Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions

.J. Mol.

Riol. (1981)

150, 341-359

Predominant End-products of Prophage Mu DNA Transposition During the Lytic Cycle are Replicon Fusions GEORGE

P.0.

CHMWSAS~.

RASIKA M. HARSHEY, SORA XXD AHMAD I. BVKHARI

SARVETXICK

Cold Spring Harbor Laboratory Box 100. Cold Spring Harbor. S.Y. 11724. (Received

19 January

TT.f?.A3.

1981)

We present biochemical and genetic experiments which strongly suggest that the net result of transposition of prophage Mu and internally deleted Mu derivatives (mini-Mu’s) during the lytic cycle is replicon fusion. When a Mu prophage located on pSClO1, a low copy number plasmid, is induced, virtually all of the pSClO1 : : Mu plasmid copies enter the host chromosome within 33 minutes. By cleaving the t,otal host DN.4 with restriction endonucleases and by hybridization with 32P-labeled pSc’lO1 DNA, we have found that the fused structures contain directly repeated copies of Mu at each junction of the plasmid and host DNA. These fused structures, called cointegrates, have been seen with Mu as low level genetic events and with almost all other transposable elements. Genetic analysis of mini-Mu transposition from pSClO1 to an F’pro’lac episome has also shown t,hat transposition invariably is associated with fusion of the whole plasmid with the episome ; no simple linear insertions of a mini-Mu molecule into the F’ episome were recovered. Our results also indicate that jumping of Mu from a chromosomal location onto an F’ episome rarely results in the linear insertion of a single copy of Mu DNB; instead. the episome apparently first undergoes Mu-mediated fusion with the chromosome followed by release of Mu-containing episomes that carry extensive deletions or insertions. Integration of Mu into an F’ episome during lysogenization, however. leads to simple point insertions of the Mu genome. Our data strongly suggest that the end-products of Mu DKA transposition during prophage induction and during lysogenization are not the same. We infer, therefore, that the process of Mu DNA transposition can occur by either of two alternate pathways which differ with respect to the end-products they generate.

1. Introduction The temperate bacteriophage Mu is a giant transposon. Transposition of Mu DIVA is an integral part of the Mu life cycle and occurs at a frequency of about lo2 events per cell during the viral lytic cycle (see Bukhari. 1976: Chaconas et al., 1981b for discussion). Mu can catalyze a variety of DNA rearrangements including replicon fusions. inversions, deletions and heterogeneous circle formation (Toussaint et al.. t Present address: Cancer Research Unit and Department of Biochemistry. Ontario, London. Ontario. N6A 5B7, Canada.

University

of Western

341 0022-2836/81/230341-19

$02.00/O

0 1981 Academic

Press Inc. (London)

Ltd.

312

(:.

(‘HB(‘ON,zS

87‘

.-ll.

1977). Mu has also bee11 shown to itlduce the duplication of five basr-pairs of host DSA4 at’ the site of insertion like other transposable elements (see C’alos & Milirr. 1980: Starlit1ger. 1980 for reviews). The Mu pwphage US-A ca11 also be exvisrd precisely and imprecisely at a low fwquency (Bukhari. 107.5: Khat,uon r,t r~l.. 1979). It is thus clear that Mu is endowed with all the versatile genetic properties exhibit’ed by transposable elements. Because Mu carries out its reactions at ~11~~11a high frequency within a single host generation, and because Mu DNA4 transpositiorl cat1 be triggered at will. Mu provides a. valuable model s+em for studies on thcl mechanisms of transposition and DSA rearrangements. We have previously reported that prophagr Mu DKA is not’ excised at a high frequency upon induction and therefore Mu 1)X-A t,ransposition must iuvotve its 1977). The idea that, transposition rna~ replication (Ljungquist & Bukhari. generally be accompanied by replication of the movable element has been sustained by studies of other transposable elemetlt,s such as Tn3 and by further analysis of t,he Mu lyt,ic cycle. Our recent work (Chaconas ef 01.. 1980.19816) with plasmids containing Mu or mini-Mu (int,ernally deleted Mus) has shown that (1) JIu sequences a,re nob detached from the plasmid during Mu replicat,ion ; (2) inst*ead t.he plasmid associates physically with the host chromosome: and (3) this physical int.ttraction requires the funct.ioning of the _-I and H genes of Mu. (lenrtic and biochemical studies reported in this paper show that association of Mwcontaining plasmids with the host chrotnosome entails fusion of t,he plasnlids with the host DSA. At each junction of the fused plasmid and host DS=\ is a col1~ of Mu DSA. The Mu sequences flanking the plasmid are in t,hc san1e orientatio11. This t,ype of replicon fusion. well known as a cointegrate st~ruct~ure. was originall!. described for Mu by Toussaint & Faelen (1973) and has beet1 studied extensively in t,he ampicillin-resistant’ transposon Tt13 (Gill of 01.. 1978). The experitnents discussed in this paper indicate t,hat under t,he experimental conditions used. replicon fusion is the primary consequence of JIu DNA transposition emanat’inp from a Mu or mini-Mu prophage during the Iytic cycle. The Mu and min-AI11 plasmids used here form cointegrates almost exclusively. suggesting that fpw. if’ any. simple linear insertions of Mu DNA occur during lytic transposition from the prophage state. Moreover. when a Mu prophage located on the b'schwichin coli chromosome is transposed onto an F’pro+lac episome, the episomes transferred to an F- strain invariably contain deletions or insertions. However. when JI 11 insertions into an F’pro+lac are isolated directly by lysogenizing t)he cells with Mu. most) of the prophages are simple linear insertions (t’ha,t is. no deletions or insertions in the episome are found). Because the end-product8s of Mu 1)X=\ transpositiotl emanating from the prophage st.ate are apparently different, from those formed b!lysogenization we infer that) Mu DNA transposition can owur by tnore than o11e pathway.

