Vol. 173, No. 13
JUlY 1991, p. 4027-4031 0021-9193/91/134027-05$02.00/0 Copyright © 1991, American Society for Microbiology
JOURNAL OF BACTERIOLOGY,
Efficient Excision of Phage from the Escherichia coli Chromosome Requires the Fis Protein CATHERINE A. BALL' AND REID C. JOHNSON 12* Molecular Biology Institute, University of California, Los Angeles,' and Department of Biological Chemistry, University of California, Los Angeles, School of Medicine,2 Los Angeles, California 90024 Received 27 December 1990/Accepted 27 April 1991
The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of X excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold infis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.
Site-specific DNA recombination reactions can demonstrate a great deal of variation in the complexity of substrate and accessory factor requirements (for a review, see reference 5). Although the complexities of DNA substrate and accessory protein requirements range from the rather simple to the very complex, these types of reactions have several characteristics in common. Unlike homologous recombination reactions, which require extensive sequence homologies, site-specific recombination reactions depend on specialized recombinase enzymes that are independent of the homologous recombination machinery to catalyze the reactions. In addition, the formation of highly ordered nucleoprotein structures during synapsis appears to be a hallmark of many site-specific recombination reactions. These nucleoprotein structures are often assembled via specific interactions between DNA, recombinase enzymes, and accessory proteins. The Escherichia coli protein Fis is known to function as an accessory protein in site-specific inversion reactions catalyzed by the Hin protein and related recombinases (11, 13, 14). We chose to investigate the in vivo role of Fis in a site-specific recombination system native to E. coli, the excision reaction of bacteriophage X. The integration and excision reactions of phage X have been extensively studied and together form one of the best-characterized genetic recombination systems (for a recent review, see reference 15). Once it has infected a cell, X can follow one of two pathways: lytic or lysogenic. In the lytic pathway, the phage replicates its chromosome, packages new phage, and lyses the cell, releasing new phage particles into the media. In the lysogenic pathway, the phage chromosome integrates into the bacterial chromosome via a site-specific recombination reaction and remains there quiescently until induced to excise and follow the lytic pathway. After injection into the cell, the X chromosome circularizes, and, if the lysogenic pathway is taken, the 240-bp supercoiled phage attachment site, attP, is bound by both host and phage proteins to form a precisely ordered nucleoprotein complex termed the inta*
some (3, 20). The intasome seeks out the 25-bp bacterial attachment site, attB, that is the target for integration (21). Recombination between attP and attB integrates the phage DNA into the chromosome and generates the two hybrid recombination sites, attL and attR (Fig. 1). A subsequent recombination event between attL and attR results in recircularization (excision) of the prophage. Both integration and excision require the phage-encoded recombinase Int and the E. coli accessory protein integration host factor (IHF), which (18) has been implicated in many other cellular processes of E. coli (8). In addition to Int and IHF, excision is dependent on the phage-encoded accessory protein, Xis (1). Of the nine Int-binding sites on attL and attR, it is only at the four core binding sites (shown as BOC' and COB' in Fig. 1B) where Int catalyzes DNA strand cleavage during excision. IHF binds one site on attL (H') and two sites on attR (Hi and H2) (6), although Hi is not required for excision (4, 9, 28). In vitro experiments have demonstrated that Xis has two adjacent binding sites, Xl and X2 (29), that are present on attR in the integrated phage chromosome. Thompson et al. have shown that Fis can bind to a site, named F, approximately 65 bp from the region of strand exchange in K attR (27). The presence of Fis was found to stimulate excisive recombination in vitro up to 20-fold at low Xis concentrations but to have no effect on integration. The Fis-binding site, F, overlaps and excludes Xis from binding to the Xis-binding site X2. Indeed, Fis bound at attR F can cooperatively enhance Xis binding to Xl (27). Together, Fis and Xis can bend the DNA within this region in a manner indistinguishable from Xis binding to both the Xl and X2 sites (25). In this work we report that in vivo, Fis has a direct and more dramatic effect on A excision than that predicted from the in vitro work. MATERIALS AND METHODS
Strains, media, and enzymes. Bacterial and phage strains, as well as plasmids used in this study, are described in Table 1. The fis::767 mutation (12) was introduced into different strains by Plvir-mediated transduction. Except where noted, cells were grown on either solid or liquid LB as described
Corresponding author. 4027
4028
BALL AND JOHNSON
J. BACTERIOL. TABLE 1. Bacterial and phage strains used
A.
Bacteria MC1000
RJ1617 RW842 RJ1784 RJ1654
x
Bacterial Chromosome
gal
Integration
LE392
Int T IHF
Int IHF
RJ1785
bio
attB
(Fis)
Excision
Xis Fis
gal
Genotype
Strain Phage x Chromosome
x
attl,
bio
attR
B. attL
B
0
C'
P'1 P'2 P'3 H'-.
.................................................................. CP1
P2
-
HI
COB'
X1X2
attR
--0 F
H2
FIG. 1. (A) Schematic representation of X recombination reactions. Integration is a conservative, site-specific DNA recombination reaction that occurs between the phage attachment site attP and the bacterial attachment site attB, producing the recombinant phage-host junctions attL and attR. It requires the phage-encoded Int protein and the E. coli protein IHF and is moderately stimulated by Fis in vivo, especially in the absence of Xis (2). The excision reaction takes place between attR and attL to yield attP and attB. In addition to Int and IHF, excision requires the phage protein Xis and is stimulated by Fis in vitro (27) and in vivo (see the text). (B) Relative locations of protein-binding sites on attP as determined by nuclease protection and mutational analysis (reviewed in reference 15). The Int-binding sites, of which B, C', P'1, P'2, P2, C, and B' are required for excision, are shown (_). Excision also requires IHF bound to H' and H2 (121) and Xis binding to Xl (E:]). Efficient excision in vivo requires Fis bound to F ( m ), which overlaps the X2 Xis-binding site. Xis can bind to both Xl and X2 in vitro to promote excision in the absence of Fis. Arrows indicate the relative orientations of the binding sites.
previously (17). Titers of phage lysates were determined, and the lysates were prepared by using T top agar and plates (23). RJ1654 and RJ1785 were constructed by subjecting either RW842 or RJ1784 (RW842 transduced tofis::767 with Plvir) to a red-plaque assay with XY1 (7). Resulting gal' colonies lack the defective prophage integrated in the gal locus and were shown to be lysogens of XY1 by their sensitivity to high temperature (42°C) and the presence of a single, integrated prophage in a Southern blot. XRJ996 was constructed as described in the accompanying paper (2), using a plasmid provided by J. Thompson and A. Landy. Restriction enzymes and DNA polymerase were purchased from Boehringer Mannheim.
