Vol. 139, No. 2

JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 597-607

0021-9193/79/08-0597/11$02.00/0

Isolation and Characterization of Replication-Deficient Mutants of ColE1 Plasmids TAMOTSU HASHIMOTO-GOTOHt AND JOSEPH INSELBURG* Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received for publication June 1979

Replication-defective mutants of plasmid ColEl were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColEl mutants isolated were all spontaneous deletion mutants that had lost the ColEl replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColEl plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColEl mutants was able to replicate in the presence of chloramphenicol. The expression of ColEl incompatibility was either markedly reduced or not detectable in the replication mutants isolated. Plasmid ColEl usually exists in Escherichia coli as an autonomously replicating, monomeric, circular, stably maintained DNA with a molecular weight of about 4.2 x 106. The plasmid genetic information that confers the replicative properties of ColEl on the plasmid DNA has been localized in a region that comprises about 10% of the total DNA (3, 15; unpublished data). This region, referred to as the "replication region," includes the replication origin and about 600 contiguous base pairs (bp) between the origin and the region coding for the colicin El immunity function (see Fig. 1). The nucleotide sequence of that region has been determined (3, 23; H. Ohmori and J. Tomizawa, personal communication). To analyze the replication region of ColE1, a chimeric plasmid, pHSG124, was constructed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1 (10), and the ColEl plasmid derivative, pDMS630 (13), that contains a Tn3 transposon (17) (Fig. 1). The Tn3 transposon, which carries a fi-lactamase gene, confers ampicillin (Ap) resistance on the host carrying it. While that newly formed chimera, pHSG124, would normally exhibit temperature-resistant replication due to the ColElmediated replication process, a mutation affecting ColEl replication would cause its replication to become temperature sensitive. Mutations affecting ColEl DNA replication were isolated and examined. All of the mutants to be described in this report contain deletions of the replication origin and terminate at different points in the

replication region of ColEl. The mutations block autonomous ColEl-moderated DNA replication. They also block the chloramphenicol (Cm)-resistant replication exhibited by a pSC101-ColEl chimera that is mediated by plasmid ColEl (4, 5). The effects of these mutations on the expression of ColEl incompatibility will be described.

t Present address: Max-Planck-Institut fiir Molekulare Genetik, D-1000, Berlin, Germany.

597

MATERIALS AND METHODS Bacterial strains and plasmids. The E. coli strains used were Om84 [tyr(Am) trp(Am) thy his ilv supD] (9); JC7623, a recB recC sbcB mutant strain from A. J. Clark (14, 21); and P678-54 (thr leu thi rpsL) (1, 12). All bacteria were grown at 30°C unless otherwise stated. The plasmids used are described in Table 1. pHSG1, previously named pHS1 (10), is a temperature-sensitive DNA replication mutant of pSC101 that contains genetic information conferring tetracycline (Tc) resistance. pDMS630, previously named 6-30 (13), is a ColEl derivative carrying the Tn3 transposon. It is colicin producing (Col+) and colicin El immune (Immn) and confers ampicillin resistance. pDMS6641, previously named d6-6d4-11 (15), is a deletion mutant of pDMS630. pHSG124, a chimera constructed during this work, contains pHSG1 and pDMS630 linked at their EcoRI endonuclease-sensitive sites. Derivatives of it are illustrated in Fig. 2. pSC134 is a composite plasmid containing both pSC101 and ColEl linked at their EcoRI endonuclease-sensitive sites (22); it was kindly provided by Y. Masamune. ColE1-Tn5 is a ColEl derivative that is Col- Imm+ and contains the Tn5 transposon, which confers kanamycin (Km) resistance (2); it was kindly supplied by D. Berg. pHSG101 is a chimeric plasmid composed of pSC101 and a kan-carrying EcoRI fragment derived from pML21. This plasmid is probably identical to pSC105 (7). pHSG100 is a spontaneous mutant of pHSG101

598

HASHIMOTO-GOTOH AND INSELBURG

Plasmid

Parental pHSG1 pDMS630 pHSG124

pSC101 replication type pHSG401 pHSG202 pHSG2O6 pHSG210 pHSG211 pHSG212 pHSG214 pHSG220 pHSG221

pHSG222

J. BACTrERIOL.

TABLE 1. Plasmids used and their derivatives Selective markerb Size (kbp) Repc AMpd Tc

Ap

Col

Imm

9.6 11.6 21.2

R S R

S R R

+ -

+ +

S R R

15.3 8.5 17.4 19.8 18.1 17.7 17.7 18.6 17.9 20.1

S R R R R R R R R R

R S R R R R R R R R

-

-

+ + + + + + + +

-

+

S S S S S S S S S S

Incompatibility with':

