Vol. 172, No. 3

Integration Host Factor Plays a Role in IS50 and Tn5 TranspositionJOHN C. MAKRIS,t PATRICE L. NORDMANN,t AND WILLIAM S. REZNIKOFF*

Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison,Madison, Wisconsin 53706

Received 23 June 1989/Accepted 7 December 1989

In Escherichua coli, the frequencies of ISSO and TnS transposition are greater in Dam- cells than in isogenicDam' cells. IS50 transposition is increased approximately 1,000-fold and TnS transposition frequencies areincreased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for bothintegration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and TnS approximatethose found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. Theseresults suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that areobserved in Dam- cells. It is also shown that the level of expression of ISSO-encoded proteins, P1 and P2,required for IS50 and TnS 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 geneexpression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pairsegment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHFbinding site remains to be determined.

Transposon Tn5 is a composite of two inverted IS50sequences that flank a unique DNA sequence encodingresistance to several antibiotics (Fig. 1) (1, 13, 16, 23, 33).Each IS50 is defined by two end sequences, the inside end(IE) and the outside end (OE), which are required for IS50transposition (33). Tn5 utilizes two inverted OE's for its owntransposition. Only IS5OR encodes the transposase proteinrequired for transposition of both IS50 and Tn5 (13, 15, 16,33). Host-encoded factors also promote and modulate thelevels of IS50 and Tn5 transposition. The DnaA proteinpromotes (31, 40) and Dam methylase decreases (21, 24, 39)IS50 and TnS transposition through specific interactions withIS50 DNA. Other host factors appear to influence the levelof IS50 and Tn5 transposition through either indirect ornonspecific interactions with IS50. Some of these factors arelonA (35), sulA (35), gyrA (14), polA (34, 37), and lexA andrecA (J. C. Makris, M. D. Weinreich, and W. S. Reznikoff,manuscript in preparation). Host factors involved in modu-lating transposition may do so by either modulating theactivities of factors required for transposition, such as thelevel of P1 transposase expression or the activity of the IEthrough methylation, or by fulfilling a structural requirementfor transposition. This report presents evidence that theprotein known as integration host factor, or IHF, is involvedin modulating the level of IS50 and Tn5 transposition in cellswhich lack the GATC Dam methylase activity. IHF appearsto act as an accessory factor that is not required fortransposition but that can enhance it under certain condi-tions.IHF is a histone-like protein with a molecular weight of

about 20,000 that consists of an alpha and a beta subunit,each with a molecular weight of about 11,000, which areencoded by himA and himD, respectively (6, 26). IHFprotein binds to specific DNA sites with a consensus ofYAANNNNTTGATW (10, 20), although it has also been

* Corresponding author.t Present address: Department of Molecular Biology, Genentech

Inc., South San Francisco, CA 94080.T Present address: Laboratoire de Bact6riologie-Virologie, H6p-

ital de la Piti6-Salpdtri&re, 75641 Paris Cedex 13, France.

found to bind to specific sites that bear no homology to thisconsensus (19a). In most cases IHF has been shown to bendDNA upon binding (34).IHF is involved in many cellular processes. It regulates

the level of expression of several genes. These genes includeilv (7, 9), pifCAB (17), and pip of phage Mu (18). It isinvolved in many site-specific recombination reactions. Ex-amples of these reactions include those associated withbacteriophages lambda (25) and +80 and P22 (20). IHF playsa role in many DNA replication systems. The gamma originof replication of plasmid R6K requires IHF for initiation ofDNA replication (5). Involvement of IHF has been shown tobe required for maintenance and replication of bacteriophageFl through a direct interaction with the Fl origin of replica-tion (12). DNA replication from the E. coli oriC is enhancedin vitro in the presence of IHF (2). IHF has been studied inrelationship to three transposition systems: IS10, IS1, andMu. It has been shown to affect the level and distribution ofIS10-mediated circle formation in vitro in a way that isdependent on the state of Dam methylation of the inside endof IS10 (29). A sequence with homology to the canonicalIHF binding site was found in IS10. Through deletionanalysis, this sequence was shown to play a role in thisreaction (29). IHF has been shown to bind to and bendspecific sequences on the ends of IS1, although the role ofIHF in IS1 transposition has not been determined (10).Finally, IHF facilitates Mu transposition through a super-coiling relief activity. This function occurs as a consequenceof IHF binding to a site approximately 950 base pairs (bp)from one end of Mu; thus, it acts as a distance (36). In manyof the systems presented here, the common function of IHFappears to be in assisting bending and wrapping ofDNA in away favorable for the reaction desired (8).

