JOURNAL OF BACrERIOLOGY, Oct. 1973, p. 163-174Copyright 0 1973 American Society for Microbiology

Vol. 116, No. 1Printed in U.S.A.

Plasmids of Shigella dysenteriae Y6R: a

Defective Col FactorBRUCE W. PORTER,' RICHARD KOLODNER, AND ROBERT C. WARNER

Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92664

Received for publication 30 April 1973

The six plasmids of Shigella dysenteriae Y6R were separated by sucrose

gradients into five fractions containing deoxyribonucleic acid (DNA), havingcontour lengths (expressed in units equal to the fraction of the length ofthe replicative form of 4X174), respectively, of 0.29, 0.35, 0.74, 1.08, and a

mixture of 5.7 and 7.2. DNA-DNA hybridization on nitrocellulose filters betweeneach of the plasmids and between plasmid-free S. dysenteriae Y6R host DNA andplasmids was investigated. There was a high degree of homologybetween the 0.29- and 0.35-unit plasmids. No significant homology was foundbetween any of the other pairs of plasmids. Homologous DNA to the extent of 2.4copies of the 1.08-unit plasmid was found in the host genome. Homology betweenthe other plasmids and the host genome is very slight, but appears to besignificant. About 0.7 of the 1.08-unit plasmid is homologous to the ColEl faqtorof Escherichia coli JC411 (ColEl). This plasmid may be defective ColEl factorwith the immunity function intact, but with a defect in the gene leading to theproduction of active colicin. Electron microscope examination of heteroduplexesformed between the two smallest plasmids and between the 1.08-unit plasmidand the ColEl factor yielded independent determinations of the extent ofhomology in agreement with the values determined by hybridization. In thelatter case, two nonhomologous regions of substitution of DNA were detected.

In previous work Shigella dysenteriae Y6Rwas found to contain six distinctly differentplasmids (16). They fall into three general sizeclasses in which the circular deoxyribonucleicacid (DNA) of each species has the followingcontour length: small, 0.29 and 0.35 units;intermediate, 0.74 and 1.08 units; large, 5.7 and7.2 units. Contour lengths, following the sugges-tion of Davis et al. (4), are expressed in 4XRFunits, i.e. the ratio of the contour length to thatof the replicative form of kX174 DNA. Apreliminary survey (16) failed to find any anti-biotic resistance that might be associated with aplasmid, and no other functional significance oftheir presence is known. In other cases, a varietyof functions have been associated with plasmidsof the size of the intermediate and large classes(11), although none has apparently been foundfor "minicircular" plasmids (12). The related-ness of these plasmids to each other, to the hostDNA, and to ColEl DNA by the methods ofDNA-DNA hybridization and of heteroduplexformation is reported here.

I Present address: Department of Biochemistry, Universityof Wisconsin, Madison, Wis. 53706.

MATERIALS AND METHODS

S. dysenteriae Y6R was obtained from I. Tessman.It was made thymine requiring by the method ofStacy and Simson (18). Escherichia coli C was ob-tained from R. L. Sinsheimer, and E. coli strainsJC411 (ColEl), YS40/El,V(ColV), and WS3110(ColE2) were obtained from D. R. Helinski. Themethods employed for analytical centrifugation werepreviously described (6, 17). The buoyant densityreference was denatured Micrococcus luteus DNA (p= 1.741 g/ml). Preparative separations were carriedout on alkaline sucrose gradients (5 to 23% sucrose, 1M NaCl, 2 mM ethylenediaminetetraacetate [EDTA],pH 13) and neutral sucrose gradients (5 to 23% su-crose, 1 M NaCl, 2 mM EDTA, pH 8) in the SpincoSW40 rotor. DNA was examined in the electron mi-croscope by methods previously used (6) and by themethod of Davis et al. (4) for preparation of hetero-duplexes. DNA was prepared for spreading by nickingwith a calibrated gamma-ray source (3'"Cs) so thathalf of the molecules would contain one nick and veryfew would contain two nicks. Contour lengths are re-ported as ratios to the length of the replicative form(RF) of rX174 DNA. The standard DNA, either*X174-RF or that of the 0.29-unit plasmid, was co-spread with the unknown and measured on photo-graphs from the same grid. Length measurements on

