Lethal Transposition of Mud Phages in Rec- Strains of Salmonella Typhimum'um

Lethal Transposition of Mud Phages in Rec- Strains of Salmonella typhimum'um

Ramesh V. Sonti,' David H. Keating and John R. Roth'

Department of Biology, Universityof Utah, Salt Lake City, Utah 84112 Manuscript received December 5, 1990 Accepted for publication September 25, 1992

ABSTRACT Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonellatyphimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.

RANSPOSITION is an integral part of the life transposition event remains unclear. Although normal T cycle of bacteriophage Mu (TAYLOR1963; BUK- levels of both replicative and conservative transposi- HARI 1976; TOUSSAINTand RESIBOIS 1983).Upon tion require both the Mu A and B functions,both infection, the Mu genome transposes from the infect- functions can occur at a reduced level with only the ing DNA fragment and integrates into thehost chro- Mu A function (CHACONASet al. 1985). The twotypes mosome. This initial transposition event is conserva- of transpositionevents may occur by substantially tive; thewis minimal replication of Mu sequences and differentmechanisms (CHACONAS1987) or may be the Mu sequences are released from their association variations of a single process (CRAIGIEand MIZUUCHI with flanking donor sequences (reviewedby HARSHEY 1985). It has been proposed that conservative trans- 1987).After integration into the chromosome, the positionrequires participation of a tail proteinof Mu genome transposes replicatively to new chromo- phageMu, which is injected with theDNA upon somal sites (reviewed by PATOand WAGGONER 1987). phageinfection (GLOORand CHACONAS1986). We Duringthe period of replicativetransposition, the believe that the data presented here can best be ex- Ixlcterial chromosome is subjectedto multiple re- plained in terms of this injected protein. ;Irrangenlentsincluding deletions, duplications and Bothconservative and replicative modes of Mu inversions (TOUSSAINTand RESIBOIS 1983). A general transposition are thought to be independent of the model for the process of replicative transposition has hostrecombination system (recA.B.C). Therefore it been proposed by SHAPIRO (1979) and in vitro exper- was surprisingto observe that MudI, a transposon iments have demonstrated and elaborated upon this derivedfrom phage Mu, shows anapparently de- model (CRAIGIE andMIZUUCHI 1985; for reviews see creasedability to transpose from a P22-transduced MIZUUCHI and CRAIGIE 1986; MIZUUCHI and HIGGINS fragmentinto the chromosome when therecipient 1987). carries a rec mutation (HUGHES, OLIVERAand ROTH The detailed mechanism of the initial conservative 1987). Since this effect of Rec deficiency on lysogen recovery was not observed when the MudI genome ' Presentaddress: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307. was injected by a Mu virion, it appeared that some ' Presentaddress: Department of Microbiology,University of Illinois, (:hampdign-Urbana, Illinois 61801. aspectof virion structure might contribute to the ' To whom correspondence should be addressed. process of Mu transposition or lysogenization.

Genetics 133: 17-28 (January, 1993) 18 R. V. Sonti, D. H. Keating and J. R. Roth

TABLE 1 carbon source was added to a concentration of 0.2%. The complexmedium was nutrient broth (0.8%; Difco) with Strain list added sodium chloride (0.5%). MacConkey-indicator plates (Difco)were supplemented with 1% D-galactose.Auxo- Strain' Genotype trophic supplements were added to minimal media at con- Source: Laboratory collection centrations described by DAVIS,ROTH and BOTSTEIN(1 980). L:r 2 Wild type Unless otherwise indicated antibiotics were used at the fol- TT97.58 hisC46 recB503::TnIU lowing concentrations in minimal and complex media, re- TT7216 his-YY44::Mudl spectively: kanamycin: 125 pg/ml and 50 pg/ml; ampicillin: TT80.50 ilvAYY pyrF231malAllO gal-851 metA27 trpE2 15 Pg/ml and 30 pg/ml. D-galactose and all antibiotics were hisF100Y rspL2OI xyl-I musAl Mucts62 Mud1 obtained from Sigma Chemical Company. TT8353 pl.PIO3-63-3(MuA+B+) Transductional methods: The high frequency general- TT8785 nadA2IY::MudA ized transducing mutant of bacteriophage P22 HT105/1 TT10199 nadA56/F'152-2 int-201 (SCHMIEGER1972) was used for all transductional TT10288 hisDYY53::MudJ his-YY4l::MudI crosses. P22 transducing lysates were grown on Gal- P22- TT10377LT2/pLP103-32-2 (Mu A+) resistant strains by introducing an E. coli F' Gal+ plasmid TT10838 recA1 (F' 152-2) which provides the missing gal functions. In trans- TTI 1 183 sd-203::Tn10d-Cdm duction crosses involving selectionfor drug resistance, equal TT 10502 musA 1 volumes of saturated cultures of recipient cells and donor TT 10503 musA 1 recA I lysates were mixed and preincubated in liquid medium at TT 10504 musA I recB503::TnIU 37" before spreading onto selective medium. The pre- TT10505 musAl recA1 recB503::TnIU incubation period was20 min when ampicillin resistance TT12116 nadA213::TnlO/F'152-2nad+ zzf-1873::MudF was selected and 45 minwhen kanamycin resistance was Source: This work selected. Transductants were first purified selectively and TT14547 purF2054::MudJ musAl rxx-3675::MupApl phage-free colonies were identified as light coloniesformed TT14548 musAl rxx-3675::MupApl following streaking on green indicator plates (CHANet al. TT14549 musAl/pLP103-32-2(Mu A+) 1972). Phage sensitivity of transductants was confirmed by TI'14.5.50 musAl recAl/pLP103-32-2(Mu A+) cross streaking with P22 H5 (a clear plaque mutant of phage TT14.5.55 recAl P22). Transduction crossesusing Mucts62 and MupApl TT145.56recB503::TnIU were done according to BERMAN,ENQUIST and SILHAVY ~lT14.5.58 recA1 recB503::TnIU (1 98 1). TTI.5216 recAI/pLP103-32-2(Mu A*) Identification and analysis of nadA-gal deletions: Gal- TT1.5220 recB503::TnIU/pLP103-32-2(Mu A+) strains were tested for Nad- and Bio- phenotypes on mini- Tl1.5221 recA1 recB503::TnlU/pLPlO3-32-2(Mu A+) mal plates to which nicotine acid or biotin or both were 1'1'15881 ara-663::Mud111681 added. The aroG phenotype of the nadA-gal deletion mu- TT17337 musAl rxx-3675::MuPAplaru-662::MudII1681 tants was determined according to ALPER andAMES (1 975). All nadA-gal deletion strains isolated are aroC- andthe a All strains are derivatives of S. typhimurium LT2. majority are phenotypically Dhb- (deficient in synthesis of dihydroxybenzoate). Growth of Dhb- strains is inhibited by citrate, because these strains are deficient in transport of Wehave pursued these observations and suggest iron (ALPERand AMES 1975). All Dhb- deletions are also here that the reduced recovery of Mud lysogens fol- Bio-. lowing P22 transduction of Mu-derived elements into Determination of the viability of rec strains: The optical Kec- hosts is due to increasedhost killing rather than density of overnight cultures of ret+, recA, recB, or recA recB strains of S. typhimurium was measured using a Klett meter. ;I failure of transposition. We propose that lethality is The cultures were diluted and plated onto complex medium due tounrepaired breaks in the hostchromosome and the number of viable cells/ml/Klett unit of the culture which occur when the phage genome transposes rep- was estimated. The viability of the rec+ strain was defined licatively(instead of conservatively) from the linear as 1. The viability of the recA strain was generally 0.66; the injected fragment. Results are interpreted to suggest recB strain was 0.29 and the recA recB double mutant was 0.2 thatconservative transposition requires a protein which is normally injected with the Mu genome; in- RESULTS jection of thisprotein requires a Mu virion. The injection or function of the proteinmay require asso- Transduced mini-Mu elements require host rec ciationwith a particular Mu DNA sequence that is function for lysogeny: Previously reported effects of missing or improperly positioned in the smaller mini- host rec mutations onMud phage inheritance involved Mu elements. experiments with the MudI(Ap Lac) element (38 kb) derived from phage Mu by CASADABANand COHEN (1979). It was found that host rec mutations reduce MATERIALS AND METHODS the recovery of lysogens formed by Mud1 elements Bacteria: All strains used are derived from Salmonella injected by P22; no such Rec-effect was seen for the typhimurium LT2. A list of strains is shown in Table 1. same Mud element injected by a Mu virion (HUGHES, Media: Minimal medium was eitherthe E mediumof VOCELand BONNER(1 956) or NCE medium (BERKOWITZet OLIVERA,and ROTH 1987). al. 1968). The E medium was supplemented with glucose. To determine whether smaller derivatives of phage The NCE medium was supplemented with D-galactose. The Mu are alsosubject to thisRec-effect, we tested a Lethal transposition of Mud phages

