JOURNAL OF BACTERIOLOGY, Aug. 1985, p. 629-6340021-9193/85/080629-06$02.00/0Copyright © 1985, American Society for Microbiology

Vol. 163, No. 2

Reexamination of Phenotypic Defects in rec-] and rec-2 Mutants ofHaemophilus influenzae Rd

ROBERT BAROUKI* AND HAMILTON 0. SMITHDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine,

Baltimore, Maryland 21205

Received 15 March 1985/Accepted 28 May 1985

Radiolabeled donor DNA is efficiently taken up into competent H. influenzae Rd rec-2 mutant cells but doesnot undergo the rapid degradation observed in wild-type cells. Furthermore, donor label is not recovered in thechromosome even after 1 h. The donor DNA appears to remain in a protected state in a compartment that canbe separated from the rest of the cell. We interpret this as a failure of the donor DNA to be translocated outof the transformasome. In contrast, rec-l cells translocate labeled donor DNA normally. The donor labelaccumulates in the recipient chromosome, but, as expected for cells with a recombination defect, there is nopreferential localization of the label in sites homologous to the donor DNA. In addition, we have observed twoenzymatic activities that act on transformasome-associated DNA of rec-2 cells, an endonuclease which may playa role in the translocation of cloped circular DNA and a phosphatase.

The rec-J and rec-2 mutants of Haemophilus influenzaeRd take up donor DNA in normal quantity, but are verydeficient in transformation (22). The rec-l mutant (strainDB117) has a phenotype analogous to that of recA mutantsof Escherichia coli. It is deficient in transformation, phagerecombination, plasmid recombination, and induction ofprophage and is sensitive to UV light (4, 17, 22). Thephenotype of the rec-2 mutant is different in that it is onlydeficient in phage recombination (22), but not in intracellularplasmid recombination (17). Also, UV sensitivity and pro-phage induction are normal (22).The fate of donor DNA in both mutants was examined by

Notani et al. (18), using radioactive and density-labeleddonor DNA. Treatment of rec-2 cells with digitonin afterDNA uptake selectively released much of the donor DNAfrom the cells, suggesting a periplasmic location of the DNA,whereas in wild-type and rec-J cells the donor DNA re-mained with the cell after such treatment. Donor DNA labelbecame associated with the recipient chromosome in wild-type and rec-l cells but not in rec-2 cells. It was not clearhow a defect in recombination could account for the ob-served phenotype of rec-2 cells.

In an attempt to clarify the nature of the rec-2 phenotype,we have reexamined the fate of donor DNA in rec-l andrec-2 cells by using recently developed techniques (1, 9) andhave interpreted the results according to the recently pro-posed transformasome model (10) for transformation in H.influenzae (Fig. 1). The essential feature of this model is theexistence of a two-stage entry process for donor DNA. DNAis first taken up into membranous surface structures, thetransformasomes, and then is translocated to the cytoplasmwhere it undergoes recombination. The parts of the donorDNA molecule that are not recombined undergo degrada-tion, and the products become randomly incorporated intothe recipient chromosome. On the basis of the results ofNotani et al. (18) and the results presented in this paper, wepropose that the transformation deficiency in rec-2 cells isprimarily due to a defect in the translocation process.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. Wild-type cells wereH. influenzae Rd KW22 (K. W. Wilcox, Ph.D. thesis, JohnsHopkins University, Baltimore, Md., 1975). Transformation-deficient strains were H. influenzae DB117 (rec-1) and H.influenzae Rd(DB117)rec- (rec-2). These were obtained fromJ. Setlow, and their derivation and phenotypes have beendescribed previously (2, 22, 23). Cells were grown in 2.5%heart infusion broth (Difco Laboratories) supplemented with10 ,ug ofhemin per ml (Eastman Chemical Products, Inc.) and2 pug ofNAD per ml (Sigma Chemical Co.). Cells were madecompetent by the M-IV procedure of Herriott et al. (8) andeither used fresh or stored in 20% glycerol at -70°C. PlasmidspEUP1 and pCML6 were isolated from E. coli HB101 (hsdSrecA) and MM294 (endA hsdR), respectively, by the alkalineprocedure of Birnboim and Doly (3) and further purified byone or two CsCl-ethidium bromide centrifugations (19).Plasmid pEUP1 contains an 11-base-pair uptake sequencecloned into the EcoRI site of pBR322 (6). Plasmid pCML6contains a 10-kilobase (kb) insert of H. influenzae Rd DNAin the BamHI site of pEUP1 (9).

