PLASMID

26,20-29 ( 199I)

Mutational Analysis Site-Specific

of att554, the Target Transposon Tn554

of the

ELLENMURPHY,ELLENREINHEIMER,ANDLESLIEHUW~LER The Public Health Research Institute, 455 First Avenue, New York, New York 10016

Received January 22, 1991; revised May 14, 1991 Tn554 is a high-frequency, site-specific transposable element ofStaphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, S-GATGTA-3’ (nucleotides numbered -3 to +3). Most of the time (r99%) insertion occurs immediately 3’ to this sequence;the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5’ to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the let?and ending at - 15or from the right ending between +8 and + 12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active aft554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequencesat the junction of aft554 and the 3’ end of Tn554. These data indicate that att554 contains a short nucleotide sequenceessential for transposition and flanking sequences that greatly increase the frequency of recombination. 0 1991 Academic

Press, Inc

Tn554 is a high frequency, site-specific transposable element found in Staphylococcus aureus (Phillips and Novick, 1979). The element exhibits two unusual properties, the absence of terminal repeats and the failure to generate a duplication of a target sequence upon transposition (Murphy and Lofdahl, 1984). Tn554 is 669 I-bp long, specifies resistance to erythromycin and spectinomycin, and contains three transposition genes called tnpA, tnpB, and tnpC (Bastos and Murphy, 1988; Murphy et al., 1985). tnpA and tnpB are essential for transposition; their precise roles are not known, but both genes specify proteins that are members of the Int family of site-specific recombinases that mediate bacteriophage integration and other site-specific recombination reactions via a 3’ phosphotyrosine intermediate (Argos et al., 1986). tnpC is involved in insertion orientation specificity

and is not absolutely required for transposition to occur; in its absence, transposition occurs at a frequency of 0.1-l% that of wildtype Tn554 (Bastos and Murphy, 1988). Insertion of Tn554 into the S. aureus chromosome occurs at a single site, designated att554 (Krolewski et al., 1981; Murphy and Lofdahl, 1984; Phillips and Novick, 1979). In the prototype, or (+) type of insertion, Tn554 is flanked by the hexanucleotide sequence 5’GATGTA-3’. These flanking sequences are not the result of a target duplication, since insertions at at054 in the opposite orientation, designated (-), or at secondary insertion sites, are not flanked by direct repeats (Murphy and Lofdahl, 1984). To better understand the requirements for Tn554 transposition to occur, we have carried out a mutational analysis of atf554. This analysis indicates that a fully functional att.554 site is between 4 1 and 53 bp in length and that partial activity is associated with a central region between 14 and 3 1 bp in length.

Portions of this work were reported previously in preliminary form (Murphy, 1990). 0147-619X/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.

20

MUTATIONAL

MATERIALS

ANALYSIS

AND METHODS

Bacterial Strains and Plasmids All strains of S. aureus are derivatives of NCTC 8325 (Novick, 1967). RN2863 (Phillips and Novick, 1979) is the prototype strain carrying a Tn554 insertion in S. aureus 83254; it was used as the donor for transduction of Tn554. RN4470 contains a deletion of an 0.8-kb chromosomal AvaI-Hind111 fragment containing att554. In place of this fragment RN4470 carries a 1.4-kb Hind111 fragment carrying the chloramphenicol acetylase gene of plasmid pC221, obtained by competent cell transformation with a linearized plasmid carrying the CAT-containing fragment inserted in place of att554 within a 3.8-kb chromosomal EcoRI fragment. The replacement of the chromosomal copy of att554 by CAT and the absence of vector sequences in the chromosome of RN4470 were confirmed by nucleic acid hybridization with appropriate probes; in addition, the Cm’ marker in RN4470 is allelic with Tn554 in genetic crosses(data not shown). pEM9701 is a 4.6kb plasmid derived from pT 18l-cop615 (copy number approximately 200 (Carleton et al., 1984) into which a 171-bp TaqI-Mb01 fragment containing att554 was cloned (see Results for details); it was used for construction of att554 mutations. Plasmid DNA was prepared by CsCl density gradient centrifugation (Clewell and Helinski, 1969; Murphy, 1983); minipreps (Bimboim and Doly, 1979) were prepared from lOlo cells and centrifuged in 2.2 ml gradients in a Beckman TLlOO centrifuge at 459,000g for 4- 16 h in a TL100.2 rotor. Protoplast transformation was as previously described (Chang and Cohen, 1979; Murphy, 1983). Nucleotide sequences were determined using linearized, double-stranded template DNA using the dideoxy chain termination method @anger et al., 1977) modified for use with [“S]dATP (Biggin et al., 1983). Hybridizations were performed according to the method of Southern (Southern, 1975) using nick-translated or end-labeled probes.

