Cell, Vol. 25, 729-736, September 1981, Copyright 0 1981 by MIT

A Novel Role for Site-Specific Recombination in Maintenance of Bacterial Replicons

Stuart Austin, Marcia Ziese and Nat Sternberg Cancer Biology Program NCI Frederick Cancer Research Center Frederick, Maryland 21701

Summary

If daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell divi- sion. We present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage Pl . Asymmetric parti- tion occurs, leading to a relatively high rate of loss of the plasmid from the growing population. In con- trast, the wild-type Pl plasmid is maintained very efficiently in host cells. We show that this efficient maintenance is due to the presence of the IoxP-cre site-specific recombination system present on the intact Pl plasmid. This system promotes rapid re- combination between two /oxP sites on dimer mol- ecules, resolving them into monomeric substrates for proper partition. We suggest that bacterial replicons that are maintained with great accuracy in recombination-proficient cells might also encode high-efficiency recombination systems.

Introduction

Many of the plasmids found in Escherichia coli resem- ble the bacterial chromosome in that they are present at very low copy number, often at only one copy per cell. These “unit-copy” plasmids, which include sex factors, antibiotic-resistance factors and the plasmid prophages of certain bacteriophages, are replicated and partitioned to daughter cells with great accuracy despite their low copy number. The plasmids must, therefore, avail themselves of a highly accurate mech- anism for the control of replication and partition that is at least functionally equivalent to that which must exist for the maintenance of the host chromosome.

Despite its potential to become a virus on induction, the prophage of bacteriophage Pl has properties that closely resemble those of other, more sedate unit- copy plasmids. Pi is a circular DNA molecule with a molecular weight of 60 x 1 O6 (Ikeda and Tomizawa, 1968; Yun and Vapnek, 1977) and is maintained at one to two copies per cell of its host, E. coli (Ikeda and Tomizawa, 1968). Fewer than one of lo4 cell divisions of E. coli cells that harbor PI produce a cured cell (Rosner, 1972).

The Pl plasmid encodes its own site-specific re- combination system, the /oxP system (Sternberg and Hamilton, 1981). This recombination system, although

superficially similar to the int-attP system of phage h, performs a different physiological role, at least in E. coli. The primary role of recombination via attP in X is to integrate the h prophage into the bacterial chro- mosome (Gottesman and Weisberg, 1971). The Pl prophage, as its plasmid nature implies, normally does not integrate into the chromosome, although rare in- tegration events have been detected (Chesney et al., 1979). In seeking alternative roles for loxf recombi- nation, we have shown that, under certain circum- stances, /oxP recombination can promote (but is not required for) circularization of injected DNA and may be involved in generalized transduction (Sternberg et al., 1980). In this study, we show that loxf plays a role in the stable maintenance of the plasmid in the following way.

E. coli, like all the naturally occurring bacterial strains that have been tested, shows the capacity to promote generalized recombination between homol- ogous regions of DNA. Because replication creates homologous pairs of DNA structures within a single ceil, recombination can occur between the sister DNA strands before or on completion of the sequential replication of the chromosome. When we consider the potential consequences of such recombination for a unit-copy replicon, such as PI, a topological problem becomes evident (Figure 1). The product of a single reciprocal recombination event is a replicon dimer. A single dimer in a cell about to divide is not a substrate for replicon partition before cell division. We cannot readily measure the rates of recombination involved but, based on indirect evidence, we assume that Pl plasmid dimers would form and break down in l-l 0% of the cells in each cell generation. Thus, once a dimer is formed, cell division would have to be delayed for a time equal to many cell generations to ensure an equal genetic dowry to each cell. We present evidence that suggests that cells containing such relatively sta- ble dimers do not wait for the dimers to be resolved and do not die, but divide, giving rise to one daughter cell that lacks the plasmid (Figure 1). However, in the case of Pi and many other naturally occurring unit- copy plasmids, this curing event clearly is rare since the apparent fidelity of plasmid partition is extremely high. We propose that this fidelity is achieved, at least in the case of Pl, by a specific mechanism that prohibits the presence of stable dimers in dividing cells. The proposed mechanism is illustrated in Figure 2. The /oxP site of the Pl plasmid is a locus of recombination between any two DNA molecules that carry it (Sternberg and Hamilton, 1981). We show that this recombination is rapid and reversible. We propose that the rapid flux between monomer and dimer forms provides a source of monomers for proper partition before cell division occurs whether generalized re- combination occurs or not. Experiments performed with Pl and derivative plasmids that provide consid-

