30 IR ends (Olasz et al., 1997), in which the order,orientation and the position of the two targets were shownnot to be relevant to the choice of insertion site. Thesefindings indicated that this system is suitable for the com-parison of targets. For many independent fusion events,the distribution of insertions into the two sites was thusdetermined, reflecting the difference in their attractiveness.

The analysis of different target pairs allowed ranking ofthe hot-spots.

First, the 260-bp-long LHS and its 24-bp-long syntheticcore region, the LSHS, were compared (Table 4A). Theanalysis revealed that there was no significant differencein the utilization of the two targets. This result also con-firmed our earlier finding that the 24-bp-long core regionalone is sufficient to determine a functional hot-spot andverified that, if the two hot-spots are identical, they receivetranspositional hits almost equally. Subsequently, the tar-get regions K8HS, P1HS, LHS and POHS were chosenfor further analysis, and the comparison of their attractive-ness resulted in the following order: K8HS<POHS<

P1HS< LHS (Table 4B). As the comparison of hot-spotsrevealed that the more attractive region received almostall the insertions, every target site had to be comparedwith each other in order to establish their ranking (e.g.LHS and P1HS were both about 50–60 times more activethan K8HS, predicting that they are of the same attractive-ness; however, when they were compared with each other,LHS was six times more active than P1HS). These find-ings suggested that the attractiveness was not additivein our system.

The synthetic POHS sequence representing the CIPconsensus was not the most preferred target in the com-parative system: P1HS, LHS and LSHS proved to bemore attractive (Table 4B). These sites share only limitedsequence homology (there are only seven identical basesout of 24 in P1HS, L(S)HS and POHS). The influence of


Table 4. Distribution of IS30 insertions in the comparative targetsystem.

Number of transpositionalevents in the two target regions

Targetregion 1

Targetregion 2

Relativefrequency Plasmid

ALSHS 53 LHS 43 1.2 pFOL387

BPOHS 35 K8HS 3 12 pAW432P1HS 55 K8HS 1 55 pAW1193P1HS 53 POHS 2 26 pFOL17LHS 57 K8HS 1 57 pAW1109LHS 55 POHS 3 18 pAW1153LSHS 54 POHS 2 27 pFOL388LHS 45 P1HS 8 6 pAW1028

CGOHS 36 POHS 1 36 pFOL519GOHS 32 LSHS 2 16 pFOL518

Target plasmids contained two sites for transpositional fusion (targetregions 1 and 2). Target region 1 was defined as the hot-spot in whichthe majority of the IS30 insertions occurred. Each tested transposi-tional fusion product originated from independent experiments. Notethat, in each event, only one of the target regions was involved in thefusion reaction. Relative frequencies were calculated as the ratio ofthe number of insertions in target regions 1 and 2 respectively.



698 F. Olasz et al.

bordering sequences and the length of the hot-spot can beruled out, as both POHS and LSHS were flanked by similarsequences in the multicloning site of plasmid pFOL388,and the length of the hot-spot was 24 bp in each case.The different attractiveness of POHS and LSHS were pre-sumably caused by the presence of different bases atambiguous positions in the CIP consensus (61, þ2 6 5,¹11 and 612). This conception was also supported bythe fact that GOHS was a more attractive target thanPOHS and LSHS (Table 4C).

Examination of the DNA structure of hot-spots

As the attractiveness of IS30 targets may be influenced bytheir specific DNA structure (e.g. bent DNA, Z-DNA), LHS,P1HS and POHS were investigated with a transverse poregradient gel electrophoresis system (Buzas et al., 1994),which is suitable for demonstrating uncommon DNA con-formations. As an alternative method, LHS and its 24-bp-long core region, LSHS, were cloned into the plasmidpBEND2, which allows the rapid detection of bent DNA(Zwieb et al., 1989). Using pBEND2, numerous DNA frag-ments can be generated, in each of which a cloned hot-spot is present in circularly permutated positions alongthe length of the DNA fragment. During gel electrophore-sis, the different mobility of restriction fragments of equallength shows the possible bent structure of hot-spot DNA.In these experiments, we were unable to detect any altera-tion in the mobility of DNA fragments containing the hot-spots. Therefore, we can conclude that the investigatedhot-spots have no unusual DNA structure.

