JOURNAL OF BACrERIOLOGY, Aug. 1992, P. 4960-49660021-9193/92/154960-07$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 174, No. 15

rec-2-Dependent Phage Recombination in Haemophilus influenzaeDORIS M. KUPFER AND DAVID McCARTHY*

Department ofBotany and Microbiology, University of Oklahoma,Norman, Oklahoma 73019-7619

Received 18 February 1992/Accepted 28 May 1992

The genetic transformation mutant Rd(DB117)' has a pleiotropic phenotype that includes reduced levelsof phage recombination. Physical mapping experiments showed that this strain has a 78.5-kbp insertion in therec-2 gene. The rec-2 dependence of phage recombination was reexamined to determine whether the defectivephenotype in Rd(DB117)'r was due to the simple disruption of the rec-2 gene or whether trans-acting factorsfrom the inserted DNA were responsible. Analysis of strains with transposon insertions in the rec-2 gene showedthat they were also defective for phage recombination. Therefore, the phage recombination defect was duesolely to the disruption of the rec-2 gene. Strain KB6 is proficient for phage recombination but has a defect ingenetic transformation resembling that of Rd(DB117)'c . The transformation defect of KB6 could becomplemented by the wild-type rec-2 gene, showing that the rec-2 contributions to genetic transformation andphage recombination were uncoupled in this strain. The rec-2-dependent phenotype of KB6 suggests that therec-2 gene participates in genetic transformation and phage recombination in different ways.

The gram-negative bacterium Haemophilus influenzae hasa natural transformation system which is expressed whencells are placed under nongrowth conditions. Several genesin the transformation pathway, including rec-1, which codesfor a homolog of the Escherichia coli RecA protein (17), andrec-2, the product of which is required for translocation oftransforming DNA from DNA uptake structures on the cellsurface (transformasomes) to the interior of the cell, havebeen identified (1, 12, 15). The spontaneous transformationmutant Rd(DB117)r,C encodes a defect in the rec-2 gene thatmakes it unable to translocate transforming DNA. The rec-2mutant is proficient for recombination between residentDNAs. Plasmid recombination is normal, although catenatedplasmids accumulate in competent Rd(DB117)rCC to anabnormally high level (11). Recombination events associatedwith postreplication repair are unaffected by the spontane-ous rec-2 mutation (18). Strain Rd(DB117)reC shows otherpleiotropic effects that are not obviously related to its DNAtranslocation defect. Unlike the wild-type strain, Rd, themutant fails to introduce single-strand breaks into its chro-mosome during competence development (8, 13). The mu-tant also is more sensitive than Rd to methyl methanesulfo-nate and X-ray irradiation. This strain is also defective forphage recombination. The phage recombination defect is notdue to an inability of phage DNA to enter the cell, becausethe phage can successfully infect competent Rd(DB117)reC(1, 16). Moreover, phage recombination can occur at lowlevels in exponential-phase Rd cells which lack transforma-somes (3).

Several lines of circumstantial evidence imply that at leastsome of the pleiotropic effects of the spontaneous rec-2mutant might be due to defects in more than one gene.Rd(DB117)reC was selected by repeatedly transforming Rdwith toxic H. parainfluenzae DNA (2). Transformation-defective mutants appeared most frequently if the Rd strainwas first transformed with DNA from the rec-l-defectivemutant DB117, hence the name Rd(DB117)yeC for the spon-taneous rec-2 mutant (16). Rd(DB117)reC is not a rec-l-defective transformant, because its DNA can transform

* Corresponding author.

DB117 to a rec-l + genotype. On the basis of this informa-tion, Setlow et al. (16) proposed that Rd(DB117)r,C arosefrom a combination of a spontaneous mutation and one ormore mutations from DB117. It is possible that the sponta-neous mutation might have resulted from insertion of H.parainfluenzae sequences into the chromosome. Such aninsertion might cause a recombination defect directly bydisrupting the rec-2 gene. It also is possible that the hypo-thetical H. parainfluenzae sequences encode one or moreproducts that interfere with DNA metabolism, causing atleast some of the pleiotropic defects in Rd(DB117)r,C to beindependent of the rec-2 gene.McCarthy recently cloned the wild-type rec-2 gene on the

basis of complementation of the transformation defect ofRd(DB117)reC and constructed rec-2::mini-TnlO mutations(12). The rec-2::mini-TnlO mutations caused a defect in thetranslocation step of transformation. This result agreed withthose of similar studies of Rd(DB117)reC (1). Hybridizationexperiments with the use of a rec-2 probe showed thatRd(DB117)reC contains an extensive rearrangement in therec-2 locus (12). This result raises the possibility that theextensive pleiotropy of Rd(DB117)reC is due to multiplemutations that map outside the rec-2 gene.