2. Materials and Methods (a) hctrrial Bacterial construct,ed

strains. using

phage standard

and

plasmids techniques

stmiw,

phagp and plasmids

used here including

are listed in Ta.blr 1. The lysogenization with Mu

strains (Bukhari

were B

ENWPRODI’CTS

OF

Mu

DN.1

TRANSPOSITION

343

Ljungquist, 1977), conjugal transfer of F’ episomes (Miller, 1972) and transformation with rec.4 strains were constructed by plasmids (Cohen it al., 1972). Isogenir WC+ and allele into thy derivatives of the desired strains, by int.roduction of the ret+ or recA56 ctonjugation w&h the Hfr strains KLl6 and JC5088, respectively (Willetts et al., 1969). followed by selection of thy+ recombinants. These recombinants were tested for the presence of the WC+ or rec=l allele by their sensitivity to irradiation with ultraviolet light. Mu resistant strains (MuR) were selected as survivors of high multiplicity Mu infections. Mu resistant lysogens were prepared by high multiplicity infections of Mu lysogens with the heteroimmunr Mu-like phage D108. About 50 91~ of the D108R Mu lysogens were also MuR as det,ermined by their inability to be infected with Mu&. Unless otherwise indicated all media and conditions for cultivation of Mu were as previously described (Bukhari & Ljungquist,. 1977). The mini-Mu plasmids used are stably maintained in the absence of antibiotic selection: however, plasmids containing an intact Mu prophage are readily lost without selective pressure. As a precautionary measure, strains containing any of the plasmids listed in Tahltx I were grown in the presence of 10 pg tetracycline/ml (Sigma).

TABLE:

Bacterial

C’hromosomal HIT”021 KlT”Oil HI’%91

strains.

and episomal

1

phage and plasmids

markers

F- Aprola~ his w/d .slrA MuR F”-Aproku met recil56 stril F’pro+ lac/Aprolac leu : :Mucts62nmp met rcvA5ti slrri D108R MuR P’pro+lrc/Aprolar leu : :Muctsfi2 met wcA56 sfrrl DlO@ MuR F’pro+lar/Aprolac leu : :Mucfs62 met rerA56 sfrA D108R MuR F’pm~lacJAprolac leu : :Murt&2 met recA56 strA D108s ML? F’pro+Zac/AproZac leu : :Mu&62 met recA56 str.4 D108R MuR F-ilprolac Muc+ trp recA56 strA D108R MuR F’-Aprolar Muc+ trp strA D108!+ ML? F-Aprolnc trp &A DlOSs MuR F-Aprolac trp recA56 .&A D108s MuR F’pro+lrr~/AproZnc leer : : Muds62 met 1~~456 sfrrl DIOAR Mus Vpro+lncl”/Aprolac trp rec.456 strA F’pro +locl~~Aprolnc trp strA P’proClnc/Aprolac IPU : :Mucts62nmp met sfrA D108R MuR F’pro+lac/AproZac leu : :MuctsGZ mef &A D108R MuR

Plasmid

Origin

pMC321 pG(‘102

This work This work This work

pSClO1 pCLl51 pGC302 pMD861

This This This This This This This This This This This This

pGC501

pMD861

work work work work work work work work work work work work

Phenotype

Origin

\Vild-type Thermoinducible Virulent mut,ant ‘I’hermoindu~ible

Taylor (1963) Howe (1953) van Vliet et al. (1978) Leach & Symontls (1979)

plaque

former.

Ap’

Description/phenotype

Origin

TC’ pSi(‘lO1 pSC101 pSClO1 pSClO1 pSClO1 psClO1

Cohen 8: Charonas Chaconas Chaconas Chaconas Chaconas Chaconas

: :Muctcs62. 1’~~ : :mini-Muldlnmp, : :mini-Mul2lkan, : : miniCMu222nmp. : :Mu c end, Tc’ : :Mu H end. Tc’

Tcr. Ap’ Tc’. Km’ Tc’. Km’

(‘hang et al. el al. et ul. ef al. ef al. et al.

(1977) (198la) (1981a) (1981a) (19810) (1981a) (1981a)

Transposition of Jfu or min-Mu sequences was monitored using a cwljugation assay whic.11 measured IPlri or mini-Mu insertions into an F’pro+lac episomr. For rxpvriments involving induction. bot,h the donors and recipients were MuR and for infection c>xpvrimtwts onlv tht, recipiwts ww hluR. The donor strains were grown at WY’ in f,B broth (3lillrr. 1971\ vont,aining I rn>l-(‘a(‘l, and 25 miwJlg(‘l, (plus IO pg tctra,cyclinr/ml if a plasmid \I as present)). At a wll drnsit,g of 3 x 10’ cells/ml the donor st)rains wertl shift,ed to 13Y to induw the Mu prophages or werrl infwtrxd at a cell density of 3 x 10s rells/ml (following growth for I generation at 13°C ‘) wit,h hlu phage at a muftiplic~it~y of infection of 1. Before and at various times after iirduvtiorl or infection 0.5 ml of the donor culture wax remowd and added to 54 ml of rvripirnt ,gro\vn to 3 x 10s cells/ml. The mating mixture was briefly mixed and t,hell incubated statically at 32”(’ for I h at which time the cells wew pelh~tcd by a low S~KYVI centrifuga~tion. Thv bacteria were resuspended in 5 ml of @YO,, (w/v) Sat’1 and various dilutions were spread on minimal glucose plates comprising 119 medium cwnt)aiiiing O?,, (w/v) gluc~osc~. I mwMgS0,. .? pg thiamine. hydroc~hloridt~/ml ant1 10 pg tryptophan/ml. tcl wlwt for JIM + exe-onjugants. Dilutions wfw also spread 011 minimal glucosc~ platc>b cont~ainirlg tryptophan and rithw ampicillin (Sigma) or kanamyin (Sigma) at 3,; &ml to wlert for /ITO+ vxvonjugant,s containing Muamp. mini-Ifrran~~~ or mini-Jluko~/. The prw~n~~ of totrayclirw at I pg/rnl in th(l mating mixture did not affect the freqwn~~y at \\-hic,ll cointcyratw \vere wc*ovvred,