FIGURE 1 .-The genetic. maps of Mu andthe Mud phages that were ij *ool-=.?S used in this work: all of these pllages contain the rts62 mut;ltion. The map A 1 E 9 Clys DEH FG I TJK L M Y NP artR of Mu was ;td;q)ted from HOWE MupApl (37kb) 8rrL $ (1987).~l~en1;1psofMu~~Apl,~~udl, MudlIl68I and MudJ were ad;tpted from FAELEN(1 987). The mq) of MudF IGIS deduced from the maps of Mudl ;tnd a nlini-Mu lar transposon that was constructed 11) CHACONASPI al. (1981). The filled in boxes repre- 8rrR sentnontransposable Tnjr sequences. Mud I (37 kb) attL In MupApI and Mudl thesese- ~p lacA YZ rrp quences encode Ap". The MudF Re- nonle containsa truncated portion of the Ap" detertninant of Tn3 and does not encodeAp". The boxes with z horizontalstripes represent non- Mud111681 (14.5kb) alK arrR transpowblel'n5 sequences. The Kan lacA Y 2 mottled boxes represent E. roli lar operonsequences. MudF encodes the entire lac operon of E. coli: the lac sequencespresent in Mudl and MudJ do not include the lar promo- tor but can generate lar operon fusions promoters located near the MudJ (11 kb) 811L arrR to insertion side. The open boxes rep- Kan lacA Y Z trP resent E. coli frp sequences. The box with vertical stripes representsan IS121 insertion in Mudl.

MudF (14.5 kb) BttL artR Kan l8CA YZOpl

mini-Mud elementconstructed by CHACONASet al. prevent inheritance of MudF by homologous recom- (1 98 1) and modified by R. V. SONTIand J. R. ROTH bination. To permit transposition of the transduced (unpublished results).This small (1 4 kb) transposition- MudF element, the recipient strain carries a plasmid defectiveelement, referred to asMudF, carries a with the MuA transposase gene expressed at a consti- complete lac operon, a kanamycinresistance deter- tutive level (VAN LEERDAM,KARREMAN and VAN DE Ininant (KmR),about I kb of Mu sequences, including PUTTE 1982; M. HOWE,personal communication). the c gene (at the left end) and about 1.5 kb at the The Mu ner gene is not included. This plasmid pro- right (S) end; it does not include the Mu A and B vides a low level of transposition proficiency. Selection transposition functions. The genomes of MudF and was madefor Lac+ transductants.Since theMudF other Mu derivatives used here are diagrammed in element includes an entirelac operon (with promoter), 1;igul-e I. all transductants which inherit MudF will show ;I Lac+ To test the effect of host rec mutations on MudF phenotype. Because recombinationalinheritance is transposition, we performed P22-mediated transduc- prevented by lack ofsequence homology, MudF is tion crosses. The donor strain (TT12 1 16) carries inherited only by transposition from the transduced MudF inserted in an E. coli F' plasmid carried by a S. fragment.This is demonstrated by the absence of typhimurium strain; the recipientis a Salmonella strain Lac+ transductants in rec+ hoststrains lacking the lacking the F' plasmid. The Escherichia coli sequences MuA plasmid. flanking the donorMud F insertion siteare sufficiently Transpositional inheritance of the mini-Mu, MudF different from S;~lmonella chromosomal sequencesto is greatly reduced in recA, recR and recA recR recipient 20 R. V. Sonti, D. H. Keating and J. R. Koth TABLE 2 transduction cross selecting for inheritance of KmR, Effects of rec mutations on recovered lysogens of mini-Mu we can follow the integration of the MudJ element; phages injected by P22 virions ApR transductantshave acquired the MupAp1 phage. In Table 4, it can be seen that the number of KmR Transpositiotl No. of MudF Relative transductants is reduced 20-fold in the recA mutant Recipient Genotype functions in lysogens (lac+ frequency strain no. of recipient recipienta tramductants) of lysogens recipient. None of the KmR transductants occurred L’l-2 rec+ None 0 0 by homologous recombination, since no KmR trans- 1’1’10377 ret+ M uA 31 32 1 ductants were found in ret+ recipients that lacked the