pSC1O1

ColEl

+ +

++ ++

++ ++

-

-

++ ++ ++ ++ ++ ++ ++ ++ ++

-

++

+ + + NT NT NT NT

-

-

ColEl replication type pHSG402 pHSG201

S S + 5.9 + R ++ S + 3.7 R + R ++ S pHSG2O8 3.6 + R + R ++ S pHSG2O9 4.5 + R + R ++ S 2.6 + pHSG215 R + R ++ S + pHSG216 3.0 R + R ++ S + R + pHSG217 2.6 R ++ 4.4 R + + S pHSG218 R ++ S + + 2.7 R R pHSG219 ++ aApproximate size, in kilobase pairs (kbp), determined by comparing the electrophoretic mobilities of endonuclease-cleaved DNA fragments of known and unknown DNAs. b Tc, Tetracycline; Ap, ampicillin; Col, colicin El production; Imm, colicin El immunity; R, resistance; S, sensitivity; +, either colicin production or immunity; -, the absence of either colicin production or immunity. c Rep, Replication property; S and R, temperature-sensitive and -resistant replication, respectively. d Amp, Amplifiability (i.e., long-term continued replication of the plasmid in the presence of chloramphenicol [180 ,ug/ml]). ' ++, Level of incompatibility similar to or greater than that of the wild-type plasmid; +, four- to sixfold reduction in the level of incompatibility between the plasmid and ColEl; -, no detectable incompatibility; NT, not tested. pSC101 or ColEl replication type refers to the replication characteristics of the plasmid. that is Tc5 (T. Hashimoto-Gotoh and K. Matsubara, unpublished data). Media. LS medium contained 10 g of peptone (Difco Laboratories), 5 g of yeast extract, 1 g of glucose, and 2.5 g of NaCl per liter (adjusted to pH 7.5). LS agar is LS medium containing 1.3% agar. M9 (6) medium supplemented with thymine and Casamino Acids (Difco) was used for growth of Om84. The concentrations of antibiotics were: ampicillin, 100 pg/ml; kanamycin, 10 pg/ml; and tetracycline, 5 ug/ml. Plasmid DNA isolation. Plasmid DNA was isolated exactly as described previously (15) with or without chloramphenicol treatment. RNA was removed from DNA preparations by passing them through a Bio-Gel A-15 m column. Covalently closed circular plasmid DNA was isolated by banding it in a cesium chloride-ethidium bromide density gradient

(15). Transformation. Transformation with isolated plasmid DNA was done without a heat pulse, as previously described (10).

Enzymatic treatment of DNA. Enzymatic digestion of DNA with BamHI, EcoRI, and HaeH endonucleases (obtained from New England Biolabs) was previously described, as were the agarose and polyacrylamide gel electrophoresis analyses of endonuclease-generated fragments (15). DNA ligation of endonuclease-cleaved fragments was performed with T4 DNA ligase obtained from New England Biolabs. The reactions were performed at 15°C for 4 h in a reaction mixture with a total volume of 17 pl. The mixtures contained about 200 pg of plasmid DNA per ml, 15 mM dithiothreitol, 88 ,uM ATP, 0.90 mM Tris-hydrochloride (pH 7.5), and 10 mM MgSO4. Temperature-sensitive-plasmid test. Cells containing a plasmid chimera were grown on LS agar at 430C without antibiotic. The drug resistance of cells derived from colonies picked from those plates was tested by plating them at 30°C on LS agar in the presence and absence of antibiotics (9). The original ColEl-pHSG1 chimera, treated in the same way, was used as a control.

REPLICATION-DEFICIENT ColEl MUTANTS

VOL. 139, 1979

RESULTS Both plasmid pDMS630 and pHSGl DNAs have a single EcoRI and a single BamHI endonuclease-sensitive site (Fig. 1). A chimeric plasmid, pHSG124, was constructed by cleaving each DNA with EcoRI endonuclease, ligating the linear monomers together with T4 ligase, and transforming E. coli cells with that DNA. Recipient cells that were Tcr Apr Imm+ ColTronsposition

Col El

'

ApH20H:

Tn3

pHISG1 A

mulagenesis

t

Rldrd-19

Amp', RepR

Amp,RepS

EcoRl T4-figose pHSG124 Amp+, Rep

pDMS630

pHSGI

pHSG402 Tn3

BamHI Tc

T4-ligose +

Amp

R Rep

Transformation of

\

/

H

exo I-

recBrecCsbcB cells

exoo

Stoble plasmids Deletion mutants involving Col El replication origin. (pSCIOI replication type Amp-, Reps)

Deletion mutants involving pSCIOI replication process. (Col E, replication type

Amp+, RepR)

FIG. 1. Schematic diagram showing the geneology of plasmid pHSG124 and deletion mutants derived from it in which autonomous ColEl-moderated DNA pHSG1 gereplication is blocked. Symbols: ColEl genome; iTn3 transposon, with nome; the region carrying the /3-lactamase gene that confers ampicillin (Ap) resistance identified by cross-hatching; mm, region of pHSGI carrying the gene(s) that confers tetracycline (Tc) resistance; imm, region containing the ColEI immunity gene; V, replication origin of pHSG1; V, replication origin of ColEI; rep, ColEI "replication region"; x, ColE) relaxation nick site; RepR and Reps, temperature-resistant and temperature-sensitive plasmid DNA replication, respectively; Amp, amplification of plasmid copy number due to continued plasmid replication in the presence of chloramphenicol; +, presence of amplification; absence amplification; 1, EcoRI endonuclease-sensitive site; 3, BamHI endonuclease-sensitive site; exo, enonuclease. "ColE) replication type" or "pSC101 replication type" refers to the characteristics of the replication process that a plasmid mutant derived from pHSG124 exhibits. See Table 1 and Fig. 2 for details ofplasmid mutants isolated from pHSG124 by this procedure. -