Preparation of radioactively labeled DNA. Nick translationof pEUPi and pCML6 DNA was carried out as described byKahn et al. (9), using either [a-32P]dCTP (3,000 Ci/mmol;Amersham Corp.) or [5-3H]dCTP (25 Ci/mmol, ICN Phar-maceuticals, Inc.). Average DNA specific activities were 2x 107 and 1 x 106 cpm/,ug, respectively. For 32P-5'-endlabeling, linear DNA was first dephosphorylated on the 5'termini in a reaction mixture (50 RI) containing 10 mMTris-hydrochloride (pH 8), 1 mM disodium EDTA, 1 ,ug ofDNA, and 10 U of bacterial alkaline phosphatase per ml(Worthington Diagnostics). Incubation was carried out at65°C for 60 min. The DNA was repurified by three phenolextractions, butanol extraction, and ethanol precipitation.The DNA was resuspended in 50 RI of reaction buffercontaining 50 mM Tris-hydrochloride (pH 8), 10 mM MgC92,5 mM dithiothreitol, 5% dimethyl sulfoxide (20), 50 puCi of[,y-32P]ATP (3,000 Ci/mmol; Amersham) and 2 U of T4polynucleotide kinase (New England BioLabs). The reactionwas carried out for 30 min at 37°C. Unincorporated label

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630 BAROUKI AND SMITH

04E

0AVtC.On.

I binding II uptake III transocation IV nucleation V IntegrationFIG. 1. Model for DNA entry and integration in H. influenzae. Biochemical and morphological data obtained recently (10) have led to the

elaboration of a hypothetical model for DNA entry and recombination in H. influenzae. DNA is taken up into small vesicles at the surfaceof the cell (transformasomes) and then is translocated into the cytoplasm. DNA first interacts reversibly with specific receptors present at thesurface of the transformasome. This binding leads to the irreversible uptake of the double-stranded DNA into the vesicle. At this stage, DNAis in a protected state; it is resistant to extemal DNases and internal restriction-modification enzymes. DNA exits the transformasome froma free end. The 5' strand is completely degraded. The 3' strand is partially degraded on entry into the cytoplasm. When homology is found,however, this strand integrates into the chromosome. If donor DNA is heterologous, the 3' strand is completely degraded. We show in thisarticle that the rec-2 mutant is blocked at the step of translocation, whereas the rec-J mutant is deficient in homologous integration.

was removed by chromatography over Sephadex G25(Pharmacia Fine Chemicals). The specific activity was ap-proximately 106 cpm/,ig.DNA uptake and reisolation. Radiolabeled DNA (30 to 100

ng) was added to ca. 2 x 109 competent cells in 1 ml of M-IVmedium in a 1.5-ml Eppendorf tube and incubated forvarious times. After this pulse period, further uptake oflabeled DNA was inhibited by addition of a 20-fold excess ofcold pCML6 DNA, and incubation was continued as speci-fied in individual experiments. The reaction was terminatedby chilling the cells, centrifuging, and washing with 1 ml of1.5 M CsCl in solution 21 (8). The cells were further washedwith TE buffer (10 mM Tris-hydrochloride [pH 8.0], 1 mMEDTA) and resuspended in 300 i,l of TE buffer. The cellswere lysed by addition of 30 ,ul of 10% sodium dodecylsulfate followed by incubation for 15 min at 37°C. The lysatewas treated with proteinase K (100 ,ug/ml; Boehringer Mann-heim Biochemicals) for 1 h at 37°C. After two phenolextractions and two butanol extractions, the DNA wascollected by ethanol precipitation and redissolved in 20 ,ul ofTE buffer. DNA was then analyzed by agarose gel electro-phoresis and autoradiography.

