Transduction of Low-Copy Number Plasmids by Bacteriophage P22

Brandon A. Mann and James M. Slauch Department of Microbiology, University of Illinois, B103 Chemical and Lfe Sciences Laboratory, Urbana, Illinois 61801 Manuscript received March 26, 1996 Accepted for publication March 13, 1997

ABSTRACT The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSClOl derivatives. We show that P22HT transduces pSClOl derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phagepackages a linear concatemercreated during rolling circle replication of pBR322, similar infashion to phagegenome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteinsin plasmid/plasmid and plasmid/chromosomeinteractions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.

HE temperate bacteriophage, P22, mediates gener- been seen in several other systems in both gram nega- T alized transduction in Salmonella typhimurium tive and gram positive species (VIRET et al. 1991). (ZINDERand LEDERBERC 1952).Wild-type P22 and mu- In contrast to pBR322, low-copynumber plasmids are tant derivatives are valuable and versatile tools used in not efficiently transduced. Previous evidence suggested Salmonella genetics. P22 packages its genome starting that these plasmids required homologous recombina- at a pac site on a linear multimer created by rolling tion with the host chromosome to be packaged by a circle replication (TYE et al. 1974; reviewed in POTEETE bacteriophage. For example, TRUN andSILHAW showed 1988). The linear phage DNAis cut into 44kb seg- that a pSClOl derivative containing homology with the ments, referred to as a headful, the minimum amount Escherichia coli chromosome could be transduced by bac- of DNA required for packaging (?LE et al. 1974). Ithas teriophage P1 (TRUN andSILHAW 1987).Apparently, been shown that P22 can also mediate plasmid trans- the phage packages a chromosomal fragment that con- duction, or the movement of nonchromosomal DNA tains a plasmid that has integrated into the chromo- from one cell to another (ORBACHandJACKsoN 1982; some by homologous recombination. This integrated SANDERSONand ROTH 1988; GARZONet al. 1995). In plasmid recircularizes inthe recipient. Thus, phage studies using wild-type P22, a concatemer of plasmid packaging essentially traps the intermediateof a double pBR322 could be packaged when the vector contained recombination event between the plasmid and the bac- a P22 pac site or a pac analogue from the bacte- terial chromosome (Figure 1A). Indeed, these authors rial chromosome(SCHMIEGER 1982; SCHMIDTand used this recombination-dependenttransduction to SCHMIECER1984; VOGEL and SCHMIEGER1986). The map mutations within the cloned chromosomal frag- high transducing mutant of P22, P22HT, can transduce ment. We have previouslyprovided similar results show- pBR322 independent of a wild-type pac site because ing that transduction of the R6K derivative pGP704 by the phage apparently has reduced stringency for pac P22HTint was dependent on the plasmid containing a recognition (MARGOLIN 1987). It has beenproposed cloned chromosomal fragment. Again, the phage ap- that P22 infection allows for productive rolling circle parently packages a chromosomal fragment containing replication of pBR322 and this provides a substrate for an integrated plasmid that resulted from homologous headful packaging into the phage head (Figure 1C). recombination with thechromosome (MAHAN et al. Indeed, this phenomenon of plasmid transduction has 1993). Induction of rolling circle replication of pBR322 has CorrPsponrlingautho~;JamesM. Slauch, Departmentof Microbiology, been seen in a variety of conditions. A common prereq- University of' Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. uisite in these cases is the inhibition of the RecBCD E-mail:[email protected] exonuclease activity that would normally prevent the