599

were selected, and a chimera, pHSG124, was chosen with which to work. The orientation of the genes of both plasmids in pHSG124 is shown in Fig. 1. It is important to note that the information for tetracycline resistance is adjacent to that for colicin immunity, and the ColEl region that has been found to be important for that plasmid's replication can be readily excised from pHSG124 by digesting the DNA with BamHI endonuclease. Plasmids pHSG401 and pHSG402 (Fig. 2; Table 1) were derived from pHSG124 by digesting it with BamHI endonuclease, circularizing the linear DNA with T4 ligase, and selecting transformants transformed with that DNA. pHSG401 exhibited temperature-sensitive replication and ampicillin resistance, whereas pHSG402 (Fig. 1) exhibited temperature-resistant replication and chloramphenicol-resistant plasmid DNA replication and confered immunity to colicin El on its host. These two plasmids were used as controls in subsequent experiments. Isolation of replication-deficient mutants of ColEl. Ream et al. (21) observed that E. coli JC7623, which is a recB recC sbcB mutant, segregated plasmidless progeny when carrying ColEl or its derivatives. Inselburg (14) subsequently observed that the presence of ColEl or its derivatives in JC7623 caused a reduced growth rate of that strain and also had a lethal effect on it. These effects contributed to the enrichment of plasmidless cells in a growing cell population. We recently observed that both pDMS630 and pHSG1 were segregated from JC7623 cells grown in LS medium at 30°C (Fig. 3a). Chimeric plasmid pHSG124 exhibited an instability that was far greater than that of either of its components under the same growth conditions (Fig. 3a). We reasoned that if a mutation blocked either the ColEl- or pHSG1-mediated replication of pHSG124, then that plasmid mutant would become more stable in JC7623. The enhanced stability of such a plasmid and the marked instability of pHSG124 should make it possible to select such mutants by extended growth in nonselecting conditions. A colony of JC7623(pHSG124) was picked from an agar plate containing ampicillin, tetracycline, and colicin El protein; inoculated into LS medium at about 106 cells/ml; and grown in the presence of either tetracycline or ampicillin for 24 h. These cells were then diluted into LS broth without antibiotic and grown for about 50 to 60 generations. They were diluted about 250-fold every 24 h. The progeny were periodically tested for all combinations of ampicillin resistance, tetracycline resistance, and colicin El immunity to determine the rate of segregation of the plasmid

600

HASHIMOTO-GOTOH AND INSELBURG

J. BACTERIOL.

BomHI EcoRI

HoeJi sites

+

II

pHSG1 *-aI

imm

BomHI

-185 +175 t1.

fr~~~loctomose

~~

p DMS630

pHSG

201.209

,, I_ 13e'^l _217 Col Eiltype 218 208,215 .M~. .__

PSCI0Itype1

20'2 206 2102

" -.' :7

I

212

220 221 222

Control

401

f

99*

F2

B -I

G

A

0

+

99

99

H

C I +2

9

k.b.p.

EF +3

+4

+5

+6

FIG. 2. Structure of deletion mutants of a ColEl replication type and pSC101 replication type derived from pHSG124. The diagram principally shows an expanded part ofpHSG124 (see Fig. 1), between the two BamHI sites, that contains the ColE) replication region. The locations of endonuclease-sensitive sites used to determine the extent of some deletions are shown. Symbols: T, HaeII endonuclease-sensitive sites; 3, BamHI sites; 1, EcoRI site. The convention of Tomizawa et. al. (23), indicating that the base pair at the replication origin (I) is +1, was used. As the replication of ColE1 is unidirectional from the origin, those base pairs adjacent to the origin and in the direction of replication are identified as plus. Those base pairs on the opposite side ofthe origin are identified as minus (23). The bp +175 and -185 define the HaeII-sensitive sites at the ends of the HaeII fragment E of ColE1 (23), which is the HaeII G fragment ofpHSG402 (Fig. 1). imm, , Approximate extent of a deleted region; ----, ends of deletions, Region affecting colicin immunity. indicating the range in which they may fall; mm DNA present in a deletion mutant. pHSG401 and pHSG402 (bottom) were constructed by BamHI cleavage ofpHSG124 (Fig. 1) and religation of the fragments. pHSG401 carries the pSC101 replication origin and is apSC1Ol replication-type plasmid, whereas pHSG402 carries the ColE1 replication origin and is a ColE1 replication-type plasmid. The HaeII fragments of pHSG402 are shown and marked A through I. A distinction between the locations of H and I has not been made. Fragments F) and F2 are produced by double digestion of circular pHSG402 with both HaeII and BamHI. HaeII fragments B and G, respectively, ofpHSG402 correspond to fragments A2 (plus apart ofpSC101 genome) and E of ColE) (23). The sizes (in base pairs) of the endonuclease-generated fragments were determined by measuring their electrophoretic mobility in either 1.4% agarose or 5% polyacrylamide slab gels: (A) 1,512; (B) 1,260; (C) 882; (D) 598; (E) 582; (F)) 283; (F2) 220; (G) 360; (H) 130; (I) 120. The sizes of endonuclease-generated fragments of ColE) previously reported (15, 20) were used as standards. k.b.p., Kilobase pairs.