MATERIALS AND METHODS

Bacterial strains. All bacterial strains used are listed inTable 1. Isogenic strains used to evaluate the effects of ahimD deletion on IS50 and Tn5 transposition were derivedfrom strains GM1859 and GM3830. These strains are iso-genic except for a dam-3 allele present in GM3830. Intothese strains were transduced, via P1 phage, wild-type galK

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JOURNAL OF BACTERIOLOGY, Mar. 1990, p. 1368-13730021-9193/90/031368-06$02.00/0Copyright C) 1990, American Society for Microbiology

IHF ROLE IN 1S50 AND Tn5 TRANSPOSITION 1369

P4o - Neo, Str, Ble 0 P2P3 a a* P1

OE IE IE OE

IS50 L IS50 RFIG. 1. Schematic representation of composite transposon Tn5.

Tn5 consists of two IS50 inverted repeats, the IS50L and the IS50R,which flank a unique sequence that encodes several antibioticresistance genes (Neo, neomycin; Str, streptomycin; Ble, bleomy-cin). IS50 transposition requires an outside end (OE) and an insideend (IE) inverted in orientation to each other. This is studied asOE-IE transposition. TnS transposition requires two inverted OEsand is studied as OE-OE transposition. Only the IS50R encodes thefunctional transposase protein, P1, and the transposition inhibitorprotein, P2. A nonsense codon in IS50L causes the expression oftruncated versions of P1 and P2; these are proteins P3 and P4. Theseproteins have no known function.

and galE alleles from strain M8302 to generate strainsJCM100 and JCM101 and a himD deletion from strainRW1717 (38) to generate strains JCM101 and JCM103.Strains JCM109 and JCM11O were produced by mating thepOX38-gen episome (16) into strains JCM100 and JCM101.The series of isogenic strains used to evaluate the role of

the himA deletion in IS50 and Tn5 transposition werederived from parent strains K-37 and K-2691 (11). StrainK-2691 contains the himAASma deletion of the carboxyterminus of himA (11). A lac deletion linked to a Kanrmarker was transduced, via P1 phage, into strains K-37 andK-2691. Lac- candidate strains were screened for on Mac-Conkey-lactose media containing kanamycin (40 ,ug/ml). Thestrains created in this way are JCM104 and JCM105, respec-tively. Strains JCM106 and JCM107 were derived fromJCM104 and JCM105 by transduction of dam::Tn9 from thestrain 48.01 (21, 39).To ensure that strains had the correct Dam methylase

phenotype, plasmid pRZ1495 was prepared from each strainand the sensitivity to restriction endonucleases MboI andDpnI was determined (3).To determine whether a strain was deficient for the IHF,

two in vivo tests were used. Bacteriophage Mu plaques willnot form on a lawn of cells deficient for IHF protein

TABLE 1. Bacterial strains used in this study

Strain Relevant genotype Source (reference)

GM1859 AlacZJ74 galK gaIE4 rpsL purF M. MarinustonA2 tsx-J thi-I

GM3830 GM1859 dam-3 M. MarinusM8302 MO AlacZ thi-J W. ReznikoffRW1717 hfrH Str' sup' A(gal-bio)592 D. Greenstein (38)

A[himD]: :catJCM100 GM1859 Gal' This studyJCM101 GM3830 Gal' This studyJCM102 JCM100 A[himD]::cat This studyJCM103 JCM101 A[himD]::cat This studyK-37 gaIK2 supo strA M. FilutowiczK2691 K-37 himAASma M. FilutowiczJCM104 K-37 A(lac pro) Kanr This studyJCM105 K2691 A(lac pro) Kanr This studyJCM106 JCM104 dam-13::Tn9 This studyJCM107 JCM105 dam-13::Tn9 This studyJCM109 JCM100(pOX38-gen) This studyJCM110 JCM101(pOX38-gen) This study

pRZ1495 pRZ1496 pRZ1497

FIG. 2. Representations of plasmids pRZ1495, pRZ1496, andpRZ1479. tetR and Ori represent the tetracycline resistance gene andthe replication origin, respectively, of these plasmids. lac Z repre-sents the reading frame of the lacZ gene. This lacZ gene is fused toan IS50 OE in order to determine the transposition phenotype of thetransposon via the papillation assay. These plasmids are not drawnto scale. Plasmids pRZ1495 and pRZ1496 are identical except for a35-base-pair sequence proximal to the 1S50. In pRZ1495 this se-quence is an IS50 OE. In pRZ1496 this sequence is an IS50 IE.Plasmid pRZ1495 is used to assay OE-OE transposition, and plas-mids pRZ1496 and pRZ1497 are used to assay OE-IE transposition.Plasmids pRZ1496 and pRZ1497 contain the same DNA sequencesexcept that the orientation of the DNA containing the tet genebetween the OE and the IE is reversed with respect to these endsequences.

