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Site-specific recombination in bacteriophage lambda
J. Mol. Biol.
(1977) 113, 385-40
Site-specific Recombination JEFFREY
ENGLER
in Bacteriophage Lambda AND Ross B. INMAN
Biophysics Laboratory of the Graduate School and Biochemistry Departm,ed of thP College of Agricultural and Lifp Scie~xes University of Wisconsin Madison, Wise. 53706: U.S.A. (Received 29 October 1976, and in revised form 17 February
1977)
Lambda attB-&P is a derix,ative of bacteriophage lambda t,hat contains both for integrative recombination. Lambda attBattB and at@, two sites required attP can undergo &-mediated recombination to yield progeny phages whose DNA is 1574 shorter than that of the parental phage. We have studied intracellular phage DNA from an infection of lysogenic bacteria with hattB-attP in the presence of int gene product, rifampicin and chloramphenicol. The majority of the intracellular phage DNA consisted of circles with lengths of 17.51, 15.09 and 2.38 pm. Partial dena.turation mapping confirmed that the 1509 and t,he 2.3%pm molec111os arose by an &-mediated intramolecular recombination reaction of the type predicted by the Campbell (1962) model. A minor proportion of the circles (30/b) were much larger (33.9, 30.2 and 4.7 pm) ; in t’hese cases denaturation mapping indicated that both intra- and inbermolrcular recombination could take place.
1. Introduction Lambda, a temperate bacteriophage of Escherichia coli, can insert its DNA into the bacterial genome under certain conditions. Our current understanding of this process is that this insertion occurs by a reciprocal recombination event at a specific attachment s&e on both the bacterial and the bacteriophage DNA molecules. This process also requires the product of the hint gene (for a re ?iew of int-mediated recombination, see Gottesman & Weisberg, 1971). A variant of X which arose by an illegitimate recombination event and which contains both the bacterial attachment site (attB) and the bacteriophage attachment site (attP) has been isolated @ash, 1974). The plaque-forming ability of this phage. called hattB-attP, is sensitive to chelating agents such as sodium pyrophosphate and EDTA. In the presence of int gene product, XattB-attP yields a high frequency of progeny phage whose plaque-forming ability is more resistant’ to sodium pyrophosphate. In the absence of the int gene product, these pyrophosphate-resistant phages are not formed. The Ret recombination pathway of E. toll: cannot. by itself, produce these pyrophosphate-resistant phages. Formation of pyrophosphate-resistant phages is independent of the presence of the xis gene product. These findings suggest that zkt-mediated recombination allows hattB-attP to lose DNA. presumably the DN,4
3citi
,J. EN(:I,EH
;\Sl)
R. B. INMAS
between attB and attP. Roth contour lengt,h measurements and CWI density cquilibrium centrifugation of AattB-attP and the p?-rophosphatc,-resistnllt phage show that t’he latter contains about 15”;, less DNA t)han does AaffR-attP (Nash (1974) and confirmed in this paper). In a subsequent paper, Nash (1975) demonst,rated that’ rccombinantj phagr: DNA first appears 20 to 30 minutes after infection at 37°C. R’o recombinant DNA can bo found if the int gene product’ is defective or if cyanide or dinitrophenol are present. The delay in the appearance of recombinant DNA molecules can be eliminated by interfering with the expression of the infecting phage genome. either by t,he use of inhibitors (rifampicin and chloramphenicol) or by the binding of h repressor to the phage DNA (infection of a bacteria lysogenic for lambda by a homoimmune AattBnttP). Nash’s work suggested that XattB-attP might be an ideal model for the study of the fate of phage DNA during in&mediated recombination in vivo. In this paper, we present an electron microscopic study of the intracellular DNA products that result from infection with AattB-attP. We have identified XattB-attP DNA molecules as well as the two product molecules expected from &t-mediated recombination. Our studies show that these products are free circles and that they are not catenated. We have also found dimer length molecules that arise at low frequency by intmediated recombination. These studies should form the basis for a future investigation of the intermediates in this interesting reaction.
2. Materials and Methods (;I) Bacteria
and
bacteriophage
strains
were
used: MVlrecA - sup’, pl~age TI-resistant The following strains of E. coli K12 (derived from strain 152, Valenzuola et al. (1976), SH28tp~~(C. Cross via E. Calva), and HN216 (N99 (Ahint-c,,,cI857), from H. Nash). The following strains of bacteriophage X were usrti: AattU-attl’ int2 sisl red114 immidd clear (Nash, 1975), hgaZ49 intd xisl red114 imm434 clear and Ab522 (all from H. Nash). recombination between attB and attL and attR are the 2 sites produced by integrative at@ and correspond to the attachment, sites found at the left, and right-hand ends of the int2 xisl red114 imm434 clear is derived from prophage, respectively. Rocause hgalll~ XattB-attP by integrative recombination and contains attL (Nash, 1974), this phage will be referred to as XattL in the text. (b) Media
and plates
M9 minimal medium consists of 20 ml of salt mixture (70 g Na,HPO,, 30 g KH,P04, 5 g NaCl, and 10 g NH,Cl/l H,O), 20 ml 4% sugar (glucose or maltose), 2 ml CaCl, (0.01 %I), 2 ml MgSOl (0.1 M) and 156 ml H,O. T-broth consists of 5 g NaCl and IO g Bacto-tryptone/l are TB-plates containing 2.68 to HzO; TB-plates are T-broth plus 1% agar. TE-plat’es 2.75 mM-EDTA (pH 8); X&B-attP phage do not grow on TE-plates while XattL phage plate well. Tris/Mg medium is 0.1 M-Tris (pH 7.2), 0.01 hl-MgSO.,. TBY, TBS, TBSS media and SE buffer are described by Nasli (1975). 2H medium is IO ml phosphate buffer (0.2 M 1 ml in 2Hz0, pH 7.2), 0.1 ml MgS04 (1 M in ZH,O), 0.01 ml FeSOa (0.016 M in 2H,0), (15NH4)sS0, (5% solution in 2HZO), 5 ml 5% srlgar (glucose or maltose in 2H,0), and 84 ml 2HZ0. (c)
Preparation
of bacteriophages
For unlabelled phage stocks, E. coli strain MVl was grown at 37°C to o,D.~~~ z 0.75, and infected with phage (multicentrifuged, resuspended in 0.1 vol. Tris/Mg medium, plicity of infection (m.o.i.) = 0.1). After 10 min on ice and 10 min at 37”C, the infected
int RECOMBINATION
IN PHAGE
LAMBDA
387
cells were diluted in fresh, prewarmed T-broth at 37°C and incubated until lysis of the infected bacteria was complete, as judged by O.D.,,,. The lysate was treated with ehloroform and centrifuged at 10,000 g for 10 min. 0.1 vol. 5 M-N&l followed by 10% (w/v) Carbowax 6000 (Schwartz/Mann) was added to the supernatant. The mixture was swirled at 4°C until dissolved and allowed to stand overnight at 4°C. This mixture was centrifuged at 10,000 g for 15 min. The pellet was resuspended in Tris/Mg medium. The phage were purified from this pellet by CsCl banding; this stock was dialyzed in Tris/Mg medium and stored at 4°C. For preparation of 3H-labelled bacteriophage, strain SH28 was grown at 37°C to o.n+s,, = 0.75 in M9 (maltose), 1:/o Casamino acids, Bi (10 pg/ml) and thymidine (10 fig/ml). The bacteria were centrifuged, resuspended in 0.1 vol. Tris/Mg medium. alld infectfed with phage (m.o.i. = 2 to 3). After 10 min on ice and 10 min at 37”C, the infected bacteria were diluted into warmed MS medium (glucose), 1e/o Casamino acids, B1 (10 @g/ml) and [3H]t,hymidine (O-023 pg/ml, spec. act. = 200 mCi/mg; New England Nuclear). Cell lgsis was generally complete within 90 min after dilution into fresh MS medium. The phage were purified in the same way as unlabelled phage. Specific activities of IO-6 to 8 x 10 -a cts/min per plaque-forming unit were achieved. (d) Heteroduplex mapping Tllcx methods described by Dalris & Parkinson (1971) as modified by Chattoraj & Inman (1974) were followed. Het’eroduplox mapping of Ab522 was consistent with results presentod by Davidson & Szybalski (197 1). (0) Spheroplast
transjections
The isolation of intracellular DNA for use in spheroplast transfections and the procedure for spheroplast transfection were both described by Nash (1975). Stocks of rifampicin (2 mg/ml, from Calbiochem, prepared according to Weiner & Weber (1973)) and chlorsmphenicol (10 mg/ml in 95% ethanol, from Calbiochem) were prepared fresh for each experiment and kept on ice away from light. Bacteria were incubated with rifampicin and chloramphenicol (final concns 200 pg/ml and 100 pg/ml, respectively) for 30 min at 30°C in the dark before infection. (f) Ieolation of intracellular phage DNA for electron microscopy Strain HN216 was grown for 3 to 4 generations at 30°C in 2H medium (maltose), ZH,O-algal hydrolysate (O*O75o/o,Merck, Sharp and Dohme), B, (10 pg/ml), and thymidine = 0.75, the bacteria were centrifuged and resuspended in 0.1 vol. (10 pg/ml). At o.D.~~~ 0.01 M-MgSO, in 2H20. Rifampicin (prepared with ‘H,O instead of H,O) and chloramphenicol were added to final concns of 200 pg/ml and 100 rg/ml, respectively. The bacteria were incubated for 30 min at 30°C in the dark. 3H-labelled XattB-attP was added (m.o.i. = 10) and adsorbed for 10 min in ice and 10 min at 30°C. The infected bacteria were diluted into 2H medium (glucose), 2H20-algal hydrolysate (O*075°h), B, (10 pg/ml), and thymidine (10 pglml), containing rifampicin and chloramphenicol at the same final concns a.s above and prewarmed to 33°C. After 60 min at 33”C, 12-ml samples of the infected bacteria were poured int.0 12 ml of cold 30% (v/v) pyridine in 0.05 M-KCN, 0.05 M-NaNs, 0.01 M-EDTA that had previously been poured over 8 g of crushed ice. After 2 to 5 min at 4”C, the bacteria were centrifuged and resuspended in 1.5 ml of 0.05 M-KCN, 0.05 XNaN,, 0.01 M-EDTA. 15 ~1 of a freshly prepared solution of lysozyme in water (50 mg/ml) was added: the suspension was frozen and thawed 3 times, followed by incubation on ice for 20 min. 25 ~1 of 30% Sarkosyl were added and the suspension incubated at 4°C for 15 min and then 5 min at 50°C. 50 ~1 of Pronase (20 mg/ml in 0.005 M-EDTA, self-digested for 4 h at 37°C) were added and incubation was for 2 to 4 h at 37°C with slow shaking. The lysate was cooled and adjusted to a density of 1.67 g/cm3 with saturated CsCl and centrifuged at 39,000 revs/min for 3 days at 9°C in a 60 Ti rotor. The gradient was fractionated from the top at. a rate of O-5 ml/min in 0.2-m] fra&ions. Fractions containing 3H counts were found at the LL (light-light) position in the gradient: these fractions were pooled, made up to a density of 1.67 g/cm3, centrifuged for 3 days at 30,000 revs/min in a 60 Ti
3XX
.J. ESGLElC
rot,or, and refract,ionated. 0.0% M-NaC1/0.005 M-EDTA Inmali & S&n& (1970). (g)
Electron
;\NI)
It.