Gel electrophoresis, autoradiography, restriction enzymes,and DNA fragment purification. DNA was fractionated byelectrophoresis on 1% agarose gels in Tris-EDTA-acetate(TEA) buffer for 2 h at 7 V/cm. Gels were dried andautoradiographed with Kodak XAR film and Dupont CronexQuanta III intensifying screens (16). Restrictionendonucleases used to analyze donor and incorporated DNAwere from New England BioLabs or Boehringer Mannheimand were used as specified by the manufacturer. Covalentlyclosed circular DNA was purified by electrophoresis in 1%low-melting-point agarose (International BiotechnologiesInc.) in TEA buffer in the presence of ethidium bromide (2jig/ml). The band was excised from the gel, and DNA wasrecovered by melting the gel at 65°C followed by repeatedphenol extractions at room temperature, butanol extraction,and ethanol precipitation.

Extraction of cells with organic solvents to assaytransformasome-associated DNA. After allowing uptake ofDNA for 60 min, cells were washed with 1.5 M CsCl in TEbuffer and resuspended in 0.5 ml of this buffer in a 1.5-mlEppendorf tube. An identical volume of phenol or phenol-

acetone (1:1, vol/vol) was added, and the mixture wasshaken gently by hand for 1 min and centrifuged for 5 min(9). The organic and aqueous phases, as well as the pellet,were separated by centrifugation, and radioactivity wasdetermined by counting Cerenkov radiation.Phage recombination. H. influenzae strains lysogenic for

the tsl and ts2 temperature-sensitive mutants of phage HP1cl (4) were obtained from J. Setlow. Phage were preparedfrom the lysogens by induction with 0.035 ,ug of mitomycin Cper ml (7). The titer of phage was approximately 2 x 1010PFU/ml. Phage recombination frequencies in wild-type andmutant cells were determined by mixed-infection experi-ments (4). Cells (5 x 108 cells/ml) were incubated with equalamounts of tsl and ts2 phage (1.5 x 109 PFU/ml of each) at34WC for 10 min in 1 ml of 3.7% brain heart infusion broth.Infected cells were then chilled, centrifuged, washed, andresuspended in 1 ml of brain heart infusion broth. They werethen diluted in this broth to a concentration of 107 cells perml and incubated for 90 min at 34°C. The amount of progenyphage was determined by plating at 34°C, whereas theamount of wild-type recombinant phage was determined byplating at 40°C. Recombination frequencies are reported as(two times the number of plaques at 40°C) x 100/(number ofplaques at 34°C).

RESULTSFate of donor DNA in rec-l and rec-2 cells. Figure 2

represents a pulse-chase experiment in which 32P-labeled,ClaI-linearized pCML6 DNA was incubated with competentwild-type, rec-1, and rec-2 cells for 5 min and then chasedwith excess cold pCML6 DNA. All three strains took upDNA to the same extent (20% of input); however, the fate ofthis adsorbed DNA differed in each case. In wild-type cells,most of the DNA was recovered from the cell in an intactform during the first 5 min, although a significant amount ofthe label was already present in degraded (faster-migrating)species. At later time points, most of the radioactivity waspresent either in degraded DNA species or in the chromo-somal DNA band. By 30 to 60 min, nearly all the radioac-tivity was at the chromosomal DNA position. In rec-J cells,the same pattern of processing of donor DNA was observedwith similar, although slightly slower, kinetics. In this typeof analysis, rec-J cells are indistinguishable from the wild