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SITEZiPECIFIC OR

t \ GENERALIZE

DIMERIC DNA

Figure 1. Hypothetical Effect of Generalized Recombination on the Maintenance of a Unit-Copy Replicon in Dividing Cells

The normal pathway for replicon partition (left pathway) is blocked by dimer formation. When the cell divides, a cured cell results (right pathway).

erable evidence for these propositions are described below.

Results

The IoxP Recombination System Can Prevent the Formation of Stable Composite DNA Structures Lambda-Pi hybrid phages, which carry any one of a number of different Eco RI fragments of Pl , have been isolated (Sternberg, 1979). These were constructed by cloning the Pi fragments into a X vector that lacks the h attachment site. As X lysogeny normally requires integration into the bacterial chromosome via this site, lysogenization frequencies were very low. However, when a PI plasmid is present in the recipient strain, lysogeny can occur. This lysogeny involves integra- tion of the hybrid phage into the Pl plasmid by gen- eralized recombination via the homology provided by the cloned fragment. The A phage is then maintained as part of the PI plasmid replicon. Of six different hybrids tested, five formed lysogens efficiently in a strain containing Pl (for example, A-PI :2, h-P1 :9, Table 1) as long as the strain was recombination- proficient. The resulting lysogens contained only plas- mids of composite size (Figure 3). The remaining X-

DIMERIC DNA

1 SITE-SPECIFIC RECOMBINATION

Figure 2. Hypothetical Effect of a Highly Active Site-Specific Recom- bination Site on the Partition of a Unit-Copy Replicon in Recombina- tion-proficient Dividing Cells

During the course of normal replication and partition (left pathway). any dimers formed by generalized or site-specific recombination are highly unstable because of the high activity of the site-specific recom- bination. Resolution into monomers rapidly provides substrates for proper partition (right pathway).

Pl hybrid, X-PI :7, failed to make detectable lysogens (Table 1). This can be explained by a model for the resolution of composite structures analogous to the model proposed for the resolution of dimeric mole- cules in Figure 2. Eco RI-7 contains an active /oxP recombination site. Thus any composite structures formed between it and the Pl plasmid would contain two such sites flanking the parental sequences. Rapid recombination between the two IoxP sites would re- solve the composite into its components. Because one of the components, X-Pl:7, cannot be maintained autonomously, it would be lost from the population and stable lysogens would not be formed.

The model predicts that the inability of h-P1 :7 to lysogenize strains containing Pl depends on both elements of the potential composite having a /oxP site. We have isolated a modified A-Pl:7, X-P1:7AloxP, from which a small (1.5 kb) Barn HI fragment (Barn HI- 9) was removed. This removes both the ioxP site and part of the recombinase gene cre, which is essential for /oxP-mediated recombination (Sternberg and Ham- ilton, 1981; Sternberg et al., 1980). A derivative of Pl , Pl dpro4a (Rosner, 1975) lacks a functional loxP site (Sternberg, unpublished data). We have also iso- lated a Pl AloxP plasmid from which the same frag- ment Barn HI-9 is deleted. Lambda lysogeny tests were performed with either h-Pl:7A/oxP as the su-

Recombination and Plasmid Maintenance 731

perinfecting phage or Pl dpro4a as the resident plas- mid, or both (Table 1). When either (or both) of the elements lacks the /oxP region, efficient lysogeny of the X-PI hybrid occurs. These lysogens are only formed in recombination-proficient hosts (Table I), and the resulting lysogens contain composite pIaS- mids (Figure 3). Similar experiments have been car- ried out with both elements containing loxf but lacking the Pl gene (cre), whose product is necessary for

Table 1. Formation of Composite Plasmids from h-Pi Hybrids and Pl

Resident Pl Infecting X- X Lysogenization Composites Plasmid Pi Hybrid Frequency x 1 O4 Detecteda