expression (27); thus, all strains were tested for their abilityto support Mu infection. The second test was based on therequirement for IHF for replication from the gamma origin ofplasmid R6K (5). All strains used in this study were assayedfor their ability to maintain the plasmid pRK526, whichcontains the R6K gamma origin for replication.

Plasmid constructs. Plasmids pRZ1495 (21), pRZ1496 (21),and pRZ1497 (22) are shown in Fig. 2. Plasmids pRZ901 andpRZ909 (39) encode lacZ fusions to the IS50 proteins (seeFig. 4).

Transposition assays. Transposition assays were per-formed through two independent methods: qualitativelythrough a papillation assay and quantitatively through amating-out assay (16, 19, 21).

Papillation assays were performed by the method of Krebsand Reznikoff (19). All strains were transformed with plas-mids pRZ1495, pRZ1496, and pRZ1497. These plasmidscontain a TnS-lacZ construct that lacks transcriptional andtranslational signals for lacZ. A strain which contains one ofthese plasmids can express 3-galactosidase as a conse-quence of the transposon transposing into an appropriatereading frame such that a ,B-galactosidase fusion protein ismade. Thus, Lac- cells bearing these plasmids will form redpapillae on lactose-MacConkey medium if transposition oc-curs. Host mutations that alter the transposition rate can beidentified by monitoring the accumulation of papillae inseveral colonies over a given time period (19, 21). By thismethod it is possible to compare the level of OE-IE and/orOE-OE transposition in a wild-type host strain to the level oftransposition in isogenic host strains with specific mutations.Since the collection of time points for each strain containingeach transposon was done simultaneously, a direct compar-ison of the level of papillation of a given transposon in adifferent strain can be made.Each papillation assay was performed three times. This

was done by plating 25 to 50 colonies on a plate and countingthe number of papillae that formed on 5 colonies on each ofthree plates over the course of several days. For each timepoint, the average numbers of papillae per colony from eachof the three plates were then averaged and a standarddeviation was determined per colony. The average standarddeviation for the number of papillae counted per colony is15%.

VOL. 172, 1990

1370 MAKRIS ET AL.

The doubling time of each strain containing these plasmidsmeasured in the presence of tetracycline (15 mg/liter)showed that all strains within an isogenic series grew at thesame rate.The mating-out transposition assay was performed by the

procedure of Johnson et al. (16). The conditions used aredescribed in Makris et al. (21). This assay allows a quanti-tative determination of the transposition frequency andpermits an independent confirmation of the transpositionphenotype as determined by the papillation assay. Unfortu-nately, strains that are deficient for IHF are unsuitable forthis assay because they are unable to maintain stably an Fplasmid episome such as pOX38-gen (10, 17). They are alsounsuitable for transposition assays that require lambdaphages which require IHF for both integration and excisionfrom the att site in the chromosome (25, 27, 38). Wecompared the papillation levels of strains with transpositionfrequencies determined by the mating-out assay with thepapillation levels of those strains in which the mating-outassay was not possible.

,I-Galactosidase assays. All f-galactosidase assays wereperformed by the method described by Miller (28).

Gel retardation assays. Gel retardation assays were per-formed by two techniques. The first technique enables thescreening of large restriction fragments, ca. 500 bp, derivedfrom IS50 for any significant alteration in their migrationwhen subjected to agarose gel electrophoresis in the pres-ence of IHF (5). Protein-DNA complexes were formed byadding dilutions of purified IHF (approximately 5 x 10-12mol; a gift from Marcin Filutowicz) to restriction fragmentsof the appropriate IS50-containing plasmid (approximately10-12 mol). These reactions were performed in TBE buffer(0.089 M Tris hydrochloride, 0.089 M boric acid, 0.002 Mdisodium EDTA) in a final volume of 20 ,ul. After preincuba-tion at room temperature for 5 min, 3 ,u of a solutioncontaining 20% (wt/vol) sucrose, 1 mM EDTA, 20 mM Trishydrochloride (pH 7.5), 0.01% (wt/vol) bromphenol blue,and 0.01% (wt/vol) xylene cyanol was added to the reactionmixture and the samples were loaded onto 1.0% agarosegels. These gels were run at 15 V/cm. After electrophoresis,the gels were stained in ethidium bromide and the DNA wasvisualized with UV light. These data are not presented.The procedure described above led to the discovery of one