I<. IXMAN
Tl~n fractions oont,aining “H counts WXIC dialyzed against (pH 8) and prepared for clcctrorl microscopy as described 111
microscopy
am!
DNA molecules were pllot.ographrd graphics calculator (Numonics Corp., 9820 calculator and 9862A plotter.
computation,
of electron
n~icrosco~x
tlato
~IKI their Icngtha measured using :L Nu~onics North Wales, Pa) interfaced to a Hewlett,-Packard
3. Results (a) Heteroduplex
analysis
and contour length of hattB-attP
The strucbure of hattB-attf’ shown in Figure 1 was determined by studying heteroduplex molecules prepared from hattB-attP and /\b522 and from hattB-attP and hattl. Lambda b522 forms a deletion loop that can be used to identify the position of attP in heteroduplexes of h phages (Davidson & Szybalski, 1971). The structure shown in Figure 1 agrees with the structure shown by h’ash (1975) and is consistent’ with the observed difference between the partial denaturation maps of X wild-type and Xatt BattP (data not shown). A
J
Of/B I
I
1 I
I II
I O*O
40.0
aft P I I I
47.6
/Jj 62-l
62.4
imm434
; I / 70.4
1 81.7
R
I I I 102.6
FIG. 1. The genetic and physical map of X&B-nttP imm434. The distances are given as % of h wild-type length which we take as 17.06 pm (Younghusband et (II., 1975). The physical dist,ances are taken from length measurements of at least 50 molecules. The lengths of double-st.ranclecl sections of the heteroduplexes were normalized so that the distance from the right-hand end of the imm434 region to the right-hand end of t,he heteroduplex molecule was 20.9% of A wiltl-typo length (Westmoreland et ul., 1969). The lengths of the double-stranded segments of the hetcroduplexes (as y0 of X wild-type length) were, st)arting at the left-hand end, 40.8 tO.9, @34+0.05, l&O&O.3 and 20.9&O+?. The positions of tho left and right-hand ends of the %mm434 region were taken from Westmoreland et al. (1969). The rect)angle in the map represents the extent of the substitution of bacterial DNA in X&B-nttP.
Under our spreading condit.ions, P4 and X wild-type phage DNA molecules have contour lengths of 4.1+0.07 pm and 17.06&0*32 pm, respectively (Younghusband et al., 1975). The contour lengths of hattB-attP and httL DNA molecules extracted from mature phage were 1761&0+21 pm and 15*02&0.34 pm, respectively, under these spreading conditions. (b) Kinetics
of recombinant DNA by int-me&ted recombination in deuterium oxide
of formation
Analysis of the kinetics of formation of recombinant DNA by &t-mediated recombination can be accomplished by a two-step procedure (Nash, 1975) which helps to simplify the analysis of in&mediated recombination by eliminating the need to produce viable phage in the same step as production of recombinant DNA. Thus, the effects of various inhibitors of bacterial processes (DNA replicat’ion. RNA synthesis, protein synthesis etc.) on &t-mediated recombination can be tested without concern for the effect of these inhibitors on the production of viable phages.
int
RECOMBINATION
IN
PHAGE
Z?H!)
LAMBDA
The effect of 2H,0 on i&-mediated recombination was tested. In this experiment., int gene product was supplied by a h prophage with an inf-constitutive mutat.ion (Shimada & Campbell, 1974); rifampicin and chloramphenicol were added to the lysogen prior to infection with h&B-&P in order to prevent the lag in appearance of recombinant DNA molecules that occurs in uninhibited lysogens (Nash, 1975). As can be seen in Figure 2, recombinant DNA can be found 15 minutes after infection in the presence of rifampicin and chloramphenicol in medium containing 2H20 ; this result is similar to that reported by Nash (1975) for experiments done in H,O at, 3SLC. although bot,h the fraction of recombinant DEB found and bhe slope of tht,
I
I
I
I
I
I
IO
20
30
40
50
60
Ttme after infection fmin)
Flc. 2. The kinetics of format,ion of recombinant DNA in H,O and in 2H,0 in the presence of rifampicin and chloramphenicol. HN216 was grown to O.D.590 = 0.75, incubated with rifampicin (200 pg/ml) and chloramphenicol(lO0 fig/ml) for 30 min at’ 3O”C, and infected with h&B-atlP as described in Materials and Methods. Samples of the infected lysogenic bacteria were killed at various time3 after infection and the DNA extracted. This DNA was used to transfect spheropla&s as dexribed by Nash (1975). The percentage of EDTA-resistant phage produced by the xpheroplasts is plotted on the ordinate. (0) Data from Nash (1975, Fig. 3) using different lysogenic bacteria (HN205) to provide int gene product by transient derepression of the prophage. The experiment wa4 done in T-broth at 37°C. (0) HN216 infected by X&B-attP in M9 medium (glucose) with rifampicin and chloramphenicol at 33°C. These results are the weighted average of 2 independent, experiments except for the point at 60 min, which is based on a single experiment. $~~I~~%16 inft,cted by XnttR-rcttP in 2H medium (glucose) with rifampicin and chloramphenicol
* I. curve are lower. We do not understand the difference between our results in H,O using HN216 (Fig. 2, open circles), and the results from Nash (1975) who used transient derepression of a temperature-sensitive prophage to supply int gene product, (Fig. 2, solid circles). According to our results, at 60 minutes after infection at 33°C in 2H20 (the time at which the bacteria were killed in the following experiment), there will be approximately 2076 recombinant DNA as compared with 400/, found in H,O at 60 minutes after infection at 33°C. (c) ElectroB microscopic (i) I.solation and study of phayle DNA
obserwalion~.~on intracellular
DNA
molecules
HN216, an E. coli strain carrying a A prophage was grown in 2H medium: treated with rifampicin
with an int-constitutive mutation. and ehloramphenicol. and infect,rd
390
J.
ENGLER
AND
R. 13. INMAX
with hattB-attP. After phage adsorption, t’he infected bacteria were diluted into fresh 2H medium and, after a further 60 minutes, t#he bacteria were killed and the phage DNA molecules extracted, purified by C&l density equilibrium gradient centrifugation, and examined in the elect,ron microscope. There was little or no shift of 3H counts toward the HL (heavy-light) density position in any of the CsCl gradients, indicating that very little if any replication of phage DNA molecules occurred; furthermore, in our electron microscopic study of this DNA sample, we have never seen a replication molecule.