Phages XY1 XRJ997 XRJ996 XRJ998 XY281
araDJ39 A(ara-leu)7697 AlacX74 galU galK strA MC1000fis::767 HfrH galT::XA(int-FII) RW842 fis::767 RW842 gal', lysogenized with XY1 RJ1784 gal', lysogenized with XY1 tonA lac Y galK metB trpR hsdR hsdM+ supE supF XcI857
xcI+ XcI857attPF XcI857Sam7 XWam4O3Eam11OOcI857
Source or reference
M. Casabadan
12 7 This work This work
This work L. Enquist R. Weisberg Laboratory collection 2 Laboratory collection 7
Phage growth. Stable lysogens were grown in LB at 30°C (for XcI857 lysogens) or 37°C (for XcI+ lysogens) to an optical density at 600 nm of 0.3. XcI857 lysogens were induced by shaking at 42°C for 30 min, followed by shaking at 37°C until lysis. XcI+ lysogens were induced by addition of mitomycin C to a final concentration of 5 jig/ml and shaking at 37°C until lysis. Lysates were cleared by addition of chloroform followed by centrifugation for 10 min at 6,000 x g. Lysate titers were determined by using LE392 as the host. For examining one-step phage yields, phage were added to a culture grown to an optical density at 600 nm of 0.3 at a multiplicity of infection of 0.1 and incubated with gentle shaking at 37°C. After 10 min of adsorption, the cells were washed and resuspended in fresh media. At intervals, samples were removed and lysates were prepared by freezing at -70°C followed by thawing in the presence of chloroform. The debris were removed by centrifugation, and the number of PFU was assayed by using LE392. Red-colony assay. Serial dilutions of XY281 were added to RW842 or RJ1784 grown to an optical density at 600 nm of 0.3, incubated for 20 min at 30°C, and plated as described previously (7) onto galactose-tetrazolium plates. Plates were incubated for 18 h at 30°C and scored for the number of red colonies on a white lawn as a function of input phage (7). Southern blot. Cells were grown to an optical density at 600 nm of 0.3 and induced by shifting to 42°C. At various times, 10-ml aliquots of cultures were chilled, harvested, washed in 50 mM Tris-HCl (pH 8.0), and resuspended in 0.5 ml of 50 mM Tris-HCl (pH 8.3)-10 mM EDTA. The resuspended cells were frozen in a dry-ice-ethanol bath, thawed on ice, and incubated for 30 min on ice in the presence of 100 ,ug of lysozyme per ml. Lysates were treated with sodium dodecyl sulfate (SDS) (final concentration, 0.2%) and proteinase K (final concentration, 100 ,ug/ml) at 65°C for 30 min. Two phenol-chloroform extractions followed by two ether extractions were performed. After addition of sodium acetate to 0.3 M, 1 volume of absolute ethanol was added and the precipitated DNA was wound onto a glass rod. The DNA was resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA, and aliquots were digested with EcoRT. Electrophoresis was performed in a 0.9% agarose gel by using Trisphosphate buffer, and transfer and hybridization were as described elsewhere (22). Total A DNA that was radiolabeled with 32P by using the random-priming method de-
EXCISION OF A FROM E. COLI REQUIRES Fis PROTEIN
VOL. 173, 1991
TABLE 2. Phage yields upon induction of A lysogens Ph
MC1000 (fis+) RJ1617 (fis)
XY1 (cI857) XY1 (cI857)
(8.7 ± 0.9) X 109 (3.7 ± 0.5) x 107 1.0 X 109b
MC1000 (fis+) RJ1617 (fis)
XRJ997 (cI+) XRJ997 (clI+)
(2.5 ± 0.5) x 1010 (1.2 ± 0.5) x 108 (3.6 ± 0.1) x 109b
MC1000 Osk) RJ1617 (fis)
XRJ996 (attRF) XRJ996 (attRF)
10.0
Phage yield (PFU/ml)a
Bacterial strain
age
4029
co
6.0
(1.0 ± 0.5) X 108 (0.9 ± 0.4) x 108
a Values represent the averages of yields from induction of four independently isolated lysogens. b These lysogens have been shown by Southern blot analysis to be double lysogens, each carrying two tandem integrated copies of the X chromosome.
scribed elsewhere (22) was used as the hybridization probe. The recA probe was generated from pBRrecA, obtained from K. McEntee. RESULTS Phage yields from a K lysogen are decreased in afis strain. To determine whether Fis plays a role in A excision in vivo, fis andfis+ E. coli strains were stably lysogenized with XY1, a phage that carries the gene encoding the cI857 temperature-sensitive repressor. The phage yields obtained upon heat induction of fourfis' lysogens and fourfis lysogens are shown in Table 2. Lysogens infis' cells gave yields of about 8.7 x 109 phage per ml. However, the lysogens in fis cells give two populations of phage yields: one at 1 x 109 phage per ml and the other at 3.7 x 107 phage per ml. Southern blots demonstrated that the lysogens infis cells that give the higher phage yield are double lysogens with two tandem copies of A integrated into the E. coli chromosome (data not shown). Double lysogens have the ability to allow phage DNA packaging through their tandem cos sites and therefore give phage yields that do not reflect the frequency of excisive recombination. Efficient packaging of polylysogens in the absence of excisive recombination has been previously observed in phages unable to excise as a result of a mutation in int (10). Yields from induction of singly integrated prophage (which are dependent on excision) in afis background show a reduction of 235-fold compared with the phage yields from induction of fis' lysogens. The higher phage yields produced from the double lysogens imply that the absence of Fis is directly affecting excision. Lysogens of XRJ997 (cI+) phage in the same strain backgrounds show a similar, 208-fold difference in phage yields when induced with mitomycin C (Table 2). Lytic growth is not affected by the absence of Fis. Although phage yields are good indicators of excision frequencies, they may also be affected by defects in other steps of lytic growth, such as phage replication or packaging. To test whether the low phage yields obtained from fis cells are due to a defect in lytic growth, we performed a one-step phage growth experiment. MC1000 and RJ1617 were incubated with XRJ998 for 10 min, washed, resuspended in fresh media, and grown lytically at 37°C. Aliquots were removed at various intervals, the cells were lysed, and the number of PFU present was assayed. The resulting phage yields from MC1000 and RJ1617 cells are plotted as a function of time in Fig. 2. The phage growth curve obtained from MC1000 is
4.0
2.0 0
50
100 Time
150
200
(min)
FIG. 2. Phage growth curve. Mid-log cultures of either MC1000 or RJ1617 were adsorbed with 4 x 106 XRJ998 phage for 10 min, washed, and grown at 37°C. The yield of phage (PFU) is plotted as a function of time. Symbols: O, phage yields infis+ cells; *, phage yields infis cells.
