Proc. Natl. Acad. Sci. USAVol. 75, No. 3, pp. 1265-1269, March 1978Biochemistry

Phenotypically cryptic EcoRI endonuclease activity specifiedby the ColE1 plasmid

(restriction/modification/DNA/recombination)

CHRISTINE A. MILLER AND STANLEY N. COHEN*Departments of Medicine and Genetics, Stanford University School of Medicine, Stanford, California 94305

Communicated by Joshua Lederberg, January 9, 1978

ABSTRACT An endonuclease having EcoRI specificityis produced by bacteria containing the ColEl plasmid. Suchbacterial cells fail to express restriction or modification func-tions in vivo, and phage or plasmid DNA obtained fromColEl-containing cells has unmodified EcoRI sites that arecleaved in vitro by purified EcoRI endonuclease or by enzymeextracted from bacteria that carry ColEl. No EcoRI DNAmethylase activity associated with ColEl has been detected.The finding of phenotypically cryptic ColEl-dependent EcoRIendonuclease activity and the absence of any detectable EcoRImodification system in ColEl-containing cells suggest a controlmechanism that appears to prevent functional expression of theColEl-determined enzyme in vivo.

The EcoRI restriction endonuclease is one of a series of site-specific endonucleases that have been used extensively for theanalysis of DNA and for DNA cloning, experiments. This en-donuclease, which was first identified in an Escherichia colistrain isolated in a clinical microbiology laboratory (1), is en-coded by genes located on a bacterial plasmid that has beendesignated pMB1 (2); a second site-specific enzyme, whichmethylates the adenosine at the center of the nucleotide se-quence comprising the EcoRI endonuclease cleavage site (3)thereby rendering the site insusceptible to cleavage, is alsospecified by the pMB1 plasmid. It has been reported that a largesegment of the pMB1 plasmid is homologous with anotherwidely studied bacterial plasmid, ColEl (2). Since ColEl DNAis cleaved in vitro by the EcoRI endonuclease (4), and becausebacterial cells carrying ColEl are not known to be associatedwith restriction activity, earlier investigators concluded thatthe EcoRI restriction and modification genes are located on a1.95-kilobase segment of DNA that is present in pMB1 but notwithin ColEl (2).

During in vitro studies of possible site-specific DNA cleavageassociated with the Tn3 transposon, we observed that bacterialcells carrying the ColEl plasmid itself produce an endonucleasehaving EcoRI restriction specificity. However ColEl-con-taining cells do not express EcoRI restriction or modificationfunctions in vivo and plasmid or phage DNA isolated from suchcells is cleaved in vitro by the EcoRI endonuclease. The presentreport describes these findings.

MATERIALS AND METHODSBacterial Strains and Plasmids. E. coli strains RY13, C600,

and HB101 (pMB4) were obtained from H. W. Boyer (Uni-versity of California, San Francisco). E. coli strain 1200 [endoI-, RNase-, rK+mK+ (5)] was provided by M. Pearson. TheColEl-K30 plasmid was obtained from D. Helinski (6), and theTn3 transposon (7) was introduced onto this plasmid from

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1265

pSC204 by the selected translocation procedure (8). The pMB3plasmid and the Ri drd-19 plasmid were isolated from E. coliRY13 and purified by a cycle of transformation (9). The pMB3and pMB4 (a spontaneous deletion mutant of pMB3) plasmids,which specify the EcoRI restriction and modification edzymes,are derived from the clinical isolate pMB1 (2). pCM100 is aspontaneously occurring mutant (isolated in our laboratory) ofpMB3 that has lost the ability to restrict or modify DNA in vvo.Bacteriophages Xmr and XCI83 (10) and plasmid pSC355 (11)were used for detection of phenotypically expressed restrictionand modification functions. Purified X DNA was isolated (12)from Xplac MS505S7 phage (13).