TABLE 1 Xize-classes of molecules observed in the electron microscope Size (w) 2.38&0.05 16.09*0.18 17.61&0.17 35.15hO.06
No. of circles
20 13 84 3
No. of linears
4 6 65 0
DNA molecules from the peak fractions of the second CsCl density equilibrium gradient were dialyzed against low-salt buffer and spread according to Inman & SchnGs (1970). Molecules were photographed at random and measured on a Numonics digitizer. All size-classes were normalized so that the length of X&B-&P circular molecules was 17.51 pm as described in the text.
Both linear and circular molecules of certain discrete lengths were observed. The contour lengths of the intracellular circles found in these LL fractions yielded three main length-classes. The ratio of the lengths was 1:036:0*14, which is exactly that expected if hattB-attP DNA molecules (1751 pm) undergo intramolecular recombination to yield hattL (15.02 pm) and attR (2.49 pm). By attR, we mean the region of hattB-attP located between attB and attP; according to the Campbell (1962) model of integrative recombination, this region would contain attR at the completion of &t-mediated recombination. Accordingly, the lengths of all of the molecules were normalized so that the major class of circles was 1751 pm. The resulting normalized lengths are shown in Table 1; the results represent 120 randomly photographed circular molecules. The identification of these classes of molecules will be further strengthened by partial denatura.tion mapping (see next section). (ii) Partial denaturation mapping of the DNA molecules The various length-classes shown in Table 1 can be positively identified by partial denaturation mapping. Figure 3 compares the histogram averages of the partial denaturation maps of hattB-attP molecules extracted from the phage (Fig. 3(a)) with the histogram averages of linear (Fig. 3(b)) and circular (Fig. 3(c)) 1751+m intracellular DNA molecules. As can be seen, the histogram averages agree very well. It was also found that the linear molecules were almost always broken at the mature ends of the phage DNA molecule, as judged by the partial denaturation patterns; only one linear molecule, out of the 22 molecules measured, was broken at some other
i,rf
RECOMBISATION
IN
I’H;\GE
I~AMBI)A
391
0.6 1
-- --
L---,
2
L-- y 2
4
6
4
6
T--m
4
’
6
8
IO
Length
12
14
16
18
(pm)
Fro. 3. A comparison of the histogram averages of the partial denaturation maps of h&B-alll-’ DNA molecules extracted from the phage with histogram averages of the partially denatured 17.51.pm linear and circular intracellular DNA molecules observed in the electron microscope. (a) The histogram average of h&B-attP DNA molecules extracted from the phage. 40 molecules were measured. (b) The histogram average of partially-denatured intracellular linear DNB molecules whose lengths were approx. 17.5 pm. 21 molecules were measured; their lengths were normalized to 17.51 pm. (c) The histogram average of partially-denatured intracellular circular DNA molecules whose lengths were approx. 17.5 pm. 30 molecules were measured; their lengths were normalized to 17.51 pm. The partial denaturation maps of t,he circular molecules were artificially broken at the point corresponding to the mature ends.
.I.
E:S(:l,F:lC
.ZiVl)
8
IO
Length
,-
Ii.
H.
INMAN
12
14
16
I2
I4
I6
18
(,um)
I
(b)
l-
,-
I-
,
2
4
6
8 Length
l.o(I a 7
IO
2 Length
(wml
T
1.0.
4 (pm)
(el
0.8 t
0.2
IL Lengtii
Quill
.I 4 Length
(urn)
FIG. 4. A comparison of t,he expected histogram averages of the partial denaturation maps of AnttL and nttR with t,he observed histogram average of partially-denatured intracellular DNA molecules observed in the eleot,ron microscope. (a) The histogram average of XattB-attP DNA molecules rxtract,ed from the bacteriophage. (b) The expected histogram average of h&l. The
irzt RECOMBIXATION
IN
PH.-\GE
LAMBl)X
3!)3
point. These linear molecules probably represent uninjected or uncircularized phagtl DKA molecules. Figure 4 shows the derivation of the histogram average of the partial denaturation maps of AatrL (b) and attR (d) from XattB-attP (a). The agreement between the derived and the actual hist,ogram averages found for t,hc int~racellular molecules (Fig. -t(c) and ((1)) is quite close. Only one linear DNA molecule was found in the 28 AattL molecules measured; the partial denaturation map of this molecule indicated that it \+‘asnot. broken at t.he mature end. Eo examples of linear aft R-lengt,h molecules \ver~ found in over 800 denatured molecules examined. On the basis of the data shoun in Figures 3 and 1. we conclude that the three major types of molecules present in the LL int~racellular DXA fractions from the second density gradient are AaffB-aftf’, hatfl, and aftR. According to Table 1. the t.uo shorter forms arc present in roughly equal numbers. Examples of t’hese three typt:s of molecules are presentt~d in E’igurc 6. (iii) I’wtial
dfmaturatio~~
of the dimeric
circles
When the undenatured intracellular DNA molecules were photographed at random. approximately 39,; exhibited a length of 35 pm (Table 1). In the process of specitically looking for partially denatured 35pm circles, we have also observed a small number of circles whose lengths differ from those listed in Table 1. The results of this non-random survey indicated that. the following circular types were present. in minor amounts: 33.9&l +? pm (13 molecules). 30.210.9 pm (5 molecules), and 4.7iO.3 pm (G molecules). It should be noted t,hat these are the lengths of partially denat,ured molecules and that 33.9&1$ pm is not significantly different from thcb m&natured length of 3515~0~06 pm list,ed in Table 1. Figure 5(a) shows the partial denaturation maps of 11 of the 33.9 pm dimers (the remaining 2 arc’ discussed below). This dcnat,uration pat,tern is most easily explsined tqr insert,ion of an additional attR monomer circle into the AattB-attP denaturation rr1a.pat the position of attB. Figure 5(c) shows the partial denaturation maps of the 392-pm dimers. These dimers appear to be composed of two units of AattL arranged head-to-tail. The 4.7-p” dimers are shown in Figure 5(e) and their partia,l denaturation pattern is consistent with two attR circles joined head-t,o-tail. The two dimers in Figure 5(g) are 33.9 pm long. The parCal denaturation maps of these molecules are different from those shown in Figure 5(a) and appear to consist, of t,wo units of AattB-attP joined head-to-tail. Figures 5(b). (d), (f) and (h) represent the tltnaturation maps expected for the above four t’ypes of dimeric structures and
cxpect.crl histogram average was con&ructed by removing the region in the XattB-nttP histogram avcrago that corresponds to the DNA between crttB and &P (region II in (a)). (c) The observed hist.ogram average of partially-denatured intracellular DNA molecules whose lengths were approx. 16 pm. 28 molecules were measured. 27 of the molecules were circles; one was a linear molecule broken approx. 2 pm from the right-hand mature end of the DNA. The partial denaturation map-; of the circular molecules were artificially broken at the point corresponding to the mature ends. (d) The expected histogram average of attR. The expected histogram average was constructed from the region of the UtB-&tP histogram average that corresponds to the DNA between att~ and rlttP (region II in (a)). (e) The histogram average of partially-denatured intracellular DNA molecules whose lengt,hs were approx. 2.4 pm. 22 circles were measured ; no linear molecules were observed. These molecules were normalized to 2.38 pm and artificially broken to give the best alignment with histogram (d).
(b) c
Cd)
(e)
(h)
0
5
IO
15
20
25
30
35
40
Length (pm) PIa. 5. The partial denaturation maps of dimeric circles observed in the sample. Each horizontal line represents one DNA molecule while the black rectangles show the position and size of the denatured sites. Denatured sites located very close together are presented as one denatured site in the map. (a) The partial denaturation maps of the first type of dimer circle observed. These partial denaturation maps have been normalized to 35.02 pm. (b) Two examples of the partial denaturation map expected for a molecule consisting of 2 units: a h&L molecule and a XnttB-uttP molecule with a second 2.38-pm circle inserted at attB. These 2 denaturation maps were constructed by pasting together randomly selected partial denaturation maps of h&L, AuttB-attP and attR. (c) The partial denaturation maps of the second type of dimer circle observed (normalized to 30.04 pm). (d) Two examples of the partial denaturation map expected for a molecule consisting of 2 h&L molecules joined head-to-tail. These 2 denaturation maps were constructed from randomly selected partial denaturation maps of X&L. (a) The partial denaturation maps of the 3rd type of dimer circle observed (normalized to 4.76 pm). (f) Two examples of the partial denaturation map expected for a molecule consisting of 2 attR molecules joined head-to-tail. These
int
RECOMBINATION
IN
I’H,\CE
were obtained by pasting together randomly selected segments. Figures 7 and 8 show electron micrographs dimeric circles discussed above.