PI /oxp+ h-P1 :2 5 Yes

Pl /oxP’ X-PI :9 4 Yes

Pl /oxp+ X-PI :7loxP+ 0.1 No

PI /oxp+ h-P1 :7AloxP 4 Yes

Pl dpro4a[AloxP] A-PI :9 4 Yes

Pl dpro4a[A/oxP] h-P1 :7/oxP+ 6 Yes

PI dpro4a[A/oxP] h-PI :7A/oxP 4 Yes

Pl AloxfiABamHl-

91 h-P1 :7/oxp+ 4 Yes

The bacterial host was N21 5fect except where Pl dpro4a was used when the host was N3072rec+Apro-lat. When an isogenic recA strain, N205, was used in analogous experiments, lysogenization frequencies were all low (< 0.1 x 1 Om4) and no stable composites were detected (data not shown). Lysogenization frequency was mea- sured by standard techniques (Experimental Procedures). All strains carrying Pl (PI Cm) were grown and tested in the presence of 25 pg/ ml chloramphenicol. a Four to six of the lysogens formed were purified by single colony isolation and subjected to agarose gel electrophoresis. Those lyso- gens containing composites form plasmid bands with a characteris- tically lower mobility than the Pl plasmid (Figure 3). Yes: all colonies contained composites. No: no colonies contained composites.

/oxP recombination. Composite formation was re- stored in this situation also (data not shown). We therefore conclude that the presence of an active /ox/J site on both components of a potential composite structure prevents the detection of the composite. We would expect that stable composites formed from identical plasmid copies would also be prohibited by the same means. Thus the formation of stable dimers from the sister copies of PI before cell division may well be prevented by the /oxP recombination system, as suggested above.

A Pl-Derived Plasmid That Lacks the /oxP Recombination Site Shows Reduced Maintenance Fidelity We isolated a X-P1 hybrid phage that contains both the information required for x phage propagation and sufficient information from Pl to maintain itself in the lysogenic state as a Pl miniplasmid. A hybrid was constructed by cloning the Eco RI fragment 5 of Pl into a X vector. The resulting hybrid does not contain sufficient information for plasmid replication. The cloned fragment was subsequently extended to in- clude part of the adjacent fragment 8 by a directed illegitimate recombination with wild-type Pl . This hy- brid, A-PI:!%, readily forms a unit-copy plasmid on infection (Sternberg and Austin, 1981; N. Sternberg, S. Austin and M. Yarmolinsky, in preparation). The X vector lacks the X attachment site, which is normally required for h to form a lysogen (Gottesman and Yarmolinsky, 1968). Thus the resulting hybrid, X-PI : 5R, forms lysogens only via the Pl -determined proc- ess that leads to the establishment of a plasmid of the PI type (Figure 4). The X-PI :5R plasmid retains most of the characteristics of the parental wild-type Pl plasmid: It is maintained at a copy number of approx- imately one per host chromosome and both exerts

Figure 3. Agarose Gel Electrophoresis of Cell Extracts Containing Composite Plasmids Formed from PI and h-P1 Hybrids

Strain N215 contained: (lane a) Pl Cm; (lanes b and c) PI Cm composite with X-PI :2; (lanes d and e) Pl Cm composite with X-P1 :9; (lanes f and g) PI Cm composite with A-P1 :7AloxP; (lanes h and 0 Pl CmAIoxP composite with X- PI :7. Strain N3072 contained: (lane j) Pl dpro4a; (lanes k, I and m) Pl dpro4a com- posite with A-P1 :7. The strains were obtained in the experiment described in Table I. Cells were labeled with ‘H-thymidine and subjected to electrophoresis as described above. The central bands are plasmid DNA of typical Pl size (Pi) or somewhat larger (composites). The lower bands are chromosomal DNA frag- ments that also appear in cells that contain no plasmid. The relative band intensities have no significance, as the material applied varied considerably in the number of 3H counts ap- plied to the gel.

a bcdefghijklm

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c d e f Figure 4. Characterization of X-PI :5R Miniplasmid and Derivatives of It That Contain a /oxP Recombination Site

(Lanes a and b) Photograph of ethidium-bromide-stained 1% agarose gel after electrophoresis of purified phage DNA cut with Barn HI endonuclease; (lane a) h-P1 :5R DNA; (lane b) X-P1 :5RBamHI-9 DNA. The small fragment is the Barn HI-9 of Pl; (lanes c-f) gel electrophoresis of 3H-thymidine-labeled whole cell lysates showing plasmid bands; (lane c) strain N205, no plasmid; (lane d) N205 (h- Pi :5R); (lane e) N205 (Pi); (lane f) N205 (composite of X-P1 :5R and h-P1 :7/oxP+).