Isolation of Enzyme. Bacterial cells used for isolation ofendonuclease and methylase were grown in L broth (14) con-taining appropriate antibiotics, at concentrations of 25 Ag/mlfor ampicillin, and 10,gg/ml for kanamycin, chloramphenicol,tetracycline, and streptomycin. Bacterial cultures of 500 ml orless were grown at 370 in shaking water baths to ODW0 = 1.0and chilled by transfer of flasks to an ice water bath. Cells werecollected by centrifugation, suspended (final concentration 1g/ml) in 20mM KH2PO4/K2HPO4 (pH 7.4), 1 mM EDTA, and2 mM dithioerythritol, and frozen quickly by addition of liquidN2. For larger preparations of enzymes, bacterial cells weregrown as described by Greene et al. (15), collected by centrif-ugation, and frozen as a paste. Cells were thawed by additionof an equal volume of cold phosphate/EDTA/dithioerythri-tol and disrupted by sonication to produce greater than 90%lysis. Enzyme was isolated as described either by Greene et al.(15) or by Modrich and Zabel (16) with minor modifications.For preparation of small batches of enzyme, protein was ab-sorbed onto P11 phosphocellulose and was eluted batchwisewith phosphate/EDTA/dithioerythritol containing 0.2, 0.4,or 0.7M KC1. One unit of enzyme was defined as the amountneeded to digest 0.1 ,ug of Xplac MS505S7 DNA to completionin 1 hr.The EcoRI methylase was isolated and assayed the procedure

of R. A. Rubin and P. Modrich (18).Other Procedures. Procedures for the isolation of plasmid

DNA (19, 20), transformation of CaCl2-treated cells withplasmids (9), enzymatic digestion of DNA with EcoRI, HindIII,or Hae II (obtained from BioLabs) restriction endonucleases(21), and electrophoresis in 0.7% agarose gels (22, 23) have beendescribed. The P1 + EKi level of containment specified in theNIH Guidelines for Recombinant DNA Research was used forwork with recombinant DNA' plasmids! used in, these studiies.

RESULTSIdentification of EcoRI Endonuclease Activity in Extracts

of Bacterial Cells Containing ColEl Plasmid. During studiesof site-specific endonuclease activity in extracts of E. coli 1200

*To whom reprint requests should be addressed.

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1266 Biochemistry: Miller and Cohen

A and pMB3

A --Aa b c d a b c d

Proc. Natl. Acad. Sci. USA 75 (1978)

Wand

ColE1 ::Tn3

a b c d

pSC355 andpSC355 pMB3

D a b a b c d

pSC355 andColE 1::Tn3

pSC355 andpCM 100

a k el A

FIG. 1. Agarose gel electrophoresis patterns ofDNA from bacteriophage Xplac MS505S7 or various plasmids analyzed after digestion with(a) purified EcoRI endonuclease; (b) purified HindIII endonuclease; (c) enzyme extract from 1200(pMB3); (d) enzyme extract from1200(CoIE1::Tn3). Both extracts were purified through the hydroxylapatite column step and contained 40,000 units of enzyme activity per ml.Reaction mixtures (23), containing 0.15 lg of-DNA and 2 units of endonuclease were incubated-for 1 hr at 37°. (A) A DNA was isolated frominduced Iysogens; the Xm+ was obtained from a lysogen carrying the pMB3 plasmid and the Xm- was obtained from a lysogen carrying Co1E1::Tn3.(B) The-DNA digested consisted of plasmid pSC355 (11), either alone or in the presence of the plasmid indicated.

carrying Co1E1::Tn3, we observed that such extracts cleave theColEl: Tn3 plasmid at a unWique site. Subsequent analysis usingeither bacteriophage.X DNA or plasmid pSC355 as substrate(Fig. 1) indicated that cleavage patterns produced. by the ex-tracts are indistinguishable from those produced by purifiedEcoRI endonuclease.or by extracts isolated from bacterial cellscarrying a plasmid (pMB3) known to code for EcoRI restrictionand modification functions. Passage of the CoIE1::Tn3 plasmidto another bacterial clone by transformation resulted in ac-quisition of the same enzymatic activity by the recipient. These

results suggested that the ColEl::Tn3 plasmid is associated withan EcoRI-like endonuclease activity.Endogenous DNA is modified at EcoRI endonuclease

cleavage sites by bacteria carrying the pMB3 plasmid (2). Sincesuch modification protects the endogenous DNA from degra-dation by the endonuclease, it has been generally believed thata DNA modification system is required for bacterial viabilityin endonuclease-producing strains. However, both the ColEland ColE1::Tn3 plasmids are susceptible to in vitro cleavageby the EcoRI endonuclease (2, 4), implying that modification

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Proc. Nati. Acad. Sci. USA 75 (1978) 1267

Table 1. In vivo restriction and modification .';, 1,I, ) Table 2. DNA methylase activity in vitroby ColEl-related plasmids - .r .