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heteroduplexes are referred to measurements ofdouble-stranded and single-stranded species of knownlength on the same grid.Bacterial growth. For the preparation of unla-

beled host genome DNA or plasmids, S. devsenteriaeY6R was grown to stationary phase in modified 3XDmedium (16). E. coli JC 411 was grown to stationaryphase in Penassay broth. For radioactive prepara-tions, thymine-requiring S. dvsenteriae Y6R or E. coliJC 411 was grown to stationary phase in a medium ofthe following composition (per liter): Na2HPO4, 7 g;KH2PO4, 3 g; NH4Cl, 1 g; MgSO4, 10-' M; FeCls, 10-6M; glucose, 4 g; vitamin-free Casamino Acids, 3 g;niacin, 10 mg; thymidine, 2 mg; and [methyl-3H]thy-midine (Schwarz/Mann, 6 Ci/mmol), 4 mCi.

Bacterial cells were washed three times with 0.15 MEDTA (pH 8.0), suspended in 100 ml of 0.05 M EDTA(pH 8.0) for each liter of culture, and stored frozen.Circular DNA, including that of the ColEl factor, wasprepared as previously described (17). All DNA prep-arations were dialyzed against 0.0015 M EDTA, 0.05x SSC (pH 8.0), unless otherwise noted (1 x SSC is0.15 M NaCl, 0.015 M Na3 citrate).

Preparation of chromosomal DNA from S. dy-senteriae Y6R. Washed bacterial cells were sus-pended at a concentration of about 1010 cells per ml in20 ml of 25% sucrose and 0.05 M tris(hydroxymethyl)-aminomethane (pH 8.0), lysed by the method ofClewell and Helinski (2), and centrifuged for 30 minat 40,000 rpm and 3 C. The resulting pellet washomogenized by hand in a glass-Teflon homogenizer,washed with the lysis buffer, and pelleted at 40,000rpm for 15 min. A second washing was made, using0.15 M NaCl, 0.1 M EDTA (pH 8.0), and centrifuga-tion at 45,000 rpm for 20 min. The pellet wassuspended in 15 ml of extraction buffer and againhomogenized. Sodium dodecyl sulfate was added to afinal concentration of 0.5%, followed by treatment at60 C for 10 min. This solution was extracted withphenol, and only the precipitate of the interface wasretained, since this procedure was shown to reducecontamination with plasmid DNA. The further stepswere those outlined by Habich et al. (9). Additionaltreatment of this DNA to remove traces of plasmids isdescribed in Results.

Separation of plasmids into size classes. Afterthe basic procedure for obtaining circular DNA,plasmid preparations were freed of traces of host DNAby repeating the reversible alkali denaturation step ofadjusting to pH 12.3, neutralizing with tris(hydroxy-methyl)aminomethane-hydrochloride, and passingthrough a nitrocellulose column. A test of this stepwith added 3H labeled host DNA showed that lessthan 0.5% remained. Since host DNA is not detecta-ble in an ultracentrifugal scan of the plasmids beforethe second alkali treatment, this step thus reduces itto well below 0.01% of the total plasmid DNA.Separation of the plasmids into individual size classeswas achieved by a series of alkaline sucrose gradients.These are much superior to neutral gradients inresolving power, but except for the 0.29-unit plasmid,result in the recovery of circular DNA in the form IVconfiguration. The designation, form IV, is used as anextension of the terminology for circular DNA confor-

mations proposed by Vinograd et al. (20) to refer tothe compact, rapidly sedimenting species in which thesecondary structure has been disrupted, but in whichthe strands remain covalently closed. Where neces-sary the form IV species were renatured to form I (W.Strider and R. C. Warner, 1971, Fed. Proc. Fed. Amer.Soc. Exp. Biol. 30: 1053; manuscript in preparation).Several of the gradient separations are illustrated inFig. 1. The purity of the fractions was determined byanalytical ultracentrifugation (Fig. 2) and electronmicroscopy.A single alkaline gradient was sufficient to separate

the two larger plasmids from the others (Fig. 1A).Subsequent separation of the 5.7- and 7.2-unit plas-mids was not attempted, and all experiments on theseplasmids were carried out with the mixture (Fig. 2C).The intermediate and small plasmids were par-

tially separated from each other on the initial gradi-ent. A second alkaline gradient of the faster fraction ofthe intermediate peak of Fig. 1A yielded pure 1.08-unit plasmid. The fractions between the two slowpeaks of Fig. IA were rerun on two gradients andyielded pure 0.74-unit plasmid (slow fractions fromthe large peak of Fig. IB).The small plasmid peak of Fig. lA was not resolved

