Proc. Natl Acad. Sci. USAVol. 78, No. 3, pp. 1503-1507, March 1981Biochemistry

Cloning and expression of the Pst I restriction-modification systemin Escherichia coli

(restriction enzymes/molecular cloning/protein overproduction/DNA sequence recognition/periplasmic proteins)

ROXANNE Y. WALDER, JAMES L. HARTLEY, JOHN E. DONELSON, AND JOSEPH A. WALDERDepartment of Biochemistry and Diabetes and Endocrinology Research Center, College of Medicine, University of Iowa, Iowa City, Iowa 52242

Communicated by Irving M. Klotz, November 24, 1980

ABSTRACT Here we report the cloning and preliminarycharacterization of the Pst I restriction-modification system ofProvidencia stuartii 164. Transformants of Escherichia coli car-rying the Pst I gene system inserted into the cloning vector pBR322were selected on the basis of acquired resistance to bacteriophageA infection. Pat I endonuclease was detected in osmotic shock fluidfrom each of the resistant clones. Plasmid and chromosomal DNAfrom these clones could not be digested by Pat I, indicating thatthe gene for the corresponding modification enzyme had also beencloned and was being expressed. The smallest recombinant plas-mid encoding both activities, pPst201, contains an insert of ap-proximately 4000 base pairs. In vitro transcription studies indicatethat this DNA fragment also contains the endogenous promoter(s)of the system. When pPst201 was introduced into a minicell-pro-ducing strain of E. coli, two new proteins, 32,000 and 35,000 dal-tons, were synthesized. We have assigned these to the Pst I mod-ification (methylase) and restriction enzymes, respectively. Theactive form of the restriction enzyme is a dimer, as determinedby gel filtration. Constructed transformants of P. stuartii 164 thatcarry the Pst I system inserted into pBR322 produce approxi-mately 10 times more Pat I endonuclease activity than does thenative strain.

Restriction enzymes have become essential tools for the ma-nipulation and characterization of DNA molecules. The restric-tion enzymes themselves, however, have not been as well char-acterized. Little is known of the molecular basis by which theseproteins recognize the specific nucleotide sequence at whichthey cleave double-stranded DNA or of the basis for control ofexpression of the genes for the restriction enzyme and its cog-nate methylase. The recognition mechanism is of particular in-terest because general features of this process may apply toproteins of diverse function that interact with DNA.

In this report we describe the cloning and preliminary char-acterization of the class II restriction-modification system ofProvidencia stuartii 164 (Pst I). Our interests in cloning the PstI system were to obtain an overproducing strain that would fa-cilitate isolation of the restriction enzyme and methylase in thelarge quantities required for detailed physical studies includingx-ray crystallographic analysis and to isolate the genes for therestriction enzyme and methylase in order to study the molec-ular mechanism by which their expression is controlled. Theselection of transformants carrying the Pst I system was basedon acquired resistance to bacteriophage A infection as used bySmith and his collaborators in cloning the Hha II system (1). Theconditions of selection were modified, however, so that onlytransformants that carried and expressed the restriction-mod-ification system were resistant to infection. The cloned genesare contained within a 4.0-kilobase (kb) HindIII fragmentwhich, from in vitro transcription studies, also appears to con-tain the endogenous promoter(s) and hence regulatory elements

of the system. Transformants of P. stuartii 164 that carry thePst I system inserted into pBR322 produced approximately 10times more of the Pst I endonuclease than did the native strain.

MATERIALS AND METHODSEnzymes and Radiolabeled Reagents. Restriction enzymes

were purchased from Bethesda Research Laboratories (Be-thesda, MD) and New England Bio-Labs. T4 DNA ligase wasobtained from Bethesda Research Laboratories. Bacterial al-kaline phosphatase was from Sigma. L-[3S]Methionine was pur-chased from Amersham and [a-32P]CTP was from New EnglandNuclear.