nearly identical to that from RJ1617 cells, indicating that X replication, packaging, and overall gene expression involved in lytic growth are unaffected by the absence of Fis. Red-colony assay for excision. The red-colony assay, developed by Enquist and Weisberg (7), provides a means of studying excisive recombination independent of the effects of lytic growth. This assay takes advantage of a strain (RW842) with a defective A prophage integrated into the galT locus of the E. coli chromosome, thus rendering the strain unable to utilize galactose. The prophage cannot excise except when supplied in trans with Int and Xis from an infecting X. When the prophage is excised, the integrity of the gal locus is restored and the gal' cells can form red colonies against a lawn of white cells on a galactosetetrazolium medium. The fis::767 mutation was transduced into the RW842 background to yield RJ1784 and used in a red-colony assay with XY281. The use of XY281 containing amber mutations in W and E ensures that no phage particles can be formed, and therefore only a single infective cycle is possible. The frequency of gal' red-colony formation was 1.02 x 10-3 per input phage in fis' cells, similar to the frequency reported by Enquist and Weisberg (7) with exponentially growing cells. Red-colony formation was reduced 16-fold to 6.37 x 10-5 per input phage in fis cells. This experiment indicates that the defect observed in phage yields fromfis X lysogens can be attributed to defects in excision. Southern blot analysis of excision. To directly assay the product of the X excision reaction, we performed a Southern blot analysis on total DNA from RJ1654 and RJ1785 lysogens that were induced for increasing lengths of time. The blot was hybridized with 32P-labeled X DNA to reveal the extent of X excision and replication (Fig. 3A) and reprobed with a 32P-labeled plasmid carrying unrelated DNA to determine the amount of chromosomal DNA loaded onto each lane (Fig. 3B). The product of excision is attP, which in this case is
4030
BALL AND JOHNSON
J. BACTERIOL.
DISCUSSION kb:
Fis + cells
a.
A. A probe
I0
-
5.8
-
a
sign.w -
0
5
cells
1530 45 60
,4
-
.
(U//Pl5.6 4.9
¶5 3045 60
5
Fis-
..
phqge/host _ to
___04W
"tS
junction -
o,//P
-phage/host junction
B. recA probe FIG. 3. Southern blot of excision time course. Lytic growth of XcI857 lysogens was induced by incubation at 42°C. DNA was isolated from cells immediately before induction and 5, 15, 30, 45, and 60 min after induction. The DNA was digested with EcoRI, electrophoresed in an agarose gel, and transferred to a nitrocellulose filter. (A) The filter was hybridized with 32P-labeled A DNA. The leftmost lane contains DNA isolated from X particles and digested with EcoRI. Appearance of the 5.6-kb attP-containing fragment that is the product of excision is dramatically reduced in fis cells compared with fis+ cells. (B) The same filter was also hybridized with a 32P-labeled plasmid carrying the recA gene, located 41 min from attB in the E. coli genetic map, to estimate the quantity of DNA loaded in each lane.
present on a 5.6-kb EcoRI fragment and is detectable in the lane containing DNA obtained from phage particles (Fig. 3A, left-hand lane). The A chromosome is present in a single integrated copy in both fis+ and fis cells before induction (t = 0). After induction of the fis' lysogen, the phage chromosome is replicated to a high copy number and the attP band is detectable within 5 min of induction and is readily apparent after 15 min. This is similar to the kinetics of excision as measured by Southern blot analysis by Ljungquist and Bukhari (16). The integrated chromosome achieves a high copy number in the fis cells, presumably owing to multiple rounds of replication initiating at the X origin within the integrated phage chromosome. However, in thefis cells, the 5.6-kb attP band is barely detectable after 60 min of induction. Therefore, we conclude that in the absence of Fis, excisive recombination is severely defective, whereas replication of the induced prophage DNA appears to be efficient. The experiment also indicates that excision in vivo is rather inefficient, even in the presence of Fis. Analysis by densitometry indicates that as much as 45% of the phage DNA remains integrated into the chromosome at 60 min after induction, just before cell lysis. Excision of attRF phage. Because the defect in excision observed infis cells could be explained by indirect effects of Fis, such as regulation of IHF, Int, or Xis levels, rather than a direct effect of binding to the F site in attR, a mutant phage (XRJ996) was constructed. XRJ996 contains the DNA sequence of the Xl Xis-binding site substituting for the X2-F region. This substitution (originally constructed in pJT111) prevents Fis binding but allows for efficient excision in vitro (26). Lysogens of this phage were induced, and excision efficiency was measured by determining phage yields. As shown in Table 2, afis lysogen yielded 0.9 x 108 phage per ml, but in this case afis' lysogen also produced a low phage yield (1.0 x 108). Thus, Fis binding to the attR F site appears to be directly responsible for most, if not all, of its stimulatory effect on X excision.