395
LAMBl)A XattB-ntfPI
of the first
AuttL and nttR three
types
of
4. Discussion In this paper, we have examined the fate of intracellular h&B-attP DNA molecules i?b viuo. We have been able to observe and study the three classes of intracellular DNA predicted by the Campbell (1962) model of &t-mediated recombination. The fa,ct that we can observe the products of int-mediated recombination in a nonreplicating system supports the suggestion by Nash (1975) that site-specific recombination is independent of DNA replication. The frequency of circular AattL and AattB-attP DNA molecules observed in the electron microscope (Table I) is consistent with the results obtained by spheroplast transfe&on ana‘lysis of intracellular DNA (Fig. 3). The frequency of AuttL and attR circles is approximately equal, as would be recombination to yield expected if one XattB-attP molecule undergoes int-mediated two product molecules. The attR circles seen in the electron microscope cannot be demonstrated by spheroplast transfection as these circles contain only attR and bio and cannot produce whole phage. It is interesting to note that i&mediated recombination in vitro results in catenat,ed DNA molecules, probably consisting of one Aaft L and one att R circle (Nash, personal communication). Most of the recombinant DNA molecules that we observe in the electron microscope are not catenated. Only rarely do we see st.ructures consistent with the catenated molecules seen by h’ash. Vsing agarose gel analysis. Kikuchi & Nash (personal communication) have also observed that in viva infmediated recombination produces free, rather than catenated, products. These results suggest t,hat some component Dhat is missing from Nash’s in, vitro recombination s>lstem normally allows separation of the catenated structures in viva. One piece of evidence that the DNA molecules that we have observed in the electron microscope are formed by &t-mediated recombination is the site-specificity of the reaction. in.t-mediated recombination is known to be site-specific (see Gottesman & W&berg, 1971). As judged by partial denaturation mapping, each hattL molecule observed was missing the same region of the DNA4 when compared with the histogram avrrapc of the partial denaturat.ion maps of XattB-aftP (Fig. 4). The histogram averagr of t,his missing region corresponds to the partial denaturation maps of the small c&R cir&s found in the experiment,. That these t,wo t>rpes of molecule ca,n be found and t,hat they always have the same partial denaturat,ion pattern demonSt,rat.Cs that the reaction is site-specific. A second piece of evidence that the molecules 2 denaturation maps were constructed from randomly srlectcd partial denaturation maps of rcttl?. (LI) The partial denaturation maps of t)he 4th type of dimer circle observed (normalized to Xi+:! pm). (h) Two examples of the partial denaturation map expected for a molecule consisting of 2 XtrttU-nttP molecules joined head-to-t’ail. These 2 denaturation maps were constructed from randomly selected partial deneturation maps of h&B-attP. The partial denaturation maps in (a), (c), (e ) and (g) are normalized to the lengths that would be expected from the model in Fig. 9. The average unnormalized lengths of the molecules in (a), (c) and (e) are all within experimental error of the normalized length. The unnormalized lengths of t,he molecules in (g) are within 2 st,andard deviations of the normalized Iengt,h; only 2 molecules were observed. The maps shown in (a), (c) and (g) havr: been art,ificially broken at the point corresponding to t,he mature ends. The maps in (e) were broken at, a point corresponding to the ends of t,he histogram in Fig. 4(d).
Length
(pm)
F‘IG. 6. Electron micrographs of partially denatured circles found among intracellular DN A mol lecules. The partial denaturet,ion maps of these molecules are reproduced for comparison wil ;h the miorogrephs. The tail of the arrow in each photograph indicates the deduced position of tl le ma1Lure ends of each molecule, while the head of the arrow indicates left to right direction alor % the molecule. Denatured sites located very close together are presented as a single denature !d site in the maps. (a) XeuB-nttP; (b) A&L; (c) CAR.
Length
(pm)
PK. 7. Electron micrograph of one type of partially denatured dimeric circle found among intracellular DNA molecules. The partial denaturation map of this molecule is reproduced for comparison with the micrograph. The tail of the arrow in the photograph indicates the deduced position of the mature ends of the molecule, while the head of the arrow indicates loft to right, rlirection along the molecule. Denatured sites located wry close together are presented as ant‘ tlouatrlwtl sitv in the map. This t,vlw of dimcr has I~rtw cvtllcvl ~r~ttL-rrtt/l-rrttR.trltI’.
398
J.
(a)
1
(b)
-
ENGLER
5
ANI)
IO
K.
15 Length
R.
20
INSI,4N
25
30
(pm)
found F ‘IQ. 8. Electron micrographs of 2 other types of partially-denatured dimeric circles DNA molecules. The partial denaturation maps of these molecules are reproamc mg intracellular with the micrographs. The tail of the arrow in each photograph in’ dicates due ed for comparison of the mature ends of the molecules, while the head of the arrow insdicates the deduced position left to right direction along each molecule. Denatured sites located very close t,oget,her are prc !sented site in the map. (a) XattP; (b) attR2. as cme denatured
int
RECOMBINATION
(a)
IN
PHAGE
LAMRl)I\ (c)
(b) AR
UflB
0
ottL UNP --
ottP
/nt
+
attL --
,ottL
mt
--UttR attB
&fP
attB
+
0
RA
ato?
ottR -0
FIG. 0. The formation of dimer circles by int-mediated recombination. (a) Two molecules of X&B-nttP. (b) The genetic structure of the first dimer formed. The expected length of these dimers would be 35.02 pm. These dimers correspond to XattL-attB-attR-attP. (c) The genetic structure of other dimers that could be formed from XnttLanttB-c&R-attP molecules by intmediated recombination. The upper molecule corresponds to 2 A&L molecules joined head-t.o-tail. The lower molecule corresponds to 2 Q&R molecules joined head-t.o-tail.
that we have studied are formed by i&mediated recombination is that hattL and attR circles are not observed among intracellular DNA molecules isolated from infection of lysogens deficient in int gene product by h&B-attP (Engler & Inman? unpublished observation). i&-mediated recombination also provides the simplest explanation for the formation of the three classes of dimers, found at low frequency and shown in Figure 5(a), (c) and (e). If an &t-mediated recombination event occurred between attB on one molecule and attP on the other molecule, the resulting molecule (Fig. 9(b)) would be a dimeric circle with a contour length of 35.02 pm. Its partial denaturation pattern would consist of two units: one unit similar to XattL and the other unit similar to Xa.ttB-c&P with a second 2.3%pm section inserted at attB. These predictions match the partial denaturation maps and contour lengths observed for 11 of the 13 33.9~pm dimers found in the DNA samples (Fig. 5(a)). These molecules contain the uttL, attB, attR and attP attachment sites and are therefore h&L-attB-attR-attP dimers. If a second i&-mediated recombination event occurs between the remaining attB and attP attachment sites on a XattL-attB-dtR-attP molecule, two product molecules would be formed (Fig. 9(c)). One of these molecules would contain two u&L attachment sites and two A-R joints; such molecules have been designated /\attL2 dimers. The contour length should be 30.04 pm. Figure 5(c) shows the partial denaturation maps of circular dimers that have the expected denaturation pattern and contour length. Similarly, the other product expected would contain two c&R attachment sites joined head-to-tail and a contour length of 4.76 pm and is shown in Figure 5(e). These molecules have been designated att R2 dimers.
400
J. ENtiLER
AND
H.
1s. ISI\Ii\N
The two molecules shown in Figure 5(g) are not predicted hy this model. ‘l’hesc molecules have partial denaturation patterns consistent \\it#h t\vo hut/ R-nttl’ molec:ulr~s joined head-t,o-tail. These moleculw might tw formed I)y t,hr wc-mediated rwonbination pathu,ay of E. coli, by joining and sealing of t,he cohesive tands of tlvo lineal X&B-nffP molecules. by attP x affP site-specific recombination. or hy some otht>r process.
Lambda attB-attP has proved to be a good syst,em for the study of i&-mediated recombination because it undergoes primarily intramolecular recombination. One of the products of this recombination event, the attR circle, can be easily distinguished from the starting DNA molecules making it relatively easy to look at DNA samples and determine whether &t-mediated recombination has occurred. We hope to exploit this system in our at.tempts to observe intermediat,es in integrative recombination. We would like to thank Dr Howard Nash for providilrg many bacteria used in this work and for his advice and criticism of thank Dr Janet Geisselsoder for her criticism of the manuscript. of MS Selma Sachs is gratefully acknowledged. This work was the National Institutes of Health (5 ROI GM1471 l- 10) and the (VC-6lE).
of the bacteriophages and the manuscript. We also Tile technical assistance support,ed by grants from Amc%rican Cancer Socict>
REFERENCES Campbell, A. (1962). ArEvan. Genel. 11, 101-145. Chattoraj, D. K. & Inman, R. B. (1974). Proc. h’at. Acad. Sci., U.S.A. 71, 311-314. Davidson, N. & Szybalski, W. (1971). In The Bacterio&ge Lambda (Hershey, A. D., ed.), pp. 45-83, Cold Spring Harbor Laboratory, New York. Davis, R. W. & Parkinson, J. S. (197 1). J. Mol. Biol. 56, 403-423. Gottesman, M. E. & Weisberg, R. A. (1971). In The Bacteriophage Lambda (Hershey, A. D., cd.), pp. 113-138, Cold Spring Harbor Laboratory, New York. Inman, R. B. & Schnijs, M. (1970). J. Mol. Biol. 49. 93-98. Nash, H. A. (1974). V’iroZogy, 57, 207-216. Nash, H. A. (1975). J. &i’oZ. Biol. 91, 501-514. Shimada, K. & Campbell, A. (1974). l;iroZogy, 60, 157- 165. Valenzuela, M. S., Freifelder, D. & Inman, R. B. (1976). ,7. Mol. Rio1 102, 569-581. Weiner, A. M. & Weber, K. (1973). J. ,woE. Biol. 80, 837..855. Westmoreland, B. C., Szybalski, W. & Kis, H. (1969). Scievnce, 163, 1343.-1348. \-ounphusband, H. B., Egan, J. B. & Tnman, R. B. ( 197.5). &lol. Cen. Tenet. 140, 101--l IO.