Genrtics 146: 447-456 (.June, 1997) 448 A. B. Mann and J. M. Slauch accumulation of linear concatemericDNA. This can be dure allowed us to directly compare transduction of the plas- accomplished in the appropriate mutant background mid (Ap') to transduction of a chromosomal marker (Pyr') to determine theefficiency of plasmid transduction (EOPT). (COHENand CLARK 1986; SILBERSTEINand COHEN Transductions into the recA, recD, and controlrecipients were 1987) or by using phage encoded functions that inhibit performed in an analogousfashion except that they were RecBCD to allow rolling circle replication of the phage plated on LB Ap and LB Kn to select for transduction of the genome (COHEN and CLARK1986; POTEETEet al. 1988). plasmid and the chromosomal marker, respectively. RecA has also been implicated in pBR322 rolling circle Determination of the frequency that a transduced marker recombines with the recipient chromosome: Transduction of replication (COHEN andCLARK 1986). Plasmid concate- plasmid pJS225 from strain JS107 results in 12% of the Ap' mers can also be generated by interplasmidic recombi- transductants being Kn'. The inheritance of the Kn' marker nation. RecF has been implicated in both interplas- could be the result of the plasmid recircularizing with the midic and intraplasmidic recombination in otherwise Kn' marker on theplasmid or, alternatively, the plasmid could wild-type cells (COHENand LABAN 1983; KOLODNERet recircularize without the Kn' marker and the remainingtrans- duced fragment could recombine with the recipient chromo- al. 1985). some. To determine the frequency of this second event, we If low-copy number plasmids require homologousre- chose 145 Ap' Kn' plasmid transductants, which were subse- combination with the chromosome to be transduced, quently streaked free of phage. The 145 samples were then this provides a mechanism to facilitate allelic exchange grouped into 29 sets of five each. Each set was grown in L between plasmid and chromosomal sequences. To de- broth with Ap. Alkaline lysis plasmid preparations (AUSUBEI. et al. 1987) were done on all 29 sets and DNA from each was termine the feasibility of this technique, we examined electroporated into strain SH6749 and diluted andplated on the mechanism of low-copy number plasmid transduc- LB Ap plates. The appropriatedilution plate was then replica tion in more detail. In this study, we show that apSClOl plated onto LB Kn Ap agar. If one of the five transductants derivative is transduced by bacteriophage P22HTint by in a set carried the Kn marker in the chromosome rather two distinct mechanisms (Figure 1). The first mecha- than the plasmid, then -20% of the Ap' transformants should be Kn'. If a set gave Kn' colonies, then the five samples in the nism requires integrationof the plasmid into the donor group were grown separately and used to make P22HTint chromosome by homologous recombination. The sec- lysates. The five lysates were then used to transduce JS112 ond mechanism is independent of chromosomal ho- (purA:: Sp), selecting for Pur+. The purAf allele is tightly mology but is apparently distinct from the mechanism linked to the Kn marker and is carried both on the plasmid involved in the transduction of pBR322. We also exam- pJS225 and in the chromosome of the donor, both of which can be trasnduced into the recipient. The Pur' transductants ine the role of various recombination proteins in the were scored for Kn'. Eight Kn' and eight Kn' colonies from generation of packageable substrates in the donor and each transduction were independently scored for Ap' and Sp'. the resolution of plasmids in the recipient. If a lysate gave Pur+ Kn' Sp' Ap' colonies (transduction of a purAf Kn' plasmid) and Pur+ Kn' Sps Aps colonies (transduction of the Kn cassette from the chromosome), thenwe con- MATERIALSAND METHODS cluded that this sample has the Kn' cassette in the chromosome. Bacterial strains and plasmids: The bacterial strains used Determination of plasmidcopy number in fyphimu- in this study are listed in Table 1. All strains are isogenic S. derivatives of the virulent S. typhimurium strain ATCC 14028 rium: The pSClOl derivative plasmid pJS225 and the pBR322 derivative plasmid pBAM362 were independently trans- (CDC 651660). formed intoJS83. These plasmids contain the identical chro- Media and chemicals: Growth media were prepared as de- mosomal insert. Total cellular DNA was isolated from over- scribed (DAVISet al. 1980).Bacteriophage were diluted in night L Ap broth cultures as described (WLOYet al. 1996). T2 buffer (~OYet al. 1996). Antibiotics were used at the A ClaI restriction enzyme digest was done on all samples, following concentrations: Ampicillin (Ap), 50 pg/ml; Kana- giving linear plasmids. ClaI cuts outside the region of homol- mycin (Kn), 50 pg/ml; Spectinomycin (Sp), 125 pg/ml; Tet- ogy in both plasmids. Three independent samples of each racycline (Tc), 25 pg/ml. Replica plates used to detect ex- plasmid were separated on a 0.8% SeaKem agarose gel. The change of markers contained Ampicillin 100 ,ug/ml and DNA was transferred to a nitrocellulose membrane and the Kanamycin 50 pg/ml, except for therecFdonor experiments membrane was probed with a 32Pa-dCTP labeled probe using where the plates contained Ampicillin 50 pg/ml and Specti- a standard Southern Blot protocol (AUSUBELet al. 1987). The nomycin 125 pg/ml. All replica plates used in testing trans- probe used is an internalPstI fragment of the region of homol- ductants contained 10 mM EGTA. ogy shared by both plasmids. Thus the region of DNA that Transductionmethods. Bacteriophage P22lysates were hybridizes to the probeis identical inboth plasmids and in the prepared according to DAVISet al. (1980). Phage were titered chromosome. The membrane was viewed using a Molecular (DAVISet al. 1980) using T2 buffer. For transductions ofJS111, Dynamics Phosphorimager and the relative intensity of the 0.1 ml of a 1:lOO dilution of phage and0.1 ml of an overnight bands was determined using the Image Quant software. We culture of JSll 1 were mixed with 1 ml of LB and incubated compared the intensity of the plasmid band us. the chromo- at 37" for 30 min. The mixture was centrifuged, the superna- somal band in each lane. Defining the chromosomal band as tant was removed and the bacterial pellet was resuspended in 1.O for stationary phase cells, we then determined theaverage 1 ml of 150 mM NaCl. The transduction mixture was centri- number of copies of a plasmid in any particular sample. fuged again, the supernatantwas removed, and the pellet was resuspended in 210 pl of 150 mM NaC1. The washing proce- dure was carried out to reduce nutrient transfer to the mini- RESULTS mal plate. Aliquots of 100 pl were plated on LB containing Ampicillin and on E minimal glucose agar to select for Ap' P22HTint transduces pSClOl derivativesand transductants and Pyr+ transductants, respectively. This proce- pBR322 by distinct mechanisms: To test the mecha- Phage P22 Plasmid Transduction 449

TABLE 1 Bacterial strains and plasmids

Salmonella strains/ plasmids/phage Genotype Origin/reference" A. Bacteria 14028 Wild type ATCC MT96 14028 oufA126: : TnuTc SLAUCH et Ul. (1995) JS83 14028 galE496 JSlOS 14028 recD542::TnlOdCm JS107 14028 rjgSlO1: : Kn JSlOS 14028 rjg8101::KnrecD542::TnlOdCm JSlO9 14028 recF522: : Tn5 JSllO 14028 recA1 JSlll 14028 pyrC691: :TnlO JSll2 14028 purA:: Sp SH6749 LT2 hisC527 ilv-452 metA22 metE551 tqB2 galE496 xy1404 HARKKIand PAINA(1984) rpsLl20 jlaA66 hsdL6 hsdSA29 B. Plasmids pWKS30 pSClOl on, Ap' WANGand KUSHNER(1991) pJS225 pWKS30, 5-kb 14028 DNA insertb pJS226 pJS225, Sp' cassette in insert pBAM26 pJS225, Kn' cassette in insert pJS204 pWKS30, 5-kb 14028 DNA insert" pAL205 pJS204: : MudJ pAL2 15 pJS204: :MugJ" pBAM31 pWKS30 : : MudJ pBAM32 pWKS30, Tc' 1.3kb from pBR322 pBR322 ColEl ori, Ap' Lab stock pBAM326 ColEl on, Ap' 5-kb 14028 DNAb pBAM362 ColEl ori, Ap' 5-kb 14028 DNA" C. Bacteriophage P22HTint HT105/1 int-201 SCHMIEGER(1972) "This study if not otherwise noted. This 5-kb region of 14028 DNA is at purA. 'This 5-kb region of 14028 DNA is at oafA. 'The MudJ insert in pAL215 is distinct from the one in pAL205. "The 5-kb region of 14028 DNA in pBAM362 is in the opposite orientation as the region in pBAM326. nism of low-copynumber plasmid transduction, we used imal medium to select for Ap' transductants and Pyr' a pSClOl derivative, pWKS30(WANG and KUSHNER transductants, respectively. This method allows us to 1991), which has no known homology with the Salmo- directly compare transduction of the plasmid (Ap') to nella chromosome. We cloned a 5-kb fragment of S. transduction of a chromosomal marker (pye)to de- typhimurium chromosome into pWKS30, yielding plas- termine theefficiency of plasmidtransduction (EOPT). mid pJS225. Both pWKS30 and pJS225 were electropor- Thus, each transduction is internally controlled for any ated into JS107, a derivative of S. typhimurium strain possible growth differences in the donor or recipient 14028 that contains a Kanamycin-resistance cassette in strain or fluctuations in phage titers. The results in the middle of the region of chromosomal homology Table 2 indicate that the plasmid without homology, shared with pJS225. This insertion mutation confers no pWKS30, was transduced with an efficiency 8% of that apparent phenotype to thecell other than Kn'. We also seen for the chromosomal marker. The plasmids con- cloned a different5-kb fragment of S. typhimurium chro- taining homology, pJS225 and pJS204, were transduced mosome into pWKS30 to make the plasmid pJS204. In at 125% and 64% of the efficiency of the chromosomal this case, plasmid pJS204 is in the strain MT96, which marker, respectively. These results were consistent with has a TnlOdI'c insertion marker in the middle of the the hypothesis thatthe transduction efficiencyof region of homology shared with the plasmid. P22HTint pWKS30 derivatives was stimulated by homology to the lysates were grownon each strain and used to transduce chromosome. strain JSlll, a pyrC auxotrophic derivative of strain As a control, a P22HTint lysate grown on wild-type 14028. Equal volumes ofthe transduction mixture were strain 14028 containing pBR322 was used to transduce plated on LB containing ampicillin and onglucose min- JSll 1 (BrQ as described above. The results show that, 450 B. A. Mann and J. M. Slauch