500-bp-long restriction fragment of IS50 DNA that wassignificantly retarded by preincubation with IHF beforeagarose gel electrophoresis. To locate the IHF binding sitemore precisely, smaller restriction fragments of this regionof IS50 were generated and end labeled with 32p. This DNAwas preincubated with protein as described above. Thesefragments were then loaded onto 5% polyacrylamide gels,prepared with TBE buffer, and run at 150 V for 1 to 3 h. Thegels were dried and the bands were visualized after a 2- to5-h exposure to Kodak XAR film.

Equivalent IHF binding results were obtained with Dam-methylated and non-Dam-methylated DNA substrates.

RESULTS

Effects of a himD deletion on OE-OE transposition. Figure3a shows the accumulation of papillae over time for theisogenic strains containing plasmid pRZ1495. This plasmidcontains a transposon which utilizes two inverted IS50 OE's,as does transposon TnS. From this plot it can be seen thatthe level of papillation was highest in Dam- cells thatproduced IHF. This agrees with previous observations (21).In Dam- himD cells, however, the level of papillation was

a)

cz

0~Q

0~

0

-oE

zc)0'

c

000)

0~0

z0)

0sc)

TIME (hours)

b~~~30- 050 9-

0

0~~~~~30 40 50 6'

TIME (hours)

* DomTlHF+

A Dom IHFA Dam+IHF+ODam+IHF+

*DomThHF+

ADam IHF

ODam+IHF+

ADam+IHF

TIME (hours)

FIG. 3. himD and TnS/IS50 transposition. The average numbersof papillae accumulated per colony at different time points areplotted. The papillae are generated by plasmids pRZ1495, pRZ14%,and pRZ1497 in strains JCM100 (Dam' IHF+), JCM101 (Dam-IHF+), JCM102 (Dam' IHF-), and JCM103 (Dam- IHF-). TheIHF- phenotype in strains JCM102 and JCM103 is caused by adeletion in the himD gene. The rate of accumulation of papillae ineach strain reflects the relative OE-OE or OE-IE transpositionfrequency in each strain. (a) Accumulation of papillae generated byOE-OE transposition events from plasmid pRZ1495. (b) Accumula-tion of papillae from IE-OE transposition events from plasmidspRZ1496. (c) Accumulation of papillae from OE-IE transpositionevents from pRZ1497.

reduced to levels similar to that found in isogenic Dam'cells. These results suggest that the presence of IHF influ-ences the level of OE-OE transposition in Dam- cells butnot in Dam' cells.

In order to relate the papillation data to transpositionfrequencies for OE-OE transposition in each strain, weassayed the level of transposition in the IHF-proficientDam' and Dam- strains JCM109 and JCM110, which alsocontain the pOX38-gen episome. In the mating-out assay thefrequency of OE-OE transposition in JCM109 was 1.1 xl0-5 ± 1.0 X 10-5, while in JCM110 the transposition

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VOL. 172, 1990 ~~~~~~~IHFROLE IN 1S50 AND Tn5 TRANSPOSITION 1371

frequency was 2.2 x i ±09x1 .These transpositionfrequencies agree with previously reported results and sug-gest by inference from the papillation data that GE-GEtransposition frequency decreases about fivefold in a Dam-himD strain compared with that in a Dam- strain (21).

Effects of a himD deletion on OE-IE transposition. The datain Fig. 3b and c represent the level of papillation observed inthe four aforementioned isogenic strains for the transposonsin plasmids pRZ1496 and PRZ1497. Plasmids pRZ1496 andpRZ1497 both contain transposons that use inverted GE-IEfor transposition, as does ISSO. In pRZ1496, however, thepromoter of the transposase gene is proximal to the IE of thetransposon, and in pRZ1497 the transposase gene promoteris proximal to the GE (22). It has been previously shown thatthe orientation of the transposase gene with respect to theIS50 ends affects the level of transposition (4, 22). From bothfigures it can be seen that the level of papillation from OE-IEtransposons was significantly higher in the Dam- strain. Inthe Dam- himD strain, however, the level of papillation wasdramatically reduced for transposons with either trans-posase gene orientation. These results demonstrate that IHFis required for the high level of GE-IE transposition that isseen in Dam- cells. In the Dam' himD strains, for eachorientation of the transposase gene the frequency of trans-position was similar to that found in the isogenic Dam'himD' strain. This suggests that IHF is not required formaximal transposition in the Dam' strain.