OF att554

21

TranspositionAssay The ability of a particular plasmid to function as a target for Tn554 insertion was initially estimated by transductional crossesin which the recipient strain contained a test att554 plasmid. All recipients carried a deletion of the chromosomal att554 site (RN4470 or derivatives). For transposition assays,recipient cells were grown to late log phase in CY broth (Novick and Brodsky, 1972) and frozen in aliquots. Frequencies were normalized for each experiment to the transduction frequency with a recipient carrying wild-type, plasmid-linked att554 (pEM970 1). The transducing phage 4 11 was used to prepare a lysate of RN2863, which carries a chromosomal insertion of Tn554 at att554. Each recipient strain to be tested (2 X 10’ cells) was mixed with 1.9 X lo7 pfu, and transductants carrying Tn554 were selected on GL agar (Novick and Brodsky, 1972) containing 5 pg/ml erythromycin. Since the Tn554 donor, RN2863, carries Tn554 inserted at att554 in the chromosome, chloramphenicol (5 pg/ml) was included in the medium to select against Em’ transductants arising via homologous recombination between sequencesflanking att554 in the donor and recipient chromosomes, an event that would result in the displacement of the Cm’ marker by Tn554. Under these conditions transduction frequencies into a given recipient varied by no more than threefold. As a second assayfor transposition the relative abundance of plasmids with and without Tn554 insertions was estimated by agarose gel electrophoresis of whole-cell lysates of transductant colonies. Cells were washed once, incubated with lysostaphin (150 &ml) and pancreatic RNAse ( 100 pg/ml) at 37°C in buffer containing 30 mMTris pH 7.8, 50 mM EDTA, 50 mM NaCl, and 20% sucrose, lysed by the addition of an equal volume of electrophoresis dye containing 2% SDS, shaken for 15 min, and frozen and thawed twice. Samples were electrophoresed in an 0.7% agarosegel at 35 V for 18 h, and DNA bands were visualized following staining with ethidium bromide.

22

MURPHY, REINHEIMER,

Construction of at054 Mutations Forty micrograms of pEM9701 DNA, cleaved at one end of the at054 insert with either BamHI or FnuDII (Fig. l), was incubated at room temperature with 4 units Ba131 nuclease (Legerski et al., 1978). Samples were withdrawn at intervals and pooled in tubes containing 0.5 M EDTA. The DNA was extracted with phenol and chloroform, precipitated with ethanol, incubated with Klenow fragment of DNA polymerase I and all four deoxyribonucleotide triphosphates, and incubated with T4 DNA ligase. Prior to transformation, the DNA was digested with BamHI or FnuDII, as appropriate, to eliminate plasmids that remained intact. Plasmids were screened by dot-blot hybridization with oligonucleotide probes complementary to the other end of the 171-bp insert to eliminate those containing deletions of the entire att554 fragment. Deletion endpoints were determined by nucleotide sequence analysis. Since some transformants clearly contained more than one plasmid species, each att554 deletion plasmid was transferred by transduction to RN4470 for clonal isolation prior to analysis. Sodium bisulfite mutations were obtained as described by Shortle and Botstein (1983). Heteroduplex DNA having a single-stranded gap corresponding to the entire 17lbp att554 insert was prepared by annealing ClaI-digested pEM9701 DNA and FnuDIIcleaved pT 181. RESULTS