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PHENOTYPIC DEFECTS IN H. INFLUENZAE MUTANTS

type. The fate of DNA is, however, very different in rec-2cells. Full-length linear pCML6 DNA molecules are stillfound even after 60 min of chase. A very slow degradation isobserved, but the most important observation is that no labelchases into the chromosome, even at very late times. Thedefect in rec-2 cells appears to occur at an earlier stage in thetransformation process than in rec-J cells, since rapid deg-radation of DNA and reincorporation into the chromosomeare not observed.Lack of detectable homologous integration of donor DNA in

rec-) cells. In the above experiment, donor DNA radioactiv-ity was efficiently incorporated into the chromosome of rec-Jcells; however, this does not necessarily imply that homol-ogous integration has taken place. To determine the extentof homologous integration, we have used the method ofrestriction analysis developed by Barany et al. (1), in whichchromosomal DNA is digested with BstEII, which cutspCML6 once near the middle of the cloned H. influenzaeDNA insert. If radioactive donor DNA has homologouslyintegrated into the chromosome, then BstEII digestion pro-duces two junction fragments, 7.0 and 12.5 kb in length (1),that can be distinguished from the unintegrated linear donorDNA fragments. Figure 3 shows the result of such anexperiment. Junction fragments are readily observed in thewild-type control cells, but are not detectable in the rec-Jcells. In addition, junction fragments are absent in bothwild-type and rec-J cells when pEUP1, the heterologousvector portion of pCML6, is used as donor DNA. Therefore,the label found in the chromosome of rec-J cells is not due tohomologous integration but rather to random reincorpora-tion into the chromosome.Donor DNA remains in the protected state in rec-2 cells.

After uptake in rec-2 cells, donor DNA radioactivity did notappear in the chromosomal band region even after 1 h (Fig.2). In the experiment shown in Fig. 4, labeled DNA isolated

w t rec-1 rec-2In 0 5 15 30 60 0 5 15 30 60 0 5 15 30 60

Chr _wigspCML6-.w4

FIG. 2. Autoradiogram showing the kinetics of DNA uptake andprocessing in wild-type (wt), rec-1, and rec-2 cells. Nick-translatedClaI-linearized pCML6 DNA (60 ng, 4 x 105 cpm) was added to 1 mlof freshly grown, competent wild-type, rec-1, and rec-2 cells at 37°Cfor 5 min (2 x 109 cells per ml). A 20-fold excess of cold linearpCML6 was then added, and 200 samples were removed at 0, 5,15, 30, and 60 min of chase. Samples were centrifuged and washedas described in the text. Total DNA was extracted and analyzed byelectrophoresis in 0.8% agarose gels and autoradiographed. InputDNA is shown in lane In. Uptake of DNA was similar for the threestrains (20 to 30% of input DNA).

abcde fgh i i kI

....z.'i .

Chr.PCML e

B.g .* -pEUP 1

FIG. 3. Autoradiogram showing the absence of homologous in-tegration in rec-l cells. Donor DNA was either ClaI-linearized,nick-translated pCML6 (lanes a through f) or ClaI-linearized, nick-translated pEUP1 (lanes g through 1). Donor DNA (50 ng) was addedto 2 x 109 cells (rec-J or wild type) for 1 h. Cells were washed, andDNA was reisolated as described in the text. Lane a: input linearpCML6. Lanes b and c: reisolated DNA from wild-type cells andrec-J cells, respectively. Lane d: BstEII digest of donor linearpCML6 DNA showing the presence of one BstEII site in pCML6and yielding two fragments, B1 and B2 (9.6 and 4.8 kb, respectively).Lanes e and f: BstEII digest of reisolated DNA from wild-type andrec-I cells, respectively. Note the presence of two junction frag-ments, J1 and J2 (-, 12.5 and 7 kb, respectively) (1), in wild-typecells that can be readily distinguished from B1 and B2 fragments. Onintegration of donor DNA, junction fragments are generated by therestriction site in the donor region and the two adjacent restrictionsites present in the flanking chromosomal DNA. The restriction mapof the chromosomal region, including the 10-kb H. influenzae DNAinsert in pCML6 and its surrounding DNA, was described byBarany et al. (1). Lane f: absence of junction fragments in rec-Jcells. The persistence of B1 and B2 fragments in lane f reflects thepersistence of donor DNA migrating as intact DNA in lane c. Inlanes g through 1, linear pEUP1 DNA was used as donor DNA.Lanes g and h: BstEII digest of reisolated DNA from wild-type andrec-l cells, respectively. Lanes i and j: reisolated DNA fromwild-type and rec-1 cells, respectively. Lane k: input linear pEUP1DNA. Lane 1: BstEII digest of linear pEUP1 DNA showing theabsence of a BstEII site.

