Mol Gen Genet (1981) 183:192-196 © Springer-Verlag 1981

Recombinational Instability of F' Plasmids in Eschevichia coli K-12 Localization of fre-Sites S.E. Bresler, S.V. Krivonogov, and V.A. Lanzov Leningrad Institute of Nuclear Physics of the Academy of Sciences of USSR

Summary. The F' plasmids ORF-1 (purE + tsx s proC + lac +) and F'14 (argE + metB + ilv +) contain active regions of recombination, fre I and fre II correspondingly. The plasmid ORF-1 is stable in recF- cells (i.e., with the RecBC pathway of recombination) and decays in rec + cells (RecBCF pathway) giving two types of product: F + and plasmid pCK-1 (tsx ~ proC + lac +) containing part of the initial D N A . They are extremely instable in the presence of the RecF pathway, (recBC- sbcB ), yielding F + and plasmid pCK-2 (proC + lac +). The instability of plasmids depends on a region of homology between the chromosome and the episome. The instability of ORF-1 shows the participation of IS3 elements (al/33 and a3/31) in the recA, recF-dependent recombinational decay and allows localization of two active sites on the chromosome: fre I1 between purE and tsx markers and fre I2 between tsx and proC. The plasmid F'14, in accordance with published data, is able to yield F + cells by recA-independent recombination. But eventually this plasmid may undergo a recA, recF-dependent decay. Genetic analysis of these events allows localization of an active point of recombination, fre II1, between argE and metB. Another active point is localized inside the F factor. The recA-dependent decay of plasmid F-14 is also excluded on the RecBC pathway (recF- strains).

Introduction

recA-dependent recombination in Escherichia coli is effected by several metabolic pathways (Clark 1974, 1979). In a recent paper (Bresler et al. 1981) we explained reasons for modification of the general scheme proposed by Clark so that three pathways of general recombination are introduced: the RecBC pathway, acting in recF- strains ; the RecF pathway functional in recBCsbcB-, cells and a mixed RecBCF pathway characteristic of wild type, i.e., rec + cells. Both the RecBC and RecF pathways are site specific. The former is initiated on the chi sites (Malone et al. 1978; Chattoraj et al. 1979; Stahl 1979). The latter requires the fre sites (Bresler et al. 1978). The chi sites are evenly and closely distributed over the E. coli chromosome and therefore are not very demonstrative in conjugational crosses. But the fre sites are not so numerous and are localized on specific regions of the chromosome. Therefore they are actively expressed sites of recombination. We found the region fre I in the lae-purE interval, the regionfre II in the ilv-argE interval, and the region Offprint requests to: Prof. S.E. Bresler, Leningrad Institute of Nuclear Physics, Gatchina, Leningrad district 188350, U S S R

0026-8925/81/0183/0192/$01.00

fre III near trp on the E. coli map (Bachmann and Low 1980). To study the site specificity of recombination we used different methods: Measurement of linkage in transconjugants; decay of F' plasmids during conjugation with the formation of deletions; and mobilization of the E. coli chromosome by the free F factor (Bresler et al. 1978, 1979). The latter phenomenon led us to the conclusion that the fre sites may be found among the IS elements responsible for the integration of F factors into the chromosome, i.e., IS2, IS3, and 76. In the present paper we report a study of recombinational decay of F' plasmids both by genetic and physical methods. We have investigated structural alterations in the decaying plasmids. Hence we have been able to localize with good precision some of the active sites in the regions fre I and fre II. Materials and Methods

Bacterial Strains are listed in Table 1. Restriction Endonuclease BamH1 was obtained from the Novosibirsk Bureau of Biologically Active Substances. The reaction conditions were the same as in the work of Alton and Vapnek (1978). Phages, Media, Conjugation Procedures and Scoringfor Recombinants were described earlier (Bresler et al. 1978). The value of the linkage coefficient g = 1 - w , was measured where w is the normalized recombination probability. The transcription P.lac+_purE=0.4(200) means the ratio of the clones selected primarily for Lac + which inherited the nonselected marker PurE +. 200 is the number of selected Lac + clones. Preparation of Plasmid DNA and Estimation of Molecular Weights were performed after Hansen and Olsen (1978). For calibration we used the known plasmids F + (63 megadaltons, MD) and RP4 (36 MD). The error was estimated as the deviation from the average value in 3-4 independent measurements. Preparation of F Factor DNA for Restriction Analysis was effected by cell lysis, alkaline denaturation, neutralization and elimination of the membrane-chromosomal complex, all procedures after Hansen and Olsen (1978). Subsequent purification of plasmid DNA included standard phenol extractions. Electrophoresis ofPlasmid DNA was performed in 9 x 12 x 0.5 cm slab gels of 0.7% agarose (BioRad, California, USA) for 3 5 h at 20 mA. Results and Discussion