Strain used for Strain used for phage growthplating phage A B C D E

A. 1200 1.0 1.0 0.8 0.8 1.2B. 1200(ColEl) 0.7 1.0 0.9 0.9 1.6C. 1200(ColEl::Tn3) 0.7 1.5 1.0 1.0 1.4D. 1200(pCM100) 1.2 1.0 0.8 1.0 1.2E. 1200(pMB3) 0.0003 0.0007 0.0003 0.0002 1.0

The values shown represent the relative efficiency ofphage platingon the strains indicated, as compared with the plating efficiency ofphage that had been both grown and plated on a single host strain.Wild-type A phage were taken from a single plaque in each instance,suspended in medium, and plated (-250 phage/plate) at 370 (12).

of endogenous DNA by the EcoRI methylase does not occur inbacterial cells carrying these plasmids.

In order to investigate further the apparent ability of theColE1::Tn3 plasmid to synthesize an endonuclease havingEcoRI specificity in the absence of modification of endogenousDNA at EcoRI cleavage sites, DNA from bacteriophage Agrown on cells containing either pMB3 or ColE1::Tn3 wastested for its ability to be cleaved by purified EcoRI restrictionendonuclease and by extracts of bacteria containing either ofthe two plasmids.

As seen in Fig. 1, DNA from X phage or plasmids grown onor propagated within pMB3-containing cells was not cleavedby the purified EcoRI endonuclease or by either of the twobacterial cell extracts. In contrast, DNA from bacteria carryingColEL::Tn3 was cleaved by both enzyme preparations, givinga characteristic EcoRI endonuclease cleavage pattern in eachcase. Cleavage by HindIII endonuclease (Fig. 1) or by otherrestriction endonucleases (data not shown) was identical for Xor plasmid DNA grown on bacteria containing either pMB3 orColE1::Tn3. The failure of extracts from ColEl-containingbacteria to cleave DNA from cells that contain the pMB3plasmid identified the nucleotide sequence recognized by theColEl-associated endonuclease as the same sites modified bythe EcoRI DNA methylase encoded by pMB3.The endonuclease activity detected in extracts of E. coli

1200(ColEl::Tn3) was purified by the procedure described byModrich and Zabel (16) for purification of the EcoRI endo-nuclease; at each step in purification, the ColE::Tn3-associatedactivity had the same fractionation characteristics as the EcoRIendonuclease; at each step in purification, the ColE1::Tn3-associated activity had the same fractionation characteristicsas the EcoRI endonuclease activity encoded by the pMB3plasmid. Although the 25-40% ammonium sulfate precipitatefraction of crude extracts of 1200(ColE1::Tn3) contained be-tween 10 and 50% of the endonuclease activity detected insimilar extracts of 1200(pMB3), the specific activity of bothenzymes was the same (i.e., -50,000 units/mg of protein) afterpurification through the hydroxylapatite column step. In twotypical preparations, 10 g of 1200(pMB3) cell paste yielded500,000 units of endonuclease, while an identical amount of1200(ColEl::Tn3) cell paste yielded 100,000 units.Absence of In Vivo Restriction or Modification by

ColEl-Containing Bacteria. Bacteria carrying the pMB3plasmid are known to restrict incoming DNA and are infectedby unmodified bacteriophage at a lower efficiency than cellslacking this plasmid; those phage that grow on pMB3-con-taining cells are modified at the EcoRI cleavage sites and canthen form plaques with normal efficiency on the same strain.No restriction of modification activity is known to be associated