on a second alkaline gradient, but residual contami-nation from intermediate species was eliminated. Thetwo sizes in this class were separated on a neutralgradient (Fig. IC) by the fortunate circumstance thatthe smaller size (0.29 unit) is renatured to form I onneutralization, while the 0.45-unit plasmid remains asform IV. This greatly increases the difference insedimentation rate between the two and allows aseparation on a single, neutral gradient. The identifi-cation of the species in Fig. IC as form IV and form Iof the respective plasmids was confirmed by addi-tional analytical centrifugation and electron micros-copy.

Hybridization. DNA-DNA hybridization was car-ried out by the method of Denhardt (5) with minormodification. The filters were incubated at 62 C for 13h in Pyrex tubes (8 mm inside diameter) stopperedwith rubber caps, in 1.2 ml of 6 x SSC containing the3H DNA. The use of small tubes containing the rolledup filters and having only a small air space greatlyimproved the reproducibility of the method. Thefilters were removed, washed on both sides with SSC,dried, and counted in a scintillation counter, using atoluene based scintillation fluid.The circular DNA was prepared for use by nicking

with gamma irradiation, using a 37Cs source at adosage of 14,100 rads. The dosage was estimated togive an average of one nick in a form IV molecule witha molecular weight of 106 on the basis of a calibrationdone in this laboratory using kX174-RF (W. Strider,unpublished data). After nicking, the DNA solutionwas brought to 0.2 N in NaOH and neutralized toinsure strand separation. The average molecularweight of the DNA was estimated to be 500,000 fromthe application of Studier's (19) formula to the S20.wdetermined by analytical zone sedimentation in 3 MCsCl. This is in agreement with that estimated fromthe degree of nicking. Plasmid DNA of this size wasused for fixing to the filters. The host DNA fixed to

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Top -*

1.0

E 0.8

0

a

0.6

z4

0

0

un 0.4t4

0.29 unitplosmidForm I

C

0.35 unitplasmidForm II

O.?.-

4 8 12 16 20 24 28FRACTION NUMBER TOP

FIG. 1. Sucrose gradient centrifugation illustrating the separation of the plasmids of S. dysenteriae Y6R.

Gradients of 13 ml were run at 4 C in the SW40 rotor at the indicated speed. Fractions are identified by thecontour lengths of the plasmids comprising them. (A) Initial alkaline gradient showing the three size classes;20,000 rpm, 10.5 h. (B) Alkaline gradient showing separation of the 0.74- and 1.08-unit plasmids; 30,000 rpm,13.5 h. (C) Neutral gradient showing separation of the 0.29- and 0.35-unit plasmids; 40,000 rpm, 13.5 h. The0.35-unit plasmid is in the form IVconfiguration, and the 0.29-unit plasmid is in the form I configuration.

the filters had an average molecular weight of 500,000.The 'H DNA used in solution was further degraded toa molecular weight of 200,000 by sonic oscillationwith a Rayson Sonicator in 0.0015 M EDTA, 0.05 x

SSC, and 0.2 M NaOH. The solution was chilled andneutralized. Lower-molecular-weight DNA in solutionwas used to reduce the amount of radioactive DNAcarried on the filter after hybridization as "tails" of

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A

FIG. 2. Analytical ultracentrifuge scans of purified plasmids. Samples were layered on neutral or alkaline 3M CsCl at 20 C. (A) 0.29-unit plasmid (form I) on neutral CsCl; 40,000 rpm, s20.w = 13.1S. (B) 1.08-unitplasmid on alkaline CsCl; 34,000 rpm, S20,W = 52S. The slow component is single-stranded DNA. (C) Mixture of5.7- and 7.2-unit plasmids (form IV) on neutral CsCl; 30,000 rpm, s20,w = 98 and 108S.

nonhomologous, nonhybridized ends attached to seg-ments that are homologous to DNA on the filter (1).Further degradation of the DNA did not reduce thiseffect. It was corrected for as explained in Results.The amount of DNA retained on filters after

hybridization was estimated by fixing 3H DNA ofeach kind to a filter at various concentrations andcarrying it through preincubation and then through ablank hybridization process using 6 x SSC. Retentionof plasmid DNA was 45 to 55% when 0.01 ug wasfiltered at a concentration of 0.02 ug/ml. When 0.1 Mgwas filtered at the same concentration, the retentionrose to 55 to 65%. When 1 Mg was filtered at 0.2 gg/ml,the retentions were 95 to 98%, 85 to 90%, and 75 to 80%for the small, intermediate, and large plasmids,respectively. The addition of 1 Mg of M. luteus DNAhad no effect on retention of plasmid DNA. Eightypercent of 5 to 20 Mg of host DNA was retained. Wherenecessary, corrections were applied on the basis ofthese figures.