Fis binding to F in the attR recombination site is required for efficient excisive recombination of phage X in vivo. Thermoinduction of temperature-sensitive prophage or mitomycin C induction of AcI+ in fis cells resulted in phage yields that are reduced by a factor of more than 200 when compared with lysogens in fis' cells. Measurements of phage yields may amplify the effect of Fis on excision frequency, since the phage DNA may replicate more efficiently when it has excised from the chromosome. However, direct examination of X DNA after induction failed to reveal a significant difference in replication efficiency in fis cells, even though only a trace of excision was detectable. The effect of Fis on phage yields is specific to the excision reaction; we find no evidence for defects in lytic growth associated with the absence of Fis. The reduced excision observed in Fis null mutants is due solely to the lack of Fis binding to the F site on attR. Mutant phage which do not contain the attR F site gave similarly low yields in the presence and absence of Fis. Thompson et al. (27) have shown in in vitro studies that Fis can cooperatively enhance Xis binding to Xl and, under certain conditions, increase the efficiency of excision. A 20-fold stimulatory effect of Fis on excision is observed in vitro only when Xis is limiting. Numrych et al. (19) have also reported that Fis stimulates excision in vitro and in experiments in vivo measuring the curing of heat-pulsed XcI857 lysogens. The effect on curing was most dramatic when using phage that contained mutations in Int arm-binding sites believed to be critical for excision. For example, curing of a phage containing a mutation in the Int P2 site located adjacent to the X1-F region in attR was reduced more than 30-fold infis strains. One model for the activity of Fis in promoting excision is that binding at attR F induces a structural change in the DNA that results in an increase in the affinity of Xis for Xl. Alternatively, protein-protein contacts may be formed between Fis and Xis that promote or stabilize Xis binding to Xl. In addition to facilitating Xis binding, the binding of Fis to attR F causes a bend in the DNA, which is believed to contribute to the stimulation of the excisive recombination reaction. Upon binding, Xis and Fis each bend DNA at an angle of approximately 90°C (25). The additive effects of Xis and Fis binding to attR create a sharper bend than the binding of either protein alone. Since both Fis and Xis bend the DNA similarly, the structure formed by the binding of Xis to Xl and Fis to attR F may be functionally equivalent to that formed by the binding of Xis to both Xl and X2 (26). The fact that the structural perturbation of DNA is similar in the presence of two Xis protomers or Fis plus Xis, combined with the fact that excision can occur efficiently in vitro given sufficiently high Xis concentrations, suggests that lambda may synthesize insufficient Xis to efficiently bind on its own to attR. The large quantity of surrounding chromosomal DNA that is present in vivo may compete with attR for limiting quantities of Xis. Fis bound to the F site at attR may direct Xis to bind to Xl. It is quite possible that high cellular Xis concentrations would alleviate the requirement for Fis, although we have been unsuccessful in overcoming the requirement for Fis by introducing multicopy Xis-overproducing plasmids (2a). However, it is not known how much Xis overproduction has been achieved in these experiments. There are additional factors that may also contribute to the greater effect of Fis measured in vivo than in vitro. Some
VOL. 173, 1991
EXCISION OF X FROM E. COLI REQUIRES Fis PROTEIN
characteristic of DNA topology or chromatin structure may accentuate the Fis requirement in vivo but not in vitro. For example, IHF stimulation of the Mu transposition is observed only at physiological supercoiling densities (24). It is also possible that levels of other proteins involved in excisive recombination are reduced in fis cells. Because the excision defect is not overcome by expression of Xis from a heterologous promoter, it is unlikely that the defect is caused by lowered Xis expression infis cells. Measurements of IHF levels in fis' versus fis cells by gel shift (2a) or Western immunoblot (6a) analysis have not revealed any significant differences. The levels of Int in the absence of Fis have not been examined, although the fact that yields of XattPF phage are not significantly lower in fis cells argues against a model in which the excision frequency is reduced because the expression or activity of Int, or the other recombination proteins, is affected by Fis. Thus, our results argue that in vivo X excision normally requires Fis bound at attR F and that Xis binding to X2 is largely irrelevant. Data in the accompanying paper are consistent with this conclusion (2). ACKNOWLEDGMENTS We thank A. Landy, H. Nash, and R. Weisberg for their interest, suggestions, and gifts of strains and phage. We are particularly grateful to J. Thompson and A. Landy for providing us with several plasmids that were made specifically for this project. We also acknowledge members of this laboratory for their help and criticism during the preparation of the manuscript. R.C.J. was the recipient of grants from the National Institutes of Health (GM-38509), March of Dimes, and the Searle Scholars Program/Chicago Community Trust. C.A.B. was supported in part by USPHS National Research Service award GM-07104.
REFERENCES 1. Abremski, K., and S. Gottesman. 1982. Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination. J. Biol. Chem. 257:9658-9662. 2. Ball, C. A., and R. C. Johnson. 1991. Multiple effects of Fis on integration and the control of lysogeny in bacteriophage lambda. J. Bacteriol. 173:4032-4038. 2a.Ball, C. A., and R. C. Johnson. Unpublished data. 3. Better, M., C. Lu, R. C. Williams, and H. Echols. 1982. Site-specific DNA condensation and pairing mediated by the Int protein of bacteriophage lambda. Proc. Natl. Acad. Sci. USA 79:5837-5841. 4. Bushman, W., J. F. Thompson, L. Vargas, and A. Landy. 1985. Control of directionality in lambda site specific recombination. Science 230:906-911. 5. Craig, N. L. 1988. The mechanism of conservative site-specific recombination. Annu. Rev. Genet. 22:77-105. 6. Craig, N. L., and H. A. Nash. 1984. E. coli integration host factor binds to specific sites in DNA. Cell 39:707-716. 6a.Ditto, M., and R. A. Weisberg. Personal communication. 7. Enquist, L. W., and R. A. Weisberg. 1976. The red plaque test: a rapid method for identification of excision defective variants of bacteriophage lambda. Virology 72:147-153. 8. Friedman, D. I. 1988. Integration host factor: a protein for all reasons. Cell 55:545-554. 9. Gardner, J. F., and H. A. Nash. 1986. Role of Escherichia coli IHF protein in lambda site-specific recombination: a mutational analysis of binding sites. J. Mol. Biol. 191:181-189. 10. Gottesman, M. E., and M. B. Yarmolinsky. 1968. The integration and excision of the bacteriophage lambda genome. Cold Spring Harbor Symp. Quant. Biol. 33:735-747. 11. Haffter, P., and T. A. Bickle. 1987. Purification and DNA-
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binding properties of Fis and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin. J. Mol. Biol. 198:579-587. 12. Johnson, R. C., C. A. Ball, D. Pfeffer, and M. Simon. 1988. Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc. Natl. Acad. Sci. USA 85:34843488. 13. Johnson, R. C., M. Bruist, and M. I. Simon. 1986. Host protein requirements for in vitro site-specific DNA inversion. Cell 46:531-539. 14. Koch, C., and R. Kahmann. 1986. Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J. Biol. Chem. 261:15673-15678. 15. Landy, A. 1989. Dynamic, structural, and regulatory aspects of A site-specific recombination. Annu. Rev. Biochem. 58:913-949. 16. Ljungquist, E., and A. I. Bukhari. 1977. State of prophage Mu DNA upon induction. Proc. Natl. Acad. Sci. USA 74:31433147. 17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Nash, H. A., and C. A. Robertson. 1981. Purification and properties of the Escherichia coli protein factor required for A integrative recombination. J. Biol. Chem. 256:9246-9253. 19. Numrych, T. E., R. I. Gumport, and J. F. Gardner. 1990. A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda. Nucleic Acids Res. 18:3953-3959. 20. Richet, E., P. Abcarian, and H. A. Nash. 1986. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell 46: 1011-1021. 21. Richet, E., P. Abcarian, and H. A. Nash. 1988. Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex. Cell 52:9-17. 22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Silhavy, T., M. L. Berman, and L. W. Enquist. 1984. Experiments in gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Surette, M. G., and G. Chaconas. 1989. A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor. J. Biol. Chem. 264:3028-3034. 25. Thompson, J. F., and A. Landy. 1988. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 16: 9687-9705. 26. Thompson, J. F., H. F. L. Mark, B. Franz, and A. Landy. 1988. Functional and structural characterization of stable DNA curvature in lambda attP, p. 119-128. In W. K. Olsen, M. H. Sarma, and M. Sundaralingam (ed.), Structure and expression, vol. 3. DNA bending and curvature. Adenine Press, Schenectady, N.Y. 27. Thompson, J. F., L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy. 1987. Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway. Cell 50:901-908. 28. Thompson, J. F., D. Waechter-Brulla, R. I. Gumport, J. F. Gardner, L. Moitoso de Vargas, and A. Landy. 1986. Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications. J. Bacteriol. 168:218-222. 29. Yin, S., W. Bushman, and A. Landy. 1985. Interaction of the X site-specific recombination protein Xis with attachment site DNA. Proc. Natl. Acad. Sci. USA 82:1040-1044.