1’T 1 52 1 6 recA M uA 482 0.15 Mu A producing plasmids. Furthermore none of the TT15220 recB M uA 73 1 0.23 KmRtransductants show the purine requirement char- TT1522 1 recA M uA 0 0 acteristic of the donor MudJ insertion, supporting the recB conclusion that all inheritance occurs by transposition. Recipient strainswere grown overnight in complexmedium Apparentlythe homologous sequences flanking the containing ampicillin (50 pg/ml). A single P22 transducing lysate grown on strain TT12116 (carrying a MudF lysogen in an E. coli F insertion are insufficient to support homologous re- plasmid) was used to infect recipient cells at a multiplicity of 2. Cells combination at a frequency detectable in this experi- were plated to select Lac+ transductants at 37”. The number of ment. At the multiplicity of infection used in Table 4, transductants was corrected fora recipientviability factor calculated as described in MATERIALS AND METHODS. one might have expectedthe coinfecting MupApl a MuA plasmid was obtainedfrom P. VAN DE PUTTE and is phages to provide transposase and allow some trans- described in VANLEERDAM, KARREMAN and VANDE PUTTE(1 982). position without the Mu A plasmid. Since no KmR transductants were seen in the recipient lacking the strains (see Table 2). Either a recA or a recB mutation Mu A plasmid, it appearsthat the preferential cis reduce the frequency of recovered lysogens by about action of Mu transposase prevents trans activation of 5-fold (after adjusting for the reduced frequency of MudJ underthese conditions. At ahigher helper viable cells in rec mutant strains). A recA recB double multiplicity, MupAp1 does provide transposase for mutant recipient strain shows a greater reduction in MudJ (see below). MudF inheritance. Results similar to these have also The Rec effects described above are not due to been obtained with the mini-Mu elements, MudJ and effects of rec mutationson the expression or copy Mud11 168 1, diagrammed in Figure 1. number of the recipient Mu A plasmid providing Transposition-proficient mini-Mu genomes show transposase function. The same cross was performed rec effects on lysogeny: The Mud I1 1681 mini-Mu with no Mu A plasmid in the recipient; transposition element (12 kb) encodes both Mu A and B functions functions were provided by addition of extra trans- (CASTILHO,OLFSON and CASADABAN 1984). Trans- position-proficient MupAp1 helper phage. Therefore position of this element does not require aplasmid to both Mu A and Bfunctions were supplied only by Mu- supply transposition functions. Inheritance of this derived genomes able to provide normal regulation. mini-Mu is reduced in Rec- strains (see Table 3), Results in Table 5 demonstrate that KmR transduc- indicating that Rec- effects on mini-Mu transposition tants are seen and their frequencyis reduced in a recA from P22-transduced fragments are seen even when recipient strain. Thusthe presence of a host recA the Mu derivative produces its own regulated trans- mutationreduces the frequency of MudJ lysogens posase; therefore the effect does not depend on pro- regardless of whether transposition functions are sup- vision of a constitutive, plasmid-borne transposase. plied by a plasmid or by ahelper Mu phage. As Mini-Mu phages show a Rec- effect even when expected from the previous results-of HUGHES,OLIV- injected by Mu virions: To test the behavior of mini- ERA and ROTH (1987), inheritance of the MupAp1 Mu phages packaged in Mu virions, a double lysogen helper phage itself is not subject to the effect of recA was constructed (TT14547) that carries the mini-Mu (see Table 6). Thus thelarge Mu genomes are them- element MudJ(Km, Lac) inserted in the purF gene of selves protected from rec effects when injected by a S. typhimurium and a plaque-forming Mu derivative at Mu virion, but they do not provide this protection in a11 unidentified site in the chromosome. This mini- trans tothe coinjected MudJ genomes, even when MudJ element is the Mud I1 1734 element constructed they provide the transposition function. by CASTILHO,OLFSON and CASADABAN (1984).The Increasing transpositjoy function reduces the in- plaque-forming Mu derivative used here is phage heritance of transduced MudF element:The behav- MupAp1, constructedby LEACH and SYMONDS (1 979).ior of injected MudF elementsin recipients expressing When a double lysogen of these two strains is induced both the Mu A andB genes suggested that lethal by shifting to high temperature (see MATERIALS AND transposition might reduce the yield of lysogens in METHODS), a mixed lysate is produced that includes Rec- strains. The Mu A function alone is sufficient Mu virions that have packaged either the MupAp1 or for transposition, but the frequency of Mu transposi- MudJ genomes. By using this lysateas donor in a tion is enhanced50-100 fold if the accessory MuB Lethal transposition of Mud phages 21

TABLE 3

Effect of rec mutations on transposition of a transposition-proficient mini-Mu prophage injectedby €22 virions

~~ ~ ~~ No. of Corrected no. Relative Recipient Total lid transpositions of frequency of strain no. genotypeRecipient transductants KmR)(Ara+, transpositionsa transposition LT2 reef 11172 10278 10278 1 TT 14555 recA 535 535 I009 0.098 TT 14556 recB 1 1 11 0.001 TT14558 recA, recB 0 0 0 0 Recipient strains were grown overnight in complex medium at 30". A single transducing lysate (P22 int, HT) was grown on donor strain TT15884 (ara-663::Mudlll681) and used to infect all recipients strains at the samemultiplicity (<2) based on culture turbidity. Transductants were selected on nutrient broth with kanamycin at 30" and transductants were replica-plated to MacConkey-arabinose-kanamycin medium to identify lysogens that had inherited KrnR by transposition (Ara+) or by homologous recombination (Ara+). a The number of transposition transductants was corrected for recipient cell viability as described in MATERIALS AND METHODS (viability of recA cells was 0.53, of red cells was 0.087 and of recA, recB cells was 0.033).