RESULTSContour length. Contour length measure-

ments on each plasmid are summarized in

Table 1. The results are expressed in OXRFunits and have been converted to molecularweight using the value 3.2 x 106 for kX174-RF(W. Strider and R. C. Warner, manuscript inpreparation). For convenience in referring to theplasmids we identified them by a rounded offvalue of their length in these units. The ratios ofthe lengths of the plasmids to each other are ingood agreement with those previously reported(16).Plasmid function. The possibility that one of

the plasmids may code for resistance to anantibiotic or metal ion was tested by comparingthe sensitivity of S. dysenteriae Y6R to that ofE. coli C to these agents. Growth was comparedat a series of concentrations of the followingsubstances incorporated into agar plates: chlor-amphenicol, sulfathiazone, tetracycline, am-picillin, streptomycin, polymycin B, kanamy-cin, neomycin, Hg2+, Co2+, Cr2+, AsO43-, CU2+,Pb2+, Ba2+, Cd2+, Zn2+, Ag+, and Al3+. Nodifference in the ability of the two bacteria to

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TABLE 1. Mean contour lengths of plasmids

Plasmid Contour length ± SDaG, Moleculardesignation (4XRF units) weight

0.29 0.29 i 0.007 0.930.35 0.349 0.012 1.10.74 0.744 + 0.054 2.41.08 1.08 0.041 3.5El 1.19 0.065d 3.95.7 5.73 X 0.25 18.07.2 7.21 X 0.31 23.0

a Number of molecules measured was 50 to 100 foreach of the smaller plasmids and 15 each for the twolargest plasmids.

b Calculated from co-spreadings of each plasmideither with OX174-RF or with the 0.29-unit plasmid.

c Based on a molecular weight for X174-RF of 3.2x 101.

dDetermined from the ratio (1.104) of the contourlength of double-stranded ColEl to that of the 1.08-unit plasmid on the same grid. The ratio of single-stranded lengths was 1.109.

survive in the presence of these agents, andthus, no apparent indication of function of thiskind for any of the S. dysenteriae Y6R plasmidswas noted. There was also no hemolytic activityobserved when S. dysenteriae Y6R was testedon blood agar plates.Attempts to cure the Shigella strain of plas-

mids by growth in the presence of acridineorange and acriflavin were unsuccessful. Indi-vidual resistant colonies often showed a reduc-tion in the quantity of plasmid, particularly ofthose of the large size. However, after repeatedtransfers in liquid media the total and relativeamounts approached those normally found.

S. dysenteriae Y6R was tested for the possibleproduction of a colicin against E. coli C. Nonewas found, but it was observed that Y6R isimmune to the action of colicin El produced byE. coli JC411 (ColEl). This was shown by thefollowing procedure, suggested by D. R. Helin-ski. The centers of bottom agar plates wereinoculated with E. coli JC411, and growth for 48h was allowed, resulting in colonies about 1 cmin diameter. Further growth was stopped byinverting the plates over a beaker of CHCl. Theplates were then overlaid with 5 ml of soft agarseeded with E. coli C or S. dysenteriae Y6R andincubated for an additional 24 h. A broad clearzone with no growth extended out from thecentral colicinogenic colony on plates seededwith E. coli C, whereas S. dysenteriae Y6R grewconfluently over and around the central colony.These results led to the inclusion of the ColElfactor in the tests for homology among theplasmids.