JUlY 1991, p. 4027-4031 0021-9193/91/134027-05$02.00/0 Copyright © 1991, American Society for Microbiology
JOURNAL OF BACTERIOLOGY,
Efficient Excision of Phage from the Escherichia coli Chromosome Requires the Fis Protein CATHERINE A. BALL' AND REID C. JOHNSON 12* Molecular Biology Institute, University of California, Los Angeles,' and Department of Biological Chemistry, University of California, Los Angeles, School of Medicine,2 Los Angeles, California 90024 Received 27 December 1990/Accepted 27 April 1991
The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of X excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold infis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.
Site-specific DNA recombination reactions can demonstrate a great deal of variation in the complexity of substrate and accessory factor requirements (for a review, see reference 5). Although the complexities of DNA substrate and accessory protein requirements range from the rather simple to the very complex, these types of reactions have several characteristics in common. Unlike homologous recombination reactions, which require extensive sequence homologies, site-specific recombination reactions depend on specialized recombinase enzymes that are independent of the homologous recombination machinery to catalyze the reactions. In addition, the formation of highly ordered nucleoprotein structures during synapsis appears to be a hallmark of many site-specific recombination reactions. These nucleoprotein structures are often assembled via specific interactions between DNA, recombinase enzymes, and accessory proteins. The Escherichia coli protein Fis is known to function as an accessory protein in site-specific inversion reactions catalyzed by the Hin protein and related recombinases (11, 13, 14). We chose to investigate the in vivo role of Fis in a site-specific recombination system native to E. coli, the excision reaction of bacteriophage X. The integration and excision reactions of phage X have been extensively studied and together form one of the best-characterized genetic recombination systems (for a recent review, see reference 15). Once it has infected a cell, X can follow one of two pathways: lytic or lysogenic. In the lytic pathway, the phage replicates its chromosome, packages new phage, and lyses the cell, releasing new phage particles into the media. In the lysogenic pathway, the phage chromosome integrates into the bacterial chromosome via a site-specific recombination reaction and remains there quiescently until induced to excise and follow the lytic pathway. After injection into the cell, the X chromosome circularizes, and, if the lysogenic pathway is taken, the 240-bp supercoiled phage attachment site, attP, is bound by both host and phage proteins to form a precisely ordered nucleoprotein complex termed the inta*
some (3, 20). The intasome seeks out the 25-bp bacterial attachment site, attB, that is the target for integration (21). Recombination between attP and attB integrates the phage DNA into the chromosome and generates the two hybrid recombination sites, attL and attR (Fig. 1). A subsequent recombination event between attL and attR results in recircularization (excision) of the prophage. Both integration and excision require the phage-encoded recombinase Int and the E. coli accessory protein integration host factor (IHF), which (18) has been implicated in many other cellular processes of E. coli (8). In addition to Int and IHF, excision is dependent on the phage-encoded accessory protein, Xis (1). Of the nine Int-binding sites on attL and attR, it is only at the four core binding sites (shown as BOC' and COB' in Fig. 1B) where Int catalyzes DNA strand cleavage during excision. IHF binds one site on attL (H') and two sites on attR (Hi and H2) (6), although Hi is not required for excision (4, 9, 28). In vitro experiments have demonstrated that Xis has two adjacent binding sites, Xl and X2 (29), that are present on attR in the integrated phage chromosome. Thompson et al. have shown that Fis can bind to a site, named F, approximately 65 bp from the region of strand exchange in K attR (27). The presence of Fis was found to stimulate excisive recombination in vitro up to 20-fold at low Xis concentrations but to have no effect on integration. The Fis-binding site, F, overlaps and excludes Xis from binding to the Xis-binding site X2. Indeed, Fis bound at attR F can cooperatively enhance Xis binding to Xl (27). Together, Fis and Xis can bend the DNA within this region in a manner indistinguishable from Xis binding to both the Xl and X2 sites (25). In this work we report that in vivo, Fis has a direct and more dramatic effect on A excision than that predicted from the in vitro work. MATERIALS AND METHODS
Strains, media, and enzymes. Bacterial and phage strains, as well as plasmids used in this study, are described in Table 1. The fis::767 mutation (12) was introduced into different strains by Plvir-mediated transduction. Except where noted, cells were grown on either solid or liquid LB as described
Corresponding author. 4027
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BALL AND JOHNSON
J. BACTERIOL. TABLE 1. Bacterial and phage strains used
A.
Bacteria MC1000
RJ1617 RW842 RJ1784 RJ1654
x
Bacterial Chromosome
gal
Integration
LE392
Int T IHF
Int IHF
RJ1785
bio
attB
(Fis)
Excision
Xis Fis
gal
Genotype
Strain Phage x Chromosome
x
attl,
bio
attR
B. attL
B
0
C'
P'1 P'2 P'3 H'-.