Functional Definition of att554 Sequence We had previously shown that pEM9605, a plasmid carrying a 3.8-kb DNA fragment containing att554, functions as a specific target for Tn554 transposition (Murphy and Lofdahl, 1984). To begin to define the extent of DNA required for transposition, a deletion analysis of att554 was performed. The initial substrate was a 171-bp TaqI-Mb01 fragment containing nucleotides -89 to +82 of att554 which was subcloned from pEM9605 into

AND HUWYLER

I

Tn554

Clal FIG. 1. Map of pEM9701. Plasmid pEM9605 carries a 3%kb S. aureus chromosomal fragment inserted at the EcoRI site of pRN8057, a temperature-sensitive derivative of pT18 1 (Murphy and Lofdahl, 1984). A 17I-bp, att554-containing TaqI-Mbol fragment from pEM9605 was inserted at the FnuDII site of pT181cop615. The cloning resulted in the regeneration of the FnuDII site at one end of the insert and creation of a BarnHI site at the other end.

the FnuDII site of pRN8020 (pT 181~0~615). This generated an insert bounded on the left by a unique FnuDII site and on the right by a unique BarnHI site (Fig. 1). This clone, pEM970 1, functioned as a specific, high-efficiency target for transposition of Tn554 (Table 1).

Analysis of Deletions of att554 The extent of deletion into att554 from either the FnuDII or BarnHI sites is shown in Fig. 2. The ability of each deleted plasmid to function as an acceptor for Tn554 transposition was assayed by introducing Tn554, by transduction, into each transformant in the RN4470 background. The frequency of Em’ colonies was taken as a rough measure of transposition. Although this assay clearly identified plasmids that lacked att554 activity, it failed to distinguish wild-type from partially active plasmids. To distinguish these phenotypes, we compared the relative amounts of 4.6-kb (target) and I 1.3-kb (target plus Tn554 insert) plasmids by agarosegel electrophoresis of whole-cell lysates of individual transductant colonies (Fig. 3). When

MUTATIONAL

23

ANALYSIS OF at054 TABLE I

PROPERTIES OFBAL3 1 DELETIONSOFatt554’

Rasmid

Strain

Controls pEM970 1 None

RN4653 RN4470

Deletions from FnuDII site (-89) pEM9832 pEM9856 pME9780 pEM9843 PEM9774 pEM9828 pEM978 1 pEM9865 pEM98 1I

RN5449 RN548 I RN5410 RN5460 RN5492 RN5445 RN541 1 RN5490 RN542 1

Deletions from BamHI site (+82) pEM9759 pEM9793 pEM98 17 pEM9813 pEM9765 pEM9814 pEM9764 pEM9796 pEM9798 pEM9790 pEM9766 pEM9815 pEM98 18 pEM9767 pEM9762 pEM9794 pEM9907 pEM9795

Deletion endpoint

Transposition frequency*

Plasmid DNA present

1.0 0.06

Insert -

-46 -28 -28 -21 -26 -15 -7 -6 -3

1.38 1.61 0.83 1.38 1.52 1.71 0.05 0.04 0.04

Insert

RN55 11 RN5413 RN5424 RN5422 RN540 1 RN5423 RN547 1

+29 +25 +25 +10 +9 +9 +10

RN5416 RN5510 RN5412 RN5402 RN5508 RN5425 RN5403 RN5399 RN5414 RN5417 RN5415

+9 +9 +8 +8 +11 +12 +10 +7 -5 -10 -11

Hybridization signal

+

Right junction sequence (No. tested)

N.A.”

GATGTA (> 10) N.A.

Mixed Target Target Target

N.D.C N.D.’ N.D. + N.D.’ + -

N.D.’ GATGTA GATGTA GATGTA GATGTA GATGTA N.A.’ N.A.’ N.A.’