Plasmids and Bacterial and Phage Strains. E. coli K-12 strainHB101 (r-m-recA) was obtained from H. W. Boyer. P. stuartii164 was from the collection of R. J. Roberts. The minicell-pro-ducing strain of E. coli K-12, xl4 1, was obtained from R. Cur-tiss III. The plasmid pBR322 and recombinant plasmids wereisolated by the CsCVethidium bromide procedure (2). PhageAcI60 was generously supplied by M. Feiss. All experimentsusing recombinant DNA were performed in accordance withthe guidelines of the National Institutes of Health.

Transformation and Colony Screening Procedures. P. stuar-tii 164 DNA (60 ,ug), isolated according to the procedure ofMarmur (3), was partially digested with 4-30 units of HindIIIfor 30 min at 37°C. The DNA from each digest was pooled andcentrifuged on a 10-40% sucrose gradient in 30 mM Tris-HCI,pH 8.1/10 mM EDTA/1 M NaCl for 20 hr at 25,000 rpm in anSW 27 rotor. Fractions collected by puncturing the bottom ofthe tube were examined by agarose gel electrophoresis, andfractions that contained predominantly fragments 3-10 kb longwere pooled and precipitated with ethanol. Hybrid plasmids ofpBR322 were constructed by ligating the sized HindIII-cut P.stuartii DNA (2.0 ,tg) to phosphatase-treated HindIII-cutpBR322 (1.6 ,ug) by using T4 DNA ligase. The reaction was al-lowed to proceed for 12 hr at 16°C in 200 ,ul of 66 mM Tris-HCl,pH 7.6/10mM MgCJ2/10mM dithiothreitoV200 ,uM ATP con-taining 50 mg of bovine serum albumin per ml.DNA from the ligation reaction (0.5 ,ug) was added to ap-

proximately 2.5 x 109 HB101 cells that had been treated withCaCl2 according to the method of Wensink et aL (4). The pro-tocol for transformation described by Bolivar and Backman (5)was followed, after which 0.9 ml of L broth was added to eachreaction mixture. After 30 min the entire reaction volumes wereplated on two 15-cm LB agar plates containing ampicillin (200,ug/ml). Approximately 2000 transformants were obtained perplate.The transformants were screened against bacteriophage A as

follows. Two milliliters of A lysate (1010 plaque-forming units/ml) were added to 1.6 x 105 cells from a subculture of the PstI genomic library in 0.6 ml of L broth containing 0.2% maltose;

Abbreviation: kb, kilobase.

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1504 Biochemistry: Walder et al.

the suspension was allowed to incubate for 10 min at 370C. Thesample was then serially diluted, and aliquots containing 104cells were layered on LB agar plates containing ampicillin (200,ug/ml). Approximately 0.1% of the transformants gave rise tonormal-appearing colonies.

Pst I endonuclease activity released by osmotic shock fromtransformed and native strains was assayed as described bySmith et aL (6). The substrate used in the assay was the plasmidp82-6B (7) which contains two Pst I recognition sites.

Analysis of Plasmid-Directed Protein Synthesis in Minicells.The minicell-producing strain of E. coli, X1411, was trans-formed with pBR322 and recombinant plasmids carrying the PstI restriction-modification system as described above for HB101cells. Small-scale plasmid isolations from the X1411 transform-ants and subsequent restriction enzyme analyses showed thatthe plasmids were not altered during their maintenance inX1411. Minicells were isolated by sucrose density gradient cen-trifugation and were incubated for 30 min at 370C with aerationto degrade endogenous mRNA (8). The proteins made withinthe minicells were labeled with L-[3S]methionine (specific ac-tivity, 800 mCi/mol; 1 Ci = 3.7 X 1010 becquerels). Cells werelysed as described (9), and lysates were run on a 10-18% gra-dient NaDodSO4polyacrylamide gel (10). Gels were stainedwith Coomassie brilliant blue, destained, dried, and exposedfor autoradiography for 12 hr at room temperature on KodakXR-5 film.