and responds to group Y-specific incompatibility typ- ical of the Pl plasmid (Hedges et al., 1975). It is not partitioned, however, to daughter cells with the great accuracy characteristic of the Pl parent. Approxi- mately 1% of all ceil-division events of X-PI :5R lyso- gens produce cured cells as judged by their lack of X immunity (Table 2) and confirmed by their inability to give a plasmid DNA band on gel electrophoresis of cell extracts (Figure 4). The data presented in Table 2 suggest that the maintenance inaccuracy of X-Pl: 5R is associated with recombination because X-PI : 5R lysogens of an otherwise isogenic recombination- deficient strain (recA3 are more stable than their ret’ counterparts. We note that X-Pl:5R does not carry the IoxP site-specific recombination region, because it is widely separated from the plasmid replication region on the Pl genetic map (Yarmolinsky, 1977).

Introduction of a Functional /oxP Site into the Pl Miniplasmid Restores Maintenance Fidelity A modified XPl:5R miniplasmid was constructed that contains an additional Pl fragment, Pl BamHI-9 (Fig- ure 5). This fragment contains a /oxP site but lacks a functional cre recombinase gene (Sternberg and Ham- ilton, 1981). Recombinational activity of the resulting hP1:5RBamHI-9 plasmid can thus be restored by in-

Table 2. Loss of h-P1 :5R Plasmid from Exponentially Growing Cultures of lsogenic ret+ and recA Strains

No. Rate of Individual Total XimmP 1- Loss/

Relevant Bacterial Lysogens Cells Sensitive Generation Genotype Tested Screened Cells x loza

ret + (h/mm2 l- PI :5R)

recA (himm2 l- PI :5R)

3 466 47 3.3

3 433 5 0.4

The ret+ and recA strains used are the isogenic pair N215 and N205, respectively. Method A was used to measure the rate of loss of the himm27-P1:5R plasmid (see Experimental Procedures). The variation in this rate for individual ret+ lysogens was low, the spread being 2.5-5 x 1 Om3. Essentially the same results were obtained when we used another isogenic pair of reef and recA strains, Q125rec+ and Qi 25recA (D’Ari et al., 1975). a Plasmid loss was measured after the cells had been grown through 30 generations without selection pressure. To determine the rate of loss per generation, we assumed that the plasmid is lost at a constant rate per generation.

t 5 t imm21

7

/I

n ’ limm21-Pl:SR

nin5

A-P1:20,BamHI-9

b538 nin5

A-Pl:CiR,BamHI-9

A-P1:5R,BamHI-9 Plasmid

Figure 5. Construction of h-PI :5R Miniplasmid Containing the BamHI-9loxP Fragment of Pl

The h-PI :SRBamHI-9 plasmid was derived from the parental phages shown by recombination in a lytic cross. The vector used to clone Barn HI-9 contained the small Eco RI-20 fragment of Pi This in- creases the size of the DNA, allowing detection of clones containing small additional fragments by our standard methods. Recombinants were selected as phages that would lysogenize N205 to give plasmids with A immunity. Phage was recovered from the lysogen by induction, and DNA was prepared from the lysates. Of four candidates, all contained Barn HI-9 as judged by digestion and gel electrophoresis (Figure 4). Arrows: Eco RI sites. Triangles: Barn HI sites.

traducing it into a strain that provides cre product in trans. The element used to provide cre product was a chromosomally integrated prophage, XimmP l-P1 : 7A101, which carries the cfe gene but lacks the /oxP site (Sternberg and Hamilton, 1981). Control strains carried Aimm2 7 -P1:7A201 that is deleted for the cre gene or Ximm21 -P1:9 that carries an unrelated frag- ment of Pl DNA (Eco W-9) derived from a region distant from /oxP on the Pl DNA. The resulting lyso- gens were analyzed by gel electrophoresis to check

Recombination and Plasmid Maintenance 733

a b C d e

Figure 6. Agarose Gel Electrophoresis of Cell Extracts Containing X-P1 :SRBamHI-9 Miniplasmids