after 15 and 60 min of uptake into rec-2 cells was found tostill be sensitive to cleavage by HindIII, i.e., it had not beenmodified by intracellular methylase. Therefore, the DNAremains in the protected state (transformasome associated),a state similar to that ofDNA in wild-type cells at early timesafter uptake (9). It should also be noted that the rapidlymigrating, partially degraded species of DNA seen at latertime points in this experiment are also sensitive to HindIllcleavage. This is most clearly seen by examining DNAspecies that are less than full length but are larger than thecleavage products. Thus these degraded forms of the donorDNA also remain in the protected state.More evidence for the localization of the DNA in the

transformasome comes from organic solvent extractions.Table 1 shows the result of an experiment in which, after 1 hof DNA uptake into either wild-type or rec-2 cells, the cellswere extracted with phenol or phenol-acetone in the pres-ence of 1.5 M CsCl. Label in the wild-type cells was largelyassociated with the pellet (chromosomal DNA), as previ-ously demonstrated (9). In contrast, label in rec-2 cells was

VOL. 163, 1985 631

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..f.

632 BAROUKI AND SMITH

In 1S 60Hindil#. - +-+ - +

..LL

FIG. 4. Autoradiogram showing the sensitivity of pCML6 DNArecovered from rec-2 cells to Hindlll cleavage. Nick-translated,

CiaI-linearized pCML6 was added to 1 ml of rec-2 cells (2 x i01 cells

per ml), and the reaction was stopped at either 15 or 60 min. Cells

were washed with 1.5 M CsCl, and DNA was extracted as described

in the text. Portions of input (In) DNA or DNA reisolated after 15

and 60 min were treated with 5 U of HindIII. Control and treated

DNA were analyzed on a 0.8% agarose gel. HindIlI cuts MiI-linearized pCML6 once and generates two fragments, of 7.6 and 6.8

kb, that can only be distinguished on very light exposure of the gel.

found mostly in fractions other than the pellet, suggestingthat it was located in a different compartment from the

chromosome. The localization in rec-2 cells is similar to that

found for donor DNA in wild-type cells at early times after

uptake (9).

Phage recombination in wild-type, rec-), and rec-2 cells.

Log-phase rec-J and rec-2 cells have already been shown to

be deficient in phage recombination (22). Since competence

increases the level of phage recombination in wild-type cells

(5), we have investigated phage recombination in competent

rec-J and rec-2 cells. Table 2 shows that recombination

between temperature-sensitive phage tsl and ts2 occurs in

wild-type cells (0.2%) and is not detectable (<10-1%) in

rec-J and rec-2 cells, in agreement with previously reportedresults (4, 22).

Enzymatic activities in the transformasome. Since donor

DNA appears unable to exit the transformasome of rec-2

cells, we were able to follow its modification with time.