and the specific characters of the plasmids that were retained (Fig. 3b). Pregrowth with tetracycline before growth in a medium without any antibiotic led to a slower rate of appearance of plasmidless progeny than did pregrowth in the presence of ampicillin (Fig. 3b). Pregrowth with tetracycline led to the greater enrichment of cells carrying plasmids containing only tetracycline resistance and colicin El immunity characters as compared with ampicillin resistance or ampicillin resistance with tetracycline resistance and/or colicin El immunity characters. This indicated that plasmids that had lost part or all of the Tn3 transposon had in some way been enriched for by this process and that they exhibited a greater stability than pHSG124. Pregrowth of cells with ampicillin did not have the same effect on the rate of loss of Apr Imm+, Apr Tcr, and Apr Tcr Immn plasmids as did pregrowth with tetracycline (Fig. 3b). The appearance of cells only carrying a very stable ampicillin resistance character was noted after pregrowth with ampicillin. Further analysis of these

cells suggested that Tn3 had become integrated into the host chromosome. Cells carrying plasmids with either the Tcr or Tcr Imm+ phenotype were isolated that exhibited a segregation rate similar to that of cells isolated after pregrowth with tetracycline. The segregation rate of those characters was similar to that found for the individual parental plasmids (Fig. 3a). Plasmid DNA was isolated from 20 independent cultures initially pregrown with either tetracycline or ampicillin, and that DNA was used to transforn E. coli Om84. Eight Tcr Apr hm., and nine Tcr Ap8 Imm+ transformants were found (Table 1). The temperature sensitivity of plasmid replication was tested as described in Materials and Methods. One of nine Tc' Ap5 Imm+ plasmids (pHSG202) and all eight Tcr Apr Imm+ plasmids expressed the same temperature-sensitive plasmid maintenance characteristics in strain Om84 as did the parental pHSGl plasmid and the pHSG401 plasmid derived from pHSG124. These mutants and derivatives will be referred to as pSC101 replication-type plas-

REPLICATION-DEFICIENT ColEl MUTANTS

VOL. 139, 1979

601

(b)

(a)

0

C) -0' 40 -J

1 10 20 34

10 20 30 40 50 60 70 80

Generation number FIG. 3. Instability of plasmids in JC7623 recB recC sbcB mutant cells. Strain JC7623 harboring each plasmid was constructed by transformation, and the transformants were 8elected on LS agarplates containing appropriate drugs. The JC7623(pHSGI24) strain was selected with aU of the selective markers (Tc, Ap, and Imm). (a) Colonies that appeared on selective plates were picked and suspended in LS broth without drug at 1 x let to 2 x le4 cells per ml and grown for 24 h. After each 24 h ofgrowth, the cells were diluted 1:250 in fresh LS broth and then grown for another 24 h. The cultures were plated on LS agar or LS agar containing tetracycline for pHSG1, ampicillin for pDMS630, and tetracycline, ampicillin, and colicin El protein for pHSG124-carrying cells. (b) Growth of JC7623(pHSG124) in LS medium with tetracycline or ampicillin for 24 h before growth without drug. The segregation kinetics in drugless medium was then followed for each combination of selective markers. The selected markers are shown at the end of each curve. Symbols: -, A, 0, a X, i, cells pregrown in tetracycline; +, A, *, x, *, cells pregrown in ampicillin. E,

mids (Table 1). All those plasmids except pHSG202 and pHSG401 contained two BamHI and two EcoRI endonuclease-sensitive sites. Furthermore, none of these temperature-sensitive plasmids could undergo extensive replication in the presence of chloramphenicol (data not shown). These results suggest that the plasmids lack a part of ColEl essential for its replication and are, therefore, replicated by the pHSG1-mediated replication system. The other eight of nine Tcr Ap8 Imm+ plasmids, as well as pHSG402, were maintained at high temperature and also replicated extensively in the presence of chloramphenicol. These mutants and derivatives will be referred to as ColEl replicationtype plasmids (Table 1). All of those plasmids contained one BamHI and one EcoRI endonuclease-sensitive site. Characterization of temperature-gsensitive and temperature-resistant replication plasmids. Plasmid DNA was isolated from the different Om84 transformants carrying temperature-sensitive replication mutants, and their sizes (Table 1) were determined by measuring *the relative mobilities of BamHI- and HaeII-

generated fragments in agarose gels (Fig. 4a and b). All of the temperature-sensitive replication plasmids except for pHSG202 and pHSG214 exhibited a deletion in the BamHI fragment, containing the ColEl replication origin, from which the plasmid pHSG402 was derived. Plasmids pHSG206, pHSG211, pHSG212, pHSG214, pHSG220, pHSG221, and pHSG222 were missing the HaeII A, B, and G fragments derivable from pHSG402 (Fig. 2 and 4b). A new HaeII fragment corresponding to a fusion between part of the HaeII B fragment and an HaeII Tn3 fragment was identified (Fig. 2 and 4b). As the new HaeII fragment in the digest of pHSG222 was identical in size to a HaeII fragment produced by digesting pHSG1, it was necessary to first isolate the BamHI fragment from the plasmid and then digest it with HaeII to identify it (data not shown). The maximnum extent of the deletion into the HaeII B fragment was estimated from the size of the new fragment generated by the fusion of the HaeII B fragment to the missing HaeII Tn3 fragment. This calculation indicated that the maxiimum extent of the deletion of the HaeII B fragment of pHSG206