The middle band has a mobility characteristic of lambda-sized plas- mids. The lower band consists of chromosomal DNA fragments. (lane a) h-P1 :5R in strain carrying a chromqsomally integrated himm27- Pi :7AlOl prophage; (lanes b, c and d) h-P1 :5RBamHI-9 in strains carrying the following integrated prophages: (lane b) himm27-Pl: 78101; (lane c) Ximm27-P1:7A201; (lane d) XimmPl-Pl:9; (lane e) strain containing integrated himm27 -Pi :7A102 that was scored as having lost its h-P1 :5RBamHI-9 plasmid in Table 3. The strains used here are examples of those used in Table 3. Several other individual lysogens of each type were also tested and gave identical results.

for the presence of the h-P1 miniplasmid (Figure 6). Neither the Barn HI-9 insertion nor the cre product supplied by an integrated prophage has any apparent effect on plasmid copy number (data not shown). Table 3 shows the maintenance fidelity of A-P1:5R and h-P1:5RBamHI-9 in these strains. The h-Pl: 5RBamHI-9 plasmid shows a 25fold increase in main- tenance fidelity when cre function is supplied in trans. The functioning /oxP site must be present in cis to the

plasmid for this effect since a IoxP’ X-PI :7 prophage does not increase the fidelity of the X-P1 :5R plasmid (data not shown). We conclude that the defect in maintenance of the h-P1 :5R miniplasmid relative to wild-type Pl is in large part due to the absence of an active /oxP site within the plasmid DNA.

This conclusion can also be drawn from observa- tions on a second /oxP-containing derivative of X-P1 : 5R. This is a plasmid chimera formed by generalized recombination between h-P1 :5R and a h-P1 hybrid phage containing Eco RI fragment 7 of Pl ; himm2 l- P1:7 (Figure 7). The resulting plasmid, hP1:5R:: Ximm2 1 -P1:7, can be detected as a covalently closed circular DNA whose size approximates the sum of its component phages (Figure 4). This plasmid contains both IoxP and cre (Figure 7). Table 4 shows that it is maintained much more faithfully than its h-P1:5R parent or analogous composites that lack /oxP func- tion. Moreover, the value obtained underestimates the increase in fidelity as the composite, unlike Pl or X- P1:5RBamHI-9, loses its loxP site during the course of the experiments. This loss is caused by the break- down of the composite into its component parts by generalized recombination followed by loss of the Ximm27-P1:7 element, which is not capable of main- tenance as an autonomous element (see legend to Table 4).

The addition of an active /oxP site greatly improved the maintenance fidelity of both the h-P1 miniplasmids described here so that the values achieved approxi- mate the value of Rosner (1972) for Pl , 1 O-4 cured cells per generation. However, our own values for the rate of loss of wild-type Pl, derived by the same indirect method used by Rosner, are lower by a factor of 20-40 (Table 5). Thus the /oxP-containing mini- plasmid may still lack another Pi element that is required for the wild-type maintenance phenotype. We can think of no obvious explanation for the differ- ence between our results and those of Rosner.

The Maintenance Fidelity of an Intact Pl Plasmid Depends upon IoxP-cre Recombination Activity We have isolated a number of mutants of PlCm that do not express a functional cre gene product (M. Ziese and N. Sternberg, in preparation). SOme of these are conditional “nonsense” (amber) mutants

whose /oxP recombination activity is restored in an amber suppressor strain. We have estimated the Pro- portion of cured cells in populations of lysogens of these plasmids by an indirect technique first described by Rosner (1972). Table 5 shows that the fidelity of maintenance of all tested Pl Cm cre- mutants is much reduced, often by a factor of 50. Amber cre- mutants can be restored to near wild-type fidelity of mainte- nance when introduced into an amber suppressor host. We conclude that cre-promoted site-specific recombination (presumably at /oxP) is required for the wild-type fidelity of maintenance of the Pl plasmid.