TABLE 4 recipient rec mutations, noted above, could be due to Effect of rec mutations on transposition of mini-Mu prophages a variety of causes. (1) The rec mutations might in- injected by Mu virions crease copy number or expression of the plasmid-born Mu A gene, leading to increased transposition and Transposition excessive killing. Alternatively alleles might reduce Recipient functions No. of MudJ Relative rec strain Genotypeprovided by frequencylysogens transposase expression to a level that limits the likeli- no. of recipientrecipient (Km' transductants) of lysogens hood of the initial transposition. We think both these TT10502 reef None 0 0 possibilities are rendered unlikely, since the effect of TT10503 recA None 0 0 rec mutations is also seen whentransposase is provided rec+ Mu A 197 1 1 TT 14549 by Mu derivatives that regulate transposase genes in TT14550 recA Mu A 103 0.05 anormal way. (2) The rec alleles might alter host Multiplicity of infection was 0.6-0.75. The donor lysate in all crosses was made by inducing strain TT14547 [purF::MudJ(Km), sensitivity to killing by the PZZ transducing phage or Mup(Ap)]. Lysates were prepared, titred and transduction crosses the several sorts of Mu phages provided. We think were performed as described in BERMAN,ENQUIST and SILHAVY this is unlikely because the same rec alleles do not (1 981). All recipients carry a mus mutation to permit infection by Mu particles. Transductions were done at 30". The numbers of impair inheritance of plasmids by P22 transduction or recombinants were corrected for the reduced viability of the recA inheritance of TnlU, TnlOdTet or Tn5by transpo- strain as described in MATERIALS AND METHODS. sition from transduced fragments (R. V. SONTI and D. H. KEATING, unpublished). Similarly, these rec alleles do not affect lysogen formation by full sized function is also provided (FAELEN,HUISMAN and Mu derivatives with normally regulated transposition TOUSSAINT 1978; O'DAY,SHULTZ and HOWE 1978; functions when theirgenome is injected by a Mu Chaconas et al. 1984). The effect of this accessory f-unction on transduced mini-Mud (MudF) genomes capsid. Thus the observation of a rec effect seems to was seen in a PZZ-mediated transduction cross. In this depend on the nature of the injecting virion and the case, the Mud F prophage in strain TT12 1 16 was size of genome injected, but not on the source of the transduced into two isogenic recipients, one express- transposase function. ing only the Mu A gene, the other expressing both These various possibilities can also be controlled by Mu A and Mu B genes constitutively from a plasmid; producing both a full-sized Mudderivative and a mini- selection was for inheritance of the KmR determinant Mud in a single donor strainand using the mixed associated with MudF. In these crosses, provision of lysate to infect a single host culture, observing the the accessory Mu B function caused a strong reduction effects of rec alleles in the recipient on inheritance of in recovery of MudF lysogens (Table 7). This is ex- the two genomes (by lysogen formation). These results pected if, under these conditions, the higher level of are presented in Table 8. A lysate was prepared on a transposition functions permit multiple roundsof rep- strain (TT17337) that carries two transposition-pro- licative transposition, which is frequently lethal to the ficient Mud derivatives, the mini-Mu Mud11168 1(Km) host cell. This result, while not surprising, suggested and the full-sized, plaque-forming phage MupApl. the possibility that thereduced frequency of KmR This lysate contains both types of genomes in Mu Mud lysogens in rec strains (described above) might capsids. As seen in Table 8, a cell culture infected by also have been due to the lethal effects of replicative this lysate yields both KmRlysogens (ofthe mini-Mud) transposition rather than reduced ability to transpose. and ApR lysogens (of the full-sized) Mu at a ratio of Explanations of the rec effects: The effects of about 1:4.6. When the same lysate infects a recA host 22 R. V. Sonti, D. H. Keatingand J. R. Roth

TABLE 5 Effect of a rscA mutation on the transpositionof mini-Mud phages in Mu virions during mixed infectionwith helper Mud phages

~ ~~~~~~ Extra Multiplicity of Recipient helper infection No. of MudJ Relative strain Genotype Mud (including lysogens frequency no. of recipient phage helper phage) (Km' transductants) of lysogens TTI 0502 rec+ Absent 0.6 0 0 TTI 0503 recA Absent 0.6 0 0 TT10502 rec+ Present 2.1 1498 1 TT10503 recA Present 2.1 191 0.13 A plaque-forming Mud phage called MupApl was used as the helper phage. MupApI was obtained from M. M. HOWE.The source of MudJ sequences was a lysate on TT14547, which carries both a MudJ(Km) and a MupAp1 lysogen. Additional helper phage functions in the transduction cross were provided by Mu particles that were obtained by inducing the MupApl lysogen (TT14548). Mu lysates were grown, titred and transductions were done as described in BERMAN,ENQUIST and SILHAVY(1981). All recipients carry a mus mutation to permit infection by Mu particles. For transduction crosses in which extra helper phage were provided, roughly two thirds of the helper Mu particles (1.5/ceII) were from the exogenous lysate and one third (0.6/cell) from the mixed lysate. The transductions were done at 30". The reduced viability of the recA strain was taken into account as described in MATERIALS AND METHODS.

TABLE 6 tively. We suggest that the default replicative trans- Effect of a recA mutation on transposition of MupApl phages position breaks the target chromosome, leaving one infected by a Mu virions copy of the Mu element at each end of the broken chromosome. The resulting breaks can be repaired Recipient No. of MupApl Relative by rec-mediated repair events. In rec mutant hosts this strain Genotype Multiplicity lysogens frequency no. of recipient of infection (Ap' transductants) of lysogens route of rejoining is impaired and many potential TT10502 ret+ 10-5 4273 1 lysogens are lost. TT 10503 recA 10-5 3244 0.76 A rec-independent route of chromosome rejoining TT 10502 ret+ 10" 82 1 isby a secondary replicative transposition of either TT 10503 recA 10-7 39 0.48 copyof the Mu elementinto sequences near the The donor strain was TT14547. MupApl lysates were prepared, opposite end of the broken chromosome; this would titredand transduction crosses were performed as described in rejoin the broken ends and would cause a deletion BERMAN,ENQUIST and SILHAVY(1981). The transductions were done at 30'. The reduced viability of the recA strain was taken into adjacentto the site of the single remaining Mud account as described in MATERIALS AND METHODS. element. We have looked for evidence of the predicted deletions. TABLE 7 Deletions are more frequently associated with insertions in Rec- recipients: It is well known that Lysogen formation by mini-Mu phages injected byP22 virions insertion of phage Mu is occasionally associated with formation of a deletion of target material adjacent to Transposition No. of MudF Relative Recipient Genotype functions in lysogens frequency the insertion site (DANIELL,ROBERTS and ABELSON, strain no. of recipient recipientQ (Kn' transductants) of lysogens 1972; HOWEand ZIPSER, 1974; CABEZONet aE. 1978; TT10377 rec+ MuA 2400 1 HOWEand SCHUMM,1981). These deletions are pre- TT8353 rec+ MuA andB 30 0.0 1 sumed to be due tosecondary replicative transposition Recipient strains were grown overnight in complex medium events occurringbefore the prophage is fully re- containing ampicillin (50 gg/ml). A P22 transducing lysate grown pressed. We have observed an increase in thefre- on strain TTl2116 was used to infect recipient cells at a multiplicity of 2. The experiment was performed at 37". quency of adjacent deletions in the lysogens formed a Plasmids expressing either MuA or MuA and B functions were by Mud elements in situations that are subject to the obtained from P. VANDE PUTTE. rec-effect described above. That is, the frequency of deletionsadjacent tothe integration site increases there is an almost 20-fold reduction in the recovery amongthe reduced number oflysogens recovered of mini-Mu lysogensand only about a3-fold reduction using rec recipients. in recovery of ApR lysogens. Although both genomes Increased deletion formation is demonstrated in an are capable of killing and both possess normally reg- experiment in which phage P22 transducesboth ulated transposition function, only the mini-Mud is MudI(Ap) and MudJ(Krn) from a single donor strain strongly affected by the recipient rec mutation. intoa recipient which cannot supply transposition We will suggest later that the rec effects described functions. The donor strain carries Mud1 and MudJ above are due to killing of the host by chromosome insertions close together within the histidine operon; breaksinduced by replicative transposition events the transposition-proficient Mud I element is oriented which occur when Mu fails totranspose conserva- with its Mu A and Mu B genes closest to the nearby Lethal transposition of Mud phages 23