Source ofpmol [3H]AdoMet

DNA used as incorporated/minenzyme substrate per 10 Ag protein

1200 Am-EcoRI 0.25 4 0.16Am+EcoRI 0.14 i 0.16

1200(ColEl::Tn3) Xm-EcoRI 0.31 + 0.10Am+EcoRI 0.28 + 0.10

1200(pMB3) Xm-EcoRI 12.4 i 0.11Xm+EcoRI 0.53 + 0.16

Values shown are the pmol of 3H-labeled S-adenosylmethionineincorporated in a 10-min assay (20) using as a recipient 5 ,g ofDNAfrom Aplac MS 505S7 (for Xm-EcoRI) or DNA from Xplac MS505S7grown in a strain containing pMB3 (for Xm+EcoRI). The enzymeextracts used were fractions eluted from the P11 phosphocellulosecolumn step in the purification procedure of Rubin and Modrich(18).

with the ColEl plasmid, which has been used extensively since1953 (24) for a wide variety of microbiological and biochemicalstudies.The ability of bacteriophage XCI83 to plate on bacteria

containing pMB3, various ColEl-derived replicons, or certainother plasmids was investigated. As shown (Table 1), bacter-iophage XCI83 plaque-forming efficiency was reduced ap-proximately %/ioo on cells carrying the pMB3 plasmid, but nosignificant change in plating efficiency resulted from thepresence of any of the other plasmids studied. We concludefrom these results that the EcoRI endonuclease activity asso-ciated with the ColEl::Tn3 plasmid is phenotypically crypticin vwo.DNA of phage grown on 1200(pMB3) is modified, as shown

by the ability of such phage to plate with normal efficiency oncells carrying the pMB3 plasmid. However, bacteria carryingCoIEl::Tn3, other ColEl-derived plasmids, or the pMB3-de-rived plasmid pCM100 (see Materials and Methods and textbelow) fail to express the EcoRI modification function in vdvo,and bacteriophage XCI83 grown on such cells is subsequentlyrestricted in its ability to form plaques on 1200 (pMB3) cells.It was of interest to determine whether a phenotypically crypticform of EcoRI DNA methylase activity is also present in bac-teria carrying the ColEl::Tn3 plasmids, despite the inabilityof such cells to express modification functions in vivo. The datashown in Table 2 indicate that no methylase was detected inenzyme extracts from these bacteria. In contrast, DNA meth-ylase activity was easily detectable in extracts prepared frombacteria carrying the pMB3 plasmid, as reported previously(15).

Phenotypically Cryptic Endonuclease Activity RequiresGenes on ColEl Itself. In order to investigate whether genespresent in E. coli strain 1200 or on the Tn3 transposon specifythe production of phenotypically cryptic EcoRI endonucleaseactivity, and to determine whether a genetic locus within theColEI plasmid itself is required for endonuclease production,extracts of bacterial cells containing either control plasmids orvarious derivatives of ColEl were tested (Table 3). In all in-stances, A DNA was used as a substrate. As shown in the table,endonuclease activity characteristic of EcoRI was detected inextracts from strains RY13, 1200(pMB3), 1200(pMB4),1200(ColE1::Tn3), 1200(ColE1), and 1200(pCM100). Insertionof an EcoRI-generated fragment of R6-5 plasmid DNA into theEcoRI site of ColEl disrupts production of colicin by theplasmid (30), but does not affect the ability of ColEl to producethe endonuclease. However, no endonuclease was detected inenzyme extracts from bacterial cells containing the ColEl de-

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1268 Biochemistry: Miller and Cohen

Table 3. Assay of E. coli strains for EcoRI endonucleaseactivity in vitro

Relevant propertiesStrain Activity* and ref.