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Table 3. Influence of cre Product in trans on a h-PI Miniplasmid That Contains the /oxP Site

Rate of Loss/ Individual Lysogens Total Cells No. h-Sensitive Generation

Plasmid Integrated X Prophage Tested Screened Cells x losa

A-P1 :5R himm27 -P1:7A101[Cref] 4 409 31 2.5 -P1:78201 [Cre-] 4 421 31 2.5 -P1:9[Cre-] 4 356 26 2.4

X-P1:5RBamHI-9 Ximm27 -P1:7AlOi [Cre’] 8 868 3 0.1 -P1:7A201[Cre-] 5 500 52 3.5 -P1:9[Cre-] 4 306 26 2.8

The spread of values for rate of loss with individual lysogens was small: for h-P1 :5RBamHI-9 lysogens of the strain containing the Cre’ prophage A-PI :7AlOl, h-sensitive/X-resistant cells varied from O/i 24 to 1 /Ql_ For all other strains, values fell within the extremes of 5/70 and 14/l 00. All h-sensitive cells were judged to have lost their A plasmids because the cells were temperature-resistant (shows loss of the immhcl857 marker of the plasmids), and a sample of the cells gave no plasmid band on agarose gel electrophoresis (See Figure 6). B Cells were assumed to have been grown through 30 generations of cell growth without selection and to have a constant rate of loss in each generation. All strains were lysogens of strain RW842. The chromosomally integrated prophages were integrated by homology into cryptic X sequences located in the gal7 gene of this strain.

4 7 f Aatt 4 7 f Aatt imml imml 0 0 b-P1:7 b-P1:7

0 0

0 0

Aimm21 -P1:5R Aimm21 -P1:5R imm21 imm21

Aatt Aatt + 5 + 5 \ \

Figure 7. Construction of a Composite Miniplasmid Containing Both the PI :5R Plasmid Maintenance and the PI :7/oxPcre Recombination Sequences

Strain N215 containing the Ximm2 l-P1 :5R miniplasmid (derived from A-PI :5R by recombination with himm2 lhyl by standard lytic crosses) was lysogenized with h-P1 :7 selecting for both X and 21 immunities. The resulting lysogens contain composite plasmids of double lambda size (that is, of size similar to PI) as seen in Figure 4. Arrows: Eco RI sites. Triangles: Barn HI sites.

Discussion

We have demonstrated that a Pl -derived miniplasmid, X-P1 :5R, is maintained less faithfully than its Pl par- ent and that this defect is largely eliminated on intro- duction into recA (recombination-deficient) cells. This ret+-dependent defect can be abolished not only by eliminating recombination but also by increasing it. The introduction of the /oxP recombination site into the miniplasmid DNA substantially restores miniplas- mid maintenance when the potent recombinase (cre product) is supplied. We also have shown that Pi depends on the activity of its own IoxP site for accu- rate plasmid maintenance. These observations sup- port the contention (see Introduction) that rec-pro- moted dimer formation can lead to plasmid loss, and that a greatly increased rate of recombination pro-

moted via /oxP can prevent this loss by providing monomeric substrates for the partition mechanism.

Two assumptions about recombination rates were implicit in this proposition. First, it was assumed that ret-promoted recombination both forms and breaks down plasmid dimers at a modest rate such that relatively stable dimers will form in some small per- centage of the total cells before cell division. Second, it was assumed that /ox-promoted recombination is much more efficient than ret-promoted recombination such that the flux between dimeric and monomeric states of /oxP-containing plasmids always provides monomeric substrates for the plasmid partition before each cell division. The frequency of ret-promoted formation and breakdown of composite plasmids formed from two partially homologous elements is consistent with the first assumption. In most cases, composites are formed in about 0.1% of those Pi lysogens that are superinfected with a single h hybrid containing a fragment of Pl (Table 2). The majority of the composites formed survive for several cell gener- ations without ret-promoted breakdown into their component parts. However, when both elements con- tain an active IoxP site, cells containing such compos- ites cannot be isolated despite the use of powerful selection procedures. Presumably, in this case, com- posites are formed but are highly unstable, which is consistent with the second assumption. The high rel- ative efficiency of /ox-promoted versus ret-promoted recombination can also be demonstrated in suitable h lytic crosses. The presence of active IoxP sites on both parent DNAs when placed between two distant h markers increases recombination frequency 50-fold (Sternberg and Hamilton, 1981). Furthermore, we have found that intramolecular recombination be- tween two IoxP sites on a single infecting h DNA is even more efficient than the intermolecular event (N. Sternberg, unpublished observation). Thus the /oxP sites of PI may promote dimer breakdown faster than

Recombination and Plasmid Maintenance 735

dimer production, which would enhance the effective- ness of /oxP in ensuring proper plasmid partition.