TABLE 8

Effect of a recA mutation on the relative frequency of lysogens of two transposition proficient Mu derivatives, mini-Mud (MudII1681) and MupAPl

No. No. of Relative Relative Recipient of MupAp 1 Recipient Mud111681 frequency frequency relevant of MupApl strain no. (Km') lysogens of mini-Mud11 lysogens(AP') genotype (adjusted)' lysogens (adjusted)' lysogens

TT 10502 rec+ 185g6 1' 84976 1 TT 10503 recA 2942105 0.06 0.35 Recipient strains were grown overnight in complex medium at 30". The dilysogenic strain (TT17337) was induced and transductions were performed as described by BERMAN,ENQUIST and SILHAVY(1981). The multiplicity of infection was IO-'. The single suspension of infected cells was plated onto nutrient broth medium and incubated undernonselective conditions for 20 hr. Lysogens of Mud111681 and MupApf were detected by replica-printing the lawn of plated, infected cells sequentially onto medium containing either ampicillin or kanamycin. All of the KmR Mud111681 lysogens formed by transposition since none of the KmR transductants inherited the donorAra- phenotype; transductants arising by homologous recombination would be expected toshow this donor phenotype. Numbers of transductants arisingin the recA strain have been adjusted to account for the reducedviability of these strains. The relative viabilitv of recA strains is 0.59 comnared to that of an isocrenicv recA+ strain; number of recombinants has been increased by dividing the number of counted transductants by 0.59.

MudJ insertion. Transduced fragments that include of Table 9includes results seen when the larger MudI MudJ frequently include the Mu A and B genes of the element is transduced by P22. As for MudJ, rec recip- MudI element and thus are provided with regulated ients show a lower total number of ApR transductants transposition functions located cis to the MudJ ele- (unpublished results; andHUGHES, OLIVERA and ment. This donor strainhas been described previously ROTH 1987) andthe fraction of insertions having (HUGHES andROTH 1988). In this experiment, trans- adjacent deletions is increased. position functions are supplied to the defective MudJ The third section of Table 9 presents the behavior element by normal, regulated Mu A and B genes of of the same MudI element when introduced by a Mu the MudI element, present on the same transduced virion. This was achieved by constructinga donor fragment. To test MudI transposition, P22 transduc- strain (TT8050) which carries both MudI and aMucfS ing lysates were grown on strain TT7216, which has prophage. At high temperature, amixed lysate can be only the his::MudI insertion and doesnot carry a madethat includes both MucfSgenomes and MudI MudJ element. genomes, within Mu capsids. This lysate was used to To detectdeletion mutants among transposition transduce recipient strains that are sensitive to Mu transductants, selection was madefor either KmR infection by virtue of a mus mutation (FAELENet al. (MudJ) or ApR (MudI) transductants (lysogens) on 198 1). Two points should be noted. First, the fre- MacConkey galactose plates. On these plates, lysogens quency of ApR transductantsis affected very little by that have acquired an insert (or a deletion) within the thepresence of a rec mutation in the recipient galactose operon form white colonies while transduc- (HUGHES, OLIVERA ROTH and 1987; data notshown). tants with an intact galactose operon form red colo- Second, the frequency of deletions is low and there is nies. By scoring the nutritional phenotypes ofGal- no evidence for an increase in deletion frequency in transductants, one can identify deletion mutants that the rec recipient strains. are also defective for either or both of the loci (nadA and bio) which flank the galactose operon. This is a DISCUSSION ~ninimalestimate of deletion formation since it only Under several conditions, host rec mutations cause detects deletions long enough to damage at least one a reduction in the ability of derivatives of phage Mu of these nearby loci. to form lysogens. We propose that the reduction in The frequencies of deletions observed in this ex- lysogen recovery is due tophage-induced killing which periment are presented in Table 9. The first section can be avoided in cells having normal recombination presents the results when MudJ is transduced by P22. functions. More specifically we suggest that thephages In rec-deficient recipients, the total number of KmR showing a rec effect all share a defect in performing transductants is reduced (5-fold lower in recA or recB Mu's initial conservative transposition. Because of this strains and 50-fold lower in recA recB strains; data not defect, they transpose replicatively fromthe linear shown) and the frequency of deletions is increased. injected genome; this causes achromosome break, The majority of the deletions (6/7 tested)exhibit which is a potentially lethal but repairable damage. 100% linkage with the MudJ; supporting their being Normally a newly injected Mu genome is thought formed by the integration event. The second section to transpose conservatively into thehost chromosome. 24 R. V. Sonti, D. H. Keating and J. R. Roth

TABLE 9 Frequency of extended deletions among Gal- insertion mutants

MudJ introduced by P22 particlesaMudl introduced by P22 particles"Mudl introduced by Mu particlesasb

Genotype of FrequencyFrequency Frequency Frequency Frequency FrequencyOf recipientDonorand of Gal- Of deletionsDonorand of Gal- of deletionsDonorand of Gal- deletions recipientC coloniesd recipientfamongGal- coloniesd recipientfamongGal- coloniesd amongGal- colonies' coloniese colonies' coloniese colonies'

ret+ TT102880.1% TT72 16 0.037% 0.12%TT8050 and LT2 (35/35000) and LT2 (29/78378) and TT10502 (40/33330) recA TT102880.35% TT72 16 0.26% 0.18%TT8050 and TT14555 (29/8260) and TT14555 (28/10769) and TT10503 (37/20558) recE TT102880.37% TT72 16 0.5% 0.28%TT8050 and TT14556 (20/5333) and TT14556 (1 8/3600) and TT10504 (40/14284) recA TT10288 0.33% TT72 16 0.098% TT8050 0.1% rccB and TT14558(15/4540) and TT14558 (16/16326) and TT10505 (40/39997) " The multiplicity of infection was between 1 and 4. To permit infection by phage Mu, both donor and recipient strains carrymus a mutation. The experimentwas performed at 37". The numbers in parentheses indicate the number of Gal- colonies among the total number of Mud lysogens scored. The total scored does not reflect the frequency of KnR transductants. Since survivalof transductants is extremely low in recB and recA recE strains many more infected cells were screened to accumulate sufficient transductants [see frequenciesin Table 2 and HUGHES,OLIVERA and ROTH (1987)l. ' The numbersin parentheses indicate the number of extended gal deletions among the total numberGal- of colonies scored. fThe experiment was performed at 30" to preventlytic growth of Mudl.