1.15 1.43 1.96 2.13 1.68 1.18 0.88

Insert Insert Insert Mixed Mixed Mixed Target

N.D.’ + N.D.” + N.D.’ + +/-

0.28 0.83 1.00 1.49 1.32 0.75 0.49 0.10 0.05 0.05 0.04

Target Target Target Target Target Target Target Target Target Target Target

-

GATGTA GATGTA N.D.’ GATGTA TACATC GATGTA GATGTA TGTGTT GATGTA GATGTA GCGTTA N.A.’ N.A.’ N.A.’ N.A.’ N.A.’ N.A.’ N.A.’ N.A.’

Insert Insert

(I) (1) (1) (1) (1)

(1) (I) (4) (1) (2) (1) (1) (1) (1) (1)

a Plasmids are listed in the same order in which they appear in Fig. 2, i.e., in order of decreasing att554 activity. b Transposition frequency is the number of Em’ transductants/PFU, expressedas a fraction of the control RN4653 (3.42 x lo-’ transducants/PFU). ’ N.A., not applicable (no inserts could be recovered for sequencing); N.D., not determined.

the recipient contained a wild-type plasmid, only the 11.3-kb insert plasmid was present; if the plasmid was totally defective, only the original target plasmid was found. Partial att554 activity was indicated by a mixture of both 11.3- and 4.6-kb plasmids.

All plasmids carrying deletions on the left side of at054 up to nt -28 or on the right side up to +25 were phenotypically indistinguishable from the wild-type, pEM9701 (Table 1 and Fig. 2). All transductants contained plasmids of the size expected for transposition

24

MURPHY, REINHEIMER,

AND HUWYLER

-46-H

pEMQ832 pEM9656 pEt.49780 pEMQa43 pEt.49774 pEt49828 pEM97al pm.49865 pEMSa11

-28 -28

EsoKv pEMS759 pEM97QJ pEMQa13 pEM9765 pEt.48814 pEM9764 pEMQ796 pEMQ79?3 pEMQ790 pEM9766 pEY9815 pEMS618 ~089767 pEMS762 pEM9794 pEMSQ07 pEM9795

+29 +25 +10 +9 +Q +10 +Q +Q

+a +a +11 +12 +10 1 , I 1

, +7 1 -5 1-10 1-11

FIG. 2. Deletions of aft554. Bal31 deletions were constructed from the FnuDII or BarnHI sites of pEM970 1. Bars represent the att554 DNA between -40 and +40 retained by each plasmid. Solid bars, plasmids with transposition activity indistinguishable from wild-type; hatched bars, plasmids with partial activity; open bars, plasmids with no at054 activity.

and contained little or no unreacted target All plasmids with deletion endpoints beplasmid (Fig. 3). The Em’ marker of Tn554 tween -26 and - 15 on the left or +8 to + 12 and the Tc’ marker of the target plasmid were on the right exhibited partial att554 activity. found to be greater than 90% genetically (An exception is pEM9843, ending at -21, linked upon transduction into a new host an anomaly with wild-type activity.) Partial (data not shown), indicating physical linkage activity is defined by wild-type Em’ transposiof Tn554 and the att554 plasmid. tion frequencies in combination with the presAll plasmids in which the GATGTA core ence of plasmid DNA carrying no insert. sequence(-3 to +3) was deleted were totally Some plasmids appeared to be nearly nornonfunctional. Deletions from the left side mal, containing mostly insert DNA; some, that extend to -7 (pEM9781), -6 (pEM- such as pEM98 13 (Fig. 3) contained approxi9865), or -3 (pEM981 l), and deletions from mately equal amounts of insert and target the right extending to +7 (pEM9762) were plasmid DNAs, and some (pEM98 18, also totally defective as targets. For all of pEM9764, and pEM9767 in Fig. 3) conthese plasmids, transduction frequencies tained no detectable insert plasmid asjudged were equal to that of the negative control (Ta- by examination of ethidium bromide-stained ble l), and no 11.3-kb plasmid band was de- gels. For some plasmids, high transduction tected following agarose gel electrophoresis frequencies were observed (Table 1) but inby hybridization with a Tn554-specific sertions could be detected only by Southern probe. The few Em’ transductants obtained hybridization of these gels or by selection for were unlinked to Tc’ and were presumably linked tetracycline and erythromycin resissecondary-site chromosomal insertions. tances upon subsequent outcross to RN4470.