1200 - (14/14) Endo I-, RNase- (5)RY13 + (18/18) EcoRI-producing strain (1)1200(pMB3) + (6/6) EcoRI-producing plasmid,

ApR (2)1200(pMB4) + (4/5) Mutant of pMB3 (2)1200(ColEl::Tn3, + (18/18) From A. C. Y. Chang, ColEl

i.e., pACYC containing Tn3 from pSC50199) inserted 50% of plasmid length

from EcoRI site1200(ColEl::Tn3) + (5/8) Contains the two plasmids listed(pSC105) (25)

1200(ColE1) + (10/10) (6)1200(pCM120) + (3/4) ColEl ligated to an EcoRI

generated KmR fragment ofR6-5

1200(pCM100) + (4/6) Spontaneous mutant of pMB3that has lost the restrictionphenotype in vivo

1200(pCM110) + (3/4) Spontaneous mutant ofpMB4that has lost the restrictionphenotype in vivo

1200(pSC101) - (2/2) (26)1200(R6-5) - (3/3) (14)1200(RSF1010) - (4/4) (27)1200(pML21) - (5/5) (28)1200(pBR313) - (2/2) (29)1200(pMC8401) - (2/2) From M. Casadaban; ColEl::Tn3

derivative lacking Hae IIfragments Al and B of ColEL.

Endonuclease activity was assayed on Xplac MS505S7 DNA inagarose gels. The enzyme preparations used were either from the25-40% ammonium sulfate fraction (15) or were the peak activityfractions eluted from a P11 phosphocellulose column (15). Specificactivity at the ammonium sulfate stage of endonuclease purificationcould not be precisely measured in all cases since complete digestionof X DNA was not always attainable. Ap, ampicillin; Km, kanamy-cin.* The denominator of the number in parentheses indicates thenumber of different preparations of extracts assayed for each bac-terial strain. The numerator represents the number of times theresult shown (+ or -) was observed in such assays. +, Endonucleaseactivity of at least 10 units/mg of protein (17) (ammonium sulfatefraction) or 300 units/mg of protein (phosphocellulose fractions).-, Undetectable enzyme activity (

Proc. Natl. Acad. Sci. USA 75 (1978) 1269

is increased substantially when plasmid DNA is introduced intobacterial cells by transformation (26). After transformation of"E. coli by pMB3 DNA, we observed mutants of pMB3 thatfailed to express restriction or modification functions in vvobut that nevertheless specified phenotypically cryptic EcoRIendonuclease activity. Analysis of several of these pMB3 de-rivatives (unpublished data) indicates that different rear-rangements of genetic material have occurred in the differentisolates and that the conversion of phenotypically expressed tophenotypically cryptic endonuclease activity is not associatedwith simple deletion or insertion of DNA.The mechanism involved in the phenotypic crypticity of the

EcoRI endonuclease activity reported here is unknown.However, we have shown that the ColEl plasmid does not in-terfere in trans with expression of the restriction phenotype bya pMB3-derived EcoRI endonuclease gene cloned onto anotherplasmid in the same cell (unpublished data). Possibly, a geneticfunction specified by the 1.95-kilobase segment of pMB3lacking in ColEl and defective in the pMB3-derived mutantsconverts the EcoRI endonuclease into an active and phenoty-pically expressed form.

Instances of in vivo crypticity of bacterial gene products haveresulted previously from the absence of genes that encode ac-cessory proteins required for phenotypic expression of theprimary product (33) or from complex formation of the pri-mary product with a second protein (34). A recent report in-dicates that the Clo DF13 plasmid enclodes a site-specificendonuclease that cleaves Clo DF13 DNA in vitro, but that theClo DF13 immunity product forms a complex with the endo-nuclease to neutralize its activity in vvo (34). Other observa-tions indicate that the site-specific endonuclease made by Ba-cillus subtilis strain Marburg 168 can cleave in vitro the DNAof a bacteriophage grown on the same strain (35).Our findings suggest that a reappraisal of current concepts

relating to the biological role of site-specific restriction-modi-fication systems may be indicated. Moreover, involvement ofthe EcoRI endonuclease in site-specific genetic recombinationin vivo (11) raises the possibility that cryptic endonuclease genesassociated with the plasmid ColEl may have a role in plasmidevolution. It remains to be determined whether analogousphenotypically cryptic site-specific endonucleases are specifiedby other bacterial plasmids that do not express detectable re-striction or modification functions in vivo.

These studies were supported by National Institutes of Health GrantAI08619, National Science Foundation Grant PCM-75-14176, andGrant VC139 from the American Cancer Society.

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