602

HASHIMOTO-GOTOH AND INSELBURG (a)

ab c de f 9 h

J. BACTERIOL.

(b) a bcde f g hi j k

j

tA

B

A

B

G

FIG. 4. (a) Agarose gel (1%) ekctrophoresis of BamHI-digestedpSC0l1 replication-type mutant DNAs. The procedures are described in the text. (a) pHSG202, (b) pHSG206, (c) pHSG210, (d) pHSG211, (e) pHSG212, (f) pHSG214, (g) pHSG220, (h) pHSG221, (i) pHSG222, (j) pHSG124. (A and B) BamHI-digested pHSG401 and pHSG402, respectively. (b) Agarose gel (1.4%) ekectrophoresis of HaeII-digestedpSC101-type mutant DNAs. (a through j) Same as for part "a" (k) pSC134, (1) pDMS630. Each arrow indicates a new fragment derived by a deletion event. The new bands of pHSG212 and pHSG222 are overlapped by the original bands in pHSG124. The bands corresponding to the A, B, and G fragments in HaeII-digested pHSG402 DNA (Fig. 2) are indicated.

was about 300 bp; of pHSG211, 1,025 bp; of pHSG212, 945 bp; of pHSG220, 660 bp; of pHSG221, 315 bp; and of pHSG222, 800 bp. The

deletion in the HaeII G fragment more precisely (data not shown; 8). It lies between the bp -107, an AluI endonuclease-sensitive site in the Haell results (Fig. 4a) indicate that pHSG214 under- G fragment (H. Ohmori, personal communicawent more-extensive changes than a simple dele- tion), and bp -185, the HaeII-sensitive site detion of the HaeII B, G, and A fragments. The fining the boundary of the HaeII G and B fragDNA fragments generated by EcoRI and HaeII ments (Fig. 2) (3, 23). (We use the Tomizawa et endonucleases were analyzed further. The HaeII al. [23] convention for identifying base pairs, in digest produced two additional bands and lost at which the first DNA base pair synthesized at least one band from the pHSG1 genome (Fig. the replication origin is bp +1. Base pairs to the 4b). EcoRI digestion generated two fragments, right of that [Fig. 2], in the direction of replicaone of which was larger than pDMS630 and one tion, are "+" and those to the left of the origin that was smaller than pHSGl (data not shown). are "-.") This result indicates that the integrity Since pHSG214 still retained two EcoRI sites of part and possibly all of the DNA between bp and two BamHI sites, the possible intramolec- +1, the replication origin, and bp -185 is essenular transposition of Tn3 in the plasmid from tial for ColEl replication. the pDMS630 to the pHSG1 portion of the Temperature-sensitive replication plasmid genome, with a concurrent deletion of part of pHSG202 was Tcr Ap5 Imm+. It was found to Tn3, may have produced the plasmid. This is have sustained a deletion that includes all of the being investigated further. ColEl DNA in pHSG124 with the exception of Plasmid pHSG210, which is a temperature- part of the HaeII B fragment (Fig. 2 and 4a). All sensitive replication derivative of pHSG124, dif- of the Tn3 DNA was also deleted. The plasmid fers from the above-mentioned plasmids in that has only the one BamHI and one EcoRI endothe deletion it contains does not extend into the nuclease-sensitive site shown in Fig. 2 (see refHaeII B fragment but terminates in the Haell erence 8). G fragment (Fig. 2 and 4a and b). The missing The results obtained from studying the HaeII G and A fragments of pHSG210 are re- pSC101 replication-type plasmids indicated that placed by a new fragment which is a fusion part or the whole HaelI G fragment of pHSG402 product of part of the HaeII G fragment and the (Fig. 2) (which is the same as the HaeII E part of the Haell A fragment in Tn3 DNA. We fragment of ColEl, which carries the ColEl plashave determined the termination site of this mid replication origin) was missing from tem-

REPLICATION-DEFICIENT ColEl MUTANTS

VOL. 139, 1979

perature-sensitive replication mutants of pHSG124. The ColEl replication origin was always deleted in these mutants. This latter observation can account for, although does not necessarily provide a complete explanation for, the loss of the ColEl replication characteristics of these plasmids. The size of the temperature-resistant ColEl replication-type plasmids was determined by measuring the mobility of HaeII endonucleasegenerated DNA fragments in agarose and polyacrylamide slab gels, representatives of which are shown in Fig. 5a and b. The results are summarized in Table 1. The temperature-resistant mutant derivatives of pHSG124 were all considerably smaller than the temperature-sensitive derivatives. Although these mutants would be expected to contain the ColEl replication origin, six of eight were missing the HaeII G fragment (Fig. 2). Since all of these mutants contained a normal HaeII B fragment, it is thought that the deletion did not extend beyond the ColEl replication origin in the HaeII G fragment and that the partially deleted HaeII G fragment was fused to another DNA fragment. The relaxation nick site was probably deleted in these mutants.