In this report, we identify an insertion within the rec-2locus of Rd(DB117),eC . We also examine phage recombina-tion with the use of highly characterized transposon mu-tants, each containing a single defect in the rec-2 locus. Ourresults show that the rec-2 gene is required for phagerecombination as well as genetic transformation.

MATERIALS AND METHODS

Bacterial strains. Wild-type strain PB and transformation-defective strain KB6 were from J. K. Setlow. PB is an Rdderivative that adsorbs phage HPlcl with high efficiency.Wild-type strain BC200 is a UV-resistant Rd derivative thatcontains an inverted duplication in its chromosome. Therearrangement is greater than 200 kb from the rec-2 locusand does not involve rec-2 sequences (10). The spontaneoustransformation-defective mutant Rd(DB117)r,C (rec-2-1 rec-1+) arose from an Rd culture that was transformed withDNA from strain DB117 (rec-1). Transformation of this

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rec-2

IV vr

_ D E

I 2I1 2 3

±4 4.3 kbp

Map Strain miniTnlOkm rec-2 allele CorrespondingReference coordinate (kbp) plasmid

- PB - Wild-type pDM62A PB92 <0.1 Wild-type pDM73B PB 100 0.85 rec-2-4::miniiTnlO NAaC PB98 1.38 rec-2-5::miniTnlO pDM79D PB91 1.92 rec-2-2::miniTnlO pDM60E PB96 2.82 Wild-tpe pDM77

a Corresponding plasmid not used in this study

FIG. 1. Transposon positions in plasmids and strains used in this study. The bar represents the rec-2 gene in the 4.3-kb PstI fragment ofpDM62. The extensions from the bar represent uncertainty as to the borders of the gene. The triangles represent the positions of transposoninsertions. The letters above the triangles correlate with similarly designated rows in the table. The filled triangles represent insertions in therec-2 gene, while the unfilled triangles represent transposons that are adjacent to the rec-2 gene. The position of the mini-TnlOkm insertionis measured in kilobase pairs from the left end of the chromosomal fragment carrying the rec-2 locus. The mutations are located at the samepositions in the plasmids and the corresponding mutant strains (12). All transposon mutants in this study are derivatives of strain PB.

culture with toxic H. parainfluenzae DNA allowed selectionof transformation-defective mutants (2). Strains PB91through PB100 were constructed by transforming MIV-competent PB with cloned rec-2 DNAs that contained singlemini-TnlOkm insertions. Figure 1 shows the rec-2 allele,mini-TnlOkm map position, and corresponding rec-2 recom-binant plasmid for each of these transposon mutants. Themap in Fig. 1 corresponds to the 4.3-kbp PstI insert in thewild-type rec-2 recombinant plasmid pDM62 (12).

Media. Cells were grown in brain heart infusion broth(BHI) supplemented with hemin (10 ,ug/ml) and NAD (2,Lg/ml) (sBHI).Transformation and strain construction. Cells were made

competent for natural transformation with the use of MIVmedium as described by Herriott et al. (5). Transposonmutations from recombinant plasmids were recombined intothe cell chromosome by transforming competent cells withPstI-cleaved plasmid DNA.

Cells treated with CaCl2 were used to construct merodip-loids without changing the corresponding chromosomal al-leles. Cells grown to an optical density at 675 nm of 0.6 to 0.7were pelleted by centrifugation and resuspended in a 0.5volume of 10 mM Tris. HCl (pH 8)-50 mM CaCl2 (TCbuffer). The cells were incubated at 4°C for 20 min and thenpelleted by centrifugation. The cells were resuspended in 0.1volume of TC buffer. The cells could be used immediatelyfor transformation or could be supplemented with a finalconcentration of 15% glycerol and frozen at -70°C for futureuse. The cells were transformed by adjusting the suspensionto 14 mM Tris (pH 8)-54 mM CaCl2-4 mM MgCl2 with theaddition of plasmid DNA. Incubation for 30 min at 4°C wasfollowed by a 2-min incubation at 37°C and then a 5-minincubation at 4°C. Four volumes of sBHI was added to thetransformed cells, and the cells were incubated for 90 min at37°C. Plasmid transformants were selected on sBHI platescontaining 4 ,ug of chloramphenicol per ml.