(1977) 113, 385-40
Site-specific Recombination JEFFREY
ENGLER
in Bacteriophage Lambda AND Ross B. INMAN
Biophysics Laboratory of the Graduate School and Biochemistry Departm,ed of thP College of Agricultural and Lifp Scie~xes University of Wisconsin Madison, Wise. 53706: U.S.A. (Received 29 October 1976, and in revised form 17 February
1977)
Lambda attB-&P is a derix,ative of bacteriophage lambda t,hat contains both for integrative recombination. Lambda attBattB and at@, two sites required attP can undergo &-mediated recombination to yield progeny phages whose DNA is 1574 shorter than that of the parental phage. We have studied intracellular phage DNA from an infection of lysogenic bacteria with hattB-attP in the presence of int gene product, rifampicin and chloramphenicol. The majority of the intracellular phage DNA consisted of circles with lengths of 17.51, 15.09 and 2.38 pm. Partial dena.turation mapping confirmed that the 1509 and t,he 2.3%pm molec111os arose by an &-mediated intramolecular recombination reaction of the type predicted by the Campbell (1962) model. A minor proportion of the circles (30/b) were much larger (33.9, 30.2 and 4.7 pm) ; in t’hese cases denaturation mapping indicated that both intra- and inbermolrcular recombination could take place.
1. Introduction Lambda, a temperate bacteriophage of Escherichia coli, can insert its DNA into the bacterial genome under certain conditions. Our current understanding of this process is that this insertion occurs by a reciprocal recombination event at a specific attachment s&e on both the bacterial and the bacteriophage DNA molecules. This process also requires the product of the hint gene (for a re ?iew of int-mediated recombination, see Gottesman & Weisberg, 1971). A variant of X which arose by an illegitimate recombination event and which contains both the bacterial attachment site (attB) and the bacteriophage attachment site (attP) has been isolated @ash, 1974). The plaque-forming ability of this phage. called hattB-attP, is sensitive to chelating agents such as sodium pyrophosphate and EDTA. In the presence of int gene product, XattB-attP yields a high frequency of progeny phage whose plaque-forming ability is more resistant’ to sodium pyrophosphate. In the absence of the int gene product, these pyrophosphate-resistant phages are not formed. The Ret recombination pathway of E. toll: cannot. by itself, produce these pyrophosphate-resistant phages. Formation of pyrophosphate-resistant phages is independent of the presence of the xis gene product. These findings suggest that zkt-mediated recombination allows hattB-attP to lose DNA. presumably the DN,4
3citi
,J. EN(:I,EH
;\Sl)
R. B. INMAS
between attB and attP. Roth contour lengt,h measurements and CWI density cquilibrium centrifugation of AattB-attP and the p?-rophosphatc,-resistnllt phage show that t’he latter contains about 15”;, less DNA t)han does AaffR-attP (Nash (1974) and confirmed in this paper). In a subsequent paper, Nash (1975) demonst,rated that’ rccombinantj phagr: DNA first appears 20 to 30 minutes after infection at 37°C. R’o recombinant DNA can bo found if the int gene product’ is defective or if cyanide or dinitrophenol are present. The delay in the appearance of recombinant DNA molecules can be eliminated by interfering with the expression of the infecting phage genome. either by t,he use of inhibitors (rifampicin and chloramphenicol) or by the binding of h repressor to the phage DNA (infection of a bacteria lysogenic for lambda by a homoimmune AattBnttP). Nash’s work suggested that XattB-attP might be an ideal model for the study of the fate of phage DNA during in&mediated recombination in vivo. In this paper, we present an electron microscopic study of the intracellular DNA products that result from infection with AattB-attP. We have identified XattB-attP DNA molecules as well as the two product molecules expected from &t-mediated recombination. Our studies show that these products are free circles and that they are not catenated. We have also found dimer length molecules that arise at low frequency by intmediated recombination. These studies should form the basis for a future investigation of the intermediates in this interesting reaction.
2. Materials and Methods (;I) Bacteria
and
bacteriophage
strains
were
used: MVlrecA - sup’, pl~age TI-resistant The following strains of E. coli K12 (derived from strain 152, Valenzuola et al. (1976), SH28tp~~(C. Cross via E. Calva), and HN216 (N99 (Ahint-c,,,cI857), from H. Nash). The following strains of bacteriophage X were usrti: AattU-attl’ int2 sisl red114 immidd clear (Nash, 1975), hgaZ49 intd xisl red114 imm434 clear and Ab522 (all from H. Nash). recombination between attB and attL and attR are the 2 sites produced by integrative at@ and correspond to the attachment, sites found at the left, and right-hand ends of the int2 xisl red114 imm434 clear is derived from prophage, respectively. Rocause hgalll~ XattB-attP by integrative recombination and contains attL (Nash, 1974), this phage will be referred to as XattL in the text. (b) Media
and plates
M9 minimal medium consists of 20 ml of salt mixture (70 g Na,HPO,, 30 g KH,P04, 5 g NaCl, and 10 g NH,Cl/l H,O), 20 ml 4% sugar (glucose or maltose), 2 ml CaCl, (0.01 %I), 2 ml MgSOl (0.1 M) and 156 ml H,O. T-broth consists of 5 g NaCl and IO g Bacto-tryptone/l are TB-plates containing 2.68 to HzO; TB-plates are T-broth plus 1% agar. TE-plat’es 2.75 mM-EDTA (pH 8); X&B-attP phage do not grow on TE-plates while XattL phage plate well. Tris/Mg medium is 0.1 M-Tris (pH 7.2), 0.01 hl-MgSO.,. TBY, TBS, TBSS media and SE buffer are described by Nasli (1975). 2H medium is IO ml phosphate buffer (0.2 M 1 ml in 2Hz0, pH 7.2), 0.1 ml MgS04 (1 M in ZH,O), 0.01 ml FeSOa (0.016 M in 2H,0), (15NH4)sS0, (5% solution in 2HZO), 5 ml 5% srlgar (glucose or maltose in 2H,0), and 84 ml 2HZ0. (c)
Preparation
of bacteriophages
For unlabelled phage stocks, E. coli strain MVl was grown at 37°C to o,D.~~~ z 0.75, and infected with phage (multicentrifuged, resuspended in 0.1 vol. Tris/Mg medium, plicity of infection (m.o.i.) = 0.1). After 10 min on ice and 10 min at 37”C, the infected
int RECOMBINATION
IN PHAGE
LAMBDA
387
cells were diluted in fresh, prewarmed T-broth at 37°C and incubated until lysis of the infected bacteria was complete, as judged by O.D.,,,. The lysate was treated with ehloroform and centrifuged at 10,000 g for 10 min. 0.1 vol. 5 M-N&l followed by 10% (w/v) Carbowax 6000 (Schwartz/Mann) was added to the supernatant. The mixture was swirled at 4°C until dissolved and allowed to stand overnight at 4°C. This mixture was centrifuged at 10,000 g for 15 min. The pellet was resuspended in Tris/Mg medium. The phage were purified from this pellet by CsCl banding; this stock was dialyzed in Tris/Mg medium and stored at 4°C. For preparation of 3H-labelled bacteriophage, strain SH28 was grown at 37°C to o.n+s,, = 0.75 in M9 (maltose), 1:/o Casamino acids, Bi (10 pg/ml) and thymidine (10 fig/ml). The bacteria were centrifuged, resuspended in 0.1 vol. Tris/Mg medium. alld infectfed with phage (m.o.i. = 2 to 3). After 10 min on ice and 10 min at 37”C, the infected bacteria were diluted into warmed MS medium (glucose), 1e/o Casamino acids, B1 (10 @g/ml) and [3H]t,hymidine (O-023 pg/ml, spec. act. = 200 mCi/mg; New England Nuclear). Cell lgsis was generally complete within 90 min after dilution into fresh MS medium. The phage were purified in the same way as unlabelled phage. Specific activities of IO-6 to 8 x 10 -a cts/min per plaque-forming unit were achieved. (d) Heteroduplex mapping Tllcx methods described by Dalris & Parkinson (1971) as modified by Chattoraj & Inman (1974) were followed. Het’eroduplox mapping of Ab522 was consistent with results presentod by Davidson & Szybalski (197 1). (0) Spheroplast
transjections