Figure 2 shows that donor DNA was slowly degraded in

rec-2 cells. The amount of apparent degradation was quan-

titated by cutting out and determining the radioactivitycontained in portions of the gel corresponding to intact and

degraded molecules. At 60 min, more than half of the DNA

migrated more rapidly than did intact DNA. However, since

donor DNA was linear, degradation can be due either to an

endonuclease or to an exonuclease activity, or to both. To

examine these possibilities, the' fate of closed circular

pEUPi DNA was studied in rec-2 cells. The number of

closed circular molecules decreased with time after uptakeand open circular, linear, and lower-molecular-weight prod-ucts appeared in sequence, suggesting the existence of an

endonuclease activity (Fig. 5). By 30 min, significantamounts of linear DNA were present and lower-molecular-

weight products were beginning to appear. However, when

TABLE 1. Distribution of DNA label after extraction withorganic solventsa

Extraction medium % of label for:and phase Wild-type cells rec-2 cells

PhenolPhenol 24 55TE-1.5 M CsCl 44 26Pellet 32 18

Phenol-acetonePhenol-acetone 0.2 0TE-1.5 M CsCl 33 83Pellet 67 17a Linear nick-translated pCML6 DNA was added to wild-type and rec-2

cells for 60 min at 37°C. Cells were then washed and suspended in 0.5 ml ofTE-1.5 M CsCl. Phenol or phenol-acetone (0.5 ml) was then added, and themixture was hand shaken gently for 1 min. This was followed by centrifuga-tion for 5 min in an Eppendorf centrifuge. The organic phase, the aqueousphase, and the pellet were separated, and the Cerenkov radiation in eachphase was counted.

samples were denatured, some intact single-stranded DNAwas observed even at late times of chase.The presence of a phosphatase in the transformasome was

suspected because of the lower apparent uptake of 5'-end-labeled DNA compared with that of nick-translated DNA(M. Pifer, personal communication). To test for phosphataseactivity on the donor DNA, ClaI-linearized pEUP1 DNAwas uniformly 3H-labeled by nick translation or was 32 5-end labeled by a kinase reaction. Both DNAs were mixed,and uptake into rec-2 cells was allowed to proceed for 5 min.Cells were then washed twice with M-IV medium, sus-pended in this medium, and incubated at 37°C. Samples werewithdrawn at various times and centrifuged, and the amountof 3H and 32P radioactivity was determined in the pellets andthe supernatants. The 3H radioactivity remained constant inthe pellet, whereas 32P radioactivity decreased progressivelyin the pellet and increased in the medium. The 32P radioac-tivity in the medium was acid soluble and was not bound byNorit (Sigma) (data not shown). It therefore corresponds tofree inorganic phosphate. This experiment indicates theexistence of a phosphatase in the transformasome whichreleases the 5'-terminal phosphates from DNA. The exist-ence of phosphatase activity should not alter the interpreta-tion of previous studies ofDNA uptake when using 32P-end-labeled fragments, since incubation of DNA with cells didnot last more than 5 to 10 min in these studies (6, 25).

DISCUSSIONPrevious studies of transformation-deficient mutants in H.

influenzae have led to the detection of different classes of

TABLE 2. Phage recombination in wild-type, rec-1, and rec-2cellsa

No. of plaques/ml at: Recombination

34°C 40°C frequency (%)

Wild type 2.5 x 1010 3.2 x 1010 0.26rec-J 1 x 1010 <104 <104rec-2 1.8 X 10l° <104 <10-4

a Cells (5 x 108/ml) were incubated with tsl and ts2 phages (1.5 x 109/ml ofeach) for 10 min at 34°C, washed, diluted 100-fold, and incubated for 90 minat 34°C. PFU were determined at 34 and 40°C. Infective centers were difficultto determine exactly because of contamination by free phage even afterwashing, but the burst size was estimated to be at least 10.