f’lasmids and episomes \vert’ visualizcvi by a modification of t hv method dcsc*ribrtl I)) Barnes (1977). Bacteria ww grown in patches vowring an area of 0.5 cm* on minimal aga, plat,es. The veils ww scraped from t’he plate wit,h a t,oothpick and dispersed in IO0 ~1 ot 50 mwTris. HC’I (pH 8.0) containing lo,, (w/v) sodium dodecyl sulfate. 2 rnhr-EDT;\. 04 31 B romophenof sucrose and O~OfO,, (n/v) blue in 1.5 ml Eppendorf conical well polypropylerw tubes. The lysis buffer containing the bact,eria was mixed by flicking with the finger until an increaw in visrosit,y was observed. and t’he mixture was subseyuently incubated at room t)emperature for 15 min. The lysate was then spun for 10 min in an Eppendorf desk-top vent,rifugc. The supernat~ant fluid was removed with a 100 ~1 capiflarv and loatfed dir&l? onto a 03.V,, to OY),, horizontal agarow gel run as previously de&ibed (Chaconas rt nl 1980).

(d) Blotti)ly

crud. hybridizatiot/

Total wllular USA was ext,ract.ed as previously described (Ljungquist & Bukhari. I977 I and digest,ed nit)h restriction endonucfeases obtained from B.R.L. The digest,s were analyzed on OS0 o (y/v) agarose gels and t,he DSA transferred to nitrocellufose (Southern. 197*5). Thea (v:‘v) formamide as previousI> filters were hybridized with a nick-translated prof)e in !W, drsrribed ((‘haconas et al.. 1980).

3. Results

In

previous

9 x lo3 base of free

forms

studies plasmid of this

on the found plasmid

t~ransposition

in 1 t’o 6 copies following

Mu

from gels containing total DSA. isolated prophage on pYIC321 (pW101 : :iMucts).

of a Mu per

host

cell)

prophape we reported

located the

on

pS(‘101

(a

disappearance

(Chaconas et al.. 1980). The blots at various times after induction of the Mu were hybridized with 32P-labeled pS(‘101 induction

ENWI’KOl)I’(‘TS

OF

Mu

I)SA

3%

TI~ASSPOSI’I’IOS

1)X;\. The Mu-containing plasmids were found to migrate with E. coli DNA as the! disappeared as free forms. Within 33 minut,es aRer induction, all plasmid copies appeared t’o he associated with the host chromosome and no detachment of the pS(‘101 DSA srquences from t’he covalent,lg linked prophage Mu could he detected. \I:r have now l)iochemically analyzed the structure of the pH”101 : :hlu-host DSA wmplex formed after prophage induction. The result,s shown in Figure 1 indicate that the hIwcontaining plasmids are covalently integrated into the E. coli DKA in Mu-pS(‘IOl-Mu (Fig. 1 ). Tot,al DSA isohtcd tlw usllal aointegrat~e structure.

Kpn I

(a)

KpnI

SmaI

(b

Ho

Uncut ---

SmaI

KPflI

RHost Sl L-

FIc:. I Analvsis of cointegrate structures generated fottowinp induction of pSC101 : :Mucts (pMC3r’l). (a) Ihapram oia cointegrate structure. The thin lines represent host DSA. the thick line pSCIO1 and thr zig-zag lines Mu DSA. The left and right ends of the Mu genome are indicated as r and S, reqwtivet~ and the KpnI and SnmI cleavage sites are shown. (b) Ethidium bromide stain (left). and hybridization (right) of 32P-tabeted pSClO1 to a nitrocellulose fitter containing total DNA from B1’2021 (whic,h harbors pSIO1 : :Murts) analyzed on a OP,, agarose get. The DN.4 was isolated before prophage induction (0 min) at which time all the ptasmid was in a free form. or at 33 min after induction when virtually all the ptasmid sequences were found to comigrate with the host 1)N.A. In the 0.5”, aparose gel shown aho\-e the retawd (K) and linear (L) plasmid is welt separated from the host DNA whi1.h (wmigrates with the superhelical (S) ptasmid. CompIet,e toss of the superhelical plasmid has been shown on tower prrwntage agarose gets where the host DXX and supercoiled plasmid are welt separated. but \vhrrr the wtaxed ptasmid migrates at the same rate as the E. &i DSA. The total DNA at 0 and 33 min \\as analyzrd without digestion as welt as after cleavage with SwfI and KpnI as indicated above.

316

(:.