TABLE 2 marker was linked to the plasmid in only 6% of the Plasmid transduction from a wild-type donor transductants. This suggests that plasmids pJS225 and pJS204 are not transduced as integrants as often as ex- Size Exchange of pected and that some additional transduction mecha- Plasmid (kb) EPOT (%)" marker (%) nism may be involved. This second mechanism may pWKS30 7.95.4 ? 2.5 <0.69 explain how plasmid pWKS30, which has no homology pJS225' 10.4125.3 ? 90.0 11.9 with the chromosome, is transduced at low frequency. pBAM26' 10.7111.0 ? 58.0 4.9 The apparent low frequency of plasmid recombina- pBAM31 15.4329.2 ? 229.3 NA tion with the chromosome during atransduction could 10.463.6 ? 19.7 pJS204' 6.0 result from a selective disadvantage of having the inser- 19.4562.7 ? 151.7 1.1 ~AL205~ tion on the plasmid such that plasmids without the in- pAL215' 19.4558.6 ? 190.6 1.4 pBAM32 18.06.7 ? 8.7 NA sertion are recovered preferentially. We tested this di- pBR322 310.64.3 ? 51.3 NA rectly by exchanging the alleles between the plasmid PBAM~~~~9.3 1044 2 932 0.045 pJS225 and the donor chromosome. If there is a selec- pBAM362' 9.3 2358 5 1121 0.45 tion against recircularization of the plasmid with the

a EOPT is the efficiency of plasmid transduction and is Kn' marker, then the same result should be obtained given as average ? SD where n z 7. NA, not applicable. whether the Kn' marker is originally in the chromo- 'Plasmids that share homology with the chromosome. some or on the plasmid in the donor strain. The plasmid pBAM26 is identical to pJS225 except that it con- consistent with previous results (SANDERSONand ROTH tains the Kn' cassettein the middle of the cloned 1988), plasmid pBR322 is transduced threefold more chromosomal fragment. A P22HTint lysate grown on efficiently than a chromosomal marker. Note that the wild-type strain 14028 carrying pBAM26was used to plasmids pBR322 and pWKS30 are similar in size and transduce JSll 1 (pyre). As expected, this plasmid was neither shares any known homology with the Salmo- transduced equally aswell as the chromosomal pyrC nella chromosome. NOVICKet al. (1986) showed that marker (Table 2). However, 95% of the Ap' transduc- the frequency of transduction of plasmid concatemers tants werealso IW. In other words, the plasmidex- generated by rolling circle replication was directly pro- changed the marker with the chromosome only 5% of portional to plasmid copy number using phage $11 in the time. These results are consistent with the previous Staphylococcus. However, copy number does not ex- interpretation that the plasmid is being transduced by plain the difference in efficiency of plasmid transduc- two separate mechanisms. A proportion of the plasmids tion in our studies. We determined the copy number are transduced via integration into the donor chromo- of pSClOl derivatives to be 7.86 & 0.54 and the copy some. Because the Kn' plasmids represent 50% of the number of pBR322 derivativesto be 13.93 ? 2.0. Clearly integrants, we conclude that 10-25% of the plasmids a twofold difference in copy number cannot account are transduced as integrants. The majority of the plas- for such a drastic difference in EOPT. Taken together, these results suggest that the pSClOl derivatives and mids are transduced by a mechanism that does not involve interaction with the donor chromosome. The fact pBR322 are transduced by distinct mechanisms. Derivatives of plasmid pm30that share homology that similar results are obtained with plasmids pJS225 and pJS204 suggests that this phenomenon is not the with the chromosome are transduced by two distinct mechanisms: If transduction of pJS225 is solely depen- result of some specific sequence cloned into plasmid dent on integration of the plasmid into the bacterial pWKS30. Since the phage packages -44 kb of DNA, chromosome, then there should be significant genetic we presume that this second mechanism involves trans- linkage between the Ap' marker on the plasmid and duction of a plasmid multimer (Figure lB), as has been the Kn' cassette in the chromosome. Presumably, when observed in a variety of systems (VIRETet al. 1991). the integrated plasmid pJS225 is transduced, the trans- It is formally possible that the Kn' plasmid transduc- ducing particle contains a duplication of 5 kb with one tants ofpJS225 are not the result of transducing an copy of the duplicated region containing the Kn" cas- integrated plasmid, but rather that the plasmid under- sette. These two regions of homology recombine during went a double recombination event in the donor and plasmid recircularization in the recipient. Since the JSn' thereby obtained the chromosomal Kn' marker. Subse- cassette is in the middle of the homologous region, quently, the plasmid could be transduced by a mecha- 50% of the circularized plasmids should contain the nism that does notrequire recombination with the Kn' cassette. We scored 1200 pJS225 Ap' transductants chromosome. We tested the frequency at which the for Kn' to test the frequency that the plasmid is trans- plasmid undergoes this double recombination event by duced as a chromosomal integrant. It was shown that determining thefrequency of Kn' plasmids in the popu- the Kn' cassette was linked to the plasmid in only 12% lation. Only 1 in 12,000 plasmidsisolated from an unin- of the clones (Table 2). Similar results were obtained fected cell carried theKn' marker. These results suggest for plasmid pJS204 in thatthe TnlOdTc insertion that the 12% linkage between transduction of Ap' and Phage P22 Plasmid Transduction 45 1