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PHENOTYPIC DEFECTS IN H. INFLUENZAE MUTANTS

recombination-deficient strains. The best studied is the rec-Jmutant, which has a phenotype similar to recA mutants in E.coli (11, 12, 22). Previously reported data strongly suggestthat the rec-J mutation prevents homologous recombinationbetween donor and resident DNA (13, 22). We have con-firmed this by finding no labeled junction fragment in thechromosome of rec-J cells after uptake of 32P-labeled cloneddonor DNA. In rec-J mutants, almost all the donor DNA isdegraded by 1 h after uptake and the donor radioactivitybecomes randomly distributed in the chromosome. Previousstudies (18) suggested that association between donor andrecipient DNA in rec-J cells was perhaps due to randomincorporation of the label. This interpretation is now seen tobe correct. In fact, the fate of DNA in rec-l cells is exactlysimilar to the fate of heterologous DNA in wild-type cells. Inboth cases, translocation out of the transformasome anddegradation take place normally, suggesting that these stepsare not necessarily coupled with homologous recombination.The rec-2 mutation has been thought of as another type of

recombination defect (18). No association between donorand resident DNA is detected, and the level of transforma-tion is extremely low. Since phage recombination is alsofound to be deficient in these cells, it was thought that adefect in an initial step of recombination was responsible forthe observed phenotype (18). The results we obtained sug-gest that donor DNA and resident DNA in rec-2 cells remainin two separate compartments, the transformasome and thecytoplasm. The translocation of DNA out of the transforma-some appears to be deficient in rec-2 cells. The existence ofsuch mutants constitutes additional evidence for the validityof the two-stage model for DNA entry during H. influenzaetransformation. In these cells, uptake of DNA into thetransformasome is normal, but exit and rapid degradation ofthe DNA are lacking. Other H. influenzae mutants, the KBmutants (21), also display a similar phenotype and might bedeficient in the translocation of DNA out of the transforma-some. However, the phenotype of rec-2 cells appears to bemore complex than that of the KB mutants. Phage recombi-nation and the competence-dependent appearance of single-stranded regions in the chromosomal DNA are normal in theKB mutants and lacking in rec-2 cells (15, 21). This raises theinteresting possibility that a protein involved in translocationmight play a role in the recombination of DNA. rec-2 cellswould be completely deficient in both activities of thisprotein, whereas the KB mutant would lack the transloca-tion activity of this protein or another. Alternatively, bothmutants could have the same defect in translocation butrec-2 cells could have an additional mutation affecting re-combination.

After the uptake of donor DNA in rec-2 cells, a slowdegradation is observed which we believe occurs within thetransformasome. This degradation is separated in time fromthe rapid degradation observed in the wild type; we thereforebelieve that the two types of degradation may be mechanis-tically different. Rapid degradation is observed a few min-utes after uptake in wild-type cells, and by 30 min most ofthe donor DNA is degraded or recomnbined (1) (Fig. 2). Inrec-2 cells, approximately half the donor DNA is still in theintact form after 60 min. It is likely, therefore, that theactivity present in rec-2 cells does not contribute signifi-cantly to the degradation products observed in the wild type.Rapid degradation is postulated to be accomplished by anexonuclease activity associated with the translocation of theDNA out of the transformasome (1). In contrast, we haveshown that the degradation observed in rec-2 cells is at leastpartially due to the existence of an endonuclease activity.

native denaturedIn 0 153060901201n 0 15306090120

0C- - -

linear _Osu11.'. * -cc

-Ss

FIG. 5. Autoradiogram showing the fate of covalently closed andopen circular pEUPi DNA in rec-2 cells. pEUP1 DNA was labeledby nick translation, and closed circular molecules were gel purifiedas described in the text and used as donor DNA. Donor DNA (50 ng,80,000 cpm) was constituted of topoisomers formed when nickedDNA was sealed and of molecules migrating as open circles. rec-2cells (2 x 109 cells in 1.2 ml) were incubated in the presence of donorDNA for 8 min at 37°C. Cold pCML6 DNA (1 ,ug) was then added,and at 0, 15, 30, 60, 90, and 120 min, 0.2-ml portions werewithdrawn, cells were washed, and total DNA was extracted asdescribed. DNA uptake was constant over the incubation time (30%of input DNA). Half of each sample was boiled for 5 min beforeloading. The same amount of radioactivity was loaded in each lane(1,000 cpm). In, input DNA; oc, open circular; ccc, completelyclosed circular; ss, single-stranded.