Temperature-sensitive plasmids pHSG202

and pHSG206 and temperature-resistant plasmid pHSG201 were reintroduced into strain JC7623 by transformation, and their stability in that host was examined. All three exhibited a stability comparable to that of pDMS630 and pHSG1. As the molecular weight of pHSG206 DNA is almost the same as that of pHSG124, the increased instability of pHSG124 compared with that of its component plasmids does not appear to be simply due to the size of the plasmid. The presence of a dual replication system in the chimeric plasmid may be a significant factor in determining its instability in strain JC7623. The interpretation that the temperature-sensitive replication mutants of pHSG124 carry pDMS630 DNA that can not replicate autonomously was tested. pHSG206, pHSG210, pHSG124, and pDMS630 DNAs were completely cleaved with EcoRI endonuclease. The DNA in each digest was then ligated and used to transforn strain P678-54. Apr or Tcr transformants were selected, and the colonies were then tested for the presence of the other drug resistance marker (Table 2). If Tcr Aps transformants were found, then Tc8 Apr transform(b)

(a) ab c d e f g h

603

i

a

b c d e f g h i

j k

A B

G

FIG. 5. (a) Agarose gel (1.4%) electrophoresis of ColE) replication-type mutant DNA digested with HaeII endonuclease. The procedures for the enzymatic reaction and electrophoresis were as described in the text. (a) pHSG201, (b) pHSG208, (c) pHSG209, (d) pHSG215, (e) pHSG216, (f) pHSG217, (g) pHSG218, (h) pHSG219, (i) pHSG124. The bands corresponding to the A, B, and G fragments in HaeII-digested pHSG402 DNA (Fig. 2) are indicated. The relatively light bands, particularily in lanes c through e, represent partial digest products. (b) Polyacrylamide gel (5%) electrophoresis of HaeII or HaeII plus BamHI-digested ColEJ replication-type mutant DNA. All samples except (b) were only digested with HaeII endonuclease. (a) pHSG124, (b) HaeII- and BamHI-digestedpHSG402, (c) pHSG402, (d) pHSG201, (e) pHSG208, (f) pHSG209, (g) pHSG215, (h) pHSG216, (i) pHSG217, (j) pHSG218, (k) pHSG219.

604

HASHIMOTO-GOTOH AND INSELBURG

TABLE 2. Test of the autonomous replication of the ColEI plasmid derivatives isolated from different chimeras Transformant: Tcr Apc Apr

Plasmida

p Tc' Apr 0/24c 24 728 pHSG206 24/25b 0/24 24 24/25 533 pHSG210 23/25 1,776 24/25 920 pHSG124 25/25 1,944 0 pDMS630 a Plasmid DNA digested with EcoRI endonuclease and then ligated with T4 DNA ligase. Fraction of Tcr colonies that were Ap'. Fraction of Apr colonies that were Tc. All Apr colonies derived from pHSG206 and pHSG210 transformations were Tcr Apr. c'

ants should also be found if pDMS630-derived

DNA obtained from the chimeras was not replication deficient. The absence of Tc8 Apr transformants strongly suggests that the pDMS630 DNA derived from the chimeras is replication deficient. pHSG124 and pDMS630 were controls. The data in Table 2 sustain the interpretation that pDMS630 DNA in plasmids pHSG206 and pHSG210 is replication deficient. It shows that the EcoRI-cleaved pHSG206 and pHSG210 DNA preparations, after ligation with T4 DNA ligase, produced Tcr Ap8 transformants, as did the pHSG124 control. A replicating pHSG1 component of the chimera (Fig. 1) was therefore reformed. As Apr Tc5 transformants were only found after ligation of the pHSG124 and pDMS630 DNA preparations, the pDMS630-derived DNA in the ligation mixture of pHSG206 and pHSG210 could not sustain its own replication. Incompatibility test of plasmids containing deletion mutations. Strain Om84 harboring each mutant plasmid was separately transformed with either a pSC101 Kmr derivative (pHSG100 or pHSG101) DNA or CoIEl .Mr (ColE1-Tn5) DNA, and transformants were selected with drugs to which the resident and incoming plasmids conferred their resistance (e.g., tetracycline and kanamycin or ampicillin and kanamycin). Colonies were picked and suspended in LS broth without any drugs at about 104 cells per ml. The cultures were incubated at 30°C, and cells were periodically withdrawn and plated on LS agar to determine the total number in the culture and on LS agar with ampicillin and kanamycin or tetracycline and kanamycin to determine the number of cells carrying both plasmids (mixed-plasmid state). Growth of the cultures was continued by successive dilutions into LS medium. The results are presented in Fig. 6a for ColEl replication-type mutants (Ta-

J. BACTERIOL.

ble 1) and in Fig. 6b for pSC101 replication-type mutants (Table 1). In Fig. 6a the expression of incompatibility of ColEl replication-type mutants is represented by data obtained for mutant pHSG201. All the other ColEl replication-type mutants showed incompatibility kinetics similar to that shown between pHSG201 and ColElTn5. Incompatibility between ColE1-Tn5 and pHSG402 (data not shown) was comparable to that shown between ColE1-Tn5 and pDMS630. On the other hand, all ColEl replication-type plasmids were compatible with pHSG100 or pHSG101. Therefore, we can conclude that the ColEl replication-type mutants not only lost the replication ability of pSC101, but also lost incompatibility to that plasmid. Because the deletions in these mutants are very large (84 to 88% of pHSG124), establishing a relationship between a region affecting replication, such as the replication origin region, and an incompatibility region of pSC101 is impossible from these data. In Fig. 6b the expression of incompatibility of some pSC101 replication-type mutants with ColEl-Tn5 is shown. Three mutants, pHSG206, pHSG210, and pHSG212, showed very weak incompatibility with ColE1-Tn5 (Fig. 6b). Two plasmids, pHSG202 and pHSG21l, 'were completely compatible. The compatibility of these plasmids was confirmed by showing that the DNA of both plasmids was isolatable from 20 independently isolated colonies derived from cells taken from the cultures at the end of the experiment. The transposition of antibiotic resistance between plasmids was thus ruled out. All five mutants were incompatible with pHSG100 or pHSG101 at levels comparable to that shown by pSC101 (see pHSG202 in Fig. 6b) or pHSG401 (data not shown). When cells carried plasmid pHSG206 or pHSG212 and ColElTn5, it required 210 generations of growth to reduce the number of cells carrying both plasmids to 10% of the total cell population. When cells carried pHSG210, a comparable result was obtained after about 260 generations of growth. On the other hand, when cells carried pDMS630 or pDMS6641 and ColE1-Tn5, 46 or 40 generations of cell growth were necessary to reach the same point (Fig. 6b). The pSC101 replicationtype plasmids, pHSG206 and pHSG210, therefore exhibit about a fivefold reduction in the expression of incompatibility. This reduction may reflect the reduction of copy number due to the switch from ColEl-type replication to pSC101-type replication. The copy number dosage effect on plasmid incompatibility was reported in studies of pSC134 versus pSC101 incompatibility (4) and predicted by theoretical studies (16, 19).