A. Integration of plasmidinto donor chromosome B. lnterplasmidicRecombination C. RollingCircleReplication bla

Formation of a packageable substrate 8-A + bla Single plasmidintegrant A

Circular multimer Possible re-circularization products - a- Plasmidwith marker Plasmid without marker Single plasmids or circular multimers

FIGURE1.-Mechanisms for theformation of packageable substrates and resolution of plasmids in the recipient. (A) A plasmid that contains homology to the chromosome can integrate via homologous recombination and thus be packaged in a 4Ckb chromosomal fragment by P22HT. The transduced integrated plasmid can recircularize in the recipient to give two possible plasmid species with respect to a chromosomal marker from the donor. The resulting transducing fragments may subsequently interact with the recipient chromosome. (B) Multiple copies of plasmid can undergo interplasmidic recombination to provide a 44kb segment of DNA to be packaged. (C) pBR322 can provide a 44kb segment of DNA to be packaged by undergoing productive rolling circle replication. Transduced plasmid multimers (B and C) can be resolved in the recipient as single copies bf free plasmid or possibly'circular multimers.

Kn' is indeed the result of transducing the integrated cause of the presence of a particular sequence in these plasmid. plasmids.For example, these larger plasmids could Transduction of pSClOlderivatives is size depen- have more or better P22HT pac sitesand thus be recog- dent: If pJS225 and pJS204 are being transduced by a nized and packaged more efficiently by P22HT. This mechanism that does notinvolve homologous recombi- packaging efficiency model could account for the ex- nation with the donor chromosome, thenwhy are these tremely efficient transduction ofpBR322. CASJENS et plasmids transducedmore efficiently than pWKS30? al. (1987) have defined a 12-bp consensus P22HT pac One possibility is that transduction by the alternative sequence. We searched forthis consensus pac sequence mechanism is size dependent. To test this hypothesis, in pBR322 and found that the two best matches in the we created a large pWlG30 derivative with no known sequence correspond to the 3' end of the tet gene. To homology with the Salmonella chromosome. This plas- test the hypothesis that this sequence is responsible for mid, pBAM31, wasmade by transposing a Mug element efficient pBR322 transduction, we cloned the tet gene (CASTILHO et al. 1984) onto pWKS30, in effect tripling from pBR322 into pWKS30 to make pBAM32. A its size. This larger plasmid was transduced (into strain P22HTintlysate on wild-type 14028containing pBAM32 JSll1) threefoldbetter than the chromosomal pyrC was used to transduce JSl11 (Pyre). The results (Table marker and 42-fold better than the smaller parental 2) show that pBAM32 is transduced only slightly better plasmid (Table 2). A second test was done using two than pWKS30. Indeed, the transduction efficiency of other plasmids, pa205 and pAL215. These plasmids pBAM32 falls on the curve plotted in Figure 2. Although contain two distinct Mug insertions in the cloned S. this experiment was based only on our limited knowl- typhimum'um DNA in pJS204. These two plasmids are edge of pac site recognition, it is consistent with the over 19 kb in size and are transduced 5.5-fold more idea that plasmid size is more important than absolute efficiently thanthe chromosomal marker (Table 2). sequence. These results suggest that the second mechanism of Transduction of pBR322: Transduction of pBR322 transduction of the pSCl0l derivatives issize depen- by P22HTint is very efficient even though this plasmid dent. This size dependence of transduction of pWKS30 is only 4.3kb (approximately the same size as pWKS30) derivatives is evident in Figure 2, where the efficiency and does not contain homology with the chromosome. of plasmid transduction is plotted us. the size of the We tested the effect of adding to pBR322 the 5-kb re- pWKS30 derivative. gion of homology that pJS225 shares with the S. typhimu- Transduction of pSClOl derivatives is apparently in- m'umchromosome. In this experimentwe can also deter- dependent of sequence: It is possible that the larger mine the relative frequency withwhich this pBR322 pSCl0l plasmids are transduced more efficiently be- derivative is transduced as an integrant in the donor 452 andB. A. Mann J. M. Slauch

TABLE 3 Plasmid transduction from a red donor

PlasmidEOPT (%)" Exchange of marker (%) pWKS30 2.1 t- 1.6 NA pBAM26" <0.339.6 t- 3.3 pBAM31 37.8 t 8.9 NA pBR322 36.5 ? 9.5 NA 0E W EOPT is the efficiency of plasmid transduction and is given as average t SD where n 2 4. NA, not applicable. ' Plasmids that share homology with the chromosome.