RESULTSSelection of Clones Carrying the Pst I Restriction-Modifi-

cation System. Transformants of HB101 carrying the Pst I sys-tem were selected on the basis of acquired resistance to infec-tion by bacteriophage A. Of the E. coli transformants containingcloned P. stuartii 164 DNA, about 0.1% of the cells formednormal-appearing confluent colonies after infection in liquidculture with A at a titer of 7.7 X 109 plaque-forming units/ml.Approximately 0.4% of the cells formed flat patchy colonies,clearly heavily infected by A. Osmotic shock fluid obtained from

seven randomly selected A resistant colonies contained Pst Irestriction enzyme activity (Fig. 1). Osmotic shock fluid froman infected colony and from the nontransformed host HB101had no Pst I activity (lanes 8 and 9, respectively). Apparently,only those cells that expressed the Pst I restriction enzyme wereresistant to A at the titer used in the selection procedure.

Characterization of Recombinant Plasmids. Plasmids wereisolated from five of the recombinant clones that produced thePst I restriction enzyme and digested with HindIII to cut outthe inserted DNA fragment (Table 1). Three of the plasmids(pPstlOl, pPstlO2, and pPst 105) had identical inserts, 5.9 kblong, which contained an internal HindIII site that divided theDNA into a 1.9-kb and a 4.0-kb fragment. Both of these frag-ments were contained within the inserts of the larger plasmidspPstlO3 and pPst 104.

At this point it appeared that both the 1.9-kb and 4.0-kb frag-ments may be required for expression of the restriction-mod-ification phenotype. However, subcloning experiments showedthat the 4.0-kb HindIII fragment contained the entire Pst I sys-tem and that cells transformed with this piece of DNA were asresistant to infection by A as were cells transformed with theentire 5.9-kb insert (data not shown). A partial restriction mapof the 4.0-kb insert from the plasmid pPst201 is shown in Fig.2.

Modification of Host DNA. Recombinant clones producingthe Pst I restriction enzyme must also express a correspondingmodification activity to protect their own DNA from digestion.The cloning vector pBR322 contains a single Pst I site. Digestionof the recombinant plasmid pPstlO5 with BamHI and Pst I es-tablished that this site is protected within pPstlO5 (Fig.3).BamHI cleaves pPstlO5 at a single site located within pBR322(lane 1). The linear molecule that is produced by BamHI diges-tion is not susceptible to further cleavage by Pst I (lane 2). Thecontrol experiments in lanes 3 and 4 demonstrate that the PstI endonuclease remains active in the incubation mixture con-taining pPstlO5.

Chromosomal DNA isolated from HB101 transformed with

1 2 3 4 5 6 7 8 9 10 11 12FIG. 1. Assay of Pst I endonuclease activity in osmotic shock fluids. The substrate in the assay was the plasmid p82-6B (7) which yields two

characteristic DNA fragments (A and B) upon digestion with Pst I. The sources of the osmotic shock fluids were as follows: lanes 1-7, individualtransformants resistant to infection by A; 8, a A sensitive transformant; 9, E. coli HB101, the nontransformed host bacteria; 10, E. coli RY13, thesource of the restriction enzyme EcoRI; and 11, P. stuartii 164. Lane 12 is p82-6B after digestion with Pst I endonuclease purified from P. stuartii164. Band C in lanes 1-11 includes chromosomal DNA released by the small and variable amount of cell lysis that occurs in the osmotic shockprocedure. DNA bands were visualized by fluorescence with ethidium bromide.