PI is only one of a large number of unit-copy replicons found in E. coli that exhibit high maintenance fidelity. These include sex factors, antibiotic-resist- ance plasmids and colicinogenic factors. The E. coli chromosome itself also shows these properties. We assume that these elements also encounter and over-

Table 4. Maintenance of Composite Plasmids Formed by Recombination between Two A-PI Hybrids

Rate of Loss/ Plasmid Generation x IOza

Ximm27-Pi :BR::A-Pi :9 5

himm21-PI:5R::A-PI :7loxP+ 0.5

Aimm21-P1:5R::h-P1:7AloxP 6

Construction of composites is described in Figure 7. Each value represents the average of eight determinations on independently isolated lysogens. Fifty colonies derived from each of the eight cultures were tested for immunity (see Experimental Procedures). Approximately 15% of the colonies tested in each class had lost A immunity but retained imm27. These colonies were presumably de- rived from cells that had lost the A-P1 component of the composite by recombination. Thus the value on the second line is an overesti- mate of the rate of loss of this composite plasmid, as this population includes some cells that contain only the less stable Aimm27-PI :5R plasmid, which can then give rise to plasmid-free cells at an elevated rate. a The percentage of immh, imm27-sensitive cells produced after eight generations of growth in nonselective broth culture was determined by method A and divided by the number of generations (see Experi- mental Procedures).

Table 5. Loss of Temperature-Inducible Pl Plasmids as Estimated by Survival of Cells at 42°C

Survival Ratio Rate of

Relevant 42”C/ Loss/ Ratio

Bacterial 32°C x Generation cre-/ Genotype Prophage 104 x losa cre +

ret + sup + Pi Cmcl .l OOcre+ 0.5 0.25 -me2 20 10 40 -cre3(am) 20 10 40

recA sup + PlCmcl .lOOcre+ 2 1 -cre2 4 2 2 -cfe3(am) 3 1.5 1.5

ret+ supE Pl Cmcl .I OOcre+ 1 0.5 -me2 50 25 50 -cre3(am) 0.5 0.25 0.5

The ret+ sup+ and recA sup’ strains used were the isogenic pair N215 and N205, respectively. The ret’ supE strain used was C600. Method B was used to assess the rate of plasmid loss (see Experi- mental Procedures, Testing for Plasmid Maintenance). a The cell populations had all undergone 20 generations of growth before testing. The survival seen with wild-type Pl is an overestimate of the amount of plasmid loss because some two thirds of the survivors contain temperature-resistant mutant prophages (Austin et al., 1978). However, in the cre- cases, where loss is greater, the survival approximates the degree of loss because the majority of survivors contain no prophage (data not shown).

come the topological problem outlined above. It is therefore possible that such replicons encode recom- bination systems with similar properties to the /oxP system. We intend to look for such sequences in other plasmids by the technique used to characterize /oxP (Sternberg and Hamilton, 1981).

Experimental Procedures

Bacterial and Phage Strains The bacterial strains used in this paper are listed in Table 6. Pl is the Pl Cm of Kondo and Mitsuhashi (1964). Pl Cmcl .lOO was described by Rosner (1972) and Pl dpro4a, which lacks the /oxP recombination site because of the substitution of Pl DNA by a segment of the E. coli chromosome containing the proline operon (N. Sternberg, unpub- lished data) was described by Rosner (1975). Pl AloxP is a derivative of Pl from which the Barn HI-9 fragment that includes /oxP and part of the cre gene has been removed (Sternberg and Hamilton, 1981). This phage has an additional uncharacterized mutation that sup- presses a deleterious effect of ABam HI-9 on Pl immunity establish- ment. Pl Cmcl .l OOcreP and Pl Cmcl .l OOcre3 are derivatives of Pi Cmcl .I 00 containing missense and amber mutations, respec- tively, in the Pi lox recombinase gene, cre (Sternberg and Hamilton, 1981). The immh (W30 and W248) and imm2 1 (B 17 and B 121) clear phages are used to select immh or imm2I lysogens as described in Shimada et al. (1972) and Sternberg (1976). The A-PI hybrid phages described in this paper were isolated after insertion of Eco RI frag- ments of Pl DNA into the single Eco RI site of the AD~srlA3 vector (ADam 5 b538 srlA3 cl857(ts)nin5) and are designated, for example, A-PI:7 and A-P1 :5 (:7 or :5 refers to the Eco RI-generated Pl fragment inserted) (Sternberg et al., 1978; Sternberg, 1979). A-PI : 5R was derived from A-PI :5 as described in Results. Composites formed by recombination between two A hybrids are designated, for example, A-P1 :5R::A-PI :7.