Percent transformation was defined as the percent eryth-romycin-resistant transformants resulting from transforma-tion of MIV-competent cells with DNA from strain MAP9.

Relative transformation frequency is defined as the ratio ofthe percent transformation of a mutant to the percent trans-formation of the wild-type control.Phage recombination. The temperature-sensitive mutants

of H. influenzae phage HPlcl, tsl and ts3, were obtainedfrom J. K. Setlow. Phage stocks were prepared by inductionof lysogens. Phage titers were routinely between 4 x 1010 to8 x 10 0 PFU/ml.To measure phage recombination, competent cells were

infected with tsl and ts3 at a multiplicity of infection of 10 foreach phage. The phages were allowed to adsorb to the cellsfor 10 min at 32°C. The cells were pelleted by centrifugationand washed twice with MIV medium. The culture was thensuspended in unsupplemented BHI to a titer of 6 x 106 cellsper ml. Following 90 min of incubation at 37°C, the cellswere removed by centrifugation and the lysate was filteredthrough a 0.45-,um-pore-size polysulfone filter. A late-expo-nential-phase culture of PB was used as a bacterial lawn forphage titering. Equal volumes of diluted lysate and PB weremixed and placed at 32°C for 10 min before the sample wascombined with 2.5 ml of BHI top agar (sBHI plus 0.7% agar).Top agar was spread on an sBHI agar plate. The percentrecombination was twice the PFU/ml formed by infection at41.5°C divided by the PFU/ml formed by infection at 32°Ctimes 100. Relative phage recombination frequency is de-fined as the ratio of the percent phage recombination in amutant host to that of the wild-type host. Burst sizes of fivephages were typical. The low daughter phage yields wereexpected for infections at high multiplicity of infection (3).

Preparation of chromosomal DNA for field inversion gelelectrophoresis (FIGE). DNA in agarose beads was preparedas described by Kauc et al. (6). Extensive washing of theDNA-containing agarose beads was essential for successfulrestriction endonuclease cleavage of the DNA. The beadswere washed at least five times with TE buffer (10 mM Tris[pH 8], 1 mM EDTA). The pellets were incubated overnightat 4°C in the last wash buffer. The washed beads were storedin 500 pul of TE buffer at 4°C for several weeks. Typically, 20to 30 p.1 of DNA-containing agarose beads was digested with

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20 U of restriction endonuclease in a final volume of 100 ,ul.After 4 to 6 h of incubation, 10 U of enzyme was added, andthe mixture was incubated overnight. The digests wereheated to 62°C to melt the beads after the addition of 0.1volume of stop buffer (50% glycerol, 40 mM EDTA, 0.25%[wt/voll bromphenol blue). The melted samples were imme-diately loaded into an agarose gel.FIGE. Electrophoresis was performed with the use of a

programmable electrophoresis controller (model PPI-200;M. J. Research, Inc., Watertown, Mass.) connected in linefrom a power supply to a horizontal gel electrophoresisapparatus. Samples were loaded into a 0.95% agarose gel(electrophoresis grade; Bethesda Research Laboratories,Bethesda, Md.) containing 0.5 x TBE buffer (lx TBE is 89mM Tris-borate plus 2 mM EDTA). Electrophoresis wasperformed at 4°C over a 15.5-h period at 25 mA and 135 to140 V. The patterns of forward and reverse pulses were asdefined by the PPI-200 program 4, which allowed resolutionof DNA fragments up to 500 kb. After electrophoresis, thegel was stained with 0.5 ,g of ethidium bromide per ml for 30min. The gel was destained in distilled water for 20 to 60 min.

Southern blots. Southern blotting to nylon membranes(Nytran; Schleicher & Schuell, Keene, N.H.) was per-formed as described previously (19). Hybridizations weredone under conditions which preserved duplexes with 80%or greater sequence complementarity. The radioactive hy-bridization probe was prepared from the 4.3-kbp PstI frag-ment from pDM62 that contains the complete rec-2 gene.Incorporation of 32P-labeled nucleotides was primed with amixture of random hexadeoxyribonucleotides (Pharmacia,Piscataway, N.J.).