Proc. Natl. Acad. Sci. USA 78 (1981)

Proc. NatL Acad. Sci. USA 78 (1981) 1505

Table 1. Cloned DNA fragments from transformants producingthe Pst I restriction enzyme

Plasmid Sizes of fragments, kb

pPstlOl 4.0; 1.9pPstlO2 4.0; 1.9pPstl03 4.0; 4.0; 2.2; 1.9; 0.4pPstl04 4.0; 4.0; 1.9; 0.4pPstl05 4.0; 1.9

Recombinant plasmids isolated from transformants producing thePst I restriction enzyme were digested with HindI, and the clonedHindI fragments were sized by agarose gel electrophoresis.

pPstlO5 was also found to be protected from digestion by PstI (data not shown). By analogy with other restriction-modifi-cation systems, DNA protection is presumably due to specificmethylation of one of the residues, either C or A, within thePst I recognition sequence (11).

Transformation of P. stuartii 164: Construction of a StableOverproducing Strain. P. stuartii 164 was transformed with therecombinant plasmid pPstlO5 encoding the Pst I system by thesame protocol used for transformation of E. coli. Transformedcells were selected on the basis of acquired resistance to am-picillin. The total amount of Pst I endonuclease produced bythis clone was measured after the cells were disrupted by son-ication. The transformed cell line was found to synthesize ap-proximately 50,000 units of enzyme per g of cells, comparedto 5600 units per g produced by the native strain. * Neither thelevel of enzyme overproduction (approximately 10-fold) nor re-sistance to ampicillin was diminished with serial culturing. Re-striction enzyme analyses of pPstlO5 recovered from P. stuartiishowed that the plasmid had not been altered during its main-tenance in this cell.Gene Products of the Pst I Restriction-Modification System.

X1411, a minicell-producing strain of E. coli, was transformedwith pBR322 and with pPst201. At least two polypeptide chainswere synthesized in minicells transformed by pPst201 that werenot made in minicells containing pBR322 (Fig. 4). The presenceof the insert also appeared to affect the relative amount of syn-thesis of several proteins encoded for by pBR322, as found pre-viously by using the plasmid vector RSF2124 (12). The two newpolypeptide chains had molecular weights of approximately32,000 and 35,000 as determined by comparison to marker pro-teins. The latter comigrated with a prominent component of apartially purified sample of the Pst I endonuclease that wasenriched during the purification of the enzyme (lane 3). On thisbasis we have tentatively assigned the 35,000-dalton polypep-tide to the restriction enzyme and the 32,000-dalton polypep-tide to the methylase. The apparent molecular weight of the

* Preliminary studies indicate that transformants of E. coli that carrythe Pst I system produce less enzyme than do native P. stuartii 164(N. Pomato and D. V. Hendrick, Bethesda Research Laboratories,personal communication).

Hindill

AIBal

-~~~~~~~~~~~~~~~~~

FIG. 3. Host cell modification of plasmid DNA encoding the Pst Isystem. The Pst I recognition site within the cloning vector pBR322is protected from digestion in the recombinant plasmid pPstlO5. Lanes:1, pPstlO5 digested with BamHI; 2, pPstlO5 digested with BamHI andPst I; 3, pPstlO5 andpBR322 digested withBamHIandPst I; 4, pBR322digested with BamHI and Pst I. DNA bands were visualized by flu-orescence with ethidium bromide.

native form of the restriction enzyme, as determined by gelfiltration through Sephacryl S-200, is 69,500 at concentrationsof the enzyme between 5 and 10 kig/ml (data not shown).Therefore, under these conditions, the catalytically active formof Pst I is a dimer.

In Vitro Transcription of the Pst I Restriction-ModificationSystem. In vitro transcription was assayed by the proceduredescribed by Chelm and Geiduschek (13) in which ternary com-plexes among the DNA template (generally a specific restrictionfragment), RNA polymerase, and short, newly synthesized RNAchains are identified by electrophoresis on agarose gels. Ideally,

Ava IHi1ch XVol

Hindi 1

Bg|I B HI 11

BIll, BIll

FIG. 2. Partial restriction map of the 4.0-kb insert that contains the Pst I restriction-modification system. The hexanucleotide recognition sitesfor the restriction enzymes EcoRI, Sal I, Hpa I, BamHI, and Pvu II are not present within the DNA sequence.