The isolation of intracellular DNA for use in spheroplast transfections and the procedure for spheroplast transfection were both described by Nash (1975). Stocks of rifampicin (2 mg/ml, from Calbiochem, prepared according to Weiner & Weber (1973)) and chlorsmphenicol (10 mg/ml in 95% ethanol, from Calbiochem) were prepared fresh for each experiment and kept on ice away from light. Bacteria were incubated with rifampicin and chloramphenicol (final concns 200 pg/ml and 100 pg/ml, respectively) for 30 min at 30°C in the dark before infection. (f) Ieolation of intracellular phage DNA for electron microscopy Strain HN216 was grown for 3 to 4 generations at 30°C in 2H medium (maltose), ZH,O-algal hydrolysate (O*O75o/o,Merck, Sharp and Dohme), B, (10 pg/ml), and thymidine = 0.75, the bacteria were centrifuged and resuspended in 0.1 vol. (10 pg/ml). At o.D.~~~ 0.01 M-MgSO, in 2H20. Rifampicin (prepared with ‘H,O instead of H,O) and chloramphenicol were added to final concns of 200 pg/ml and 100 rg/ml, respectively. The bacteria were incubated for 30 min at 30°C in the dark. 3H-labelled XattB-attP was added (m.o.i. = 10) and adsorbed for 10 min in ice and 10 min at 30°C. The infected bacteria were diluted into 2H medium (glucose), 2H20-algal hydrolysate (O*075°h), B, (10 pg/ml), and thymidine (10 pglml), containing rifampicin and chloramphenicol at the same final concns a.s above and prewarmed to 33°C. After 60 min at 33”C, 12-ml samples of the infected bacteria were poured int.0 12 ml of cold 30% (v/v) pyridine in 0.05 M-KCN, 0.05 M-NaNs, 0.01 M-EDTA that had previously been poured over 8 g of crushed ice. After 2 to 5 min at 4”C, the bacteria were centrifuged and resuspended in 1.5 ml of 0.05 M-KCN, 0.05 XNaN,, 0.01 M-EDTA. 15 ~1 of a freshly prepared solution of lysozyme in water (50 mg/ml) was added: the suspension was frozen and thawed 3 times, followed by incubation on ice for 20 min. 25 ~1 of 30% Sarkosyl were added and the suspension incubated at 4°C for 15 min and then 5 min at 50°C. 50 ~1 of Pronase (20 mg/ml in 0.005 M-EDTA, self-digested for 4 h at 37°C) were added and incubation was for 2 to 4 h at 37°C with slow shaking. The lysate was cooled and adjusted to a density of 1.67 g/cm3 with saturated CsCl and centrifuged at 39,000 revs/min for 3 days at 9°C in a 60 Ti rotor. The gradient was fractionated from the top at. a rate of O-5 ml/min in 0.2-m] fra&ions. Fractions containing 3H counts were found at the LL (light-light) position in the gradient: these fractions were pooled, made up to a density of 1.67 g/cm3, centrifuged for 3 days at 30,000 revs/min in a 60 Ti
3XX
.J. ESGLElC
rot,or, and refract,ionated. 0.0% M-NaC1/0.005 M-EDTA Inmali & S&n& (1970). (g)
Electron
;\NI)
It.
I<. IXMAN
Tl~n fractions oont,aining “H counts WXIC dialyzed against (pH 8) and prepared for clcctrorl microscopy as described 111
microscopy
am!
DNA molecules were pllot.ographrd graphics calculator (Numonics Corp., 9820 calculator and 9862A plotter.
computation,
of electron
n~icrosco~x
tlato
~IKI their Icngtha measured using :L Nu~onics North Wales, Pa) interfaced to a Hewlett,-Packard
3. Results (a) Heteroduplex
analysis
and contour length of hattB-attP
The strucbure of hattB-attf’ shown in Figure 1 was determined by studying heteroduplex molecules prepared from hattB-attP and /\b522 and from hattB-attP and hattl. Lambda b522 forms a deletion loop that can be used to identify the position of attP in heteroduplexes of h phages (Davidson & Szybalski, 1971). The structure shown in Figure 1 agrees with the structure shown by h’ash (1975) and is consistent’ with the observed difference between the partial denaturation maps of X wild-type and Xatt BattP (data not shown). A
J
Of/B I
I
1 I
I II
I O*O
40.0
aft P I I I
47.6
/Jj 62-l
62.4
imm434
; I / 70.4
1 81.7
R
I I I 102.6
FIG. 1. The genetic and physical map of X&B-nttP imm434. The distances are given as % of h wild-type length which we take as 17.06 pm (Younghusband et (II., 1975). The physical dist,ances are taken from length measurements of at least 50 molecules. The lengths of double-st.ranclecl sections of the heteroduplexes were normalized so that the distance from the right-hand end of the imm434 region to the right-hand end of t,he heteroduplex molecule was 20.9% of A wiltl-typo length (Westmoreland et ul., 1969). The lengths of the double-stranded segments of the hetcroduplexes (as y0 of X wild-type length) were, st)arting at the left-hand end, 40.8 tO.9, @34+0.05, l&O&O.3 and 20.9&O+?. The positions of tho left and right-hand ends of the %mm434 region were taken from Westmoreland et al. (1969). The rect)angle in the map represents the extent of the substitution of bacterial DNA in X&B-nttP.
Under our spreading condit.ions, P4 and X wild-type phage DNA molecules have contour lengths of 4.1+0.07 pm and 17.06&0*32 pm, respectively (Younghusband et al., 1975). The contour lengths of hattB-attP and httL DNA molecules extracted from mature phage were 1761&0+21 pm and 15*02&0.34 pm, respectively, under these spreading conditions. (b) Kinetics
of recombinant DNA by int-me&ted recombination in deuterium oxide
of formation
Analysis of the kinetics of formation of recombinant DNA by &t-mediated recombination can be accomplished by a two-step procedure (Nash, 1975) which helps to simplify the analysis of in&mediated recombination by eliminating the need to produce viable phage in the same step as production of recombinant DNA. Thus, the effects of various inhibitors of bacterial processes (DNA replicat’ion. RNA synthesis, protein synthesis etc.) on &t-mediated recombination can be tested without concern for the effect of these inhibitors on the production of viable phages.
int
RECOMBINATION
IN
PHAGE
Z?H!)
LAMBDA
The effect of 2H,0 on i&-mediated recombination was tested. In this experiment., int gene product was supplied by a h prophage with an inf-constitutive mutat.ion (Shimada & Campbell, 1974); rifampicin and chloramphenicol were added to the lysogen prior to infection with h&B-&P in order to prevent the lag in appearance of recombinant DNA molecules that occurs in uninhibited lysogens (Nash, 1975). As can be seen in Figure 2, recombinant DNA can be found 15 minutes after infection in the presence of rifampicin and chloramphenicol in medium containing 2H20 ; this result is similar to that reported by Nash (1975) for experiments done in H,O at, 3SLC. although bot,h the fraction of recombinant DEB found and bhe slope of tht,
I
I
I
I
I
I
IO
20
30
40
50
60
Ttme after infection fmin)
Flc. 2. The kinetics of format,ion of recombinant DNA in H,O and in 2H,0 in the presence of rifampicin and chloramphenicol. HN216 was grown to O.D.590 = 0.75, incubated with rifampicin (200 pg/ml) and chloramphenicol(lO0 fig/ml) for 30 min at’ 3O”C, and infected with h&B-atlP as described in Materials and Methods. Samples of the infected lysogenic bacteria were killed at various time3 after infection and the DNA extracted. This DNA was used to transfect spheropla&s as dexribed by Nash (1975). The percentage of EDTA-resistant phage produced by the xpheroplasts is plotted on the ordinate. (0) Data from Nash (1975, Fig. 3) using different lysogenic bacteria (HN205) to provide int gene product by transient derepression of the prophage. The experiment wa4 done in T-broth at 37°C. (0) HN216 infected by X&B-attP in M9 medium (glucose) with rifampicin and chloramphenicol at 33°C. These results are the weighted average of 2 independent, experiments except for the point at 60 min, which is based on a single experiment. $~~I~~%16 inft,cted by XnttR-rcttP in 2H medium (glucose) with rifampicin and chloramphenicol
* I. curve are lower. We do not understand the difference between our results in H,O using HN216 (Fig. 2, open circles), and the results from Nash (1975) who used transient derepression of a temperature-sensitive prophage to supply int gene product, (Fig. 2, solid circles). According to our results, at 60 minutes after infection at 33°C in 2H20 (the time at which the bacteria were killed in the following experiment), there will be approximately 2076 recombinant DNA as compared with 400/, found in H,O at 60 minutes after infection at 33°C. (c) ElectroB microscopic (i) I.solation and study of phayle DNA
obserwalion~.~on intracellular
DNA
molecules
HN216, an E. coli strain carrying a A prophage was grown in 2H medium: treated with rifampicin
with an int-constitutive mutation. and ehloramphenicol. and infect,rd
390
J.