REPLICATION-DEFICIENT ColEl MUTANTS

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605

(a) EV

0 0-

0

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0)

-a 10 E

-I

Mo)pDMS630 -ColE1: :Tn5 Ap

0 cnl

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>.)pDMS6641-Col E1::Tn5 Km \ Ap {^)pHSG201-Col E1::Tn5 Km Tc (o)pHSGI24-Col Ei::Tn5 >l0 Ap Km

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-J

Generation number (b) _w

1

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A

_ 0

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e

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_ 87

.-_

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06

=-

-

3 50

100

IS0

200

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Generation number

FIG. 6. Incompatibility kinetics of ColE1 and pSC101 replication-type plasmids. (a) Cells carrying ColE1 replication-type plasmids [Om84(pHSG201), Om84(pHSG124), 0m84(pDMS6641), and Om84(pDMS630)] were transformed to Kmr with ColE1-Th5, and a transformant colony of each was suspended in 10 ml of LS broth without any drug at about 10' cells per ml. At intervals, samples from cultures carrying pHSG201 were plated on LS agar plates containing no drug or both tetracycline and kanamycin to determine the concentration of total cells and "mixed-plasmid-state" cells, respectively. The other cultures were plated on LS and LS plus ampicillin and kanamycin. The incompatibility ofplasmid pHSG201 with pSC101 was examined essentially as described above, except that pHSG1IO (pSC101 Km' Tc5) instead of ColEI-Tn5 was introduced into the cell. The first plasmid listed is the resident plasmid, and the second is the in-coming plasmid (e.g., pHSG201pHSG100). (b) Strain Om84 carrying each indicated pSC101 replication-type plasmid was transformed with ColE1-Tn5 DNA and studied exactly as described above except for the prolonged incubation of cells. The first component of each plasmid pair listed is the resident plasmid, and the second component is the in-coming plasmid. The antibiotic or other selective marker used to determine the number of "mixed-plasmid-state" cells is indicated below the plasmid's name.

606 HASHIMOTO-GOTOH AND INSELBURG These results indicate that deletion mutations that involve the ColEl replication origin and extend to the left of it (Fig. 1) not only affect replication, but also can affect ColEl plasmid incompatibility. A detailed examination of the relationship between the expression of ColEl plasmid incompatibility and ColEl-moderated replication is reported in the accompanying paper (8).

J. BACTERIOL.

panying paper (8) we show that part of the cause of this incompatibility reduction is probably due to the effects of the mutations on plasmid copy number. The integrity of a region of the plasmid to the left ofthe ColEl replication origin appears essential to the expression of ColEl plasmid incompatibility. ACKNOWLEDGMENTS We thank Y. Masamune and D. Berg for bacterial strains and plasmids and H. Ohmori and J. Tomizawa for generously supplying us with information about the ColEl nucleotide sequences. This work was supported by Public Health Service grant AI 08937 from the National Institutes of Health to Joseph