Densitometry. Negatives (Polaroid type 55 film) of ethid-ium bromide-stained agarose gels were scanned with aBeckman model DU8B spectrophotometer programmed toperform integration for determination of relative concentra-tion of DNA fragments or to assign sizes to DNA fragmentsby comparison with standards. The fragments in SmaIdigests of Rd were used as internal molecular weight stan-dards (9).

RESULTS

Chromosomal map position of the rec-2 locus. Pulsed-fieldgel electrophoresis was used previously to derive SmaI andApaI restriction maps of the H. influenzae chromosome (6,9). The rec-2 locus is mapped to the 79-kbp overlap betweenSmaI fragment G and ApaI fragment H (Fig. 2B) (9). Thesebands correspond to SmaI band 7 and ApaI band 8 by theassignments of Kauc et al. (6). We used H. influenzae strainscarrying transposon mutations within the rec-2 gene toposition the rec-2 locus more precisely within this region.Chromosomal DNAs from the rec-2::mini-TnlOkm mutantsPB91 and PB98 were digested with SmaI and subjected toFIGE. The SmaI G fragment was absent from digests of themutated DNAs, because each mini-TnlOkm insertion intro-duces a single SmaI site (Fig. 2A, lanes 2 and 3). The newfragments, called Ga and Gb, hybridized to the PstI rec-2probe (not shown). The Ga and Gb fragments in the PB91digest were 62 and 58 kbp, respectively, while the corre-sponding bands in the PB98 digest were 63 and 57 kbp,respectively. This strain-dependent discrepancy in fragmentsizes reflects the difference in the positions of the transposoninsertions in the mutants. To place the rec-2 locus within thepublished map, the sizes of fragments Ga and Gb werenormalized so that their sum equaled the size for fragmentSmaI-G and the transposon insert (136.6 kbp). On the basis

A1 2 3

B

79kb

H K ApaG Sma

-r--r--"rec2

-G

a-b

FIG. 2. Positioning of the rec-2 locus on the H. influenzaechromosome. (A) FIGE of two transposon-carrying strains of PB.DNAs were digested with SmaI. The positions of Ga and Gb(labelled a and b, respectively) vary slightly between lanes 2 and 3because of the different positions of the transposon in the twostrains. PB is the wild-type parent of each transposon mutant.Lanes: 1, PB; 2, PB91; 3, PB98. (B) Distribution ofApaI and SmaIsites in the rec-2 region of the H. influenzae chromosome map. Therectangle labelled 79.0 kbp represents the overlap between theApaIH fragment and the SmaI G fragment, the only fragments for eachdigest that hybridize to a rec-2 probe (7). The small horizontal linesbelow the restriction map represent the 4.3-kb PstI fragment con-taining the rec-2 locus and are shown in their alternative positions,depending on the two possible orientations of the Ga and Gbfragments. The positions of the additional SmaI site introduced bymini-TnlOkmn are shown as vertical lines through the PstI lines. Theshaded bar below the PstI lines represent the largest region (9.4 kb)in which the rec-2 locus could map. The total size represented by Gaand Gb (120 kb) is less than the sum of the published size of the135-kbp G fragment plus the 1.6-kb transposon (136.6 kbp). Thesizes of Ga and Gb were normalized proportionally to compensatefor the discrepancy between the published map and our measure-ments (10). Band sizes were normalized to 70.6 kbp (Ga) and 66 kbp(Gb) for PB91 and 71.7 kbp (Ga) and 64.9 kbp (Gb) for PB98, so thatthe rec-2 locus could be placed in the context of the published map(10).

of this calculation, we estimate that the rec-2 locus is withina 9.4-kbp segment between 63.6 and 73 kbp from the SmaIsite that defines the junction between SmaI fragments 0 andG (10) (Fig. 2B).