Biochemistry: Walder et aL

1506 Biochemistry: Walder etaLP

66,000

45,000

A....B Z..'_

amp 30,000

A

Ow- B

c

3 4

...... ...;i

':4 ;i

2

I 2 3FIG. 4. Plasmid-directed protein synthesis in minicells. Minicells

were incubated in the presence of L-[35S]methionine to label newlysynthesized plasmid-encoded proteins. Minicell lysates and a partiallypurified sample of the Pst I endonuclease were electrophoresed on a

10-18% gradient NaDodSO4/polyacrylamide gel. The gel was stainedwith Coomassie brilliant blue, dried, and exposed for autoradiography.Lanes 1 and 2 are from the autoradiogram of the gel and correspondto minicell lysates prepared from transformants containing pBR322and the recombinant plasmid pPst201 encoding the Pst I system, re-

spectively. Lane 3 is the Coomassie blue stain of the partially purifiedPst I endonuclease. Two new polypeptides (A and B) having molecularweights of 32,000 and 35,000 were synthesized in minicells containingpPst2O1 but not in minicells containing pBR322. The larger polypep-tide comigrated with a prominent component of the partially purifiedPst I restriction enzyme. The molecular weight calibration was derivedfrom electrophoresis of a standard set of marker proteins not shownin the figure. amp, Product of the ampicillinase gene of pBR322.

ternary complexes would be formed only with restriction frag-ments that contained an active promoter site. RNA synthesisis carried out in the presence of one or more [a-32P]ribotriphosphates so that the ternary complexes may be lo-

FIG. 5. In vitro transcription of the Pst I restriction-modificationsystem. Lanes: 1 and 3, ethidium bromide fluorescence of the 4.0-kbDNA fragment encoding the Pst I system and the fragment after diges-tion with Ava I, respectively. 2 and 4, autoradiograms of ternary com-plexes of E. coli RNA polymerase, DNA, and nascent RNA chainsformed on DNA templates in lanes 1 and 3, respectively. Ternary com-plexes were labeled with [a-32P]CTP. The intensity of the band due toternary complexes formed on the 2.3-kb Ava I fragment (A) is morethan 12-fold greater than that due to ternary complexes formed on the1.3-kb Ava I fragment (B), as determined by densitometric scanningof the autoradiogram at 550 nm. The 0.4-kb Ava I fragment (see Fig.2) was not detected by ethidium bromide fluorescence in this gel butdid give rise to a discernible level of transciption complex (C). The ter-nary complexes formed on the 0.4-kb and 1.3-kbAva I fragment serveas endogenous controls for the level of nonspecific initiation oftranscription.

calized within the agarose gel by autoradiography.In the experiment described in Fig. 5, the 4.0-kb fragment

carrying the Pst I restriction-modification system as well as theAva I digest of this fragment (see Fig. 2) were used as templatesfor in vitro transcription. The yield of ternary complex obtainedwith the entire 4.0-kb fragment and with the 2.3-kb Ava I frag-ment were comparable; that obtained with the 1.3-kb Ava Ifragment was much less (< 10%) (see legend to Fig. 5). The ter-nary complex formed with the 1.3-kbAva I fragment is probablydue to nonspecific initiation of transcription and, therefore,serves as an endogenous control for the amount of RNA syn-thesis directed by the other templates. These results stronglysuggest that the 4.0-kb insert contains the native promoter(s)of the Pst I system and that the promoter(s) lie within the 2.3-kb Ava I fragment. As such, it should be possible to use thecloned system to study the physiological control of the expres-sion of the restriction enzyme and methylase genes.