Indeed, linear DNA molecules are readily generated fromclosed circular molecules (Fig. 5). The existence of such anendonuclease activity in the transformasome could reconcilethe apparent contradiction between biochemical studies sug-gesting the requirement for a free end for efficient exit fromthe transformasome (1) and biological studies showing rela-tively efficient transformation by closed circular plasmidmolecules (24). In marker rescue experiments, closed circu-lar plasmid molecules transform cells containing a homolo-gous resident plasmid 30 to 50% as well as do linear DNAmolecules (M. Pifer, personal communication). After uptakein the transformasome, closed circular DNA molecules areslowly linearized, allowing them to exit the transformasomeand recombine with homologous DNA.Some properties of the endonuclease activity can be

deduced from the experiment shown in Fig. 5. In theory,linear DNA molecules can be generated from closed circularmolecules by random nicking of the DNA. In this case, onewould expect a significant buildup of open circular moleculesbefore the appearance of linear forms. Also, one would notexpect the persistence of intact strands ofDNA at late times.Obviously, this is not what is observed here. The data aremore consistent with the alternative possibility, in which asecond nick occurs preferentially opposite the first one,directly generating a linear molecule. In this case, opencircular molecules do not accumulate. Further endonucle-olytic breakswould result in lower-molecular-weight forms.From our results, we cannot determine whether nicks onboth strands occur simultaneously or are separated in time.This is due to the difficulty in the preparation of highlylabeled closed circular DNA free of open circular forms.Interestingly, in gram-positive cells, an endonuclease activ-ity has been shown to cut large fragments of DNA into

VOL. 163, 1985 633

634 BAROUKI AND SMITH

>1000 __

~~~~~~~~A

0

0~~~

0 500 A

CC

0 30

TIME (min)FIG. 6. Fate of label from nick-translated, end-labeled DNA

after uptake in rec-2 cells. A mixture of nick-translated linearpEUP1 DNA labeled with 3H (20 ng, 7,800 cpm) and linear pEUP1DNA end labeled with 32P (8 ng, 4,200 cpm) was added to rec-2 cells(109 cells, 0.5 ml) and incubated for 5 min at 37°C. Cells were thencentrifuged, washed with M-IV medium, and suspended in 0.5 ml ofthis medium. The cells were incubated at 37°C for various times,chilled, centrifuged, washed once with TE buffer containing 1.5 MCsCl, and suspended in 0.5 ml of TE buffer. The radioactivitypresent in the first supernatant and the resuspended cells was

counted by liquid scintillation. The CsCl wash released nonspecifi-cally bound DNA that was not dephosphorylated (ratio of 3H to 32pcounts was the same as input).

smaller pieces, thereby initiating the translocation of theDNA into the cytoplasm (14). The exit step of DNA out ofthe transformasome in H. influenzae appears to share a

number of similar steps with the entry of DNA in gram-

positive bacteria (26).

ACKNOWLEDGMENTSWe thank Marilyn Pifer, Marc Kahn, and David Hudacek for

helpful discussions and Mildred Kahler for expert typing.This work was supported by Public Health Service grant no.

5-PO1-CA16519 from the National Institutes of Health. H.O.S. is anAmerican Cancer Society Research Professor. R.B. is supported bya European Molecular Biology Organization postdoctoral fellow-ship.

LITERATURE CITED1. Barany, F., M. E. Kahn, and H. 0. Smith. 1983. Directional

transport and integration of donor DNA in Haemophilus influ-enzae transformation. Proc'. Natl. Acad. Sci. U.S.A.80:7274-7278.

2. Beattie, K. L., and J. K. Setlow. 1971. Transformation-defectivestrains of Haemophilus influenzae. Nature (London) New Biol.231:177-179.

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