S. dysenteriae Y6R was also tested in the

same way with E. coli strains that producecolicins E2 and V and was found to be sensitiveto these colicins.Removal of plasmids from host DNA. To

use the host DNA for hybridization with plas-mid DNA, all traces of the latter must beremoved. In the case of the large plasmids (5.7-and 7.2-unit), the effectiveness of this removalcan be monitored in the analytical ultracentri-fuge, since these plasmids have a buoyantdensity in CsCl of 1.700 as compared with 1.710for the host DNA. The amount of these twoplasmids present in lysates of cells examineddirectly in CsCl gradients was 5.6% of the totalDNA. The light component was still present inthe DNA prepared from such lysates by themethod of Habich et al. (9). It clearly decreasedin amount after successive phenol extractionsand precipitations, but was reduced to anundetectable level (less than 0.5%) only whenthe Clewell and Helinski (2) lysis procedure andother steps outlined in Materials and Methodswere used. However, when DNA prepared bythis method was banded in a preparative CsClgradient, 5% of the light component (p = 1.700)was found by subsequent analytical centrifuga-tion in the lightest 9% of the DNA from thesingle peak (mean p = 1.710) of this gradi-ent, confirming a contamination of about 0.5%in the original sample. No light componentcould be detected in the middle 32% or theheavy 59% of the DNA from the same prepara-tive gradient. It is thus indicated that themiddle 32% has been purified by an additionalseveralfold and the heavy 59% by considerablymore. This heavy fraction was used in thehybridization experiments involving the twolarge plasmids and host DNA. A conservativeestimate of the contamination of host DNA withthese plasmids is 0.04%.The four small plasmids (0.29- to 1.08-unit)

have the same buoyant density as the hostDNA, and contamination by them can neitherbe monitored nor removed by density gradients.The lysis procedure of Clewell and Helinski (2)is probably more effective in releasing smallthan it is in releasing large plasmids from thepellet of host DNA. These authors consider thatthe method gives an essentially quantitativeyield of the ColEl plasmid, although no directexamination of the pelleted host DNA wasmade. We found no satisfactory way of monitor-ing this step, and consider that a conservativeestimate is that 10% of the four small plasmidswhich are present at about 2% of the amount ofhost DNA might remain in the pellet of hostDNA or a contamination of 0.2%. This is furtherreduced in the two precipitations that follow thephenol extractions in the subsequent purifica-

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tion procedure. This was determined to be apurification of at least sixfold for each precipita-tion by adding 3H labeled 1.08-unit plasmids tothe DNA and carrying the mixture through theremainder of the procedure. In addition a su-crose gradient fractionation was used in whichthe top fractions were discarded. Monitoringof the gradient with 3H labeled 1.08-unit plas-mids indicated a reduction in specific activityof the host DNA of fivefold. We thus estimate apossible contamination of host DNA of 0.001%by all four small plasmids.Hybridization with an excess of DNA on

the filter. The possible homologies among theplasmids and between the plasmids and theColEl factor were examined under the condi-tions and with the results shown in Table 2.

TABLE 2. Hybridization between different plasmidsa

Plasmid 3H DNA Radio-DNA on filter" in solutionb activity

(unit size) bound (%)

5.7 + 7.2-unit 5.7 + 7.2 37plasmid 1.08 0.5

0.74 0.40.35 0.40.29 1.0

1.08-unit plasmid 5.7 + 7.2 1.01.08 530.74 2.10.35 0.80.29 1.2

0.74-unit plasmid 1.08 3.80.4 3.0

0.29-unit plasmid 5.7 + 7.2 1.01.08 1.00.74 1.80.35 470.29 52

ColEl 5.7 + 7.2 0.51.08 350.74 1.50.35 1.10.29 1.5

OX174-RF mixed plasmids 0.1

4X174-RF 0.74 0

none mixed plasmids 0a Hybridization under conditions of excess DNA on

the filter (see Results).'Unlabeled DNA (1 jg) on the filter and 0.2 ,g of

3H DNA in solution (specific activities: 5.7- + 7.2-unitand mixed plasmids, 23,000 counts per min per jig;1.08-unit plasmid, 1,900 to 5,300 counts per min perjg; other, 4,900 to 6,700 counts per min per gg).

Extensive homology between the 0.29- and0.35-unit plasmids and between the 1.08-unitplasmid and the ColEl factor was found. Minorhomology may exist between the other pairs ofplasmids and between plasmids and the ColElfactor. Hybridization between OX174-RF andplasmids was negligible.