The ability of LHS, K8HS and P1HS to bind the N-terminalpart of the IS30 transposase protein (Stalder et al., 1990)was also investigated. In bandshift assays, no measurabletransposase binding to the hot-spots was observed (fordetails, see Experimental procedures), while the othertype of target sites, the IRs of IS30 (Olasz et al., 1997),showed retardation in the same experiment (data notshown).

The fidelity of target duplication

It has been described previously that the length of target

duplication can vary for some IS elements (IS1: Iida andHiestand-Nauer, 1986; IS4: Mayaux et al., 1984, Haber-mann et al., 1979; IS21: Reimmann et al., 1989; IS186:Sengstag et al., 1986). On the contrary, a 2 bp duplicationof the target sequence has been found so far in all exam-ined IS30 insertions (Caspers et al., 1984; Olasz et al.,1993; and this work). Furthermore, high fidelity of targetduplication was also observed when IRs of IS30 servedas target (Olasz et al., 1997).

As shown in Fig. 5, the POHS sequence contains aPvuII recognition site exactly at the site being duplicatedduring transposition. Therefore, the insertion of (IS30)2

into POHS creates PvuII sites at both junctions, providedthat the target duplication is precise. As a consequence,the number of PvuII sites reflects the fidelity of target dupli-cation. As expected, in 60 out of 63 independent trans-positional fusion products of pAW1039 and pAW495, twoPvuII sites were found in the IS30 junctions, reflectingthe high fidelity of target duplication. The sequence analy-sis of the remaining three products revealed that, in onecase, a small adjacent deletion occurred and, in the othertwo cases, the target duplication was GG instead of GC.This latter finding raised the possibility that the GG basesare derived from donor (IS30)2, which contains GG basesbetween the joined IRs.

Discussion

Transposable elements are particular DNA segments thatcan insert into many non-homologous DNA sequences.Although they use very different target sites, their choiceis generally not random. Most of them exhibit some degreeof target specificity, which is characteristic of the element.Transposition of the E. coli insertion element IS30 alsoshows similar properties, as it always occurs in the sameposition of the hot-spots and generates 2 bp duplicationof the target sequence with high fidelity. According to theinsertion sites targeted by IS30, the specificity of the ele-ment can be characterized by a 24-bp-long nearly palin-dromic consensus sequence. No completely conservedsites were determined in the consensus, and there wereseveral positions that may be occupied by alternative


Fig. 4. The consensus sequence of IS30 insertions in the genome of E. coli.A. Sequences flanking the genomic copies of IS30 in E. coli. *A truncated IS30 copy is present in ECIS1B. ** The ECAE499 sequencesrepresent the flanking regions of the same IS30 copy, but the two joining bases corresponding to the target duplications are different, whichprobably indicates a secondary rearrangement mediated by IS30.B. Target sequences of chromosomal IS30 insertions. Capital letters correspond to bases matching the consensus (in A and B). The names ofrepresentative genomic E. coli clones in the EMBL library are also indicated as reference. The IGEN1319 sequence was not found in theEMBL database. The insertion sites, except IGEN271 and IGEN1319, were sequenced from both IS30 ends.C. The CIG consensus was deduced as described in Results.D. Comparison of CIG to CIP consensus and GOHS.E. Comparison of LSHS to GOHS and POHS. The ambiguous positions of CIP and the corresponding bases in GOHS, LSHS and POHS arein bold.F. Hypothetical ‘prohibited’ nucleotides in IS30 target sites. Capital letters correspond to bases located symmetrically. Symmetrical positions,at which the prohibited nucleotides never occurred in the 24 examined target sites, are underlined twice. The other abbreviations are the sameas in Fig. 3.


bases. Considering the 24 bp length, it cannot be expectedthat all the positions are highly conserved, because in thiscase the occurrence of a target site would be extremelyrare – in contrast to our experimental observations.

The target specificity of IS30 may be influenced by dif-ferent factors. According to our results, specific DNA con-formation and bordering sequences of the 24 bp targetsites as well as homology to the (IS30)2 intermediate(Olasz et al., 1997) seem to be irrelevant in respect of tar-get activity. On the other hand, the frequent utilization of atarget sequence can also depend on its similarity to theconsensus, for example K8HS, P1HS and LHS matchthe consensus at 15, 16 and 17 positions (Fig. 3A) respec-tively and, as a possible result of these alterations, theirattractivities differ by one order of magnitude (Table 4B).Furthermore, the GOHS sequence, which showed a highlevel of homology to both the CIP (20 matches) and theCIG consensus (22 matches; Fig. 4C), was the mostpreferred hot-spot investigated so far (Table 4C).