MUTATIONAL oEM9793

OEM981 7

tsM9ai

a

25

ANALYSIS OF att554 DEM9813

DEM9764

DEM9767 chr ‘om. 11. 3 kb 4.6 kb

+ _ pEM9875 I

pEM9876

chrom. 11.3 kb 4.6 kb

FIG. 3. Agarose gel electrophoresis of whole-cell lysates prepared from transductant colonies (seven for each recipient plasmid) showing examples of plasmids with wild-type (pEM9793, pEM9817, pEM9875, pEM9876), partial (pEM98 13), and no activity (pEM98 18, pEM9764, pEM9767). The six plasmids in the upper panel have deletions in att5.54 and the two in the lower panel carry chemically induced mutations. Lanes marked + and - are controls containing purified plasmid DNA of pEM9701 ::Tn554 and pEM970 1, respectively. A faint band with a slightly faster mobility than the 11.3-kb plasmid, visible in some of the lanes, is the open circular form of the target plasmid.

For other plasmids with high transduction frequencies, inserts could not be detected or isolated by any of these methods (Table 1). Nevertheless, these plasmids were considered to possesspartial att554 activity since their high transduction frequencies distinguish them from the totally nonfunctional targets. The apparent paradox of these high transduction frequencies in the absence of physical demonstration of Tn554 insertion into these plasmids probably reflects the fact that a single transposition event would be sufficient to produce an Em’ colony, since the level of resistance to erythromycin is insensitive to gene dosage (one copy, if constitutively expressed,renders the cell resistant to > 100 pg/ ml erythromycin). However, detection of a single copy of the 11.3-kb plasmid per cell would have been below the sensitivity of these methods. Taken together, the mutant plasmids define a region of att554 that is absolutely essential for expression of transposition activity. This region includes the core sequence GATGTA, -3 to +3 and extends upstream from the core to a point somewhere between

- 7 and - 15 and downstream to + 8. Transposition does not occur if deletions into att554 extend past these boundaries. Transposition frequency is greatly increased by the presence of additional flanking sequences at att554, and transposition into plasmids containing sequencesbeyond -28 on the left and +25 on the right is essentially normal. Thus, the DNA required for efficient recognition of att554 is not more than 53-bp long.

Analysis of Point Mutations in att554 Following transformation of bisulfitetreated pEM970 1/pT 181 heteroduplex DNA into RN4470, 200 colonies were tested for att554 activity. Eight phenotypically att554colonies were isolated, of which six contained pEM9701 carrying an average of 11 mutations per plasmid within the att554 fragment (-89 to +82). The other two att554- colonies contained wild-type pT 181 plasmid, derived from the non-att554 containing strand of the heteroduplex DNA. Ten of the remaining 192 att554+ colonies were chosen randomly for sequence analysis; of these, six contained

26

MURPHY, REINHEIMER, -20

-30

-10

AND HUWYLER +lO

+l

+20

pEM9878 ,,EMg87, pEM9878 pEM9879 pEM9701 p&,9887 p&,9888 pEMl3870 pEM9871 pEM9872 pEM9875

t30 .

.

.

A---r-.------.r-----.----~--------'-----A----.--.....-.

-----_________________________________

**A---------A---------.-...

-----------------------T-----------------.----~----.---.......