(‘HI\(‘OS.iS

/s”/’

.A/,

before. and 33 minutes aft,er induction of KY2021 (which contains pJl(‘3Pl ) \I HS digestBed with restriction endonucleases KpnI and Srntrl. independentI>,. Thv digests were subjected t,o agarose gel electrophoresis followed 1)~ transfer of the DNA to nit,roceIlulose paper and hybridization to 321’-lahelrd pS(‘lOl DSX. Before induct,ion. \vhen all of the pM(‘321 was in free form. cleavage lvith KJ~H 1. \\.hic*h makes one cut near the right end of Mu (Kahmann r,l rxl.. 1977) hut nww itt l)S(‘lOl resulted in the conversion of all the circular DN.4 to the linear form. Similarly. A’ma~I which cuts twice within a 300 base-pair region in pS(‘101 ((‘.-I’. D. Tu & S. N. Cohen. personal wmmunication), but not) in 31~. caatalyzed thth (wnvr~rxion of’ ttilx free plasmid to a linear form 300 l)asCl-pairs smaller l)rlt irldist’illg:uislrwl)le in its paI migration from the Kpwl-linearized plasmid. .At 30 minIltc,s af’t,ttr induration \~~he~r virtually all of the Mwcontaining plasmid had associated \vith t~lltb host IjS.4. cleavage wit)h Kl”/I resulted in the lil)eration of all t’he plasmid DNA from the host chromosome as the linear form. This result is expected from a cwintegrate strrtctllrrb. sinct, cleavage of each of t,he directly repeated c*opirs of Mu with K/w I would (*lit out, a linea,r plasmid. Digestion \vit,h SnrrrI. however. did not wlcasc* a discwtv lilltv~ t’ plasmid band but in&ad a smear of mat)erial larger in size than thc~b lintxa1, lAwnid form, This is also cxpect)ed from a wint~tyratc structure. sinw th(k I)SA hyl)riclizing with IN’101 after digest’ion with A’wtxI cwuld ha,vc> only lwen lilwtnt,ed 1)~ .~‘r~l cleavage of both pH(‘101 and E. coli I)SA sequences. Since Mu insert8ions throughout the host chromosome the size of the host USA at,tached to thv J11r seyucnces would be variable in size in a population of molecules. resulting ItI iI smeared pattIerr of hybridization as observed. These results demonstrate that essentially all of the p8ClOI : : Mn plasmid copies are integrated into the host 1)s A in the form of a cointegrate structure during t,ransposition triggered by I)t’ol)haytb induction. (‘it11

oc~-ur

To st,udy replicon fusion fbrther. \ve set up an experimental syst,em in which t tw fusion of IN’101 : :mini-Mu wit,h a conjugat)ive plasmid could be examined genetically. If replicon fusion \vere the predominant event during the lytic vyclt~. then all of the episomes carrying the mini-Mu sequences would also conta,in thtx plasmid. We construct,ed recombinant plasmids tha.t carry mini-Mu sequences with genetically selectable markers between the Mu ends (Chaconas d ~1.. 1981~). The mini-Mu molecules used in t,he studies reported here (all of which are c*arrietl on pSClO1) are shown in Figure 2 : they do not encode the Mu transposition funvt,ions (the .-I and R gene products) hut can be complemrrltt~d by Mu prot~rins in ftwtts t’o undergo Mu-specific transposition, replication and packaging ((‘havonas et (xl.. 198lb: M. DuBow & A. I. Rukhari. unpublished results). To monitor t,hc transposit’ion of bhese deleted Mu prophages itt c?cv. a conjugation assay was used (see Materials and Methods). Strains were construct8ed that carried a geneticall? marked min-Mu plasmid which was not conjugally t,ransferal)lc. a thermoinducihle Mu helper prophage located in the host chromosome. and an F’pro’hc episome. These st,rains awe mated before and after prophagt induc%ion. with a \-ariety of Aprolac recipients. and ~),ro+ as well as Xl)‘. /WO+ or Kmr. JWO+exconjugants

END-PKODl:CTS

OF

Mu

DNA

Mini-Mu121

TKASSPOS1TIOS amp(pMD661)

1

Mini -Mu222

I0

I 5

317

‘1

Mini - Mu121 km (pGCl02)

amp (pGC50ll

I IO

I 15

FIG:. 2. Diagram of t,he mini-Mu derivatives used in the experiments reported below. The solid blwk line indicates Mu DNA sequences and the hatched areas show insertions of foreign DNA within the miniMus. The left and right ends of the Mu genome are indicated by c and S. respectively, and the brackets denote the position of the invertible G region of Mu (part of t,he G region is missing in mini-Mul2lamp). The mini-Mu derivatives are all carried on pSClO1 and the designation for each plasmid is listed in parenthesis. The scale at the bottom is in bases x lo3 and the construction of these mini-Mu molecules has been previously described (Chaconas at al., 1981n~.

(depending upon the mini-Mu used) were selected. As shown in Table 2, at 30 to 90 minutes after prophage induction the frequency of transferred F’ episomes carrying mini-Mu insertions as judged by Apr or Km’ increased between t’wo and three orders of magnitude as compared to the pre-induction values. This increase in transposition was dependent, upon transposition functions provided in tram, since it did not occur upon temperature shift up in the absence of a helper prophage (data not shown). Upon further examination of the Apr or Km’ exconjugants it was also found that most of them were Tc’ suggesting that the plasmid vector, pSC101. which confers resistance to this antibiotic, has been mobilized. The transfer of tet’racycline resistance upon Mu induction was dependent upon the presence of both ends of the Mu genome on pSC101, since an increase in the Tc’ transfer was not observed with pSClO1 alone or when the plasmid contained only the left or right end of the Mu genome (Table 3). In addition, a second round of episome transfer from ret-4 recipients resulted in near lOO~/b cotransfer of Ap’ and Tc’. These results suggested that mobilization of the plasmid sequences was linked to mini-Mu transposition and that perhaps pSClO1 had also been integrated into the F’ episome in a cointegrate structure as described above for pSClO1 : :Mu. To determine whether the Tc’ exconjugants which we recovered conformed to this structure we analyzed both rrcA and ret+ exconjugants for the presence of mini-Mu plasmids as shown in Figure 3. Mini-Mu plasmids were observable in ret+ but not, reed exconjugant,s suggesting that they were generated by a recombination event bet)ween directly repeated mini-Mus integrated into the F’ episome, flanking pSC101. Restriction endonuclease cleavage analysis has confirmed the mini-Muplasmid&mini-Mu structure (Chaconas et al., 1981b; our unpublished results). As shown in Table 2 the frequency at which Tc’ exconjugants were recovered was dependent upon the characteristics of the recipient strain. Use of a WC-4. nonlysogenic recipient resulted in 99 to loo?;, cointegrate recovery while a WC+, non13

-----cc xw-5

-.- c zi

=F-=p c

-

.Jhsencr plnsmids

of tramposition F’pro+lar

of pSCIO1 rr~d Mlr left end nrld right cud following iuductiorl of a Mu helper prophnge

irlto

Time

Trr.