mid. In other words, at what frequency is the plasmid integrated into the donor chromosome at the time the chromosomal marker is packaged. In all three cases, this frequency was 3-6.7%. Thus, all three plasmids can be transduced as an integrant. However, the pBR322 4 6 4 8 10 12 1614 18 20 plasmids are apparently transduced by an additional Size of pWKS30 derivative plasmids (kb) mechanism that is not available to the pWKS30 derivatives. FIGURE 2.-Plasmid transduction is size dependent. Effi- Formation of packageablesubstrates in the do- ciency of plasmid transduction (EOPT) is plotted us. plasmid nor: The data presented above suggests that pWKS30 size for pwKs30 derivative plasmids. Data are from Table 2. is transduced predominantly by a mechanism that does not involve interaction with the chromosome and that chromosome by determining the linkage to the chro- this mechanism is different than that responsible for mosomal Kn' marker. The 5-kb region of homology the majority of transduction of pBR322.It seems plausi- from plasmid pJS225was cloned into pBR322, giving ble that transduction of both plasmids requires packag- two plasmids, each having the fragment in a different ing of aconcatemer or multimer (SCHMIDTand orientation as determined by restriction analysis. The SCHMIEGER1984; POTEETE1988). To understand the two plasmids, pBAM326 and pBAM362,were intro- formation of the packageable species, we examined the duced into strain JS107 (zjg8101::Kn) and P22HTint role of various recombination proteins in the donor lysates weremade and used to transduce recipient strain strain on transduction of pSCl0l derivatives. We first JSlll (PyrC). The EOPT of each plasmid (Table 2) tested the role of RecA in plasmid transduction. Four was increased dramatically over the EOPT of parental plasmids(pWKS30, pBAM26, pBAM31 and pBR322) plasmid pBR322. Indeed, the effect of increased size were electroporated intoJSll0, a recAl mutant of strain on transduction efficiency is much more drastic than 14028, and P22HTint lysates grown on the four strains that seen with pWKS30 derivatives. Moreover, whereas were used to transduce a recAf recipient strain, JSlll plasmid pJS225 recombines with the Kn' marker from (pyrc). The transduction efficiency of allplasmids the chromosome 12% of the time, plasmids pBAM326 tested, including pBR322, was reduced five- to 10-fold and pBAM362 recombine with the chromosomal by the recA mutation (Table 3). Thus RecA is involved marker only0.045% and 0.45% of the time, respec- in, but not absolutely required for, the transduction tively. These results reiterate themechanistic difference of both pBR322 and pSCl0l derivatives. Although the between transduction of pBR322 and pWKS30 deriva- pSClOl derivative plasmids were stilltransducable from tives. the recA strain, integration of the plasmids containing The plasmid pJS225 (derived from pWKS30), and homology into the donorchromosome was eliminated. plasmids pBAM326 and pBAM362 (derived from The pWSK30 derivative plasmid pBAM26 contains the pBR322) share thesame amount of sequence homology Kn'. marker in the middle of the 5-kb region of chromo- with the chromosome. Indeed, when the absolute num- somal homology. None (<0.33%) of the over 300 Ap' ber of Ap' transductants that are also Kn' is compared transductants of pBAM26 from the recA donor showed to the transduction of the pyrC marker, it is evident loss of the Kn' cassette compared to4.9% from thewild- that the number of plasmids that are transduced as an type donor. integrant in the donor chromosome is approximately The recFpathway ofrecombination has been shown to equal in each case (data from Table 2). In addition, we affect intermolecular plasmidic recombination (COHEN have determined the frequency with which each plas- and LABAN 1983; KOLODNERet al. 1985). Because this mid interacts with the chromosome by performing is a potential source of multimeric plasmid, we tested transductions where we select for the Kn' chromosomal the role of RecF in the donor strain on plasmid trans- marker and then score for coinheritance of the plas- duction. ArecF: : Tn5 strain,JS109, was constructed and Phage P22 Plasmid Transduction 453

TABLE 4 TABLE 5 Plasmid transduction from a recF donor Plasmid transduction from a recD donor

PlasmidEOPT (%)" Exchange of marker (%) PlasmidEOPT (%)' Exchange of marker (%) pW30 2.9 ? 1.5 NA pW30 86 ? 17 NA pJS226' 43.9 ? 24.5 2.3 pJS225' 1010 ? 174 10.0 pBR322 284.5 ? 61.9 NA pBR322 753 ? 191 NA " EOPT is the efficiency of plasmid transduction and is EOPT is theefficiency of plasmid transduction and is given as average 2 SD where n 2 10. NA, not applicable. given as average ? SD where n 2 5. NA, not applicable. ' Plasmids that share homology with the chromosome. ' Plasmids that share homology with the chromosome. electroporated with three plasmids: pWKS30, pBR322, plex normally inhibits the formation of packeagable and the pWKS30 derivative plasmid pJS226. The plas- substrates for the pWSK30 derivatives. It follows that mid pJS226 contains the5-kb fragment of chromosomal both mechanisms of pWKS30 derivative plasmid trans- DNA with a Sp' cassette (1.9 kb) cloned into the unique duction involve a double-stranded end. EcoRI site in place of the Kn' cassette (1.3 kb) used in Interaction of the transduced fragment with the re- previous experiments. P22HTint lysates were grown on cipientchromosome: When the pWSK30 derivative each of the strains and used to transduce theret+ recipi- pJS225 is transduced from the donorJS107, which con- ent strain JSlll (pyrC). The transduction efficiency of tains a Kn' cassette in the middle of the homology pWKS30 from the recF donor was 2.7-fold lower than shared with the plasmid, 12% of the Ap' transductants seen for there& donor but thetransduction efficiency are also Kn', apparently as a result of transducing a of pBR322 was not significantly affected (Table 4). This chromosomalfragment that contains an integrated result shows that the transductionof the low-copy num- plasmid. Thus, the inheritanceof the Kn' marker could ber plasmid pWKS30 is somewhat dependent on RecF be the result of the plasmid recircularizing with the Kn' in the donorwhile transduction of pBR322 is indepen- marker on the plasmid or, alternatively, the plasmid dent of RecF. could recircularize without the Kn' marker and the re- Multiple lysates of pJS226 in the recF donor JSl09 mainingtransduced fragment could recombine with were used to transduce the ret' recipient strain JSlll. therecipient chromosome. Todetermine the fre- Analogous to the parental plasmid pWKS30, the trans- quency of this second event, we measured the percent- duction efficiency of this plasmid was reduced an average of Ap', Kn' transductants that contained the Kn' age of 2.5-fold by the donor recF mutation, with one marker in the chromosome of the recipient. Of 145 lysate consistently giving a transduction efficiency re- Ap' Kn' transductants, 13 had the Kn' marker in the duced 6.5-fold compared to the re& donor. The plas- chromosome. Assuming that this represents 50% of the mid pJS226 can still interact with the chromosome of events, -18% of the transduced particles that contain the recFstrain and be transduced as an integrant. Scor- an integrated plasmid undergo a recombination event ing over 300 Ap' transductants forSp' showed that plas- with the recipient chromosome. This is consistent with mid pJS226 lost the Sp' cassette at a frequency of 2%. previous results for the frequency of interaction of a The RecBCD complex is an exonuclease that digests transducing particle with the chromosome (EBEL-TSIPIS linear DNA at double-stranded breaks (reviewed in KO- et al. 1972) and provides further evidence that the ge- WALCZYKOWSKI et al. 1994).It has been hypothesized netic linkage between the Kn'. and Ap' markers is the that pBR322 is packaged by P22 during rolling circle result of transduction of the integrated plasmid. replication that produces a linear plasmid concatemer Recircularization of theplasmid in therecipient: with a double-stranded end (COHENand CLARK1986; Presumably, recircularization of the plasmid in the re- SILBERSTEINand COHEN 1987).We tested the effect of cipient is mediated by homologous recombination be- a donor recD mutation on transduction of the various tween duplicated regions ( ORBACHand JACKSON 1982). plasmids. We electroporated pWKS30,pJS225, and To test if the recircularization of the pSCl0l derivative pBR322 into the recD donor, JSlOS, and P22HTint ly- plasmids was RecA dependent, the plasmid pJS225 was sates were made and used to transduce JSlll (pyrC). transduced from donor strain JS107 (contains the Kn' The results (Table 5) showed that the recD mutation marker in the chromosome)into isogenic recA and wild- caused an approximately 10-fold increase in the EOPT type 14028 strains at a MOI of 0.2. All transduction of pWKS30 derivatives while the EOPT of pBR322 in- mixtures were divided and plated on LB Ap and LB Kn creased only threefold. Interestingly, the proportion of to independently select for transduction of the plasmid the pWKS30 derivative plasmid pJS225 transduced as and the chromosomal marker, respectively. Note that an integrant in the chromosome is approximately the the chromosomal marker (Kn') can be transduced ei- same as that seen in the rec" donor. These results sug- ther by itself or in association with the plasmid. The gest that the exonuclease activity of the RecBCD com- data (Table 6) shows that the plasmid can recircularize 454 B. A. Mann and J. M. Slauch