ENGLER
AND
R. 13. INMAX
with hattB-attP. After phage adsorption, t’he infected bacteria were diluted into fresh 2H medium and, after a further 60 minutes, t#he bacteria were killed and the phage DNA molecules extracted, purified by C&l density equilibrium gradient centrifugation, and examined in the elect,ron microscope. There was little or no shift of 3H counts toward the HL (heavy-light) density position in any of the CsCl gradients, indicating that very little if any replication of phage DNA molecules occurred; furthermore, in our electron microscopic study of this DNA sample, we have never seen a replication molecule.
TABLE 1 Xize-classes of molecules observed in the electron microscope Size (w) 2.38&0.05 16.09*0.18 17.61&0.17 35.15hO.06
No. of circles
20 13 84 3
No. of linears
4 6 65 0
DNA molecules from the peak fractions of the second CsCl density equilibrium gradient were dialyzed against low-salt buffer and spread according to Inman & SchnGs (1970). Molecules were photographed at random and measured on a Numonics digitizer. All size-classes were normalized so that the length of X&B-&P circular molecules was 17.51 pm as described in the text.
Both linear and circular molecules of certain discrete lengths were observed. The contour lengths of the intracellular circles found in these LL fractions yielded three main length-classes. The ratio of the lengths was 1:036:0*14, which is exactly that expected if hattB-attP DNA molecules (1751 pm) undergo intramolecular recombination to yield hattL (15.02 pm) and attR (2.49 pm). By attR, we mean the region of hattB-attP located between attB and attP; according to the Campbell (1962) model of integrative recombination, this region would contain attR at the completion of &t-mediated recombination. Accordingly, the lengths of all of the molecules were normalized so that the major class of circles was 1751 pm. The resulting normalized lengths are shown in Table 1; the results represent 120 randomly photographed circular molecules. The identification of these classes of molecules will be further strengthened by partial denatura.tion mapping (see next section). (ii) Partial denaturation mapping of the DNA molecules The various length-classes shown in Table 1 can be positively identified by partial denaturation mapping. Figure 3 compares the histogram averages of the partial denaturation maps of hattB-attP molecules extracted from the phage (Fig. 3(a)) with the histogram averages of linear (Fig. 3(b)) and circular (Fig. 3(c)) 1751+m intracellular DNA molecules. As can be seen, the histogram averages agree very well. It was also found that the linear molecules were almost always broken at the mature ends of the phage DNA molecule, as judged by the partial denaturation patterns; only one linear molecule, out of the 22 molecules measured, was broken at some other
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Fro. 3. A comparison of the histogram averages of the partial denaturation maps of h&B-alll-’ DNA molecules extracted from the phage with histogram averages of the partially denatured 17.51.pm linear and circular intracellular DNA molecules observed in the electron microscope. (a) The histogram average of h&B-attP DNA molecules extracted from the phage. 40 molecules were measured. (b) The histogram average of partially-denatured intracellular linear DNB molecules whose lengths were approx. 17.5 pm. 21 molecules were measured; their lengths were normalized to 17.51 pm. (c) The histogram average of partially-denatured intracellular circular DNA molecules whose lengths were approx. 17.5 pm. 30 molecules were measured; their lengths were normalized to 17.51 pm. The partial denaturation maps of t,he circular molecules were artificially broken at the point corresponding to the mature ends.
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FIG. 4. A comparison of t,he expected histogram averages of the partial denaturation maps of AnttL and nttR with t,he observed histogram average of partially-denatured intracellular DNA molecules observed in the eleot,ron microscope. (a) The histogram average of XattB-attP DNA molecules rxtract,ed from the bacteriophage. (b) The expected histogram average of h&l. The
irzt RECOMBIXATION
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point. These linear molecules probably represent uninjected or uncircularized phagtl DKA molecules. Figure 4 shows the derivation of the histogram average of the partial denaturation maps of AatrL (b) and attR (d) from XattB-attP (a). The agreement between the derived and the actual hist,ogram averages found for t,hc int~racellular molecules (Fig. -t(c) and ((1)) is quite close. Only one linear DNA molecule was found in the 28 AattL molecules measured; the partial denaturation map of this molecule indicated that it \+‘asnot. broken at t.he mature end. Eo examples of linear aft R-lengt,h molecules \ver~ found in over 800 denatured molecules examined. On the basis of the data shoun in Figures 3 and 1. we conclude that the three major types of molecules present in the LL int~racellular DXA fractions from the second density gradient are AaffB-aftf’, hatfl, and aftR. According to Table 1. the t.uo shorter forms arc present in roughly equal numbers. Examples of t’hese three typt:s of molecules are presentt~d in E’igurc 6. (iii) I’wtial
dfmaturatio~~
of the dimeric
circles
When the undenatured intracellular DNA molecules were photographed at random. approximately 39,; exhibited a length of 35 pm (Table 1). In the process of specitically looking for partially denatured 35pm circles, we have also observed a small number of circles whose lengths differ from those listed in Table 1. The results of this non-random survey indicated that. the following circular types were present. in minor amounts: 33.9&l +? pm (13 molecules). 30.210.9 pm (5 molecules), and 4.7iO.3 pm (G molecules). It should be noted t,hat these are the lengths of partially denat,ured molecules and that 33.9&1$ pm is not significantly different from thcb m&natured length of 3515~0~06 pm list,ed in Table 1. Figure 5(a) shows the partial denaturation maps of 11 of the 33.9 pm dimers (the remaining 2 arc’ discussed below). This dcnat,uration pat,tern is most easily explsined tqr insert,ion of an additional attR monomer circle into the AattB-attP denaturation rr1a.pat the position of attB. Figure 5(c) shows the partial denaturation maps of the 392-pm dimers. These dimers appear to be composed of two units of AattL arranged head-to-tail. The 4.7-p” dimers are shown in Figure 5(e) and their partia,l denaturation pattern is consistent with two attR circles joined head-t,o-tail. The two dimers in Figure 5(g) are 33.9 pm long. The parCal denaturation maps of these molecules are different from those shown in Figure 5(a) and appear to consist, of t,wo units of AattB-attP joined head-to-tail. Figures 5(b). (d), (f) and (h) represent the tltnaturation maps expected for the above four t’ypes of dimeric structures and
cxpect.crl histogram average was con&ructed by removing the region in the XattB-nttP histogram avcrago that corresponds to the DNA between crttB and &P (region II in (a)). (c) The observed hist.ogram average of partially-denatured intracellular DNA molecules whose lengths were approx. 16 pm. 28 molecules were measured. 27 of the molecules were circles; one was a linear molecule broken approx. 2 pm from the right-hand mature end of the DNA. The partial denaturation map-; of the circular molecules were artificially broken at the point corresponding to the mature ends. (d) The expected histogram average of attR. The expected histogram average was constructed from the region of the UtB-&tP histogram average that corresponds to the DNA between att~ and rlttP (region II in (a)). (e) The histogram average of partially-denatured intracellular DNA molecules whose lengt,hs were approx. 2.4 pm. 22 circles were measured ; no linear molecules were observed. These molecules were normalized to 2.38 pm and artificially broken to give the best alignment with histogram (d).
(b) c
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(e)
(h)
0
5
IO
15
20
25
30
35
40
Length (pm) PIa. 5. The partial denaturation maps of dimeric circles observed in the sample. Each horizontal line represents one DNA molecule while the black rectangles show the position and size of the denatured sites. Denatured sites located very close together are presented as one denatured site in the map. (a) The partial denaturation maps of the first type of dimer circle observed. These partial denaturation maps have been normalized to 35.02 pm. (b) Two examples of the partial denaturation map expected for a molecule consisting of 2 units: a h&L molecule and a XnttB-uttP molecule with a second 2.38-pm circle inserted at attB. These 2 denaturation maps were constructed by pasting together randomly selected partial denaturation maps of h&L, AuttB-attP and attR. (c) The partial denaturation maps of the second type of dimer circle observed (normalized to 30.04 pm). (d) Two examples of the partial denaturation map expected for a molecule consisting of 2 h&L molecules joined head-to-tail. These 2 denaturation maps were constructed from randomly selected partial denaturation maps of X&L. (a) The partial denaturation maps of the 3rd type of dimer circle observed (normalized to 4.76 pm). (f) Two examples of the partial denaturation map expected for a molecule consisting of 2 attR molecules joined head-to-tail. These
int
RECOMBINATION
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were obtained by pasting together randomly selected segments. Figures 7 and 8 show electron micrographs dimeric circles discussed above.