Extensive rearrangement affecting the rec-2 locus inRd(DB117)' . Previous restriction mapping of the rec-2locus showed that this region in Rd(DB117)reC was rear-ranged compared with the rec-2 locus in BC200 (12). FIGEof SmaI digests of Rd(DB117)reC , Rd, and PB DNAs al-lowed us to characterize the rearrangement in the rec-2mutant in the context of the whole chromosome. Rd and PBDNAs had identical SmaI restriction patterns; however, theRd(DB117)reC digest showed a rearrangement in the regionthat contains the rec-2 locus (Fig. 3). The 135-kbp SmaI-Gband was replaced by two SmaI bands corresponding to 93.5kbp (band G') and 60 kbp (band I' in Fig. 3A, lane 2).Densitometer scans of a photographic negative of Fig. 3Ashowed that DNA in band I' was present at twice the

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PHAGE RECOMBINATION IN H. INFLUENZAE 4963

A B

-G _VG -

IHo--IG'-

Bg Pv

E Ev Ev EA | P

Pv

Ev E pBg Ev E

Bg

E Evp EPv p

C .......

BgWild-type

Bg

Rd(DB1 17)

2.5 kbp

D

FIG. 3. FIGE of the Rd(DB117) Ce chromosome. (A) Chromo-somal DNAs were prepared and cut with SmaI as described inMaterials and Methods. Lanes: 1, Rd; 2, Rd(DB117) eC; 3, PB. (B)Autoradiograph of the FIGE gel (A). Only band G hybridizes withthe 4.3-kbp PstI rec-2 locus probe in the digests of wild-type Rd andPB DNAs (lanes 1 and 3, respectively). Two bands, G' and I',hybridize in Rd(DB117)"eC (lane 2). The autoradiogram and theethidium bromide gel are not to the same scale.

expected stoichiometry, assuming that its size was 60 kbp.All of the bands in the lane exhibited a linear relationshipbetween photographic intensity and DNA amount. Appar-ently, SmaI fragment G acquired two additional SmaI sitesas a result of the mutation in Rd(DB117)reC . The DNA inband I' represents two comigrating fragments that accountfor a 120-kbp portion of the Rd(DB117)reC genome. Theamount of DNA represented by the G' and I' fragments is213.5 kbp, corresponding to a 78.5-kbp increase over the sizeof SmaI fragment G. The SmaI G fragment from Rd and theG' and I' bands hybridize to a rec-2 probe (Fig. 3B),suggesting that the rearrangement is a simple insertion in therec-2 locus.

High-resolution restriction maps revealed the edges of theinsertion mutation in Rd(DB117)r,C and were consistentwith the disruption of the rec-2 gene. Restriction sites to theleft of the PvuII site in the rec-2 locus were mappedpreviously for BC200 and Rd(DB117)"eC DNAs (12). Differ-ences in the restriction maps over a distance greater than 10kbp indicate that the rec-2 mutant has an extensive chromo-somal rearrangement within and to the left of the PvuII site(interpreted as the heavy line in Fig. 4B). We extended thisanalysis to the right of the BglII site at the edge of the rec-2locus. Figure 4A shows a composite restriction map ofwild-type DNA based on digests of BC200 DNA (definingthe leftward sites) and PB DNA (defining the rightwardsites). The digests were probed with the PstI fragment thatcontains the entire rec-2 locus. Figure 4B and C show therestriction map that was derived from digests ofRd(DB117)reC . In this case, most enzyme combinationsproduced two radioactive bands of different intensities (Fig.4D). This discrepancy in the amount of hybridization to therec-2 probe was probably due to the skewed position of theinsertion in the rec-2 locus. The map in Fig. 4B was based onthe mobilities of high-intensity bands, while the map in Fig.