TABLE 6 mid integrated by homologousrecombination. This Transduction of plasmid pJS225 into rec recipients structurecontains the plasmid sequences flanked by direct repeats of chromosomal DNA. We have observed Ap' transductants Kn' transductants these events by using a donor that has an antibiotic- resistance marker in the middleof the region of homol- Recipient Number" % Kn' Number" % Ap' ogy shared by the plasmid and the chromosome. In Wild type 231 14 582 6 the recipient, this structure resolves itself yielding the recA 48.1 3 0.8 100 plasmid and the remaining chromosomal transducing Wild type 87 16.5 372 4.3 fragment. Approximately 18% of the remaining trans- TPCD 133.3 19 1019 2.9 ducing fragments recombine with the recipient chro- "The number of transductants is an average per transduc- mosome. tion where N = 3 for the wild-type recipient and for the recD The second mechanism of pSClOl transduction is recipient, while N = 10 for the recA recipient. For the top two size-dependent and is apparently independent of re- lines transductions were done at an MOI of 0.2, while for the combination with thedonor chromosome.It seems bottom two lines transductions were done at an MOI of 0.04. likely that the phage packages a multimer of plasmid DNA, as seen in all others systems examined (VIRETet in the recA recipient although the frequency is down al. 1991). We believe that generation of the pWSK30 slightly. Moreover, the effect of recA differentiates the multimer is mechanistically different than the genera- plasmids transduced as anintegrant us. those trans- tion of the concatemer for pBR322. The most likely duced as a putative concatemer, in that the percentage mechanism for pWSK30 involves interplasmidic recom- of Kn' transductants is decreased from 14% to 3%. bination to give a large multimeric circle (Figure 1B). Thus, the putative concatemer is more efficiently recir- According to our model, all plasmids can undergo such cularized than the integrant in the recA recipient. As recombination events. However, the phage requires 44 expected, recombination into the recA recipient chro- kb of DNA to package. Therefore, the larger the plas- mosome was negligible; transduction of the chromo- mid, the fewer recombination events are required to somal Kn" marker from the donor was completely de- give a sufficiently large circle. The pSClOl derivatives pendent on transduction of the plasmid. arepresent in approximately eight copies per cell. Finally, we examined what effect a recipient recD mu- Therefore, in the case of pWKS30 (5.4 kb), even if all tation had on transduction of pWKS30 derivatives. The of the plasmids recombine, then theresulting multimer plasmid pJS225 was transduced from donor strain JS107 is only 43 kb and is insufficient for packaging. If the (contains the Kn' marker in the chromosome) intoiso- sizeof the plasmid is increased to 10.4 kb (pJS225), genic recD uS106) and wild-type 10428 at an MOIof then the large concatemer could potentially be 83 kb 0.04 selecting for plasmid transduction (Ap') or chro- and is now packageable. This apparent transition be- mosomal transduction (Kn'). Table 6 shows that trans- tween efficient and nonefficient transduction can be duction of the plasmid is increased slightly in the recD seen in the graph in Figure 2. recipient whereas transduction of the chromosomal The formation of the putative pSClOl circular marker is increased threefold. The relative frequency multimers by interplasmidic recombination is sup- of transducing the putative multimer and anintegrated ported by the fact that a mutationin recFreduces trans- plasmid is similar to that seen in a rec' recipient. duction of pWKS30 derivatives approximately 2.5-fold but does not significantly affect transduction of DISCUSSION pBR322. Thus RecF is involved, but not absolutely required, for creationof the packageable pSClOl species, The high-transducing mutant of bacteriophage P22 but is not significantly involved in the formation of the is capable of carrying out generalized transduction of pBR322 concatemer. This result is consistent with two plasmids in S. typhimum'um. For eolEl plasmids, such as different mechanisms being involved in the transduc- pBR322, it has been proposed that productive rolling tion of pBR322 us. pSClOl derivatives. circle replication of the plasmid in the donor allows Absence of RecD in the donor causes a dramatic in- packaging of the concatemeric DNA molecule by P22 crease in thetransduction of pSClOl derivatives. (SCHMIDTand SCHMIEGER 1984).Low-copy plasmids, Whether this is a result of amplifylng the predominant such as pSClOl derivatives, are also transduced by pathway involved in the formation of packageable sub- P22HT. We provide evidence that this transduction strates or inducing a newpathway is unclear. Is the takes place by two separate mechanisms, both of which pSClOl derivative now being packaged via rolling circle differ from transduction of pBR322 (Figure 1). replication in the recD strain? We think this is unlikely The first mechanism of pSClOl transduction involves because of the fact that transduction via integration those plasmids that have homology with the bacterial into the donor chromosome still represents -10% of chromosome(Figure 1A). In these cases, thephage the events. This is in contrast to the analogous pBR322 packages a chromosomal fragment containing a plas- derivatives that have homology with the chromosome. Plasmid TransductionPhage P22 Plasmid 455