DISCUSSIONThe Pst I restriction enzyme initially was thought to be encodedon a plasmid because it was found in only one clinical isolate,P. stuartii 164, and not in several other strains of P. stuartiiexamined (6). However, more recent studies indicate that P.stuartii 164 does not contain a plasmid (C. I. Kado, personal

I

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Natl. Acad. Sci. USA 78 (1981) 1507

communication). Therefore, the Pst I restriction-modificationsystem must be located on chromosomal DNA and is presum-ably present in only one copy per cell. The transformants of P.stuartii that we have constructed that carry the Pst I system onthe multicopy plasmid pBR322 produce a 10-fold greateramount of the restriction enzyme than that made in the nativestrain. With further manipulation, it should be possible to con-struct clones that produce even greater yields of the enzymeto facilitate further the large-scale purification of the protein.For example, from the autoradiogram shown in Fig. 4 it wouldseem that, if the gene for the restriction enzyme could be linkeddirectly to the end of the coding region for the signal sequence(14) of the ampicillinase gene of pBR322, the synthesis of therestriction enzyme would be markedly increased. In addition,this should greatly simplify the purification of the enzyme be-cause most if not all of the protein would then be secreted intothe periplasmic space (15).

As in the native organism, a fraction of the Pst I restrictionenzyme produced in the transformants is transported into theperiplasmic space and can be released by osmotic shock of thecells. The signal sequence (14) which codes for the transport ofthe enzymes into the periplasmic space of P. stuartii 164 there-fore must also be recognized at the cytoplasmic membrane ofE. coli. The periplasmic space is clearly a strategic location forthe restriction enzyme to intercept foreign DNA. The restric-tion enzyme located within this extracytoplasmic compartmentmay be particularly important for expression of the restrictionphenotype and may be required for resistance against the veryhigh titer of phage used in the selection procedure.

At the titer of A used for selection, only those transformantsthat carried and expressed the restriction-modification systemwere resistant to infection. Colonies were also formed fromabout 0.4% of transformants that did not carry the Pst I system,but these were clearly heavily infected by A and readily distin-guished from the normal-appearing colonies that arose fromcells having the restriction-modification system. Subsequently,we have found that the efficiency of plating of transformantscontaining the Pst I system is approximately 0.4 in the presenceof A at the titer used in the selection procedure.

There are two conditions that must be met in order for a re-striction-modification system to be cloned and expressed in anew host. It is essential that the genes for the restriction enzymeand methylase be very closely linked and that, after the trans-

formation, the methylase be expressed in advance of the re-striction enzyme to protect the DNA of the host. Three cellularrestriction-modification systems have been shown to satisfythese criteria: (i) EcoRI which occurs naturally on a plasmid (16),(ii) Hha II cloned by Smith and his collaborators (1), and (iii) PstI. The close linkage of the genes in these systems may be re-quired for their coordinated expression and suggests that theyare transcribed as a polycistronic message. If the sequentialexpression of the genes after transformation is related to thephysiological control of their expression, one would anticipatethat other restriction-modification systems would be organizedsimilarly.We thank Dr. Mike Feiss for generously supplying us with the bac-

teriophage Ac160 from his collection and Mrs. Tamara Gregori andDeborah Siegele for technical assistance in various aspects of this work.We also thank Mr. Kenneth Chen for assistance in preparation of theillustrations. This investigation was supported in part by National Sci-ence Foundation Grant PCM 76-13461 and National Institutes ofHealth Grant GM-21696 to J. E. D. and by National Institutes of HealthDiabetes and Endocrinology Research Center Grant AM 25295.

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S. (1974) Cell, 3, 315-325.5. Bolivar, F. & Backman, K. (1979) in Methods Enzymol. 68,

245-267.6. Smith, D. I., Blattner, F. R. & Davies, J. (1976) Nucleic. Acids.

Res. 3, 343-353.7. Hartley, J. L. & Donelson, J. E. (1980) Nature (London) 286,

860-865.8. Reeve, J. (1979) Methods Enzymol 68, 493-503.9. Miller, D. L., Gubbins, E. J., Pegg, E. W., III & Donelson, J.

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