395
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4. Discussion In this paper, we have examined the fate of intracellular h&B-attP DNA molecules i?b viuo. We have been able to observe and study the three classes of intracellular DNA predicted by the Campbell (1962) model of &t-mediated recombination. The fa,ct that we can observe the products of int-mediated recombination in a nonreplicating system supports the suggestion by Nash (1975) that site-specific recombination is independent of DNA replication. The frequency of circular AattL and AattB-attP DNA molecules observed in the electron microscope (Table I) is consistent with the results obtained by spheroplast transfe&on ana‘lysis of intracellular DNA (Fig. 3). The frequency of AuttL and attR circles is approximately equal, as would be recombination to yield expected if one XattB-attP molecule undergoes int-mediated two product molecules. The attR circles seen in the electron microscope cannot be demonstrated by spheroplast transfection as these circles contain only attR and bio and cannot produce whole phage. It is interesting to note that i&mediated recombination in vitro results in catenat,ed DNA molecules, probably consisting of one Aaft L and one att R circle (Nash, personal communication). Most of the recombinant DNA molecules that we observe in the electron microscope are not catenated. Only rarely do we see st.ructures consistent with the catenated molecules seen by h’ash. Vsing agarose gel analysis. Kikuchi & Nash (personal communication) have also observed that in viva infmediated recombination produces free, rather than catenated, products. These results suggest t,hat some component Dhat is missing from Nash’s in, vitro recombination s>lstem normally allows separation of the catenated structures in viva. One piece of evidence that the DNA molecules that we have observed in the electron microscope are formed by &t-mediated recombination is the site-specificity of the reaction. in.t-mediated recombination is known to be site-specific (see Gottesman & W&berg, 1971). As judged by partial denaturation mapping, each hattL molecule observed was missing the same region of the DNA4 when compared with the histogram avrrapc of the partial denaturat.ion maps of XattB-aftP (Fig. 4). The histogram averagr of t,his missing region corresponds to the partial denaturation maps of the small c&R cir&s found in the experiment,. That these t,wo t>rpes of molecule ca,n be found and t,hat they always have the same partial denaturat,ion pattern demonSt,rat.Cs that the reaction is site-specific. A second piece of evidence that the molecules 2 denaturation maps were constructed from randomly srlectcd partial denaturation maps of rcttl?. (LI) The partial denaturation maps of t)he 4th type of dimer circle observed (normalized to Xi+:! pm). (h) Two examples of the partial denaturation map expected for a molecule consisting of 2 XtrttU-nttP molecules joined head-to-t’ail. These 2 denaturation maps were constructed from randomly selected partial deneturation maps of h&B-attP. The partial denaturation maps in (a), (c), (e ) and (g) are normalized to the lengths that would be expected from the model in Fig. 9. The average unnormalized lengths of the molecules in (a), (c) and (e) are all within experimental error of the normalized length. The unnormalized lengths of t,he molecules in (g) are within 2 st,andard deviations of the normalized Iengt,h; only 2 molecules were observed. The maps shown in (a), (c) and (g) havr: been art,ificially broken at the point corresponding to t,he mature ends. The maps in (e) were broken at, a point corresponding to the ends of t,he histogram in Fig. 4(d).
Length
(pm)
F‘IG. 6. Electron micrographs of partially denatured circles found among intracellular DN A mol lecules. The partial denaturet,ion maps of these molecules are reproduced for comparison wil ;h the miorogrephs. The tail of the arrow in each photograph indicates the deduced position of tl le ma1Lure ends of each molecule, while the head of the arrow indicates left to right direction alor % the molecule. Denatured sites located very close together are presented as a single denature !d site in the maps. (a) XeuB-nttP; (b) A&L; (c) CAR.
Length
(pm)
PK. 7. Electron micrograph of one type of partially denatured dimeric circle found among intracellular DNA molecules. The partial denaturation map of this molecule is reproduced for comparison with the micrograph. The tail of the arrow in the photograph indicates the deduced position of the mature ends of the molecule, while the head of the arrow indicates loft to right, rlirection along the molecule. Denatured sites located wry close together are presented as ant‘ tlouatrlwtl sitv in the map. This t,vlw of dimcr has I~rtw cvtllcvl ~r~ttL-rrtt/l-rrttR.trltI’.
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30
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found F ‘IQ. 8. Electron micrographs of 2 other types of partially-denatured dimeric circles DNA molecules. The partial denaturation maps of these molecules are reproamc mg intracellular with the micrographs. The tail of the arrow in each photograph in’ dicates due ed for comparison of the mature ends of the molecules, while the head of the arrow insdicates the deduced position left to right direction along each molecule. Denatured sites located very close t,oget,her are prc !sented site in the map. (a) XattP; (b) attR2. as cme denatured
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RECOMBINATION
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FIG. 0. The formation of dimer circles by int-mediated recombination. (a) Two molecules of X&B-nttP. (b) The genetic structure of the first dimer formed. The expected length of these dimers would be 35.02 pm. These dimers correspond to XattL-attB-attR-attP. (c) The genetic structure of other dimers that could be formed from XnttLanttB-c&R-attP molecules by intmediated recombination. The upper molecule corresponds to 2 A&L molecules joined head-t.o-tail. The lower molecule corresponds to 2 Q&R molecules joined head-t.o-tail.
that we have studied are formed by i&mediated recombination is that hattL and attR circles are not observed among intracellular DNA molecules isolated from infection of lysogens deficient in int gene product by h&B-attP (Engler & Inman? unpublished observation). i&-mediated recombination also provides the simplest explanation for the formation of the three classes of dimers, found at low frequency and shown in Figure 5(a), (c) and (e). If an &t-mediated recombination event occurred between attB on one molecule and attP on the other molecule, the resulting molecule (Fig. 9(b)) would be a dimeric circle with a contour length of 35.02 pm. Its partial denaturation pattern would consist of two units: one unit similar to XattL and the other unit similar to Xa.ttB-c&P with a second 2.3%pm section inserted at attB. These predictions match the partial denaturation maps and contour lengths observed for 11 of the 13 33.9~pm dimers found in the DNA samples (Fig. 5(a)). These molecules contain the uttL, attB, attR and attP attachment sites and are therefore h&L-attB-attR-attP dimers. If a second i&-mediated recombination event occurs between the remaining attB and attP attachment sites on a XattL-attB-dtR-attP molecule, two product molecules would be formed (Fig. 9(c)). One of these molecules would contain two u&L attachment sites and two A-R joints; such molecules have been designated /\attL2 dimers. The contour length should be 30.04 pm. Figure 5(c) shows the partial denaturation maps of circular dimers that have the expected denaturation pattern and contour length. Similarly, the other product expected would contain two c&R attachment sites joined head-to-tail and a contour length of 4.76 pm and is shown in Figure 5(e). These molecules have been designated att R2 dimers.
400
J. ENtiLER
AND
H.
1s. ISI\Ii\N
The two molecules shown in Figure 5(g) are not predicted hy this model. ‘l’hesc molecules have partial denaturation patterns consistent \\it#h t\vo hut/ R-nttl’ molec:ulr~s joined head-t,o-tail. These moleculw might tw formed I)y t,hr wc-mediated rwonbination pathu,ay of E. coli, by joining and sealing of t,he cohesive tands of tlvo lineal X&B-nffP molecules. by attP x affP site-specific recombination. or hy some otht>r process.
Lambda attB-attP has proved to be a good syst,em for the study of i&-mediated recombination because it undergoes primarily intramolecular recombination. One of the products of this recombination event, the attR circle, can be easily distinguished from the starting DNA molecules making it relatively easy to look at DNA samples and determine whether &t-mediated recombination has occurred. We hope to exploit this system in our at.tempts to observe intermediat,es in integrative recombination. We would like to thank Dr Howard Nash for providilrg many bacteria used in this work and for his advice and criticism of thank Dr Janet Geisselsoder for her criticism of the manuscript. of MS Selma Sachs is gratefully acknowledged. This work was the National Institutes of Health (5 ROI GM1471 l- 10) and the (VC-6lE).
of the bacteriophages and the manuscript. We also Tile technical assistance support,ed by grants from Amc%rican Cancer Socict>
REFERENCES Campbell, A. (1962). ArEvan. Genel. 11, 101-145. Chattoraj, D. K. & Inman, R. B. (1974). Proc. h’at. Acad. Sci., U.S.A. 71, 311-314. Davidson, N. & Szybalski, W. (1971). In The Bacterio&ge Lambda (Hershey, A. D., ed.), pp. 45-83, Cold Spring Harbor Laboratory, New York. Davis, R. W. & Parkinson, J. S. (197 1). J. Mol. Biol. 56, 403-423. Gottesman, M. E. & Weisberg, R. A. (1971). In The Bacteriophage Lambda (Hershey, A. D., cd.), pp. 113-138, Cold Spring Harbor Laboratory, New York. Inman, R. B. & Schnijs, M. (1970). J. Mol. Biol. 49. 93-98. Nash, H. A. (1974). V’iroZogy, 57, 207-216. Nash, H. A. (1975). J. &i’oZ. Biol. 91, 501-514. Shimada, K. & Campbell, A. (1974). l;iroZogy, 60, 157- 165. Valenzuela, M. S., Freifelder, D. & Inman, R. B. (1976). ,7. Mol. Rio1 102, 569-581. Weiner, A. M. & Weber, K. (1973). J. ,woE. Biol. 80, 837..855. Westmoreland, B. C., Szybalski, W. & Kis, H. (1969). Scievnce, 163, 1343.-1348. \-ounphusband, H. B., Egan, J. B. & Tnman, R. B. ( 197.5). &lol. Cen. Tenet. 140, 101--l IO.