Not only overall similarities to the consensus are required,but certain positions should be occupied by the ‘appropriate’bases. For instance, GOHS was a more attractive targetthan POHS (Table 4C). However, it differed from POHSjust at some of the ambiguous CIP positions (61, þ2,65 and 612). The ‘natural’ hot-spot L(S)HS showedmore similarity to GOHS (seven out of nine) than toPOHS (three out of nine) at these positions (Fig. 4E),and the differences in the homology were in good agree-ment with the observed ranking of these targets: LSHSwas more attractive than POHS but less active thanGOHS. These results indicate that the occupation of oneor more of the positions 61, þ2, 65 and 612 stronglyinfluences the attractiveness of the hot-spot. At thisstage in the experiments, it is difficult to determine whichis the ‘optimal’ base in a certain position, but the sequenceof the most effective hot-spot GOHS may approach it.

The fact that the half POHS sequences (Fig. 3D) were

not recognized as target suggested that the hot-spotsmay function as a bipartite binding site of a head-to-headdimer protein (transposase oligomer?). This hypothesisis supported by the more or less symmetrical nature ofindividual targets and the pronounced symmetry of theconsensus sequences. Therefore, not only a high degreeof similarity to the consensus is required for being a hot-spot, but the distribution of matches is also significant. Itis interesting to note that the sequence 1/2POHS682was not an active target either, despite the fact that thelevel of homology (71%) to the consensus and the distribu-tion of matches were similar to that of the active hot-spotK8HS. It could be assumed that the structure of DNA orthe presence of hypothetical non-permitted base(s) in cer-tain positions prevents the utilization of this sequence astarget. This feature is also similar to that of the bipartitebinding sites, whose function can be destroyed by justone base change. Comparing the 24 insertion sites, it isstriking that certain bases were very rare or missing atgiven positions, so they were considered as ‘prohibited’.The ‘optimal’ occupation of a sequence enhances theattractivity of a hot-spot, while the presence of ‘prohibited’residues may decrease, or perhaps ruin, the target activ-ity. The possible prohibited nucleotides (zero or one occur-rence in the 24 known targets) are shown for each positionin Fig. 4F. The obvious symmetry of these bases strength-ens the suggested hypothesis. The G and C bases at posi-tions 610 represent residues that were not found in any ofthe 24 target sites, and 1/2POHS682 containing the pro-hibited base C at position þ10 (Fig. 3D) was inactive astarget. However, its similarity to the consensus was thesame or even higher than that of efficient target sitesK8HS, K11TS, pAW83TS, pAW341TS, pMAD33TS,ECIS2IS30, IGEN221 or IGEN271. Extensive mutationalanalysis of attractive hot-spots may provide greater insightinto the role of prohibited nucleotides.

We examined whether some other IS elements possess


Fig. 5. Target duplication in thetranspositional fusion products of the donorplasmid pAW1039 and target plasmidpAW495. Capital, small and italic letters in thesequences represent IS30, POHS and thespacer region between the IRs of IS30elements respectively. The IRs are numbered,and the PvuII recognition sites are shownabove the sequence. The spacer region (lostupon transposition) and the 2 bp undergoingtarget duplication (boxed) are separated bygaps from other parts of the sequences.Other symbols and abbreviations are thesame as in Fig. 1.

700 F. Olasz et al.

an IS30-like target specificity, i.e. if they insert into nearlypalindromic sequences flanking the site of target dupli-cation. The criteria for identifying possible consensussequences were the same as in the case of IS30. Consen-sus sequences could be determined for IS3 (Spielmann-Ryser et al., 1991), IS6 (Mollet et al., 1985) and IS21(Reimmann et al., 1989) (Fig. 6). Conserved bases werefound both in the site of target duplication and in the flank-ing sequences. The length of the consensus in the flankingregion varied from 5 bp (IS6) to 10 bp (IS21). In the con-sensus sequence for IS6, the majority of the conservedbases were found in the target duplication, while in theother cases they were in the flanking sequences. Withthe available data, we could not find any consensus forthe target sequences of IS1 (Galas et al., 1980), IS2(Sengstag et al., 1983) and Tn3 (Tu and Cohen, 1980).