In order to approximate the transposition frequency foreach GE-IE transposon in the himD strains, mating-outassays were performed. It was determined that in JCM109the transposition frequencies from the transposons on plas-mids pRZ1496 and pRZ1497 were 1.4 x 16±11x1-and 1.6 x 10-5 ± 1.5 x i0-5, respectively. In strain JCM110the transposition frequencies were 1.1 X 10-3± 1.0 X 10-3for the transposon on pRZ1496 and 9.5 x 10-3± 4.6 x 10-3for the transposon on plasmid pRZ1497. These transpositionfrequencies agree with previously reported values for thesetransposons (21) and suggest by inference from the papilla-tion data that GE-IE transposition decreases more than100-fold in Dam- IHF- cells compared with the levels inDam- IHF' cells.

Effects of a himA deletion on OE-OE and OE-IE transposi-tion. Deletion of either himA or himD leads to an IHF-phenotype. When GE-GE and GE-IE transposition wasexamined by the papillation assay in isogenic strains whichwere Dam' himA', Dam' himA, Dam- himA', or Dam-himA, results essentially identical to those yielded by for thehimD experiments were obtained (J. C. Makris, Ph.D.thesis, University of Wisconsin-Madison, 1989).

Effects of a himD deletion on ISSO protein expression. Thedata in Fig. 4 show the level of expression of P-galactosidasefusions to the carboxy termini of both the P1 transposase andthe P2 inhibitor or to the P1 transposase protein alone. Aspreviously reported, the level of 1S50 protein expressionincreased in the Dam- cells owing to an increased level ofexpression of the P1 transposase protein. These data showthat the level of IS50 protein expression does not decrease inhimD strains but in fact increases by approximately twofold.This is true both for the expression of P1 and P2 proteinstogether and for P1 expression alone. These data demon-strate that the decrease in papillation seen in Dam- himDstrains compared with Dam- himD' strains is not caused bya decrease in the expression of the IS50-encoded trans-posase.IHF protein can specifically bind to IS50 DNA. Figure 5

shows that restriction fragments derived from the outside

Plasmid

Pi P2

Fusion Proteins B -galactosidaseConstruction Expressed Units

2 Dom+ Dom- Dom+ IHF- DomTlHF-

pRZ901OE IS50 Inc Z

P2 Inhibitor p-gal

Pl1+P2 169±66 399±62 273±37 369±50Pl Tronsposase p8-gal

P1 P2rw r

pRZ9O9IOE IS50 lbc Z

I ~~~~~~~Pi76±35 134±16 104±20 174±37

Pi Tronsposase p-gal

FIG. 4. Effect of himD on IS50 gene expression. PlasmidspRZ9O1 and pRZ9O9 contain lacZ gene fusions to the IS50 genesfrom P1 and P2 proteins. P1 protein is the 1S50 transposase requiredfor 1S50 and Tn5 transposition. Plasmid pRZ9O1 can express 13-galactosidase fusion proteins of both P1 and P2 proteins. PlasmidpRZ9O9 expresses only the Pl-13-galactosidase fusion protein. Thelevel of expression of the P3-galactosidase fusion proteins from eachplasmid was assayed in strains JCM100 (Dam'), JCM101 (Dam-),JCM102 (Dam' IHF-), and JCM103 (Dam- IHF-). Strains JCM102and JCM103 contain deletions in the himD gene.

one-third of IS50 are specifically retarded on polyacrylamidegels in the presence of purified IHF. Data from this gelsuggest that IHF can specifically bind to a site between theBglI restriction site at base pair 201 of IS50 and the AvaIrestriction site at base pair 490 of 1550. Contained within thissequence is at least one putative IHF binding site with partial