DISCUSSION In this paper we have described the isolation and partial characterization of deletion mutations that affect replication of plasmid ColEl. Inselburg. The selection of mutations affecting the CoIEl LITERATURE CITED replication capacity by screening for the stable maintenance of a chimera in strain JC7623 1. Adler, H., W. D. Fisher, A. Cohen, and A. A. Hardigree. 1967. Miniature Escherichia coli cells deficient should provide a useful means for further anain DNA. Proc. Natl. Acad. Sci. U.S.A. 57:324-326. lyzing the plasmid replication process. The find- 2. Berg, D. E. 1977. Insertion and excision of the transposing that the ColEl replication-deficient mutants able kanamycin resistance determinant Tn5, p. 205-212. In A. L. Bukhari, J. A. Shapiro, and S. L. Adhya (ed.), were caused by deletions involving the replicaDNA insertion elements, plasmids, and episomes. Cold tion origin suggests that the origin is not replaceSpring Harbor Laboratory, Cold Spring Harbor, N.Y. able by another site remaining in the deleted 3. Bolivar, F., M. C. Betlach, H. L Heyneker, J. Shine, ColEl DNA. The importance of other nucleotide R. L Rodriguez, and H. W. Boyer. 1977. Origin of replication of pBR345 plasmid DNA. Proc. Natl. Acad. sequences to CoIEl replication cannot be ruled Sci. U.S.A. 74:5265-5269. out by these findings. Mutant pHSG210 was 4. Cabello, F., K. Timmis, and S. N. Cohen. 1976. Replifound to have sustained a deletion that extended cation control in a composite plasmid constructed by in to the left of the replication origin to a point vitro linkage of two distinct replicons. Nature (London) 259:285-290. between bp -107 and -185. A deletion mutant, D. B. 1972. Nature of ColEl plasmid replication isolated by H. Boyer and co-workers (personal 5. Clewell, in Escherichia coli in the presence of chloramphenicol. communication), that had lost all but a few J. Bacteriol. 110:667-676. nucleotides to the right of the replication origin 6. Clowes, R. C., and W. Hayes. 1968. Experiments in microbial genetics, p. 187. John Wiley and Sons Inc., replicated normally and was amplifiable in the New York. presence of chloramphenicol. As shown by mu- 7. Cohen, S. N., A. C. Y. Chang, H. W. Boyer, and R. B. tant pHSG210, the intergrity ofthe ColEl region Helling. 1973. Construction of biologically functional between the replication origin and some point bacterial plasmids in vitro Proc. Natl. Acad. Sci. U.S.A. 70:3240-3244. between bp -107 and -185 is essential for auT., and J. Inselburg. 1979. ColEl tonomous ColEl replication. The results also 8. Hashimoto-Gotoh, plasmid incompatibility: localization and analysis of suggest that the instability of chimeric plasmid mutations affecting incompatibility. J. Bacteriol. 139: pHSG124 in strain JC7623 is due to the presence 608-619. of two plasmid-mediated replication processes 9. Haahlroto-Gotoh, T., and M. Sekiguchi. 1976. Isolation of temperature-sensitive mutants of R plasmid by rather than the large size of the plasmid. It in vitro mutagenesis with hydroxylamine. J. Bacteriol. appears that, whereas pHSG124 replication gen127:1561-1563. erally displays preferential use of the ColEl- 10. Hashimoto-Gotoh, T., and M. Sekiguchi. 1977. Mutations to temperature sensitivity in R plasmid pSC101. mediated replication process (4), some aspect of J. Bacteriol. 131:405-412. the pSC101-mediated process may still be ex- 11. Heffron, F., P. Bedinger, J. Champoux, and S. Falpressed, leading to instability. The mechanism kow. 1977. Deletions affecting transposition of an anof this instability will be explored. tibiotic resistance gene, p. 161-167. In A. I. Bukhari, J. A. Shapiro, and S. L. Adhya (ed.), DNA insertion eleIt has been reported that the presence of ments, plasmids and episomes. Cold Spring Harbor transposon Tn3 may stimulate deletion events Laboratory, Cold Spring Harbor, N.Y. (11, 18). Our results appear to confirm those 12. Inselburg, J. 1970. Segregation into and replication of observations. plasmid deoxyribonucleic acid in chromosomeless segregants of Escherichia coli. J. Bacteriol. 102:642-647. The examination of incompatibility exhibited J. 1977. Isolation, mapping, and examination by CoIEl replication-deficient mutants indi- 13. Inselburg, of effects of TnA insertions in CoIEl plasmids. J. Baccated that mutations inhibiting the replication teriol. 129:482-491. of ColEl can also reduce or eliminate the expres- 14. Inselburg, J. 1978. CoIEl plasmid mutants affecting growth of an Escherichia coli recB recC sbcB mutant. sion of plasmid incompatibility. In the accom-

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REPLICATION-DEFICIENT ColEl MUTANTS

J. Bacteriol. 133:433-436. 15. Inselburg, J., and P. Ware. 1977. Isolation and genetic analysis of deletion mutants of colicin El plasmids carrying a TnA insertion. J. Bacteriol. 132:321-331. 16. Ishii, K., T. Hashimoto-Gotoh, and K. Matsubara. 1978. Random replication and random assortment model for plasmid incompatibility in bacteria. Plasmid 1:435-445. 17. Kopecko, D. J., and S. N. Cohen. 1975. Sites specific

recA-independent recombination between bacterial plasmids: involvement of palindromes at the recombinational loci. Proc. Natl. Acad. Sci. U.S.A. 72:13731377. 18. Nisen, P. D., D. J. Kopecko, J. Chou, and S. N. Cohen. 1977. Site-specific DNA deletions occurring adjacent to the termini of a transposable ampicillin resistance element (Tn3). J. Mol. Biol. 117:975-998.

607

19. Novick, R. P., and F. C. Hoppensteadt. 1978. On plasmid incompatibility. Plasmid 1:421-434. 20. Oka, A., and M. Takanami. 1976. Cleavage map of colicin El plasmid. Nature (London) 264:193-196. 21. Ream, L W., N. Crisona, and A. J. Clark. 1978. ColEl plasmid stability in Exol ExoV- strains of Escherichia coli K-12, p. 78-80. In D. Schlessinger (ed.), Microbiology-1978. American Society for Microbiology, Washington, D.C. 22. Timmis, K., F. Cabello, and S. N. Cohen. 1974. Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons. Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560. 23. Tomizawa, J., H. Ohmori, and R. E. Bird. 1977. Origin of replication of colicin El plasmid DNA. Proc. Natl. Acad. Sci. U.S.A. 74:1865-1869.