FIG. 4. Restriction mapping of the wild-type and Rd(DB117)"eCrec-2 loci. The rectangles in panels A to C represent all or part of thePstI segment cloned into pDM62. (A) Composite partial restrictionmap of the region surrounding the wild-type rec-2 locus (rectangle).Sites to the left of the locus were mapped previously for BC200 (13).Sites to the right were mapped for PB in this work on the basis ofSouthern blots, using the PstI rec-2 locus as a probe. Restrictionsites: E, EcoRI; Ev, EcoRV; Bg, BglII; P, PstI; Pv, PvuII. (B)Composite partial restriction map of the region surrounding therightward end of proposed insertion (heavy line), including anundetermined portion of the rec-2 locus (open-ended rectangle) andthe region to the right of the rec-2 locus (thin line) presumed to bewild-type. (C) Composite partial restriction map of the left end of theproposed insertion. The thin line represents the wild-type region.The open-ended rectangle represents a portion of the rec-2 locus.The heavy line represents the proposed insertion. For panels B andC, the sites to the left of each partial rec-2 locus were determinedpreviously for BC200 (12). The sites to the right were mapped in thiswork. The size of the insert was determined by FIGE to beapproximately 78.5 kbp (Fig. 3). The lines at the ends of therectangles represent uncertainty about the location of the junctionbetween the insert and the rec-2 gene. (D) The 4.3-kbp PstI fragmentcontaining the rec-2 locus served as probe for the hybridizationwhich was used to construct the maps in panels A to C. The DNAsare Rd(DB117) Cc (lanes 1, 3, 5, 8, and 10) and PB (lanes 2, 4, 6, 7,and 9). The restriction enzymes used were BglII (lanes 1 and 2),BglII-EcoRV (lanes 3 and 4), BglII-EcoRI (lanes 5 and 6), BglII-PvuII (lanes 7 and 8), and PstI (lanes 9 and 10).

4C was based on the mobilities of the low-intensity bands.The order and spacing of restriction sites to the right of therec-2 gene appeared to be the same in PB and Rd(DB117)recDNAs. The discrepancies were within the range of experi-mental error. The wild-type pattern of spacing betweenrestriction sites on the left side of the rec-2 locus also couldbe reconstructed for the rec-2 mutant. The reconstruction ofidentical restriction patterns flanking the rec-2 gene, forwild-type and Rd(DB117)"CC DNAs, suggests that the mu-tant suffers a simple insertion in the rec-2 gene. This inter-pretation is consistent with the results of the SmaI digestionsof PB and Rd(DB117)rcc.

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TABLE 1. Correlated transformation and phage recombinationphenotypes associated with selected mini-TnlO mutations

Relative frequency

straina % Transfor- % Phage (mutant/PB)6mation cnation Genetic trans- Phage recom-

formation bination

Expt 1PBc 1.45 0.13 1.0 1.0PB91 1.75 x 10-3 5.9 x 10-3 1.2 x 10-3 0.045PB98 1.62 x 10-5 3.8 x 10-3 1.1 X 10-5 0.029PB100 9.88 x 10-6 2.1 x 10-3 6.8 x 10-6 0.016

Expt 2PB 0.562 0.14 1.0 1.0PB92 0.59 0.12 1.05 0.85PB96 1.33 0.19 2.35 1.36

a See Fig. 1 for positions of the transposon in strains.b Determined as described in Materials and Methods.c Wild-type control.

rec-2-dependent phage recombination. The results of therestriction mapping experiments suggested two possible inter-pretations for the pleiotropic phenotype of Rd(DB117)rec .

First, the defects might be due solely to the disruption of therec-2 gene. Second, the pleiotropic defects in Rd(DB117)"Cmight be due to a combination of chromosomal changes,including the disruption of the rec-2 gene by the insertion, theproduction of trans-acting factors from the insert, as well asmutations acquired from DB117. Consequently, we reexam-ined the inference that phage recombination is dependent onthe rec-2 gene. Strain PB was the wild-type host in our phagerecombination studies, because it absorbed phage HPlclbetter than did Rd and BC200. Five transposon mutationsfrom the rec-2 locus of BC200 were transformed into PB (Fig.1). The resulting mutants were tested for the ability topromote phage recombination and to undergo genetic trans-formation. The host strains that contained mini-TnlOkm in-sertions outside the rec-2 gene (PB92 and PB96) showedwild-type levels of both phage recombination and transforma-tion (Table 1). On the other hand, the rec-2::mini-TnlOkmnmutants PB91, PB98, and PB100 each showed reduced levelsof phage recombination and transformation relative to PB.We confirmed the rec-2 dependence of phage recombina-

tion by performing complementation tests with the use ofplasmids that contained transposon mutations in the rec-2locus. Table 2 shows that pDM62, which contains thewild-type rec-2 locus, complemented both the transforma-tion defect and the phage recombination defect of each rec-2mutant. Plasmids containing mutations in the rec-2 genewere unable to complement the transformation and phagerecombination defects of the rec-2 mutant hosts. The inabil-ity of plasmids containing rec-2::mini-TnlO mutations tocomplement the phage recombination defect was not a resultof polarity from the plasmid linked rec-2 mutations. Trans-poson mutations that flanked either side of the rec-2 genewere able to complement the phage recombination defect ofthe rec-2 hosts as effectively as did pDM62 (Table 2, pDM73and pDM77).Complementation of the genetic transformation defect of