In the case of the well-studied mobile elements IS4(Mayaux et al., 1984), IS5 (Engler and van Bree, 1981)and IS10 (Foster, 1977; Halling and Kleckner, 1982),where consensus has been derived, its length is lessthan 10 bp, and the positions are relatively highly con-served. On the contrary, the pronounced target specificityof IS30 is determined by an extremely long and moredegenerate target sequence. The variance in the lengthand stringency of consensus sequences indicates that themobile elements may apply different strategies for targetselection. The spectrum of alternative possibilities rangesfrom the very loose target specificity of Mu (Castilho et al.,1984; Castilho and Casadaban, 1991) to the stringent site-specificity of Tn7 (Craig, 1991). On the basis of the avail-able data, we suggest that, in this more or less continuousrange, there are four main strategies in target selection.One of them is a preference for targets characterized bya very short and less defined consensus like that of Mu(Mizuuchi and Mizuuchi, 1993), which allows insertionsalmost randomly. The other extremity, the nearly exclusiveusage of a long and strict target site that may even restrictthe transposition, is represented by Tn7 (Lichtenstein and

Brenner, 1981; 1982). The two further alternatives are theselection of short, but well-determined sequences (e.g.IS4, IS5 and IS10) or, on the contrary, usage of longerbut less conserved sequences, as is the case with IS30and, maybe, with IS3, IS6 and IS21. In the first case, theshortness, and in the last case, the low stringency of theconsensus may provide the appropriate number of poten-tial target sites for insertion and may regulate the trans-position activity through target specificity.

Experimental procedures

Enzymes, chemicals and DNA techniques

Restriction endonucleases and T4 DNA ligase were obtainedfrom Boehringer Mannheim and Minotech. Antibiotics wereused at final concentrations of kanamycin (Km) 20 mg ml¹1,chloramphenicol (Cm) 20 mg ml¹1 and ampicillin (Ap) 150 mgml¹1. a-[35S]-dATP for sequencing was obtained from Amer-sham. Commonly used DNA techniques were performedaccording to Sambrook et al. (1989). Sequencing was doneby the chain termination method of Sanger et al. (1977),using the T7 sequencing kit from Pharmacia or the Sequen-ase version 2.0 sequencing kit from US Biochemicals. Com-puter analysis was performed with the GCG software package(Devereux et al., 1984).

Plasmids and bacteriophages are listed in Table 1.

Detection of simple transposition of the compositetransposon Tn2706

The lgt11 lysogenic JM109 strain was transformed with theCmRKmR plasmid pAW332 (harbouring the IS30 compositetransposon Tn2706) and selected for the CmR marker ofthe transposon. Independent colonies were grown overnightin Cm LB. Cultures were diluted (1:100) and grown underthe same conditions – this step was repeated twice. Theprophage was induced by heat treatment at 428C to obtain pri-mer phage lysate. JM109 (Yanisch-Perron et al., 1985) wasinfected with the primer phage lysate (multiplicity of infectionof 5) and spread on Cm-supplemented LB plates. The


Fig. 6. Possible consensus sequences forinsertion sites of IS3, IS6, IS21 and IS30.A. Schematic presentation of the hypotheticalstructure of target sequence, in which thebases being duplicated are flanked bysymmetrical sequences.B. Consensus target sequences of theanalysed insertions. The sequence of targetduplication is separated from the flankingsequences, which are presented with only onehalf according to the presumed symmetricalstructure. The consensus was deduced asdescribed in Results. In the case of IS21, thefew available sequences allowed only a weakdetermination of the consensus. Theabbreviations are the same as in Fig. 3 withan exception: S represents alternatively G orC bases.


frequency of CmR-transducing phages was determined as aratio of CmR transductant colonies/titre of phages. CmR trans-ductants were tested for resistance or sensitivity to Km andphage production. Finally, DNA was isolated from CmRKmS

phage-producing colonies and analysed by restriction clea-vage. Insertions in K8HS, K11TS, LHS and P1HS werecloned into pEMBL19 and sequenced using oligo primerscompatible with IS30: left end primer 58-TCTGCTGTA-AATGTT-38, right end primer 58-CAACAGACCGAGAAA-38.

Transpositional fusion of plasmids

The method for detecting transposition with the aid of mobiliz-able target plasmids and the helper phage R408 has beendescribed by Olasz et al. (1993). In these experiments, the(IS30)2 donor plasmid pAW1039 was introduced into therecA¹ JM109 host already harbouring a target plasmid. After12–16 h incubation in LB medium, bacteria were infectedwith the helper phage R408 and target plasmids and theirrearranged derivatives were packed into phage particlesused to infect new JM109 host. Applying appropriate selection(donor plasmid: KmR; target plasmids: ApR), fusion productswere detected as KmRApR colonies. The frequency of therearrangements was calculated as the ratio of KmRApR/ApR

transductant colonies.