In this case, transduction of the integrant represents a fold more likely to giverise to stabile plasmid than very small fraction of the total. Indeed, the fact that the transduced integrant.The difference between these both mechanisms of transduction of the pSClOl deriva- molecules is that the integratedplasmid has one region tive are equally increased in the recD donor suggests of duplication that is internal to the molecule, whereas that both are products of recombinational events initi- the concatemer has a number of tandem duplications ated by double strand breaks. both within the molecule as well as on the ends. The RecA is involved in the formation of all of the pack- fact that the concatemer is more efficient implies that ageable substrates. A donor recA mutationreduces much of the productive recircularization occurs close transduction of both pBR322 and the pSClOl deriva- to the ends, giving rise to a circular multimer that, in tives approximately five- to 10-fold. This does not distin- apparent contrast to pBR328, leads to stabile inheri- guish formation of the pBR322 concatemer and the tance of the plasmid. pSClOl multimer. However, the recA mutation com- Mostof the plasmids, including sufficiently large pletely blocks plasmid transduction via integration into pSClOl derivatives, pBR322,or pBR328, are transduced the donor chromosome. These results support the hy- with an efficiency that is equal to or greater than a pothesis that two distinct mechanisms are involved in chromosomal marker.When the pSClOl derivatives are the transduction of pSClOl derivatives that share ho- transduced integrated into thedonor chromosome, the mology with the bacterial chromosome. transducing fragment that remainsafter plasmid resolu- We do not mean to imply that pBR322 cannot be tion recombines with the recipient chromosome- 18% transduced by the same mechanisms aspSC101. In- of the time. This is similar to previous results showing deed, in the case of the pBR322 derivatives, pBAM326 that -10% of standard transducing fragments recom- and pBAM362, that contain 5 kb of homology with the bine into the recipient chromosome (EBEL-TSIPISet al. chromosome,transduction via integrationinto the 1972). This implies that the majority of plasmids that chromosome occurs at the same rate as for the analo- are injected into the recipient resolve and replicate. It gous pWKs30derivative pJS225, when compared to follows that although the overall transduction of the transduction of the chromosomal marker. However, in plasmids can exceed that of a chromosomal marker, contrast to pJS225, the pBR322 derivative plasmids can the frequency is in large part due to the events in the also be transduced by a much more efficient mecha- recipient. Thus, plasmida that is transduced atthe same nism: generation of a concatemerby rolling circle repli- frequency as a chromosomal marker is packaged at a cation. frequency of only 10-20% of the chromosomal marker, When any of the plasmid transducing particles are suggesting thatthe chromosome is thefar superior injected into the recipient, the plasmids must recircu- packaging substrate. larize to be stably maintained. GARZONet al. (1995) In the uninfected cell, the plasmid pJS225 recom- have shown that the recircularization of a pBR328 con- bines with the chromosome at a frequency of 1/6000, catemer can be mediated by either RecA or Erf, pro- as measured by the numberof plasmids that obtain the duced from a co-infecting phage particle. They also chromosomal Kn' marker. However, when we select for have shown that RecB is required to resolve both the transduction of the chromosomal Kn' marker, the plas- plasmid concatemer as well asa plasmid integrated into mid integrant is cotransduced at -5%. If the plasmid the P22 phage genome by homologous recombination. is present at eight copies per cell, then the integrated However, RecB is not required for circularization of plasmid represents -0.6% of the total plasmid popula- P22 phage DNA. They argue that recircularization of tion. This implies that this plasmid-chromosome recom- the plasmid requires recombination within internal re- bination event, and perhaps recombination in general, peats, whereas circularization of the phage must take is increased at least 37-fold in the P22 infected cell. place on the terminally redundant ends of the phage Note that the recombination between the plasmid and genome. This implies that recircularization of the the chromosome isRecA dependent and, therefore, pBR328 plasmid concatemer by recombination at the this increased recombination cannot be due solely to ends of the fragment does not lead to stabile plasmid phage recombination proteins. inheritance, perhapsbecause a circular multimeris un- Is low-copy plasmid transduction a feasible method stable (SUMMERSand SHERRATT1984). of allelic exchange? Given the fact that recombination In the experiments presented here, the pSClOl plas- is dramatically increased in the donor, plasmid trans- mids are capable of recircularizing in the absence of duction is an ideal method to move alleles from the RecA. Although this is relatively efficient at an MOI of chromosome to the plasmid. Depending on thesize of 0.2, theexperiments presented here do not directly the plasmid, recombinants are a significant fraction of address the potential role of Erf in this process. The the transduced plasmids. Thus, even if the recombinant plasmid can recircularize when transduced either as an plasmid must be identified by restriction mapping, for integrant in a chromosomal fragment or as a putative example, it is feasible. Moving alleles from the plasmid concatemer. However, our results indicate that in the to the chromosome is slightly more problematic. This absence of RecA, the concatemer is approximately five- is because the plasmid is efficiently transduced indepen- 456 B. A. Mann and J. M. Slauch dently of the chromosome and, even when the plasmid EBEL-TSIPIS,J., M. S. FOXand D. BOTSTEIN,1972 Salmonella typhimurium. 