strain KB6. Beattie and Setlow (2) isolated a collection ofN-methyl-N-nitro-N'-nitrosoguanidine-induced transforma-tion-defective mutants with the use of the H. parainfluenzaeDNA transformation selection. KB6, a representative mu-tant from this collection, is able to take up transformingDNA but fails to integrate it into the chromosome (2). This

TABLE 2. Genetic complementation of strains carryingtransposon insertions within the rec-2 locus

Relative frequencybStrain" Plasmid" Genetic trans- Phage recom- tationc

formation bination

PB91 pDM62 0.97 1.0 +pDM73 0.70 0.62 +pDM79 0.085 0.08 -pDM60 0.021 0.004 -pDM77 0.81 0.46 +

PB98 pDM62 0.59 2.0 +pDM73 0.58 1.06 +pDM79 2.7 x 10-6 0.01 -pDM60 4.0 x 10-4 0.045 -pDM77 0.37 0.615 +

aSee Fig. 1 for positions of the transposon in strains and plasmids.b PB[pDM621 served as the wild-type control with normalized frequencies

of phage recombination and transformation of 1.c +, complementation for both phage recombination and transformation; -,

complementation for neither phenotype.

defect closely resembles the transformation phenotype ofRd(DB117) Cc . KB6 differs from Rd(DB117)rCc by beingproficient for phage recombination (14). Table 3 shows theresults of complementation tests using Rd(DB117)reC andKB6 as hosts for plasmids containing the cloned rec-2 locus.The rec-2+ plasmid pDM62 restored wild-type levels ofgenetic transformation and phage recombination to Rd(DB117)rec . This result is consistent with the pattern ofcomplementation of the rec-2::mini-TnlO mutants (Table 2).KB6[pDM62] also exhibited wild-type levels of genetictransformation, indicating that the transformation defect inKB6 was due to a mutation in the rec-2 gene. This inferencewas confirmed by the inability of pDM79 (rec-2-5::mini-TnlO) to complement the transformation defect of KB6. Thedivergence of the phenotypes for transformation and phagerecombination in KB6 implies that the rec-2 gene contributesto genetic transformation and phage recombination in differ-ent ways.

DISCUSSION

Recent physical mapping of the rec-2 locus fromRd(DB117)reC shows that this strain contains a rearrange-ment in the rec-2 region (12). In this report, we show that themutation is a simple insertion of 78 kbp in the rec-2 gene.This result has important implications for the interpretationof the relationships among the multiple phenotypic defects ofthis mutant. It is possible that all of the defects are the resultsolely of the disruption of the rec-2 gene by the insertion.In this case, we would expect that the rec-2-1 allele ofRd(DB117)reC would be phenotypically equivalent to therec-2::mini-TnlO insertions. An alternative possibility is thatsome of the defects of Rd(DB117)rCc are due to the expres-sion of genes in the 78 kbp of DNA that are inserted in therec-2 gene. Setlow et al. (16) suggested that the insertedDNA is an H. parainfluenzae sequence that entered the Rdchromosome during the selection for Rd(DB117),Cc . DNAmetabolism gene products from the putative H. parainfluen-zae insert could functionally displace H. influenzae ho-mologs without interacting productively with the H. influen-zae recombination machinery. In this situation, thephenotypic defects of the rec-2-1 allele would not be equiv-alent to those of the rec-2::mini-TnJO mutations. We couldtest these interpretations for any phenotypic defect by

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TABLE 3. Genetic complementation of the transformation and phage recombination defects of Rd(DB117)r,C and KB6

Relative frequency (mutant/Rd)Straina Plasmid % Transformation % Phagerecombination Genetic Phage

transformation recombination

Rd PDM62 2.9 x 10-2 0.104 1.0 1.0Rd(DB117)reC None 1.19 x 10-5 2.6 x 10-3 4.1 x 10-4 0.025

pDM62 6.4 x 10-2 0.208 2.2 2.0pDM79 1.0 X 10-6 rqDb 3.4 x 10-5 ND

KB6 None 1.89 x 10-6 0.3 6.5 x 10-5 2.88pDM62 2.7 x 10-2 0.32 0.93 3.07pDM79 4.8 x 10-6 0.27 1.65 x 10-4 2.6

a Rd(DB177) Cc and KB6 are derived from Rd (wild type).b ND, not done.