Generating and sequencing genomic IS30 insertions

Genomic insertions were generated in the recA¹ E. coli strainTG2 (Sambrook et al., 1989) by an in vitro constructed trans-poson (J. Kiss, unpublished). The trans system (Farkas et al.,1996) was applied to integrate the CmR IS30-derived artificialtransposon. In eight independent experiments, competentTG2 cells harbouring an ApR transposase producer plasmidpJKI132 (Farkas et al., 1996) were transformed with 1.5–1.5 mg of DNA containing the transposon and selected onCm LB plates. Genomic DNA was purified from at least twoCmR colonies in each experiment, and 10–50 ng of DNAwas digested overnight with 20 units of HindIII (having norecognition site in the transposon) and ligated into pBluescriptSK (Stratagene). Plasmid DNA was extracted from CmRApR

transformants, and the insertion sites were sequenced withprimers compatible with IS30 ends directed outwards fromthe element. IS30L primer: 58-GTTCGTCTCATTCAA-38;IS30R primer: 58-CCGAAAGAGATAATT-38.

Examination of the physical properties of hot-spot DNAs

For the transverse pore gradient gel electrophoresis, hot-spotswere isolated as short linear fragments: LHS was isolated asthe XhoI–Sal I fragment of pJKI40, which was constructed bycloning of the 260 bp XhoI–Sal I fragment of l DNA into theXhoI–Sal I site of pBluescript SK; P1HS was isolated as a104 bp BamHI fragment of pAW1016; POHS was a Bgl I–Pst I fragment of pAW495. The hot-spot fragments isolatedfrom agarose gel were phenol–chloroform extracted and fil-tered on a Sephadex G50 column, dried under vacuum anddissolved in TE in a final concentration of approximately1 ng ml¹1. DNA (3–5 ng) was loaded on the transverse poregradient gel, and electrophoresis was carried out as described

by Buzas et al. (1994) with the Phast system (Pharmacia)electrophoresis apparatus.

For bent DNA analysis, LHS was isolated as a 266 bp Sal I–XbaI fragment of pAW1023 (identical to pAW782 but containsthe LHS in the opposite direction) and inserted into the Sal I–XbaI site of pBEND2, which resulted in pFOL274. The blunt-ended EcoRI–Sal I fragment of pFOL201 containing LSHSwas cloned into pBEND2, which was digested with Sal I–XbaIand filled in with Klenow polymerase, yielding pFOL275. Theplasmids pFOL274, pFOL275 and pBEND2 as negative con-trol were digested with Bgl II, XhoI, PvuII or BamHI sepa-rately. The samples were phenol–chloroform extracted andfiltered on a Sephadex G50 column, dried under vacuumand dissolved in TE. DNA (3–5 ng) were loaded and run onPhast system electrophoresis apparatus.

Bandshift assay

Linear DNA fragments (300–600 ng) containing IS30 hot-spotswere isolated as follows: K8HS as a 658 bp EcoRI–HindIIIfragment of pAW780; LHS as a 284 bp Sal I–HindIII fragmentof pAW782; and P1HS as a 160 bp EcoRI fragment ofpAW1016. After dephosphorylation, DNA was labelled withg-[32P]-dATP and filtered on a Sephadex G50 column. DNAwas dissolved in A buffer (Stalder et al., 1990) and adjustedto 10 000 c.p.m. activity. Then, 5 ml of the solution was incu-bated with 0, 1 or 2 ml of IS30 transposase extract (N-terminalpart: 7 mg ml¹1; Stalder et al., 1990) in the presence of 2 ml of0.1 M MgCl2 and 2 ml calf thymus DNA (25 mg ml¹1) in a totalvolume of 20 ml. The samples were run on 8% PAGE, and thegel was visualized by autoradiography.

Acknowledgements

We are grateful to Professor Laszlo Orosz for his support, andto Rosemarie Hiestand-Nauer, Agota Bakos-Nagy and IlonaKereszturi-Konczol for their highly skilled technical assistance.This research was supported by grants OTKA 221/90,T019365, T6054 and F017090 of the Hungarian Academyof Sciences.

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