(min)

pro+/pro+

esconjupant~

Ihnor

Plasmid

B1’2099 B1’2099

psc101 psc101

0 40

<“.:!

1~~2100 Hr11OO

pCL151 pCLl51

0 40

<1.5x 1or4
KL’L’IOl HI-2101

pGC302 pGC302

0 90

2% x 1w4 <2%x 10-i

(ix 1w4 x lorb

‘I‘hr, transposition of pSClO1 and plasmids containing the right (pCL151) or left end (pGC302) of thtx 11~ ph;tge grnomr* into F’proiZac was monibred using the conjugation assay described in Materials and anti the portions wew Methods (also see Table 2). The recipient strain was BIJ503ti (rep’. Muc+) removrd for mating &fore (0 min) and at the onset of lysis (40 to 90 min after induction. depending upon the strain used).

Iysogen or a WC~~. Muc+ lysogen as recipient resulted in only 91 t)o SP,, cointegrates. .\ further decrease in cointegrate frequency to 80 t’o W$, was obsrrvrd \vhen the recipient strain was a ret+, Muc+ lysogen. These results suggested t,hr possibility of deletion formation in the recipient st’rains as an explanation of the variability in the recovery of Tc’ exconjugants. To explore this possibility the size of the F’ episomes in a number of Tc? exconjugants was analyzed 011 @3.V,, agarose gels as shown in Figure 4. Indeed, most of the F’ factors \vere noticeably smaller than expected. In addition there was a heterogeneous size distribution which was not observed in the case of Tc’ exconjugants (Fig. 3). From thrsc~ results we infer that mini-Mu transposition occurs by a mechanism generating only cointegrate structures. This is supported by findings from another laborator! using mini-Mus which contain the Mu A and R genes. Howe & Schumm (1981) constructed these deleted prophages using a phage A ve&or and report’ed that in W’,, of the transposition events the h sequences were found to cointegrate with the min-Mu. It would also appear that cointegrate structures are substrat,es for deletion formation requiring the host recA protein or a Mu-encoded function. The prrsencta of hot h the wc.4 protein and a Mu prophage in the recipient has an additive effect. Thta presence or absence of the recA protein in the donor has no effect’ on the cointegrat,c frequency, nor does the presence or absence of the invertible Mu (: region on thca mini-Mu or the select’able marker bet#ween the ends of Mu in the minMu (Tabk 2). Additionally. t,he manner in which t,he lytic pathway is initiatrd (induc$ion tir. infection) does not affect’ the frequency at which cointrgrates are rrcovt~rrd. (c) Iuteqration (i)

7’~ trspositiorr

qf Mu

from

of thr

the Mu

qertome irtto an F’ Ppiwvnr

h,ost chromosome

Previous experiments which showed difCrt,nt sites on t,he host genomr during

to thp episonre rlurinq

that Mu the IFtic

the lytic

cyclr

D?;A is integrat’ed at many cycle involved insertion of Mu

-

YIC.. 3 ~I~~~~t,-u1,11o1~rti~~ analysis of mini-Mu plasmidn in Apr. Tc’, oyc .4 I 60 mirl atirr irvlr~c~tion of BIT2102 (see Table 2). The desired sr~pe~l oi’l’:~n~l ly~cl. The cleared lysatea were analyzed on a 035Oh v ith cthidiurn bromide followed by irradiation w-ith ultraviolet light. ans I I IW ~~i!l~cxi tr<).t IIS.\ found ill the cleared Iysates as well as

PM 08 861 -

E . ccIli

F’

pro+ exconjugants from the mating of BU2102 (donor). with RU5039 (ret+) ant1 B1’.7040 exconjugants were patched on minimal plates containing ampicillin and the bac,ttka wcw agarose gel its described in Materials and Methods, and the DKA was visualized Kay staining The position of the F’ factors (which are more obvious in the WC + mww~ thr rwrt it rai~l.j the relaxed form of’ mini-Mu plarmiti pMIXX1 are intlic.atecl.

F’pro %c

from the mating of BU2102 (donor), with #‘IO. 4. Elec*trophoretic analysis of F’ episomes containing mini-Mu insertions in Ap’, Tc‘, pro+ exconjugants of BU2102 (see Table 2). The desired exconjugants were patched on BU5039 (VW’), BU5034 (recA, Mue+) and BU5036 (ret+, Muc+) 60 min after induction minimal plates containing ampicillin and the bacteria were scraped off and lysed. The cleared lysates were analyzed on a 035oi, agarose gel as described in Materials and Methods. The DNA was visualized by staining with ethidium bromide followed by irradiation with ultraviolet light. The slots containing F’pro+lnc and F’pro+Zac : : Mu markers are indicated.

E. coli-

recA, Muc+

F’pro + Ioc: : Mu

352

DKA

(:.

onto

an F’pro’la,c

(‘HAC’OSAS

episomr

15.7’ .-1/A

af’tcr prophagc

inducth

(Razzaki

& Hukhari.