--- A-------------------------------A-------~.--------.--..-ATCGCAGTTCATAATCATCCATCCGGTGATGTAACGCCCTCACAAGAAGATATCATAACA -T--TT--------------T-~TT-T~--------..--. -_-______----T-----T--TT---T--------------.-----------........ _________ ______________---__---

--AA-------------------A--A--A------.-..-

-T--------.T--T----.------T--TT----TT..--.---.--......-..-------- - -A--------------------A-~--..----.---.--..-A--A-..-....... -T-----T--TT--T------.-----.TTT---..-..--..---...... --------

FIG. 4. Mutations induced in att554 by sodium bisulfite mutagenesis. Only the region -32 to +32 is shown; all plasmids contained additional mutations in the nonessential portions of the 171-bp FnuDIIBamHI fragment (-89 to +82). Four att554+ plasmids (listed above the sequenceof wild-type att554) had transposition frequencies ranging from 93 to 23 1% of wild-type. Six att554- plasmids (listed below the wild-type) had transposition frequencies (as in Table 1) equal to that of a negative control.

wild-type parental att554 sequences, while the remaining four carried an averageof eight mutations per plasmid. Thus, plasmids carrying multiple mutations but having wildtype activity were about 10 times more frequent than plasmids deficient for att554 activity. However, the distribution of mutations differed significantly between the two groups: the average density of hits within the region -30 to +30 was three times higher for the att554- plasmids than for the att554+ plasmids (Fig. 4), emphasizing the importance of this central region. Because all the plasmids carried multiple mutations, only limited conclusions can be drawn with respect to particular nucleotides. However, att554 appears to tolerate a significant amount of mutation; mutations at -7, + 1, and +6 within the essential region, and at -27, -24 and +16 in the intermediate zone (Fig. 4), had no effect on att554 function.

Novel Junctions Generatedby Insertion into Partially DefectiveTargets At least one Tn554 insertion into each deleted plasmid with partial or wild-type att554 activity was analyzed to verify that the original target mutation was present and to determine whether transposition into the mutant att554 occurred at the usual location between +3 and +4 in the (+) orientation or between -3 and -4 in the (-) orientation. Both the left and right junctions were sequenced,using

primers complementary to nt 59-43 and 661 l-6627 of Tn554 (Murphy et al., 1985). All insertions examined were found to be correctly located, and the att554 mutations were present as expected. However, in three insertions (into pEM9765, pEM9790, and pEM9764) the junction between the right end of Tn554 and the mutant target deviated from the wild-type sequence (Table 1); all of the left junction sequenceswere normal. In two casesthe abnormal sequencesreplacing GATGTA are present within att554: the pEM9765 insertion contains 5’-TACATC-3’, the complement of the wild-type sequence5’GATGTA-3’, and the pEM9790 insertion contains 5’-GCGTTA-3’, the complement of nucleotides +2 through +7 of att554. Thus site and orientation specificity of transposition were unaffected by these deletions, but aberrant right junction sequenceswere common. The possible origin of thesejunctions is discussed below. DISCUSSION

We have used in vitro mutagenesis to begin to define the transposition target of Tn554, a site- and orientation-specific transposable element. Nucleotides were deleted on either the right or left side of the 6-bp core sequence, 5’-GATGTA-3’ (-3 to +3). Deletions ending between -7 and +7 abolish target activity of att554. Those that stop outside -28 or +25 have no effect. Thus both arms are required

MUTATIONAL

ANALYSIS

for att5.54 activity, a result that contrasts with findings from a similar study of the attachment site for another site-specific transposon, Tn7. In that case the required sequenceslie entirely to one side of the insertion site and do not include the site itself (Gringauz et al., 1988). Deletions ending between - 15 and -26 or between +8 and +12 reduced transposition by varying degrees. For mutants displaying partial activity, there was no clear correlation between the length of the remaining att554 sequences and transposition frequency. For example, pEM9843 (endpoint at -21) was indistinguishable from wild-type while pEM9774 and pEM9828 (endpoints at -26 and - 15, respectively) both exhibited somewhat reduced activity. Similarly, a cluster of 11 deletions ending between +8 and + 12 displayed transposition activities ranging from nearly normal to virtually undetectable. The simplest explanation for this variability is that each plasmid carries different sequences adjacent to att554, due to the varying extent of Ba131 deletion into the vector in each case (data not shown). For example, pEM98 13 (endpoint + lo), among the most active ofthe intermediate class, has the best fit ( 1l/ 15 nt) to the wild-type sequence in the region + 11 to +25. In contrast, pEM9767, with the same endpoint but different flanking sequences,is among the least active plasmids. However, this explanation does not hold for all plasmids: pEM9765 (+9), with a very poor fit (5/ 15 nt) to nt + 11 to +25, has approximately the same activity as pEM98 13. It is probable that individual matched or mismatched nucleotides within this intermediate region of att554 play a role in determining the phenotype of each plasmid, but the data do not suggest a consensus sequence. The absence of a sharp boundary between recombination-proficient and -deficient targets implies that att554 contains multiple or complex protein binding sites, a feature common to other site-specific recombination systems such asthose of bacteriophage X integration or Tn3 resolvase (Bushman et al., 1985; Grindley et al., 1982). Both are known to in-