Region fre I The plasmid ORF-1 transfers during conjugation a chromosome fragment involving the whole or the major part of the fre I

193 Table 1. Bacterial strains

Strain

Genotype

Source

RP4/J53

RP4: Tc Km Ap N m / F - : p r o

Dr. N. Datta

met

F+:

WA 993 LC 179 F': Z517 GC 417 533 Hfr: P 72 F

gal m e t h s d M hsdR thr leu lac thyA mal dnaA ilv thi

Dr. B. Wolf Dr. L. Caro

F'(ORF-1): p u r E + - l a c ÷ / F - : ( p u r E - l a c ) F'14 : argE+-ilv +/F - : (argE-ilv) p r o A lac gal his strA thi F'13 :lac +-purE÷/F - :azi tsx strA m e t

Dr. R. Curtiss Dr. J. George Dr. W. Hayes

m e t B thi

Dr. F. Jacob

l a c Z p r o C tsx trp strA thr ara leu p r o A tsx lac his strA argE thi as AB 1157 but recA13 as AB 1157 but recB21 recB22 sbcB15 as AB 1157 but recF143 as JC 7623 but arg + x y l + rnetB recF143 as ECK 033 but recBC ÷ as JC 7623 but nalB f e c a l 3

Dr. F. Jacob Dr. P. Howard-Flanders Dr. P. Howard-Flanders Dr. A.J. Clark Dr. A.J. Clark This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory F+/LC 179 x ECK 061 F +/LC 179 x ECK 064 P 72 x ECK 070 P 72 x ECK 071 F +/LC 179 x JC 9239 P 72x ECK 074

:

X 5036 AB 1157 AB 2463 JC 7623 JC 9239 ECK 033 ECK 035 ECK 041 ECK 061 ECK 064 ECK 067 ECK 070 ECK 071 ECK 072 ECK 073 ECK 074 ECK 075

as AB 1157 but pro ÷ as JC 7623 but pro ÷ ara leu lac p r o C p u r E gal trp strA m t l x y l ilv m e t A thi as AB 1157 but pro + x y l + ilv as JC 7623 but pro + x y l + ilv as ECK 070 but thr + leu + m e t B as ECK 071 but thr + leu + m e t B as JC 9239 but x y l + ilv as ECK 074 but thr + leu + m e t B

IS3

IS3 lac

,,~,,,; r~;rql..~

~.

~

pro C

tsx

purE

...... r~ovr, ,,-...... m m I n ~

freI2 freIl Fig. 1. Structure of a part of the chromosome of Hfr OR11 (Hu et al. 1975; Hadley and Deonier 1979). Full line: F factor; Wavy line: chromosome. Shady box: IS3 ; empty box : IS2; empty circle: fre-site

Fig. 2a-c. Electrophoretic patterns of plasmid DNA. a Plasmid ORF-l,transferred into strain AB 1157 immediately after conjugation, b F + plasmid in strain WA 993. c Original plasmid ORF-I in strain 7~517

a

b

c

region with the sequence of markers p u r E + - t s x s - p r o C +-lac +. This plasmid originates from the d o n o r O R 11 by integration of the F factor into an IS3 element (see Fig. 1). The stability of this plasmid in the initial strain ORF-1/X517 is guaranteed by deletion of a corresponding part of the chromosome, homologous to the c h r o m o s o m a l piece of D N A in the plasmid. Electrophoretic analysis of D N A from transconjugants (products of the cross ORF-1/)~517 t s x s stW x AB 1157 lac t s x r rec + str r) manifests three zones of plasmid D N A (Fig. 2, lane a). One is due to the original plasmid ORF-1 (Fig. 2, lane c), a second to the F factor (Fig. 2, lane b), a third to a new plasmid, n o m i n a t e d pCK-1 (85_+4 MD). After subsequent cultivation of transconjugants F ' / A B 1157 (7-9 generations) the zone of the original plasmid disappears. Presumably the transconjugants contain two plasmids within the genetic b a c k g r o u n d of strain AB 1157; a free F factor and a short plasmid, pCK-1. These strains are able to transmit the F factor with high probability into several recipients (AB 1157, E C K 062, X5036). This was established by the acquisition of new properties by these cells, sensitivity to phage MS2 and ability to mobilize the chromosome. The fact that the F factor is excised from the ORF-1 plasmid was shown by restriction analysis. F factor D N A was prepared from the strain F + / W A 993 and served as a control. Both sets of restriction fragments from F +/WA 993 and F +/AB 1157 were found to be identical. A similar excision was observed of the free F factor from the plasmid F'13 in a wild-type b a c k g r o u n d (data not presented). In our previous paper (Bresler et al. 1978) genetic analysis showed decay of plasmid ORF-1 by the RecF pathway of recombination. The plasmid pCK-1 is not transmissi-