11. Mechanism of integration of transducing DNA. J. Mol. is transduced as an integrant, the resulting transducing Biol. 71: 449-469. particle does not always recombine with the recipient GARZON,A., D. A. CAN0 and J. CASADESUS, 1995 Role of Erf'recombi- chromosome. Moreover, these transduction events are nase in PPBmediated plasmid transduction. Genetics 140: 427- 434. phenotypically indistinguishable. These complications HARKKI, A,, and E. T. PAWA, 1984 Application of phage lambda can be avoided by selecting for chromosomal transduc- technology to Salmonella typhimurium. Construction of a lambda- tion from a plasmid-bearing strain. For example, we sensitive Salmonella strain. Mol. Gen. Genet. 195: 256-259. KOLODNER,R., R. A. FISIIEI.and M. HOWARD,1985 Genetic recombi- have efficiently movedthe MudJ (Kn') insertions in plas- nation of bacterial plasmid DNA effect of RecF pathway muta- mids pAL205 and pAL215 into the chromosome by the tions on plasmid recombination in E.schm'chin coli. J. Bacteriol. following method. We constructed astrain that contains 163: 1060-1066. KOU'ALC~OM~SKI,S. C., D. A. DIXON,A. K. E(:GI.ESTOX,S. D. LAL:I)ER a TnlOnc in the chromosome that is genetically linked and W. M. REHRAUEK, 1994 Biochemistry of homologous re- to, but not in the chromosomal fragment that corre- combination in Eschm'chia coli. Microbiol. Rev. 58: 401 -465. sponds to the insert in the plasmids. Each of the plas- WN,M. J., J. M. SIAUCH and J. J. MEKAIANOS, 1993 Bacterio- phage P22 transduction of integrated plasmids: single-step clon- mids was introduced into this strain and lysates were ing of Salmonella typhimurium gene fusions. J. Bacteriol. 175: grown and used to transduce the wild-type recipient 7086-7091. strain for Tc', selecting for transduction of the chromo- MAI.OY,S. R., V. L. STEWART and R. K.TAYI.OR,1996 Grnrtic Analyi.r of Pathogenic Bartm'a: A Laboratmy Manual. Cold Spring Harbor somal marker. Approximately 3% of the transductants Laboratoly Press, Cold Spring Harbor, NY. also inherited a plasmid that had integrated into the MARGOLIN,P., 1987 Generalizedtransduction, pp. 1154-1168 in donor chromosome during phage propagation. Of the Esrherichia coli and Salmonella Qphimurium, edited by F. C. NEIL). HARDT. American Society for Microbiology, Washington, DC. plasmid-bearing transductants, up to 50% contain the NOVICK,R. P., I. EDELMAN, andS. LOFDAHI., 1986 Small Staphylococ- MudJ recombined into the recipientchromosome. The cus aureus plasmids are transduced as linear multimers that arc formed and resolved by replicative processes. J. Mol. Biol. 192: actual percentage is dependent on the position of the 209-220. Mud in the chromosomal insert. Even if the frequency ORBACH, M.J., and E. N. JAC:KSON, 1982 Transfer of chimeric plas- is SO%, these strains can be dramatically enriched for mids among Salmonella typhimurium strains by P22 transduction. J. Bacteriol. 149: 985-994. by pooling the Tc', Kn' transductants, making a P22HT POTEETE,A. R., 1988 Bacteriophage P22, pp. 647-681 in Thr Back- lysate, and transducing the wild-type strain for Tc" and riophages, Vo. 2, edited by R. CAIXNIMR.Plenum Press, NewYork. subsequently screening forKn' and Ap'. Any MudJ that POTEETE,A. R., A. C. FENTONand K. C. MLIRPHY,1988 Modulation of Escherichia roli RecBCD activity hy the bacteriophage lambda was in the chromosome in the donor is transduced at Gam and P22 Abc functi0ns.J. Bacteriol. 170: 2012-2021. high frequency dependingon the linkage of the SANDERSON,K. E., and J. R. ROTH, 1988 Linkage map of Sulmondla TnlOnc. Thus,the two steps required to exchange typhimurium, edition. VII. Microbiol. Rev. 52: 485-532. SCHMIDT,C., and H. SCHMIEGER,1984 Selective transduction of re- alleles from plasmid to chromosome (a double recom- combinant plasmids with cloned puc sites by Salmonella phage bination event with the chromosome and subsequent P22. Mol. Gen. Genet. 196 123-128. loss of the plasmid) are facilitated by performing two SCHMIEGER,H., 1972 Phage PP2-mutants with increased orde- creased transduction abilities. Mol. Gen. Genet. 119: 75-88. concurrent P22 transductions. SCHMIECER,H., 1982 Packaging signals for phage P22 on the chromosome of Salmonella typhimurium. Mol. Gen. Genet. 187: 516- We thank GREGORY PHILLIPS,STANLEY MALOY, JEFFREY GARDNER, 518. THOMASZART, ANGELA LEE and the members of our laboratory for SILBERSTEIN,Z., and A. COIIEN, 1987 Synthesis of linear multimers helpful discussions. Thisresearch was supported by US. Public of OriC and pBR322 derivatives in Eschm'chin coli K-12: role of Health Service grant AI37530, American Heart Association-Illinois recombination andreplication functions.J. Bacteriol. 169: 3131- affiliate grant 96-GB-01, and a Junior Faculty Research award from 3137. the American Cancer Society. SLALKH,J. M., M. J. MAHAN,P. MICHETTI, M.R. NEL~TRA andJ.J. MEMANOS,1995 Acetylation (0-factor 5) affects the structural and immunological properties ofSalmonella typhimurium lipopoly- saccharide 0 antigen. Infect. Immun. 63: 437-441. LITERATURE CITED SUMMERS,D. K., and D. J. SHERRATT,1984 Multimerization of high copy number plasmids causes instability: CoIEl encodes a deter- AUSUBEI.,F. M., R. BRENT, R. E. KINGSTON, D. D. 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