OF

aft.554

27

volve complex binding sites for one or more proteins that extend well beyond the actual site of recombination. Unlike h integration, however, Tn554 transposition does not appear to have an absolute requirement for a short region of sequence homology between the recombining partners. The bisulfite-induced mutation at +l in att554 is particularly significant because it introduces a mismatch into the homologous core (GATGTA + GATATA) without detectably affecting transposition frequency. In contrast, in the X, Cre, andflp site-specific recombination systems(all of which, like Tn554, utilize recombinases belonging to the Int family [Argos et al., 1986]), mutations in the core are best tolerated when both partners are affected; sequence homology, rather than specific sequence or length of the core, is the more important variable affecting recombination proficiency (Bauer et al., 1984; De Massy et al., 1984; Hoess et al., 1986; Ross and Landy, 1983; Senecoff et al., 1985). In addition to increasing the recombination frequency, DNA sequencesflanking the essential core region of att554 appear to be important for the generation of the correct junction sequences.Despite that fact that insertions into truncated att554 sites retain site and orientation specificity, aberrant junction sequences are frequently found. These always occur in the right end of Tn554, regardless of the orientation of insertion; a different 6-bp sequence, sometimes identical to nearby sequences within att554, is substituted for the GATGTA core. Analysis of serial transposition events indicates that the hexanucleotide sequence at left junction sequence of a new insertion is derived from the hexanucleotide at the insertion site. The right end of the new insertion is derived from the left junction of the parent insertion, which is itself a remnant of the previous transposition target (Murphy, 1990; E. Murphy, L. Huwyler, L. Tillotson, and D. Dubin, in preparation). This is formally similar to the situation with Tn9 16 and Tn 1545, except for two important mechanistic differences: Tn554 junction sequence translocation proceeds unidi-

28

MURPHY, REINHEIMER,

rectionally whereas that of Tn9 16 and Tn1545 occurs in either direction (Caillaud and Courvalin, 1987; Clewell et al., 1988) and the two conjugative transposons utilize a free circular intermediate (Gawron-Burke and Clewell, 1982; Scott et al., 1988) while Tn554 excision occurs on the order of 10e7 per donor molecule (Murphy et al., 1981). Thus one explanation for the aberrant junctions of the insertions into pEM9790, pEM9764, and pEM9765 is that each of these insertions are derived from an intervening secondary-site insertion, their right junctions accurately reflecting the left junctions of the putative parental insertions. (In the case of pEM9765, the putative insertion would be a (-) insertion at att554). Another explanation is that the abnormal junction sequences reflect some type of misalignment during recombination resulting from the absence, in the deleted at054 sites, of flanking DNA-protein contacts that normally serve to stabilize the recombination complex. The net result of this misalignment would be an incorrect transfer of sequence information from the 5’junction of the donor to the 3’junction of the new insertion. For the insertion into pEM9790, at least, the new information would appear to be derived from nearby att554 sequences. ACKNOWLEDGMENTS We thank Donald T. Dubin, Karl A. Drlica, and Steven J. Projan for helpful discussions and critical comments on the manuscript. This work was supported by Grant GM27253 from the National Institutes of Health.

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AND HUWYLER

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