-4--

FIG. 5. IHF protein-induced retardation of a DNA fragment ofIS50 DNA. The lane on the right shows the polyacrylamide gelelectrophoretic migration of restriction fragments of 1S50 DNAwithout preincubation with IHF protein. The lane on the left showsthe electrophoretic migration of the same IS50 restriction fragmentsin the presence of IHF protein. All restriction fragments wereproduced by cleaving IS50 DNA with restriction enzymes AvaI andBgll. The IS50 DNA fragments with little or no altered electropho-retic mobility in the presence of IHF protein are a 78-base-pairfragment at the bottom of the figure and a fragment at the top of thefigure which is 377 base pairs long. The fragment with alteredelectrophoretic mobility in the presence of IHF protein lies betweenthe BglI site at position 201 and the Aval site at position 490 of the1550. This DNA fragment is 289 base pairs long and contains onesequence with homology to the consensus IHF protein binding site.Under the conditions used in this assay, no DNA restrictionfragments containing other regions of IS50 DNA were found to havealtered electrophoretic mobility in the presence of IHF protein (datanot shown).

VOL. 172, 1990

1372 MAKRIS ET AL. J. BACTERIOL.

homology to the consensus IHF binding sequence. Thissequence is shown below:

Putative IHF binding site 386CACctctTTGAGT398Consensus IHF binding site CAANNNNTTGATT

IHF binding to other regions of IS50 DNA was not detected(data not shown). Regions which were examined include theIE.

DISCUSSIONFrom the data presented in this study, it is shown that IHF

plays a role in IS50 and Tn5 transposition in strains deficientfor Dam methylation. In strains in which Dam methylation isunimpaired, deletion of either himA or himD does notdramatically affect the frequency of IS50 and Tn5 transpo-sition. Since transposition still occurs in the absence of IHFexpression, IHF appears to modulate the transposition fre-quency only under special circumstances. It is also shownthat the level of expression of IS50-encoded proteins doesnot decrease when the cell is unable to produce IHF. Thisdemonstrates that IHF in dam cells is not involved inmodulating either IS50 transposition or TnS transposition byincreasing transposase expression. Since it is known thatIHF can bend DNA upon binding (30), it is possible that IHFplays some structural role in the transposition process.

Preliminary evidence is also presented in this report thatIHF can specifically bind in vitro to a region of IS50 DNAthat contains a sequence with homology to the consensusIHF binding site. However, the physiological role of this sitehas not been determined, and the exact points of contact ofIHF will have to be defined further through DNA footprint-ing. Once this is accomplished, a genetic study of this siteshould reveal its role in vivo.

It has been reported that DNA sequences with homologyto the consensus IHF binding site are present in the IS50 IE(21). Since, under the conditions tested, IHF was not foundto retard the electrophoretic mobility of DNA fragmentscontaining the IE, it is possible that direct IHF-IE interac-tions are not required for modulation of transposition. Thesedata agree with the observations of Makris et al. (21) and ofDodson and Berg (4a) that putative IHF binding sites foundat the IS50 IE may not play a direct role in OE-IE transpo-sition. That is, sequence changes in the presumed IE IHFsites do not affect transposition in the expected fashion indam hosts.The analysis of the role of IHF in Mu transposition (36)

suggests a possible model for its effect on IS50/Tn5 transpo-sition. IHF is thought to facilitate Mu transposition bycompensating for an insufficient negative superhelicity in thedonor DNA molecule. This supercoiling relief activity ofIHF occurs as a result of its binding at an interior site in theMu genome. How might this mechanism be applicable to theIS50/TnS system? We would need to hypothesize that thelack of Dam methylation has three effects: increase intransposase synthesis (39), increase in transposase recogni-tion of IE (21, 39), and change in the superhelical density ofthe donor vector. This last hypothetical activity might serveto counteract the transposition-enhancing effects of the othertwo known Dam effects unless a supercoiling relief activitysuch as IHF were present. This model is currently beingtested.A comparison of the IS50 and IS0 transposition systems

yields some interesting similarities. In both systems the roleof IHF protein is strongly influenced by the methylationstate of the DNAs (29). In both systems the IHF protein actsas an accessory protein which, under some conditions, can

strongly stimulate the reaction being assayed. Finally, inboth systems the site on the insertion sequences with whichIHF has been implicated in interacting in vitro is closer tothe OE than to the IE (29) despite the fact that the GATCdam methylase recognition sites known to influence the levelof transposition of both insertion sequences are found in theIE's (21, 29).Many factors are common to the functions of both oriC

and IS50. These factors include dnaA protein, Dam methyl-ation, and IHF (2, 32). These factors may have similar rolesin promoting the respective functions of each system. Onepossible role for IHF in each system is to assist in specificDNA wrapping and/or bending that promotes the requiredinteractions for the subsequent function of each DNA se-quence (8, 30).