194

a

b

c

Fig. 3a-e. Comparative electrophoresis of restriction fragments (endonuclease BamHI) of F plasmids from different sources, a Wild-type plasmid from strain WA 993; b F plasmid originating by decay of ORF-1 in the recipient AB 1157; c F plasmid formed by cleavage of ORF-1 in the strain JC 7623

a

b

c

d

e

f

Fig. 4a-f. Electrophoretic pattern of plasmid ORF-I harbored by strains with different recombination pathways: a ORF-1/AB2463 recA; b ORF-1/JC 9239 recF; e ORF-1/ECK 035 recF; d ORF-1/ ECK 033 recBC sbcB recF; e control ORF-1/Z517; f control F+/ WA 993 •

Fig. 5a-d. Electrophoretic pattern of plasmid ORF-1 DNA from different sources : a transconjugants AB 1157 after cultivation for 7 8 generations; b, e independent clones of transconjugants Lac + Tsx' Str' after a cross ORF-1/Z517 x JC 7623 ; d transconjugant Lac + Tsx s Str s from the same cross

a

b

c

d

ble but propagates in cells during their division. It inherits the markers lac+-proC+-tsx ~ but not the subsequent p u r e +. This was confirmed by the following data: (1) After a cross ORF-1/ )~517 x E C K 067 full linkage was found between selected PurE + and nonselected Lac + markers, ~tpurE+.lac=1 (200 clones analyzed) but practically no linkage between these markers after selection of Lac + recombinants: gloc+_purE=0.58(200). Hence it is apparent that the linkage between the proximal marker PurE + and the distal marker Lac + on the episome is interrupted. Subsequent cultivation of the products of these crosses showed that the PurE + marker is totally lost in 90% of the cells. This means that the PurE + locus of the plasmid ORF-1 is degraded and lost after transmission into rec + recipients. (2) After performing beforehand a cross ORF-1/Z517 x AB 1157 its product was used as a donor with the recipient E C K 067. We found 100% linkage between the markers Lac +, ProC +, Tsx s ( g = 1) but total absence of linkage between Lac + and PurE + : glac*_purE=0.4(200), gpu,.E+.lae =0.53(200). In this case the marker PurE + is obviously absent from the episome and can originate only from the chromosome of the recipient ceil (AB 1157) by F-mobilization. Comparing our new plasmid pCK-1 with the genetic map of Hfr O R t l (Fig. 1) it may be assumed that excision of the D N A fragment containing PurE is performed with the participation of the f r e I1 site and the IS3 element located inside the F factor. The instability of the plasmid ORF-1 in rec + strains is an incontestable recombination phenomenon because it is absolutely dependent on the presence of homologous sequences in the plasmid and chromosome and on the functional integrity of the RecA and RecF genes. In Fig. 4 is given direct proof of this last statement. The electrophoretic patterns of D N A from transconjugants in the case of recipients of type recA, recF or r e c B C sbcB recF show only one zone. The corresponding cells harbor only one stable pIasmid, ORF-1. To study the genetic determination of the decay of the ORF-1 plasmid a cross was made with a recipient possessing the RecF recombination pathway ORF-1/)~517 s t r S x JC 7623 r e c B C sbcB tsx r str r. After a primary selection for Lac + Str + it was found that up to 90% of the transmitted plasmids decayed with the loss of the dominant marker Tsx ~ (Bresler et al, 1978). The electrophoretic patterns of these altered plasmids contained two zones (Fig. 5, lanes a and b). One very intense zone corresponds to a new plasmid of 54_+ 2 M D pCK-2, a second much weaker zone is presumably the same pCK-1 (88_+2 MD) found earlier in recipients with the RecBCF pathway. In this case no free F factor was revealed but the cells were preferentially Hfrs. It can be predicted that they are mostly Hfr C because their point of origin is one of the most active points in the .free I region (Bresler et al. 1979). To verify this prediction ten independent Lac + Tsx r transconjugants were studied. (1) All of them transmitted the Lac ÷ marker into the recipient ECK 067 with high efficiency (proximal marker). (2) The corresponding secondary transconjugants were stable to the phage MS2, i.e. were free of F factors. (3) These transconjugants revealed the following linkage values: g lac+.p~re= 0.9(200) ; Ixlac+.proC= gproC* lac= 1(200) ; gp.rE+_lac = 0.53(200). These figures are characteristic of Hfrs transmitting the Lac + marker distal to the PurE +. After cultivation of the mixed clones pCK-2/pCK-1/JC 7623 (7-8 generations) no more of the pCK-1 zone was found in their electrophoretic patterns. Probably pCK-2 is a descendent of pCK-1. Transfer of the plasmid pCK-1 from the strain F+/ pCK-1/AB 1 t57 into the recipient JC 7623 is accompanied by the loss of the dominant marker Tsx S and electrophoresis of the product shows that it contains only pCK-2. Additional information can be obtained by analysis of the