comparing Rd(DB117)"CC with any of the rec-2::TnlO mu-tants. Such a comparison shows that the defect in translo-cation is due solely to the disruption of the rec-2 gene. Thisdefective phenotype is not dependent on theparticular insertthat disrupts the rec-2 gene in Rd(DB117)reC (12). The sameconclusion can be drawn about the phage recombinationdefect based on the defective phenotype of the rec-2::mini-TnlO mutants.A second source of uncertainty regarding the determi-

nant(s) of the pleiotropy of Rd(DB117)reC is the likelihoodthat this strain has one or more mutations from strainDB117. Transformation of Rd with DNA from DB117 was aprerequisite for the isolation of Rd(DB117)reC . The methylmethanesulfonate-sensitive phenotype of Rd(DB117)reCprobably is due to the acquisition of a mutated mex genefrom DB117. This mex allele has no effects on transforma-tion frequency or on phage recombination, although thesingle mex-defective mutant has not been examined for thepresence of competence-specific chromosomal strand breaks(16). It is possible that Rd(DB117)reC has additional muta-tions from DB117; however, these hypothetical mutationsprobably do not account for the transformation and phagerecombination phenotypes of Rd(DB117)reC because therec-2 gene alone can complement these defects. Moreover,these phenotypes can be produced in an Rd background bythe rec-2::mini-TnlOkm alleles.The requirement for rec-2 in phage recombination and

genetic transformation in our complementation experimentsdemonstrates rec-2 involvement in two recombination path-ways. The phenotype of KB6 implies that rec-2 is notinvolved in these pathways at a common step. The rec-2defect in transformation is at the translocation step in all ofthe rec-2 mutants that have been examined (1, 12, 15). Phagerecombination does not seem to include DNA translocation.Transformasomes are not required for phage infection, andone would expect that any phage chromosomes that wereinjected into transformasomes of wild-type cells would enteras single-stranded chromosomes that would be rapidly de-graded. We discount the possibility that phage recombina-tion depends on a small number of phage chromosomes thatenter the cell by way of transformasomes and act analo-gously to transforming DNA. The inability of KB6 to pro-mote translocation while showing no defect in phage recom-bination is inconsistent with this hypothesis.Our results are consistent with three general explanations

for the rec-2 phenotypes of Rd(DB117)r,C and KB6. Onepossibility is that a single rec-2 activity is involved in genetictransformation and phage recombination, but the pathwayshave different needs for the rec-2 product. For example,

transformation might require the rec-2 product at muchhigher levels than does phage recombination. The transpo-son mutations would cause phenotypic defects in bothpathways by eliminating rec-2 product. KB6 might havereduced levels of rec-2 product that are inadequate fortransformation but are not limiting for phage recombination.An alternative is that the rec-2 gene product is a multifunc-tional enzyme. One of its activities would be required fortranslocation during transformation, while another functionwould play a role in phage recombination. The transposonmutants would be defective for both pathways because thetransposon insertions disrupt the rec-2 gene. KB6 wouldsuffer a substitution mutation that affects only the activitythat would be required for transformation. The third possi-bility is that rec-2 is a regulatory gene that affects theexpression of functions that are involved in transformationand phage recombination. The transposon mutations wouldeither block expression of functions that are needed by bothpathways or prevent the repression of interfering activitiesfor both pathways. The substitution mutation in KB6 wouldaffect the expression of only factors involved in transforma-tion. If the rec-2 gene is a regulatory function, it wouldcontrol the expression of only a subset of competencefunctions, because the rec-2 mutants are able to maketransformasomes that are functional for DNA uptake. Theavailable data do not allow us to distinguish among thesepossibilities; however, they are all amenable to experimentaltests.

ACKNOWLEDGMENTS

We thank J. K. Setlow for KB6 and the phage mutants that wereused in this study.

This work was supported by a grant from the Oklahoma Centerfor the Advancement of Science and Technology (Health ResearchProgram grant HR9-060).

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