ACKNOWLEDGMENTS

We thank Marcin Filutowicz for the gift of IHF, G. Marinus forproviding us with strains GM1859 and GM3830, and David Green-stein for strains RW1717 and P011734.

This work was funded by Public Health Service grant GM-19670from the National Institutes of Health. P.L.N. was supported in partby the Elf-Aquitaine and Sanofi Companies (France).

LITERATURE CITED1. Berg, D. E. 1989. Transposon TnS, p. 185-210. In D. E. Berg

and M. M. Howe (ed.), Mobile DNA. American Society forMicrobiology, Washington, D.C.

2. Bramhill, D., and A. Kornberg. 1988. Duplex opening by dnaAprotein at novel sequences in initiation of replication at theorigin of the E. coli chromosome. Cell 52:743-755.

3. Davis, A. 1980. Advanced bacterial genetics. Cold Spring Har-bor Laboratory, Cold Spring Harbor, N.Y.

4. Dodson, K. W., and W. Berg. 1989. Factors affecting transpo-sition activity of IS50 and TnS ends. Gene 76:207-213.

4a.Dodson, K. W., and W. Berg. 1989. Saturation mutagenesis ofthe inside end of insertion sequence IS50. Gene 85:75-81.

5. Filutowicz, M., and K. Appelt. 1988. The integration host factorof Escherichia coli binds to multiple sites at plasmids R6K'sgamma origin and is essential for replication. Nucleic AcidsRes. 16:3829-3843.

6. Flamm, E. L., and R. A. Weisberg. 1988. Primary structure ofthe hip gene of E. coli and of its product the beta subunit ofintegration host factor. J. Mol. Biol. 183:117-128.

7. Friden, P., K. Voelkel, R. Sternglanz, and M. Freundlich. 1984.Reduced expression of the isoleucine and valine enzymes inintegration host factor mutants of Escherichia coli. J. Mol. Biol.172:573-579.

8. Friedman, D. I. 1988. Integration host factor: a protein for allreasons. Cell 55:545-554.

9. Friedman, D. I., E. J. Olson, D. Carver, and M. Geliert. 1984.Synergistic effect of himA and gyrB mutations: evidence thathim functions control expression of ilv and xyl genes. J. Bacte-riol. 157:484-489.

10. Gamas, P., M. Chandler, P. Prentki, and D. Galas. 1987.Escherichia coli integration host factor binds specifically to theends of insertion sequence IS1 and to its major insertion hotspotin pBR322. J. Mol. Biol. 195:261-272.

11. Granston, A. E., D. M. Alessi, L. J. Eodes, and D. I. Friedman.1988. A point mutation in the Nul gene of bacteriophage lambdafacilitates growth in Escherichia coli with himA and gyrBmutations. Mol. Gen. Genet. 212:149-156.

12. Greenstein, D., N. D. Zinder, and K. Horiuchi. 1988. Integrationhost factor interacts with the DNA replication enhancer of thefilamentous phage Fl. Proc. Natl. Acad. Sci. USA 85:6262-6266.

13. Isberg, R. R., A. L. Lazaar, and M. Syvanen. 1982. Regulationof TnS by the right repeat proteins: control at the level of thetransposition reaction? Cell 30:883-892.

14. Isberg, R. R., and M. Syvanen. 1982. DNA gyrase is a host

VOL. 172, 1990 IHF ROLE IN IS50 AND TnS TRANSPOSITION 1373

factor required for transposition of Tn5. Cell 30:9-18.15. Johnson, R. C., and W. S. Reznikoff. 1984. Role of ISSOR

proteins in the promotion and control of Tn5 transposition. J.Mol. Biol. 177:645-661.

16. Johnson, R. C., J. C. P. Yin, and W. S. Reznikoff. 1982. Controlof Tn5 transposition in Escherichia coli is mediated by a proteinfrom the right repeat. Cell 30:873-882.

17. Kennedy, M., M. Chandler, and D. Lare. 1988. Mapping andregulation of the pifC promoter of the F-plasmid. Biochim.Biophys. Acta 950:75-80.

18. Krause, H. M., and M. P. Higgins. 1986. Positive and negativeregulation of the Mu operator by Mu repressor and Escherichiacoli integration host factor. J. Biol. Chem. 261:3744-3752.

19. Krebs, M. P., and W. S. Reznikoff. 1988. Use of a Tn5 derivativethat creates lacZ translational fusions to obtain a transpositionmutant. Gene 63:277-285.