This leaves a cell with an intact bacterial chromosome the site of Mu insertion (Figure 2). We propose that which can survive as a lysogen (if no subsequent trans- in a rec mutant host a larger fraction of recovered position occurs) or can lyse following a series of sec- lysogens would be formed by this secondary transpo- ondary rounds of replicative Mu transposition. If the sition event. The predicted increase in the frequency initial conservative transposition event were pre- of deletion mutants was found among the lysogens vented, and a replicative transposition occurred from recovered in rec-deficient hosts. the injected linear molecule, a break would be caused Reduced lysogen recovery in hosts with rec muta- in the host chromosome, leaving one copy of the Mu tions (due, we argue, to the failure of conservative prophage at each broken end (see Figure 2). Unless transposition) was shown by Mu derivatives under repaired, such breaks would be lethal. Repair could several circumstances (Figure 3). Sensitivity to a host occur by homologous recombination between the two rec defect was seen for all tested Mu derivatives when Mu genomes to regenerate a complete circular host their genome was injected by a virion of phage P22; chromosome containing asingle added Mu prophage. this included both transposition-proficient Mu deriv- Such repair would be stimulated by the host recA, B atives and defective elements whose transposition and C functions. We suggest that under some of the function was provided by a plasmid carrying the Mu conditions of Mu infection described here, the infect- A gene. Mini-Mu phages showed a rec effect following ing Mu genome is unable to transpose conservatively. injection by either Mu or P22 virions. Lysogeny by Replicative transposition, we suggest, thenoccurs, Mud1 elements as well as theplaque-forming Mu inducinga break in the host chromosome. Under derivative, MupApI (and presumably wild-type phage these conditions, arec-deficient host shows a decrease Mu), is unaffected by host rec mutations when these in the frequency of surviving lysogens. elements are injected by a Mu virion. In the absence of rec functions,the Mu-induced The fact that genomes delivered by a P22 virion chromosome breaks described above can be rejoined are subject to rec effects suggests thata function if one of the Mu prophages at the chromosome break (presumably a protein), normally injected by the Mu points performs a second replicative transposition to virion, cannot be providedby the P22 virion and thus a target site near the opposite chromosome end. If cannotprevent the rec sensitivity. Since mini-Mud strand exchange during transposition occurs in one of phages show rec sensitivity regardless of the injecting the twopossible orientations andthe target site is virion, we suggest that they may lack or improperly sufficiently close to the broken end,this transposition position some part of the Mu genome required for event would reformthe circular host chromosome injection or action of this protein. It is interesting to and leave a viable strain with a deletion of chromo- notethat in experiments with mixed infection of somal material adjacent to the addedMu prophage at MupApI and Mini-Mud (Tables 5 and 8), these two Lethal transposition of Mud phages 25

Replicative transposition from a linear fragment

4 Mudl 4 Do nor Mudl -(infecting DNA) f-7 replicative transposition-

FIGURE2.-Consequences of Recipient Bacterial chromosome replicative transposition of a P22 (Bacterial is linearized packaged Mud1 element onto the chromosome) bacterial chromosome. (1) The bacterial chromosome is broken In r#+ cells the linearized chromosome can be re-circularized by homologous recombination during replicative transposition of betveen the tvo copies of Mudl sequences Mudl MudI from P22a transduced fragment. (2) In rec+ cells the broken chromosome can be re-circularized by homologous recombination between the MudI elements. (3) In rec cells the broken Simple chromosome can be re-circular- insertion ized by a secondary transposition of Mudl event involving one of the Mud elements. The genetic material between the Mud insertion and the target site is deleted. The Bacterial small arrows represent sites at chromosome which Mu transposase introduces In r# cells the linearized chromosome can be re-circularized only by a second replicative either single strand nicks or dou- transposition0- event 0 ble strand breaks in DNA.

Deletion of bacterial sequences adjacent to Mudl insertion

Mudl Sequence cannot replicate and is lost after cell division genomes are replicated and packaged into Mu virions The function hypothesized above shares many fea- by the same donor cells. The progeny phages inject tures with a known Mu protein that has been previ- into thesame host cell and (at high helper multiplicity) ously studied in considerable detail. The Mu N gene transposition functions are provided in trans to the encodes a Mu virion protein that is injected into the defective Mu derivative (Table 5). Nevertheless, the recipient cell along with the Mu DNA (GLOORand frequency of mini-Mud lysogens is reduced in rec- CHACONAS1986). The Mu N gene is one of the late deficient hosts. This suggests that a plaque-forming genes of Mu and was originally identified as being MupApl genome cannot provide, in either the donor essential for the synthesis of Mu tails (GRUNDY and or recipient cell, the function needed to protect mini- HOWE 1985). Besides its role intail formation, the Mud phages from Rec effects. This leads us to suggest that mini-Mud phages lack genomic features required Mu N gene product can protect transfecting Mu DNA either for injection or action of the protein that pro- from degradation by exonuclease V (CHASEand BEN- motes conservative transposition. We predict that nor- ZINGER 1982; and HARSHEY and BUKHARICon- 1983). mally the protein is tightly associated with the injected sistent with the ability to recognize a specific site in genome and cannot actin trans to aid other genomes DNA is the fact that the amino acid sequence of the in the new host. N protein includes a helix turn helix motif common 26 R. V. Sonti, D. H. Keating and J. R. Roth

Mudl mini-Mu FIGURE3.-Model to explain the Mu CAPSIDMu CAPSID Mu effect of the packaging virion on the Mudl mini-Mu recovery of Mud phage lysogens. In P22 CAPSID P22 CAPSID thefigure solid black genome seg- mentsindicate Mu sequences;adjacent while sequences arechromo- somal sequences. The small shaded Sequence Sequence sequence is ahypothesized binding No Protein No Protein site for the injected protein. It is proposed that a Mu-encoded tail protein normally associates with a specific sequence at the right end of the Mu genome during phage assembly and fu leftend MU left end MU left end is injected in association with that 1 I 1 sequence. Once injected, the protein Mu left end Mu right end contributes to conservative transposition of the Mu or Mud genome. Mu right end This protein can be injected by Mu Mu right end Mu right end virions which carry a Mu or a full sized Mud genome; in mini-Mu PROTEIN+DNA INJECTED DNA LACKS phages (dueto headfulpackaging) COMPLEX IS COHPLEXED PROTEIN the binding site is located within the INJECTED capsid and cannot associate with the I I v 4 tail protein. An alternative model (not depicted) is that the critical se- Conservative Replicative transposition causes quence is missing from the mini-Mu transposition chromosome breaks phages tested. In either model, P22 1 1 virions do not inject the needed pro- Rec- defect does not Rec- defect reduces tein.Without thisinjected protein, affect lysogeny recovery lysogenrecovery the initial transposition event is replicative and causes abreak in the recipient chromosome.