1975: Schriider S van de Putte, 1974). The realizat)ion that replicon fusion is the predominant event after induction of a Mu prophagr on pS(‘101 prompted us t,o c>xamincthe nature of’ Mu DNA transposition from t’he chromosome to the t~pisornt~. If cointegrates were indeed end-products of t,ransposition then simple Mu insert’ions into an intact F’ factor would not be expected. Mu instlrtions into F’pro+/ac were isolated by inducing strains containing the F’ c~pisomcarid ii Muctsanb;c-prophage located in the leucine operon. Following therrrloillductioll the, strains were mated with isogenic rec=l, ret+ recipients and pro+ A f tlscoll,j upmts were s&&d. As shown in Table 4, Mu insertions were found in about~4()(, of’ t Irra TABLE 4 Trmsposition chromosome ftelevant genotype

into

of Muctsamp located F’pro+lac during @tic

Time

(min)

in

the growth

Ap’.

E.

c-oli qf MU

pro + :}lrll +

IA<.

f(‘,$ !

The transfwsition of’ a thermoinducible Munmp prophage into an Fpro+Iw ef)isome was monitort~rl using the conjugation assay described in Materials and Methods (also see Table 2). The rec+ient strain (B1’6034) cv~ntained a Mue+ prophage in a recA background. The La? phenotype was determinrtl b,v replica-plating the rxconjugants onto MacConkey/lnc*tose indicator plates cwntaining :IO/+ ampicillin/ml.

tmnsferred rpisomes after Mu induction. This frequency is comparablt~ to that observed for mini-Mu insertions into F”pro+lac (Table 2). More than 2S0,, of thv exconjugants carrying Mu insertions in F’pro+lac. however, displayed a I,at. phenotype. Since the Zac operon constitutes ouly a very small portion of F’~No+Inc. the frequency of lac- episomesresulting from Mu insertional inactivat,ion shoutd 1~ less than So/“. In the case of mini-Mu cointegrate insertions into F’pro’Iac. only I.7o/o of the 400 insertions recovered were Zac-. The SO-fold increase itr /UC rpisomes, when the original Mu prophage was located in the host chromosome. suggested that) F’ episomes carrying the Mu insertions were deleted forms of J’ generated during Mu DNA t,ransposition. This was further supported by the finding that, about 20t;h of the episomes carrying Muamp insert’ions NYW also Tra To test, directly for deletions. the episomes cont,aining M11m7p insertions ill Al)’ exconjugants were analyzed by etrctrophorrsis in low t~oncrlltrat,ior1 agarosr gels as shown in Figure .5(a). The results of t,his type of analysis revealed het,erogeneit,y in the sizes of the episomesregardless of t,he WCgLrnotypv of thr donor and rtacipient strains or the time aft,er induction (between 15 ad 60 min) \vhchnthe mat,ing pairs were formed. X heterogeneous size distribution of’ F’ t~f)isorncv taarrying Mu

l3SILPROI>C’CTS

OF

Mu

DN.4

1’R.~BSPOSI1’IOS

353

insertions was also observed during lytic growth following Mu infect’ion (dat’a not shown). *As shown in Figure 5, episomes carrying ?uluamp insertions were found to contain deletions and insertions as judged by comparison with t,he migration of an authentic F’pro’Zac : :Mu episome. In general most of the Tra+. Lacf episomes were larger than F’pro+Zac : :Mu while most of the episomes which were Traor I>ac - were smaller. The generation of F’ episomes carrying deletions and insertions since episomes which did not contain Jlu \vas dependent upon Mu insertion, insertions but which were transferred out of the donor strains following Jlu induction did not show any size heterogeneity. Similarly, conjugal transfer of 17’ episomes carrying previously characterized Mu insert’ions did not result in deletions or insertions in the episomes. suggesting that these events occurred during transposition rather than during or after episomal transfer. While man\- delet~ions were evident from size analysis of the episomes from Ap’ exconjugants. these deletions did not extend into the Mu prophage. since all deleted episomes were found to contain prophages capable of producing viable Mu phage (data not sllo\r-n).

111 csontrast to the heterogeneous size distribut’ion of ,IIu-containing episomrs gellerated during the lytic cycle, Mu insertions into F’pro+lac Ga the lysogenic pathway in ret+ or red strains resulted in a homogeneous pat,tern of episomes. The F’1)ro+lclc strains were infected with Mu and lpsogens carrying Mu insertions in the lcrcl gene on the episomes were recovered. Figure 5(b) shows a gel analysis of these cpisomes. Within the limits of the gel system all the episomes exhibitred the same size aw an authent’ic F’pro+ZacZ : :Mucts episome. containing a point) insertion of Mu jvithout a deletion. When the insertions formed by lysogenization were located in the ItxcZ gene, most, could be shown to revert to Lac+ zjia the MuX pathway (Hukhari, 1975). demonstrating that during lysogenization. delet’ions did usualI> not occur at t’he sit,e at which t,he Mu genome was inserted.

4. Discussion \l’r hart1 shown in this paper that, a plasmid containing a Jlu prophage or a miniMu is fused with the host chromosome upon prophage induction. This replicon fusiotl involves a duplication of the Mu sequences such that one copy of Mu is I)rcbsent at each junct,ion of the plasmid and the chromosome. These replicon fusions are similar to t)he cointegrate structures seen when fusion of two DNA4 molecules is mediat)cd 1)~ IS elements or transposons (see (‘alas K: Miller, 1980; Starlinger, 1980). ( ‘ointrprat,es containing Mu DNA were originally proposed by Toussaint & Faelen (1973) and more recently cointegrat,es cont’aining mini-Mu DXA1 hare been oljserved in several laboratories (Chaconas of ~1.. 1981b: Howe P: Schumm. 1981 ; Xlaynard-Smith it ~1.. 19X0). Evidence presented in this paper. however. st*rongl> suggests that cointegrate formation is not merely one of the events occurring during transposition from the prophage stat’e: it is apparent,ly the predominant evcbtrt and results in the ill toto insertion of Mu-containing plasmids int,o the