.................................................................. CP1
P2
-
HI
COB'
X1X2
attR
--0 F
H2
FIG. 1. (A) Schematic representation of X recombination reactions. Integration is a conservative, site-specific DNA recombination reaction that occurs between the phage attachment site attP and the bacterial attachment site attB, producing the recombinant phage-host junctions attL and attR. It requires the phage-encoded Int protein and the E. coli protein IHF and is moderately stimulated by Fis in vivo, especially in the absence of Xis (2). The excision reaction takes place between attR and attL to yield attP and attB. In addition to Int and IHF, excision requires the phage protein Xis and is stimulated by Fis in vitro (27) and in vivo (see the text). (B) Relative locations of protein-binding sites on attP as determined by nuclease protection and mutational analysis (reviewed in reference 15). The Int-binding sites, of which B, C', P'1, P'2, P2, C, and B' are required for excision, are shown (_). Excision also requires IHF bound to H' and H2 (121) and Xis binding to Xl (E:]). Efficient excision in vivo requires Fis bound to F ( m ), which overlaps the X2 Xis-binding site. Xis can bind to both Xl and X2 in vitro to promote excision in the absence of Fis. Arrows indicate the relative orientations of the binding sites.
previously (17). Titers of phage lysates were determined, and the lysates were prepared by using T top agar and plates (23). RJ1654 and RJ1785 were constructed by subjecting either RW842 or RJ1784 (RW842 transduced tofis::767 with Plvir) to a red-plaque assay with XY1 (7). Resulting gal' colonies lack the defective prophage integrated in the gal locus and were shown to be lysogens of XY1 by their sensitivity to high temperature (42°C) and the presence of a single, integrated prophage in a Southern blot. XRJ996 was constructed as described in the accompanying paper (2), using a plasmid provided by J. Thompson and A. Landy. Restriction enzymes and DNA polymerase were purchased from Boehringer Mannheim.
Phages XY1 XRJ997 XRJ996 XRJ998 XY281
araDJ39 A(ara-leu)7697 AlacX74 galU galK strA MC1000fis::767 HfrH galT::XA(int-FII) RW842 fis::767 RW842 gal', lysogenized with XY1 RJ1784 gal', lysogenized with XY1 tonA lac Y galK metB trpR hsdR hsdM+ supE supF XcI857
xcI+ XcI857attPF XcI857Sam7 XWam4O3Eam11OOcI857
Source or reference
M. Casabadan
12 7 This work This work
This work L. Enquist R. Weisberg Laboratory collection 2 Laboratory collection 7
Phage growth. Stable lysogens were grown in LB at 30°C (for XcI857 lysogens) or 37°C (for XcI+ lysogens) to an optical density at 600 nm of 0.3. XcI857 lysogens were induced by shaking at 42°C for 30 min, followed by shaking at 37°C until lysis. XcI+ lysogens were induced by addition of mitomycin C to a final concentration of 5 jig/ml and shaking at 37°C until lysis. Lysates were cleared by addition of chloroform followed by centrifugation for 10 min at 6,000 x g. Lysate titers were determined by using LE392 as the host. For examining one-step phage yields, phage were added to a culture grown to an optical density at 600 nm of 0.3 at a multiplicity of infection of 0.1 and incubated with gentle shaking at 37°C. After 10 min of adsorption, the cells were washed and resuspended in fresh media. At intervals, samples were removed and lysates were prepared by freezing at -70°C followed by thawing in the presence of chloroform. The debris were removed by centrifugation, and the number of PFU was assayed by using LE392. Red-colony assay. Serial dilutions of XY281 were added to RW842 or RJ1784 grown to an optical density at 600 nm of 0.3, incubated for 20 min at 30°C, and plated as described previously (7) onto galactose-tetrazolium plates. Plates were incubated for 18 h at 30°C and scored for the number of red colonies on a white lawn as a function of input phage (7). Southern blot. Cells were grown to an optical density at 600 nm of 0.3 and induced by shifting to 42°C. At various times, 10-ml aliquots of cultures were chilled, harvested, washed in 50 mM Tris-HCl (pH 8.0), and resuspended in 0.5 ml of 50 mM Tris-HCl (pH 8.3)-10 mM EDTA. The resuspended cells were frozen in a dry-ice-ethanol bath, thawed on ice, and incubated for 30 min on ice in the presence of 100 ,ug of lysozyme per ml. Lysates were treated with sodium dodecyl sulfate (SDS) (final concentration, 0.2%) and proteinase K (final concentration, 100 ,ug/ml) at 65°C for 30 min. Two phenol-chloroform extractions followed by two ether extractions were performed. After addition of sodium acetate to 0.3 M, 1 volume of absolute ethanol was added and the precipitated DNA was wound onto a glass rod. The DNA was resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA, and aliquots were digested with EcoRT. Electrophoresis was performed in a 0.9% agarose gel by using Trisphosphate buffer, and transfer and hybridization were as described elsewhere (22). Total A DNA that was radiolabeled with 32P by using the random-priming method de-
EXCISION OF A FROM E. COLI REQUIRES Fis PROTEIN
VOL. 173, 1991
TABLE 2. Phage yields upon induction of A lysogens Ph
MC1000 (fis+) RJ1617 (fis)
XY1 (cI857) XY1 (cI857)
(8.7 ± 0.9) X 109 (3.7 ± 0.5) x 107 1.0 X 109b
MC1000 (fis+) RJ1617 (fis)
XRJ997 (cI+) XRJ997 (clI+)
(2.5 ± 0.5) x 1010 (1.2 ± 0.5) x 108 (3.6 ± 0.1) x 109b
MC1000 Osk) RJ1617 (fis)
XRJ996 (attRF) XRJ996 (attRF)
10.0
Phage yield (PFU/ml)a
Bacterial strain
age
4029
co
6.0
(1.0 ± 0.5) X 108 (0.9 ± 0.4) x 108
a Values represent the averages of yields from induction of four independently isolated lysogens. b These lysogens have been shown by Southern blot analysis to be double lysogens, each carrying two tandem integrated copies of the X chromosome.
scribed elsewhere (22) was used as the hybridization probe. The recA probe was generated from pBRrecA, obtained from K. McEntee. RESULTS Phage yields from a K lysogen are decreased in afis strain. To determine whether Fis plays a role in A excision in vivo, fis andfis+ E. coli strains were stably lysogenized with XY1, a phage that carries the gene encoding the cI857 temperature-sensitive repressor. The phage yields obtained upon heat induction of fourfis' lysogens and fourfis lysogens are shown in Table 2. Lysogens infis' cells gave yields of about 8.7 x 109 phage per ml. However, the lysogens in fis cells give two populations of phage yields: one at 1 x 109 phage per ml and the other at 3.7 x 107 phage per ml. Southern blots demonstrated that the lysogens infis cells that give the higher phage yield are double lysogens with two tandem copies of A integrated into the E. coli chromosome (data not shown). Double lysogens have the ability to allow phage DNA packaging through their tandem cos sites and therefore give phage yields that do not reflect the frequency of excisive recombination. Efficient packaging of polylysogens in the absence of excisive recombination has been previously observed in phages unable to excise as a result of a mutation in int (10). Yields from induction of singly integrated prophage (which are dependent on excision) in afis background show a reduction of 235-fold compared with the phage yields from induction of fis' lysogens. The higher phage yields produced from the double lysogens imply that the absence of Fis is directly affecting excision. Lysogens of XRJ997 (cI+) phage in the same strain backgrounds show a similar, 208-fold difference in phage yields when induced with mitomycin C (Table 2). Lytic growth is not affected by the absence of Fis. Although phage yields are good indicators of excision frequencies, they may also be affected by defects in other steps of lytic growth, such as phage replication or packaging. To test whether the low phage yields obtained from fis cells are due to a defect in lytic growth, we performed a one-step phage growth experiment. MC1000 and RJ1617 were incubated with XRJ998 for 10 min, washed, resuspended in fresh media, and grown lytically at 37°C. Aliquots were removed at various intervals, the cells were lysed, and the number of PFU present was assayed. The resulting phage yields from MC1000 and RJ1617 cells are plotted as a function of time in Fig. 2. The phage growth curve obtained from MC1000 is
4.0
2.0 0
50
100 Time
150
200
(min)
FIG. 2. Phage growth curve. Mid-log cultures of either MC1000 or RJ1617 were adsorbed with 4 x 106 XRJ998 phage for 10 min, washed, and grown at 37°C. The yield of phage (PFU) is plotted as a function of time. Symbols: O, phage yields infis+ cells; *, phage yields infis cells.