195 ilv ~'b

rrrrrrm 1wTfr

T' |

~'0 •

argE

LqT umJ

metB

freli1

Region fre H

(841) (84/8g.2)(89) (88.3) Fig. 6. Structure of plasmJd F'14 (Ohtsuba et al. 1974a). Wavy line: bacterial component. • element 75; x the point of circularization of the plasmid. All other symbols as Fig. 1. Numbers in brackets: localization of the corresponding marker on the map in min (Bachmann and Low 1980)

Table 2. Genetic instability of the plasmid F'14: argE + metB + ilv* (+ + +) after its cultivation in recipients possessing different pathways of recombination Recombination pathway (genotype)

Composition of daughter cells (%) F + cells, Cells with genotype recombinant (---) genotypes (+ +-), (+--) and

Cells with nonrecombinant genotypes (+ + +)

(+) RecBCF (rec +) RecBC (recF) RecF a (recBC sbcB)

70 80 30

10 <1 70

factor and the Tsx ~ locus from the ORF-1 plasmid into the chromosome.

20 20 0.5

a Approximately half of the transconjugants with the RecF pathway appeared to be Hfrs of genotype + + +. They were absolutely stable (data not presented)

transconjugants Lac + Tsx Swhich are formed with low frequency in the Cross ORF-1/Z517 x JC 7623. The electrophoretic pattern of these clones reveals two plasmids (Fig. 5, lane d). One (64 + 1 MD) is the F factor (as established by restriction analysis, see Fig. 3), the second corresponds to pCK-2 (55_+ 2 MD). The resulting strains F + / p C K - 2 / J C 7623 are good donors of the F factor and transmit the markers Lac ÷ and ProC + to the recipient ECK 067 with a 50-fold higher efficiency than PurE-- : besides, the linkage glac+_proC=gproC+_lac= 1(200). Hence we conclude that both markers Lac and ProC are transferred by the plasmid pCK-2, which is a derivative of pCK-1 with the Tsx s region entirely deleted. However this latter D N A particle was not lost because the resulting strain F +/pCK-2/JC 7623 became sensitive to phage T6. Presumably the particle was integrated into the chromosome by recombination. By genetic and electrophoretic methods some independent series of Lac + S t r r transconjugants were studied, products of genetic crosses O R F - 1 / Z 5 1 7 x A B l l 5 7 and ORF-1/Z517x JC 7623. In all cases the same products were obtained F +/pCK1/AB 1157, pCK-2/Hfr JC 7623, and F+/pCK-2/JC7623. Obviously there is strict site-specificity of cleavage of the plasmid ORF-1 into F +, pCK-1 or pCK-2. For the formation of pCK-2 a special point, fre 12, between ProC and Tsx is important (Fig. 1). Therefore we can localize two active points of recA, recF-dependent recombination, both belonging to fre I. Excision of the F factor shows the participation of the element IS3, in accordance with the observations of Hopkins et al. (1980) and our earlier suggestions (Bresler et al. 1979). The recombinational cleavage of plasmid ORF-1 into pCK-1 or pCK-2 requires a region of homology between the plasmid and the chromosome. Formation of the shorter plasmid pCK-2 is connected with two independent recombinational events, the integration of the F