IO many regulatory proteins (GLOORand CHACONAS, SCHNOSand HOWE, 1976; BREEPOELet al.1976). 1988).Once bound to DNA, N proteincannot be Because of Mu’s headful packaging mechanism which transferredto another DNA molecule in vitro (G. starts at the left end of the genome, amini-Mu with a GLOORand G. CHACONAS,personal communication). small genome would have its right end located within In recipient cells, the Mu N gene product circularizes the capsid unassociated withtail proteins. If the N the infecting DNA by binding at or close to the ends protein must associate with the right endof Mu in the of the DNA in a non-covalent fashion (HARSHEYand course of phage assembly, the location of the right UUKHARI1983; PUSPURS,TRUN and REEVE1983; and end within the capsid of mini-Mud phages would GLOORand CHACONAS1986). Most importantly, it has prevent N protein association and result in a failure been suggested that this protein plays a role in con- of N protein to be injected into the new host. servative transposition (GLOORand CHACONAS1986). The observations reported here point out a poten- These properties of the Mu N gene product suggest tial danger and a useful property of mini-Mud phages it is the function that facilitates recovery of Mud when used in genetic analysis. These phages have an lysogens ih Rec- strains. increased probability of generating deletions at the If the Mu N protein explains the results presented site of insertion, particularly when insertions are iso- here, ourresults, although indirect,may shed light on lated inrec-deficient hosts (see Table 9). This can several aspects of the function and mode of injection cause a problem when fusion insertions are formed to of the Mu N protein. If the defect in conservative study gene regulation. The phenotype by which an transposition of mini-Mu genomes (packaged in a nor- insertion mutant is isolated could reflect material re- mal Mu virion) is due to a lack of N protein, these moved by an adjacent deletionand the geneor operon phages must be defective in either injection or re- fusion providing lac expression may involve an unre- sponse to theMu N function. This defect couldreflect lated gene at the end of the deletion. The propensity sequences missing fromthe mini-Mud genome or, of mini-Mu phages to make deletions in rec-deficient more likely, sequences that are improperly positioned hosts may be avaluable means of generating deletions in the mini-Mu virion. (Figure 3 describes the latter since the frequency of deletions among surviving ly- possibility.) The right end of a normal Mu genome is sogens approaches 100%; these deletions are particu- known to be associated with the Mu tail (INMAN, larly valuable since they are associated with the select- Lethal transposition of Mud phages 27 able drug resistance determinant of the mini-Mud DANIELL,E., R. ROBERTSand J. ABELSON,1972 Mutations in the lactose operon caused by the insertion of bacteriophage Mu. J. phage and thuscan be introduced selectively into new Mol. Biol. 69 1-8. genetic backgrounds. DAVIS,R. W., D. BOTSTEINand J. R. ROTH, 1980 Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring This work was supported by U.S. Public Health Service grant Harbor, N.Y. GM 27068 to J.R.R. from the National Institutes of Health. The FAELEN,M., 1987 Useful Mu and mini-Mu derivatives, pp 309- idea that host recombination functions might repair chromosome 316 in Phage Mu, edited by N. SYMONDS,A. TOUSSAINT,P. breaks caused by replicative Mud transposition was suggested by VAN DE PUTTEand M. M. HOWE.Cold Spring Harbor Labo- JAMES SHAPIRO.GEORGE CHACONAS suggested that mini-Mud ratory, Cold Spring Harbor, N.Y. phages might improperly localize a sequence needed for Mu N FAELEN,M., 0. HUISMANAND A. TOUSSAINT,1978 Involvement protein injection. of phage Mu-1 early functions in Mu mediated chromosomal rearrangement. Nature 271: 580-581. LITERATURECITED FAELEN,M., M. MERGEAY,J. GERITS,A. TOUSSAINTand N. LE- ALPER,M.D., and B. N. AMES, 1975 Positive selection of mutants FEBVRE, 1981 Genetic mapping of a mutation conferring with deletions of the gal-chl region of the Salmonella chromo- sensitivity to bacteriophage Mu in Salmonella typhimurium LT2. some as a screening procedure for mutagens that cause dele- J. Bacteriol. 146: 914-919. tions. J. Bacteriol. 121: 259-266. GLOOR,G., and G. CHACONAS,1986 The bacteriophage Mu N BERKOWITZ,D., J. HUSHON, H. WHITFIELD,J. R. ROTH and B. N. gene encodes the 64-kDa virion protein which is injected with AMES,1968 Procedure for identifying nonsence mutations. and circularizes infecting Mu DNA. J. Biol. Chem. 261: 16682- J. Bacteriol. 96: 2 15-220. 16688. BERMAN,M. L.L., W. ENQUIST and T. J. SILHAVY, GLOOR,G., and G. CHACONAS,1988 Sequence of the bacterio- 198 1 Experiments with Gene Fusions. Cold Spring Harbor Lab- phage Mu N and P genes. Nucleic Acids Res 16: 521 1-5212. oratory, Cold Spring Harbor, N.Y. GRUNDY, H.W., and M.M. HOWE,1984 Morphogenetic struc- BREEPOEL,H., J. HOOGENDORP,J. E. MELLEMAand C. WIFFLEMAN, tures present in lysates of amber mutants of bacteriophage Mu. 1976 Linkage of the variable end of bacteriophage Mu DNA Virology 134: 296-317. to the tail. Virology 74: 279-286. HARSHEY,R.M., 1987Integration of infecting Mu DNA, pp. BURKHARI,A. I., 1976 Bacteriophage Mu as a transposition ele- 11 1-135 in Phage Mu, edited by N. SYMONDS,A. TOUSSAINT, ment. Annu. Rev. Genet. 10 389-412. P. VAN DE PUTTEand M.M. HOWE.Cold Spring Harbor CABEZON,T., F. VAN GIJSEGEM,A. TOUSSAINT,M. FAELEN and A. Laboratory, Cold Spring Harbor, N.Y. BOLLEN, 1978 Phage Mu-1 mediated transposition: a tool to HARSHEY,R.M., and A. I. BUKHARI,1983 Infecting bacterio- study the organization of ribosomal protein genes in Escherichia phage Mu DNA forms a circular DNA-protein complex. J. Mol. coli. Mol. Gen. Genet. 161: 291-296. Biol. 167: 427-441. CASADABAN,M. J., and S. N. COHEN,1979 Lactose genes fused Howe, M. M., 1987 Phage Mu: An overview, pp. 25-39 in Phage to exogenous promoters in one step using a Mu-lac bacterio- Mu, edited by N. SYMONDS,A. TOUSSAINT,P. VAN DE PUTTE phage: In vivo probefor transcriptional control sequences. and M.M. HOWE. Cold Spring HarborLaboratory, Cold Proc. Natl. Acad. Sci. USA 76: 4530-4533. Spring Harbor, N.Y. CASTILHO,B. A,, P. OLFSONand M. J. CASADABAN, 1984Plasmid HOWE,M. M., and J. W. SCHUMM,1981 Transposition of bacte- insertion mutagenesis and lac gene fusion with mini-Mu bac- riophage Mu-properties of lambda phages containing both ends teriophage transposons. J. Bacteriol. 158: 488-495. of Mu. Cold Spring Harbor Symp. Quant. Biol. 45: 337-346. CHACONAS,G., 1987 Transposition of Mu DNA in vivo, pp. 137- HOWE,M. M., and D. ZIPSER,1974 Host deletions caused by the 157 in Phage Mu, edited byN. SYMONDS,A. TOUSSAINT,P. integration of bacteriophage Mu-1. Abstr. Annu. Meet. Am. VAN DE PUTTE,and M. M. HOWE.Cold Spring Harbor Labo- Soc. Microbiol. 208: 235. ratory, Cold Spring Harbor, N.Y. HUGHES,K. T., B.M. OLIVERA and J.R. ROTH, 1987 Rec de- CHACONAS,G., F. J. DEBRUIJN,M. J. CASADABAN,J. R. LUPSKI,T. pendence of Mu transposition from P22-transducing frag- J. KWOH,R. M. HARSHEY,M. S. DUBOWand A. I. BUKHARI, ments. J. Bacteriol. 169: 403-409. 198 1 In vitro and in vivo manipulations of bacteriophage Mu HUGHES,K. T., and J.R. ROTH, 1988 Transitory cis complemen- DNA: cloning of Mu ends and construction of mini-Mu’s car- tation: a method for providing transposition functions to de- rying selectable markers. Gene 13: 37-46. fective transposons. Genetics 119 9-12. CHACONAS,G., G. GLOOR,J. L. MILLER,D. L. KENNEDY, E. B. INMAN,R. B., M. SCHNOSand M. HOWE,1976 Location of the GIDDENSand C. R. NAGAINIS,1984 Transposition of bacte- “variable end” of Mu DNA within the bacteriophage particle. riophage Mu DNA: expression ofA and B proteins from lambda Virology 72: 393-40 1. PL and analysisof infecting Mu DNA. Cold Spring Harbor LEACH,D., and N. SYMONDS, 1979The isolation and character- Symp. Quant. Biol. 49: 279-284. ization of a plaque-forming derivative of bacteriophage Mu CHACONAS,G., E. B. GIDDENS,J. L. MILLER and G. GLOOR, carrying a fragment of Tn3 conferring ampicillin resistance. 1985 A truncated form of the bacteriophage Mu B protein Mol. Gen. Genet. 172: 179-184. promotes conservative integration but not replicative transpo- MIZUUCHI,K., and R. CRAIGIE, 1986Mechanism of bacteriophage sition of Mu DNA. Cell 41: 857-865. Mu transposition. Annu. Rev. Genet. 20 385-429. CHAN,R. K., D. BOTSTEIN,T. WATANABEand Y. OGATA, MIZUUCHI,K., and N. P. HIGGINS, 1987 Transposition-replication 1972 Specialized transduction of tetracycline resistance by of Mu in vitro: Use of cell free reaction systems for the study phage P22 in Salmonella typhimurium. 11. Properties of a high of the reaction mechanism, pp. 159-1 75 in Phage Mu, edited frequency transducing lysate. Virology 50: 883-898. by N. SYMONDS,A. TOUSSAINT,P. VAN DE PUTTEand M. M. CHASE,C. D., and R. H. BENZINGER,1982 Transfection of Esch- HOWE.Cold Spring Harbor Laboratory, Cold Spring Harbor, erichia coli spheroplasts with a bacteriophage Mu DNA-protein N.Y. complex. J. Virol. 42: 176-185. DAY, K. J., D. SHULTZand M. M. HOWE,1978 Search for CRAIGIE,R. M., and K. MIZUUCHI,1985 Mechanism of transpo- integration-deficient mutants of bacteriophage Mu, pp. 48-59 sition of bacteriophage Mu: structure of a transposition inter- in Microbiology-1978, edited byD. SCHLESSINGER.American mediate. Cell 41: 867-876. Society for Microbiology, Washington, D.C. 28 R. V. Sonti, D. H. Keating and J. R. Roth