nearly identical to that from RJ1617 cells, indicating that X replication, packaging, and overall gene expression involved in lytic growth are unaffected by the absence of Fis. Red-colony assay for excision. The red-colony assay, developed by Enquist and Weisberg (7), provides a means of studying excisive recombination independent of the effects of lytic growth. This assay takes advantage of a strain (RW842) with a defective A prophage integrated into the galT locus of the E. coli chromosome, thus rendering the strain unable to utilize galactose. The prophage cannot excise except when supplied in trans with Int and Xis from an infecting X. When the prophage is excised, the integrity of the gal locus is restored and the gal' cells can form red colonies against a lawn of white cells on a galactosetetrazolium medium. The fis::767 mutation was transduced into the RW842 background to yield RJ1784 and used in a red-colony assay with XY281. The use of XY281 containing amber mutations in W and E ensures that no phage particles can be formed, and therefore only a single infective cycle is possible. The frequency of gal' red-colony formation was 1.02 x 10-3 per input phage in fis' cells, similar to the frequency reported by Enquist and Weisberg (7) with exponentially growing cells. Red-colony formation was reduced 16-fold to 6.37 x 10-5 per input phage in fis cells. This experiment indicates that the defect observed in phage yields fromfis X lysogens can be attributed to defects in excision. Southern blot analysis of excision. To directly assay the product of the X excision reaction, we performed a Southern blot analysis on total DNA from RJ1654 and RJ1785 lysogens that were induced for increasing lengths of time. The blot was hybridized with 32P-labeled X DNA to reveal the extent of X excision and replication (Fig. 3A) and reprobed with a 32P-labeled plasmid carrying unrelated DNA to determine the amount of chromosomal DNA loaded onto each lane (Fig. 3B). The product of excision is attP, which in this case is
4030
BALL AND JOHNSON
J. BACTERIOL.
DISCUSSION kb:
Fis + cells
a.
A. A probe
I0
-
5.8
-
a
sign.w -
0
5
cells
1530 45 60
,4
-
.
(U//Pl5.6 4.9
¶5 3045 60
5
Fis-
..
phqge/host _ to
___04W
"tS
junction -
o,//P
-phage/host junction
B. recA probe FIG. 3. Southern blot of excision time course. Lytic growth of XcI857 lysogens was induced by incubation at 42°C. DNA was isolated from cells immediately before induction and 5, 15, 30, 45, and 60 min after induction. The DNA was digested with EcoRI, electrophoresed in an agarose gel, and transferred to a nitrocellulose filter. (A) The filter was hybridized with 32P-labeled A DNA. The leftmost lane contains DNA isolated from X particles and digested with EcoRI. Appearance of the 5.6-kb attP-containing fragment that is the product of excision is dramatically reduced in fis cells compared with fis+ cells. (B) The same filter was also hybridized with a 32P-labeled plasmid carrying the recA gene, located 41 min from attB in the E. coli genetic map, to estimate the quantity of DNA loaded in each lane.
present on a 5.6-kb EcoRI fragment and is detectable in the lane containing DNA obtained from phage particles (Fig. 3A, left-hand lane). The A chromosome is present in a single integrated copy in both fis+ and fis cells before induction (t = 0). After induction of the fis' lysogen, the phage chromosome is replicated to a high copy number and the attP band is detectable within 5 min of induction and is readily apparent after 15 min. This is similar to the kinetics of excision as measured by Southern blot analysis by Ljungquist and Bukhari (16). The integrated chromosome achieves a high copy number in the fis cells, presumably owing to multiple rounds of replication initiating at the X origin within the integrated phage chromosome. However, in thefis cells, the 5.6-kb attP band is barely detectable after 60 min of induction. Therefore, we conclude that in the absence of Fis, excisive recombination is severely defective, whereas replication of the induced prophage DNA appears to be efficient. The experiment also indicates that excision in vivo is rather inefficient, even in the presence of Fis. Analysis by densitometry indicates that as much as 45% of the phage DNA remains integrated into the chromosome at 60 min after induction, just before cell lysis. Excision of attRF phage. Because the defect in excision observed infis cells could be explained by indirect effects of Fis, such as regulation of IHF, Int, or Xis levels, rather than a direct effect of binding to the F site in attR, a mutant phage (XRJ996) was constructed. XRJ996 contains the DNA sequence of the Xl Xis-binding site substituting for the X2-F region. This substitution (originally constructed in pJT111) prevents Fis binding but allows for efficient excision in vitro (26). Lysogens of this phage were induced, and excision efficiency was measured by determining phage yields. As shown in Table 2, afis lysogen yielded 0.9 x 108 phage per ml, but in this case afis' lysogen also produced a low phage yield (1.0 x 108). Thus, Fis binding to the attR F site appears to be directly responsible for most, if not all, of its stimulatory effect on X excision.