F’pro+

lac:

: Mu Tra+,

F’pro+kx: Lac-

Tra+,

:Mu

Lac+

Tra-,

Lac-

----I (0)

E. coli -

F’pro

‘/ac: :Mu

Plc:. ,Y. Elwt r~l~hor~ti~~ analysis of F’ episomes containing Muclsuln/p insrrtions generated during I>-1 w growth (a) or by lywgmizat& (1)) of Mu. (a) Insertions of Muc~srrvt~~ into an F’~~ro+/rrc el)iscmw following intlnc+ion of a t~hermoinduc4~lr Munmp prophage in thv E. di c~hromosnme w-ew isolatrti as des~~ribetl in Table 4 using KU2091 as the donor strain ant1 BV.5034 as the rwipi~nt. The Apr. ,AW’ csconjugants were grown on minimal plates containing ampicdlin and the hxcteria wew scralwtl clff. lywtl. and the cdrared Iyates analyzed on a 0.35”,, agsrose gel its dewrilwl in Materials and Mrthotlh. ‘I%~ 1)X-A \+a~ visualized by staining with ethitiium hromi~lc followetl by irradiation with ultrariolrt light. The Jots wnt,aining authentic, F’pro’lnr : :Mu DS.4 marker ns well as thaw containing Tra + l,a~,+. ‘1‘~ + Lat.- and Tra- IAH- episomes a~-~~indicated (h) Insertions of Mur/scrnn~~ in thr 1er.I genr of an b”?~ro’/~~I cpisomra werr isolated 1)~ spotting a phagt‘ Iysatr on lawns of HI’5048 plated 011 hlac,(‘onke\-/lac~tosr indicator plates. Survivors of the infdion which wew IAC~~ were streaked for sinplr cdonies on I\lnc,(‘onke~/lac~tose plat,es containing ampidlin and wcw rhevketi for their abilit?: to conjugally transfer the Ap’ marker and the Mu prophage when mat.4 with thr q~propriatr rrt~lplent strains. The lysogens with Muctscrmp insertions in the lrrcl gene wwe groan on minimal plates wntaining ampicillin and the hactrria were wraped off. lysetl. anti thP c~leartd Iysates analyzed as dwc~ribetl in (a j. The slot containing an authent,ic F’pro+lrrc : :Mn marker q)isomr is inrlicatd

ESL)-PRODU('TS

OF Mu DNA TRANSPOSITION

3.55

chromosome during lytic development of Mu. We have shown this by biochemicalI> analyzing the structure of the pSC101 : :Mu-host DNA complex formed after prophage Mu induction. The observed cointegrate formation appears to be a unidirectional process. The plasmid DPL’A is fused with the host chromosome, but is not dissociated from it; no detectable equilibrium between free plasmid and integrated plasmid forms appears to be established. This result further sustains the earlier proposal that integrative recombination of Mu is a duplicative process requiring Mu DSA synthesis. and that Mu sequences are not’ excised during the transposition process (Ljungquist oi Bukhari, 1977 : Bukhari. 1977). The unidirectionality of the pSClO1 : : Mu insertion into the E. coli chromosome indicates that t,here is no efficient recombination function to resolve the Mugenerated cointegrates following prophage induction. This conclusion is furthet strengthened by experiments on transposition of mini-Mu sequences that contain selectable markers between the Mu ends. Mini-Mu transposition from pS(‘101 to a wrrjugally transferable episome during the Iytic cycle invariably results in the fusion of the plasmid and episomal DNA thereby generating mini-Mu-pS(‘lOIP min-Mu cointegrates. These cointegrates are much more stable in wc;I than in WC+ cells. Mobilizat~ion of the mini-Mu plasmids by the episome after Mu induction sometimes results in deletion of t)he plasmid sequences if the recipient is recC and Iysogenic~ for wild-type Mu. The deletions result in heterogeneity in the sizes of the episornes transferred and are clearly not’ simple resolutions of t’he cointegrates. This obscure effect of t’he rec=l function and the Mu prophage in the recipient st,rain is not understood. These findings do. however. suggest, caution in the study of cointegratr resolution. since deletions emanating from cointegrates are detectable at a high frequency and ma\- he genetically indistinguishable from resolved cointegratr structures. It is well known that a Mu prophage located on the E. coli chromosome can be transposed t,o an F’pro+lac episome after induction (Razzaki & Bukhari. 1975). Examination of the episomes onto which such transposition of Mu has occurred has resulted in the surprising finding t’hat few. if any. integration events are simple linear insertions of Mu DNA. The episomes generally contain large deletions or insertions of DS.4 in addition to the Mu genome. The deletions frequent’ly remove the ltrc or tm genes. To explain these results we postulate that Mu does not “jump” dirert’ly ont’o an episome but that the episome is first, fused with the chromosome (that) is, the chromosome and the Mu prophage are cointegrated int,o the episome). Since there is no high frequency cointegrate resolution function operating upon induction of the lytic cycle of Mu. t,he episome cannot be simply recombined out of the chromosome. Instead. a subsequent transposition event would release heterogeneous circular forms of the episomes containing Mu. This mwhanism is shown in Figure 6. If t,he heterogeneous circle released contains the whole cointegrate structure and some adjacent E. co/i DXA then the episomes would be larger t’han expected. The larger episornes can also arise by two separate events: a heterogeneous circle containing Mu is first formed and this circle is then fused with the F’ episome. It should be noted that isolation of Mu-containing plasmids aft,er prophage Mu induction provides a good met,hod for isolating deletions in conjugative plasmids.