PATO,M. L., and B. T. WAGGONER,1987 Replication of Mu DNA ments. Proc. Natl. Acad. Sci. USA 76 1933-1937. in vivo, pp. 177-189 in Phage Mu, edited by N. SYMONDS,A. TAYLOR,A. L., 1963 Bacteriophage-induced mutation in E. coli. TOUSSAINT,P. VAN DE PUTTEand M. M. HOWE.Cold Spring Proc. Natl. Acad. Sci. USA 50 1043-1051. Harbor Laboratory, Cold Spring Harbor, N.Y. TOUSSAINT,A., and A. RESIBOIS,1983 Phage Mu: transposition I’USPURS,A. H., N. J. TRUNand J. N. REEVE,1983 Bacteriophage as a lifestyle, pp. 105- 158 in Mobile Genetic Elements, edited by Mu DNA circularizes following infection of Escherichia coli. J. A. SHAPIRO.Academic Press, New York. EMBO J. 2: 345-352. VAN LEERDAM,E., c. KARREMANand P. VAN DE PUTTE, SANDERSON,K. E., and J. R. ROTH, 1988 Linkage map of Salmo- 1982 Ner, a Cro like function of bacteriophage Mu. Virology nella typhimurium, Edition VII. Microbiol. Rev. 52: 485-532. 123: 19-28. SCHMIEGER,H., 1972 Phage P22- mutants with increased or VOGEL,H. J., and D. M. BONNER,1956 Acetyl ornithase of decreased transduction abilities. Mol. Gen. Genet. 119 75-88. Escherichia coli: partial purification and some properties. J. Biol. SHAPIRO,J. A., 1979 Molecular model for the transposition and Chem. 218: 97-106. replication of bacteriophage Mu and other transposable ele- Communicating editor: N. KLECKNER