Fis binding to F in the attR recombination site is required for efficient excisive recombination of phage X in vivo. Thermoinduction of temperature-sensitive prophage or mitomycin C induction of AcI+ in fis cells resulted in phage yields that are reduced by a factor of more than 200 when compared with lysogens in fis' cells. Measurements of phage yields may amplify the effect of Fis on excision frequency, since the phage DNA may replicate more efficiently when it has excised from the chromosome. However, direct examination of X DNA after induction failed to reveal a significant difference in replication efficiency in fis cells, even though only a trace of excision was detectable. The effect of Fis on phage yields is specific to the excision reaction; we find no evidence for defects in lytic growth associated with the absence of Fis. The reduced excision observed in Fis null mutants is due solely to the lack of Fis binding to the F site on attR. Mutant phage which do not contain the attR F site gave similarly low yields in the presence and absence of Fis. Thompson et al. (27) have shown in in vitro studies that Fis can cooperatively enhance Xis binding to Xl and, under certain conditions, increase the efficiency of excision. A 20-fold stimulatory effect of Fis on excision is observed in vitro only when Xis is limiting. Numrych et al. (19) have also reported that Fis stimulates excision in vitro and in experiments in vivo measuring the curing of heat-pulsed XcI857 lysogens. The effect on curing was most dramatic when using phage that contained mutations in Int arm-binding sites believed to be critical for excision. For example, curing of a phage containing a mutation in the Int P2 site located adjacent to the X1-F region in attR was reduced more than 30-fold infis strains. One model for the activity of Fis in promoting excision is that binding at attR F induces a structural change in the DNA that results in an increase in the affinity of Xis for Xl. Alternatively, protein-protein contacts may be formed between Fis and Xis that promote or stabilize Xis binding to Xl. In addition to facilitating Xis binding, the binding of Fis to attR F causes a bend in the DNA, which is believed to contribute to the stimulation of the excisive recombination reaction. Upon binding, Xis and Fis each bend DNA at an angle of approximately 90°C (25). The additive effects of Xis and Fis binding to attR create a sharper bend than the binding of either protein alone. Since both Fis and Xis bend the DNA similarly, the structure formed by the binding of Xis to Xl and Fis to attR F may be functionally equivalent to that formed by the binding of Xis to both Xl and X2 (26). The fact that the structural perturbation of DNA is similar in the presence of two Xis protomers or Fis plus Xis, combined with the fact that excision can occur efficiently in vitro given sufficiently high Xis concentrations, suggests that lambda may synthesize insufficient Xis to efficiently bind on its own to attR. The large quantity of surrounding chromosomal DNA that is present in vivo may compete with attR for limiting quantities of Xis. Fis bound to the F site at attR may direct Xis to bind to Xl. It is quite possible that high cellular Xis concentrations would alleviate the requirement for Fis, although we have been unsuccessful in overcoming the requirement for Fis by introducing multicopy Xis-overproducing plasmids (2a). However, it is not known how much Xis overproduction has been achieved in these experiments. There are additional factors that may also contribute to the greater effect of Fis measured in vivo than in vitro. Some
VOL. 173, 1991
EXCISION OF X FROM E. COLI REQUIRES Fis PROTEIN
characteristic of DNA topology or chromatin structure may accentuate the Fis requirement in vivo but not in vitro. For example, IHF stimulation of the Mu transposition is observed only at physiological supercoiling densities (24). It is also possible that levels of other proteins involved in excisive recombination are reduced in fis cells. Because the excision defect is not overcome by expression of Xis from a heterologous promoter, it is unlikely that the defect is caused by lowered Xis expression infis cells. Measurements of IHF levels in fis' versus fis cells by gel shift (2a) or Western immunoblot (6a) analysis have not revealed any significant differences. The levels of Int in the absence of Fis have not been examined, although the fact that yields of XattPF phage are not significantly lower in fis cells argues against a model in which the excision frequency is reduced because the expression or activity of Int, or the other recombination proteins, is affected by Fis. Thus, our results argue that in vivo X excision normally requires Fis bound at attR F and that Xis binding to X2 is largely irrelevant. Data in the accompanying paper are consistent with this conclusion (2). ACKNOWLEDGMENTS We thank A. Landy, H. Nash, and R. Weisberg for their interest, suggestions, and gifts of strains and phage. We are particularly grateful to J. Thompson and A. Landy for providing us with several plasmids that were made specifically for this project. We also acknowledge members of this laboratory for their help and criticism during the preparation of the manuscript. R.C.J. was the recipient of grants from the National Institutes of Health (GM-38509), March of Dimes, and the Searle Scholars Program/Chicago Community Trust. C.A.B. was supported in part by USPHS National Research Service award GM-07104.
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binding properties of Fis and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin. J. Mol. Biol. 198:579-587. 12. Johnson, R. C., C. A. Ball, D. Pfeffer, and M. Simon. 1988. Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc. Natl. Acad. Sci. USA 85:34843488. 13. Johnson, R. C., M. Bruist, and M. I. Simon. 1986. Host protein requirements for in vitro site-specific DNA inversion. Cell 46:531-539. 14. Koch, C., and R. Kahmann. 1986. Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J. Biol. Chem. 261:15673-15678. 15. Landy, A. 1989. Dynamic, structural, and regulatory aspects of A site-specific recombination. Annu. Rev. Biochem. 58:913-949. 16. Ljungquist, E., and A. I. Bukhari. 1977. State of prophage Mu DNA upon induction. Proc. Natl. Acad. Sci. USA 74:31433147. 17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Nash, H. A., and C. A. Robertson. 1981. Purification and properties of the Escherichia coli protein factor required for A integrative recombination. J. Biol. Chem. 256:9246-9253. 19. Numrych, T. E., R. I. Gumport, and J. F. Gardner. 1990. A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda. Nucleic Acids Res. 18:3953-3959. 20. Richet, E., P. Abcarian, and H. A. Nash. 1986. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell 46: 1011-1021. 21. Richet, E., P. Abcarian, and H. A. Nash. 1988. Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex. Cell 52:9-17. 22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Silhavy, T., M. L. Berman, and L. W. Enquist. 1984. Experiments in gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Surette, M. G., and G. Chaconas. 1989. A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor. J. Biol. Chem. 264:3028-3034. 25. Thompson, J. F., and A. Landy. 1988. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 16: 9687-9705. 26. Thompson, J. F., H. F. L. Mark, B. Franz, and A. Landy. 1988. Functional and structural characterization of stable DNA curvature in lambda attP, p. 119-128. In W. K. Olsen, M. H. Sarma, and M. Sundaralingam (ed.), Structure and expression, vol. 3. DNA bending and curvature. Adenine Press, Schenectady, N.Y. 27. Thompson, J. F., L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy. 1987. Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway. Cell 50:901-908. 28. Thompson, J. F., D. Waechter-Brulla, R. I. Gumport, J. F. Gardner, L. Moitoso de Vargas, and A. Landy. 1986. Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications. J. Bacteriol. 168:218-222. 29. Yin, S., W. Bushman, and A. Landy. 1985. Interaction of the X site-specific recombination protein Xis with attachment site DNA. Proc. Natl. Acad. Sci. USA 82:1040-1044.