19a.Kur, J., N. Hasan, and W. Szybalski. 1989. Physical andbiological consequences of interactions between the integrationhost factor (IHF) and the coliphage X PR' promoter and itsmutants. Gene 81:1-15.

20. Leong, J., S. Nunes-Duby, C. Lesser, P. Youderain, M. Susskind,and A. Landy. 1985. The phi80 and P22 attachment sites:primary structure and interaction with Escherichia coli integra-tion host factor. J. Biol. Chem. 260:4468 4477.

21. Makris, J. C., P. L. Nordmann, and W. S. Reznikoff. 1988.Mutational analysis of insertion sequence 50 (IS50) and trans-poson 5 (Tn5) ends. Proc. Natl. Acad. Sci. USA 85:2224-2228.

22. Makris, J. C., and W. S. Reznikoff. 1989. Orientation of IS50transposase gene and transposition. J. Bacteriol. 171:5212-5214.

23. Mazodier, P., P. Cossart, E. Giraud, and F. Gasser. 1985.Completion of the nucleotide sequence of the central region ofTn5 confirms the presence of three resistance genes. NucleicAcids Res. 13:195-205.

24. McCommas, S. A., and M. Syvanen. 1988. Temporal control oftransposition in TnS. J. Bacteriol. 170:889-894.

25. Miller, H. I. 1981. Multilevel regulation of bacteriophagelambda lysogeny by the E. coli himA gene. Cell 25:269-276.

26. Miller, H. I. 1984. Primary structure of the himA gene of E. coli:homology with DNA binding protein HU and association withthe phenylalanine tRNA synthetase operon. Cold Spring HarborSymp. Quant. Biol. 49:691-698.

27. Miller, H. I., A. Kikuchi, H. A. Nash, R. A. Weisberg, and D. I.Friedman. 1979. Site specific recombination of bacteriophagelambda: the role of host gene products. Cold Spring HarborSymp. Quant. Biol. 43:1121-1126.

28. Miller, J. 1972. Experiments in molecular genetics, p. 352-355.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

29. Morisato, D., and N. Kleckner. 1987. TnlO transposition andcircle formation in vitro. Cell 51:101-111.

30. Pettijohn, D. E. 1988. Histone-like proteins and bacterial chro-mosome structure. J. Biol. Chem. 263:12793-12796.

31. Phadnis, S. H., and D. E. Berg. 1987. Identification of base pairsin the IS50 0 end needed for IS50 and Tn5 transposition. Proc.Natl. Acad. Sci. USA 84:9118-9122.

32. Russel, D. W., and M. Zinder. 1987. Hemimethylation preventsDNA replication in E. coli. Cell 50:1071-1079.

33. Sasakawa, C., and D. E. Berg. 1982. IS50 mediated transposi-tion: discrimination between the ends of an IS element. J. Mol.Biol. 159:257-271.

34. Sasakawa, C., Y. Uno, and M. Yashikawa. 1981. The require-ment for both DNA polymerase and 5' to 3' exonucleaseactivities of DNA polymerase I during Tn5 transposition. Mol.Gen. Genet. 182:19-24.

35. Sasakawa, C., Y. Uno, and M. Yashikawa. 1987. lonA-sulAregulatory function affects the efficiency of transposition of Tn5from lambda b221CI857 Pam 0am to the chromosome. Biochem.Biophys. Res. Commun. 142:879-884.

36. Surette, M. G., B. D. Lavoie, and G. Chaconas. 1989. Action ata distance in Mu DNA transposition: an enhancer-like elementis the site of action of supercoiling relief activity by integrationhost factor (IHF). EMBO J. 8:3483-3489.

37. Syvanen, M., J. D. Hopkins, and M. Clements. 1982. A new classof mutants in DNA polymerase I that affects gene transposition.J. Mol. Biol. 158:203-212.

38. Weisberg, R. A., and A. Landy. 1983. Site specific recombina-tion in phage lambda, p. 211-250. In R. W. Hendrix, J. W.Roberts, F. W. Stahl, and R. A. Weisberg (ed.), Lambda II.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

39. Yin, J. C. P., M. P. Krebs, and W. S. Reznikoff. 1988. The effectof dam methylation on Tn5 transposition. J. Mol. Biol. 199:35-45.

40. Yin, J. C. P., and W. S. Reznikoff. 1987. dnaA, an essential hostgene, and TnS transposition. J. Bacteriol. 169:4637-4645.