i

FIN.. (i. I’ro~)ostvi mr~chanisrn tl~r the yweration of F’ vpiwnws c’ay ~ng rl 1111 Ilroptl;tg~~ lib u(lli itdeletions or in,wrtions of E. co/i DS.4 during the lytic- cycle of Mu The end-products of transposition art’ xhov n as replicwtl fusionn and the general arguments for inversion and dr~lrtion fixmatirm tll~ring replicxm fusion mrdiatecl by transposable elements hax brrn discussed by Shapiro (1979). Arthur S Sherratt (19i9) and Harshey & Bukhari (1981). The thin lines represent the K. wli c~hromosumr. rhv thick linrh rpisomal DX.4 and the zig-zag lines bacteriophage Mu 1)X,\. The left ~ncl right end% oft he 31~1 genome are denoted b,v c and S. respectively, and the filled circles at the junction points of 1111 ant1 E. coli repwwnt the 5 base-pair tluplic~ations of host DNA found at thr insertion sitr of’Mu I~~ogrn~. ‘l’hrx letten on thtx rl~iwmal and E. coli DNA1 shot+ the position of’ arbitrary rrgicw. In the tirst ,tqr ii c~hromohomally located 11~1 prophage integrates into an episomr rrsultiny in fusion of’t he plasmid wncl R c,oli DS.4 sequences. .I swond Mu insertion from the wp~ of Mu lwated near the A region of’ the t~pisom~~ to a position between L) and E c*ould hare two possible outwmes. One of these (B) would be the formation of a deleted F’ factor c,arrying a Mu insertion and the other would result in the inrrrsion ~,f’th~~ tINA4 betwwn the donor and target site i.4’). Similarly a sevnntl Mu insertion from the copes of 3111 located near the A region of the episome to a position betnren T and Z in the chromosomal I)S;\ u-o~~l~l have 2 altrrnativw In one of these (B) an F’ episome cwryinp 2 tlirwtly repeated c,opier of Mu flanking ;i region of the host c~hromosomr woultl be generated and in the other (H’) an inversion of thrs 1)S.I between the clonor and target site mould owur. Heteroyrneous circles of the type which rwuld be generated by pathways A anti B have heen shown to exist durinp Iytic. yruu-th of Mu (\Vaggoner PI trl 1971.1977: 1)6narii rl crl.. 1977: Faelen P/ nl.. 1957). SimilarI\Mu-mediated inversions ant1 tlelrtionh haw alho bwn wported (F‘aelell R- Towsaint. 1978.1980: Towsaint rl rrl,. 19iii

ESD-PRODUCTS

OF

Mu

DNA

TRANSPOSITION

357

If there is no discernible function that can resolve the Mu-generated cointegrates at a detectable frequency during the lytic cycle, then how can one explain the simple insertion of linear Mu DNA during lysogenization? The results presented here show that in contrast to transposition of Mu from the chromosome to the eplsome. insertion of Mu into the episome during lysogenization results in a homogeneous population of episomes. Excision tests, genetic mapping (Bukhari. 1976). and DNB sequence analysis (Allet, 1979: Kahmann & Kamp, 1979) have confirmed that most of these types of insertions of Mu are faithful point insertions. Infection of host cells by the linear form of Mu DNA presents special problems. As shown in Figure 7, insertion of Mu into the chromosome by a mechanism leading to

Recipient

Mu

P

Rec!pient

DNA

Mu

_-_

DNA

MU

Mu Y

Frc;. i. Integration of Mu DNA carried on a circular plasmid, and linear Mu DNA carrying covalently linked host DNB sequences into host DSA by a replicon fusion mechanism. In the case of the circular molecule a cwintegrate st,ructure is generated. With the linear Mu DNA, however, 2 copies of Mu are inserted into the host DNA resulting in a discontinuity in the chromosome. The covalently linked host DNA sequenws would presumably remain attached to the Mu genome.

cointegrate formation would disrupt the chromosome, since the host DKB sequences at the ends of Mu are not connected (that is, Mu DNA is linear). Formation of a prophage during lysogenization can then be explained in two different ways. (1) There is a low level function that resolves linear Mu cointegrates thereby regenerating a circular chromosome containing a Mu prophage, and a free form of linear Mu DNA. This interpretation follows thlx reasoning of Shapiro (1979) who has proposed a model of transposition in which cointegrates are the obligatory intermediates of transposition. (2) Alternate modes of transposition could function during lysogenization versus transposition from the prophage state. Under appropriate conditions the cointegrate mode would switch to a simple insertion mode. In this second possibility the switch between the alternate transposition pathways could be governed by any one or a combination of the following three possibilities. (1) The differential expression of proteins involved in the integrative-replication of Mu DXA following infection versus prophage induction. (2) The differences in the physical state of infecting Mu DNA (linear) versus a resident prophage (superhelical). (3) The injection of packaged proteins involved in integrative replication from the Mu virion into the host cell along with the Mu phage DNA.

358

G. (‘H,$COh‘AS

E7’ dl,

A molecular model for t’he transposition of Mu 1)X-A which is based on th alternat,e mode hypothesis presented here has been recently proposed by Harshe) B Bukhari (1981). Experiments to det,ermine whether the difference in Mu DNA transposition from the prophage stat,e WMUS Iysogenization results from a resolution of cointegrates or an alternate mode of integration are now in progress. FVe thank Louisa Dalessandro for her excellent, typing of the manuscript. and Mike Ockler and Cindy Carpenter for their expert, preparation of t,he Figures. This work was supported b>grant,s from the National Science Foundation (PCM 7826710) and the Xational Institut,es of Health. One of us (G.C.) was a recipient of post#doct,oral fellowships from the Medical Research Council of Canada. and the Xational Institut,es of Healt)h. U.S.A. (1 F32 G>lO679.501).

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OF

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DN.\

‘l’K.-\NSPOSITIOS

359

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