General Phage Methods General phage manipulations and crosses were performed as previ- ously described (Sternberg and Hamilton, 1981).

Testing Lysogens for cre Function The function of the cre gene present in lysogens of Aimm27-P1:7 derivatives was confirmed by the ability of such strains to cause the breakdown to a smaller form of the DNA of a phage that contains two loxP sites but no cre gene (AloxP’; N. Sternberg, in preparation). AloxP’ is highly unstable on growth in cre-containing lysogens (80% breakdown in one growth cycle) but stable in otherwise isogenic lysogens that lack cre (10.5% breakdown).

Testing for Plasmid Maintenance Isolated colonies of plasmid-containing strains were obtained on suitable selection agar (EMBO agar with selector phages for fmmh or imm21 plasmids, or both: Ll agar with chloramphenicol [25p/ml] for Pl Cm or Pl Cmcre plasmids). Unless otherwise stated, cells from these colonies (two per test) were streaked to give isolated colonies on Li agar after growth at 32’C. The single colonies (four per streak)

Table 6. Bacterial Strains Used in This Studv

Strain Relevant Genotype Reference

N205 recA Sternberg and Weisberg (1975)

N215 ret + Sternberg and Weisberg (1975)

N3072 ret+ Alac-pro Rosner (1975)

RW842 ret+ cryptic A in ga/J Enquist and Weisberg (1976)

C600 ret+ supE Appleyard (1954)

Cell 736

were used to inoculate Ll citrate broth cultures. The composition of these cultures was tested in one of two ways. The cultures ware grown at 32°C with aeration to 10’ cells/ml. Suitable dilutions ware spread on Li agar to give -400 colonies per plate at 32°C. These were picked with toothpicks onto Li agar and onto selector agar (as above) to score for the presence of plasmid markers (immh or imm27, or both, or Cm’). A small sample (four to eight colonies) of those colonies scored as having lost the plasmid were evaluated by agarose gel electrophoresis. All proved to be plasmid-free in each test (method A). The other method was to spread suitable dilutions on two Ll citrate agar plates, one of which was prewarmed to 42”C, establish viable counts on growth at 32OC and 42’C and estimate the loss of the temperature-inducible marker immPl cl .I 00, and hence the loss of the PI plasmid, from the survival rate at 42’C (method 6).

Agarose Gel Electrophoresis for Plasmid Detection Agarose gel electrophoresis for detection of plasmid DNA was per-

formed as previously described except that cells were labeled by overnight growth of colonies formed by streaking cultures on agar plates containing 10 ml Ll agar, 2.5 mg deoxyadenosine and 0.1 &i ‘H-thymidine 60 Ci/mM. The radioactive gels were fixed in 10% acetic acid, 15% methanol for 1 hr, soaked in Enhance’” solution (New England Nuclear) for 1 hr and in water for 1 hr. The gels were dried onto filter paper by standard methods and exposed to X-ray film by direct contact. Film was developed by standard procedures.

Analysis of DNA by Use of Restriction Endonuclease Barn HI Methods for isolation of DNA from X-P1 hybrids and its analysis by endonuclease digestion and agarose gel electrophoresis were as described previously (Sternberg, 1979), except that Barn HI digestion was carried out in 20 mM Tris-HCI (pH 7.4), 7 mM MgC&, 2 mM p- mercaptoethanol for 1 hr at 37’C.

Acknowledgments

We wish to thank Michael Yarmolinsky for constructive criticism and Donna Follin for diligent preparation of the manuscript. Research was sponsored by the National Cancer Institute, DHHS, under contract with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US. government.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 2, 1981

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