This region partially overlaps the plasmid F'14 (204 MD), whose structure was studied in detail by physical methods (Ohtsubo et al. 1974a, b; Lee et al. 1974; Deonier et al. 1974). This plasmid originated from Hfr 313 in which the F factor was integrated by means of the insertion sequence 75. It is well known that this plasmid is extremely unstable (Pittard et al. 1963; Glandsdorf 1967). Cleavage of the plasmid F'14 is recA-independent (Ohtsubo et al. 1974a). The electrophoretic patterns of plasmid D N A from strains with different recombination genotypes reveal not only reeA, but also recF-independence of F factor excision (data not presented). On the other hand, a different type of instability is manifested in recA + recipients. The plasmid F'14 transmits its genetic markers during conjugation in the sequence ArgE +-MetB +-Ilv + ( + + +). Genetic analysis of transconjugants revealed cleavage of the plasmid on the RecF pathway. If primary selection is effected for ArgE + recombinants, we find the following combinations of genotypes : +++, 39%; + - + , 5%; + + - , 30%; + - - , 26%. The distance between the loci metB-argE or argE-ilv is about 1/7th of the distance ilv-metB, but recombination events in both regions are equiprobable. Hence we conclude that an active point is situated in the interval metB-argE (fre II1). Another active point in the region ilv-F factor-argE is situated presumably on F. It is a consequence of the observation that all stable recombination events inside this region (i.e., + + and + - - ) are Hfrs. Therefore we must assume that recombination removes or damages the sequence 7 c5 which is indispensable for excision of the F factor. Another confirmation of this fact follows from the fate of the unstable products + - + . This type of recombinant is obviously segregated without the participation of F factor genetic material. After a short period of cultivation these clones decay into F + (genotype - - - ) and Hfr ( + - - ) . The plasmid F'14 is also extremely unstable during cultuvation (Table 2). From Table 2 it can be seen that F + cells are actively segregated by all three pathways. Other recombinant types do not arise on the RecBC pathway but are seven times more frequent on the RecF pathway than on RecBCF. In summing up it can be seen that the recombination cleavage of plasmid F'14 allows localization of active site of recombination (fre I l l ) and a second one within the F factor. The latter leads, as in the case of ORF-1, to the formation of stable Hfrs. The active points are recA and recF-dependent and are most active on the RecF pathway of recombination. It is quite probable that the active points in the F factors are due to elements IS2 and IS3 (Deonier and Mirels 1977; Hopkins et al. 1980). In contrast with ORF-1, the plasmid F'14 is unable to give autoreplicative structures of type pCK besides F factors. The specific behaviour of plasmid ORF-1 forces us to suggest the existence of a site of replication initiation, ori, in the region lac-proC of E. coli, which is used in the multiplication of plasmids pCK-1 and pCK-2. Presumably this additional ori is functional only in the presence of the F factor because of complementation with its products. References

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and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J Bacteriol 135:227-238 Hopkins JD, Clements MB, Liang T-Y, Isberg RR, Syvanen M (1980) Recombination genes on the Escherichia coli sex factor specific for transposable elements. Proc Natl Acad Sci USA 77:2814-2818 Hu S, Ohtsubo E, Davidson N (1975) Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F 13 and related F-primes. J Bacteriol 122: 749 763 Lee H, Ohtsubo E, Deonier RC, Davidson N (1974) Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coll. V. Ilv + deletion mutants of F14. J Mol Biol 89 : 585-597 Malone RE, Chattoraj DK, Faulds DH, Stahl MM, Stahl FW (1978) Hotspots for generalized recombination in the Escherichia coli chromosome. J Mol Biol 121:473-491 Ohtsubo E, Deonier RC, Lee H J, DavidsonN (1974a) Electron microscope heteroduplex studies of sequence relations among plasmids ofEscherichia coli. IV. The F sequences in F14. J Mol Biol 89: 565584 Ohtsubo E, Lee HJ, Deonier RC, Davidson N (1974b) Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VI. Mapping of F14 sequences homologous to ~80dmetBJF and d?80argECBH bacteriophages. J Mol Biol 89 : 599-618 Pittard J, Loutit JS, Adelberg EA (1963) Gene transfer by F 1 strains of Escherichia coli K-12. I. Delay in initiation of chromosome transfer. J Bacteriol 85:1394-1408 Stahl FW (1979) Special sites in generalized recombination. Ann Rev Genet 13:7-24 Communicated by D. Goldfarb Received March 31, 1981