Hybridization under these conditions is sensi-tive for detecting homology, but is not suited toquantitating its extent. To estimate the degreeof homology with the limited quantities ofpurified plasmids available, we adopted a modi-fication of the usual procedures for DNA-DNAhybridization that employ DNA fixed to amembrane. It is difficult to approach saturationof the DNA on the filter or to arrange conditionsso that the major part of the DNA in solution ishybridized to the DNA on the filter (5, 21; seealso inset of Fig. 3). This is in part because ofthe competing self-hybridization of the DNA in

0 0.02 0.04 0.06DNA on the filter, pLg

FIG. 3. Hybridization of each size of plasmid onthe filter against the same plasmid in solution. Theamount of plasmid on the filter has been corrected forretention. In each case there was 0.2 ,ug of 3H DNA insolution, having the specific activity indicated inTable 1. The amount bound to the filter has beenconverted to micrograms with no correction for "tail-ing." The self-hybridization of the different plasmidswithin each size class could not be distinguished, andthe curves for each class have been drawn through themean of the values at each loading of the filter. Theplasmids comprising each size class are given inkXRF units as labels for the respective curves. Theerrors bars for each point show the standard deviationfor four experiments, two with each plasmid for theupper curve; five experiments for the middle curve;and two for the bottom curve. The inset shows thesame three curves carried out to higher values ofmicrograms loaded on the filter. The label on thecoordinates of the main figure apply also to the inset.

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solution, and can be minimized by maintaininga large excess of DNA in solution and bykeeping the concentration low. This requires theuse of low amounts of DNA on the filter, andmay present a problem when low degrees of-homology result in low amounts of radioactivityon the filter. We determined the hybridizationof each plasmid with itself in the region of lowquantities on the filter and used these curves tocorrect for the remaining effects of failure toreach saturation and of the presence of nonhy-bridized single-stranded tails on the filter.

Before adopting the procedure outlined belowa number of competition experiments weretried. Homologies were readily demonstrated bycompetition with unlabeled DNA, but thequantities of plasmids needed were too largeand the estimates of the degree of homologywere subject to too much uncertainty to employthis method.

Selfhybridization of plasmids. The hybridi-zation of 0.2 gg of each plasmid with varyingamounts of the same plasmid on the filter isshown in Fig. 3. The curves approach linearityat low amounts of DNA on the filter, but fall offsharply above 0.1 jg on the filter as shown in theinset. The effect of the complexity of the DNA,in the sense used by Wetmur and Davidson(22), on the extent of hybridization is clearlyevident. However, the curves for the closelyrelated plasmids could not be distinguished,and they have been averaged as shown in Fig. 3.The lower initial slope of the hybridizationcurves, for the intermediate and large plasmidscan be attributed to their increasing molecularcomplexity. If they do not contain repeatedsequences, the complexity will be proportionalto the molecular weight and the concentrationof hybridizable sequences will be inversely pro-portional. The quantitative relation among thethree curves on Fig. 3 can be approximatelyaccounted for on this basis, if it is assumed thatthe curve for the small plasmids is close tosaturation of the DNA on the filter and theapproximation is employed that the basic sec-ond-order kinetics of a single renaturationreaction (1, 22) is reduced to a first-orderprocess because of the large excess of DNA insolution. The detailed calculation to supportthis conclusion is not presented because weemployed the curves of Fig. 3 empirically indetermining the extent of hybridization. Itshould be recognized, however, that the validityof this empirical calculation is based on thedependence of the change in slope from onecurve to another on the second-order nature ofthe reaction, even though a single curve followsa first-order course.

In the cases of the small and intermediateplasmids, the amount of DNA bound to thefilter is greater than that initially fixed. Weattribute this to tailing and note that its extentis roughly consistent with the estimate of thiseffect by Britten and Kohne (1). The method ofcalculation which we used corrects for tailingbecause it employs a comparison of the hybridi-zation of one DNA to another on the filter withthe hybridization of the same DNA preparationto itself.Homologies among different plasmids. Hy-

bridization curves obtained under the sameconditions as those in Fig. 3 for the two cases inTable 2 in which pairs of plasmids were found toshow homology are presented in Fig. 4. Eachpair was tested with each member on the filterand the other in solution. The amount of DNAof the type in solution homologous to thequantity of DNA on the filter was calculated byinterpolation from the appropriate curve in Fig.3. The amount of DNA hybridized, e.g., at 0.005Mg on the filter from a curve from Fig. 4, wasused on the curve from Fig. 3 for the same DNAin solution to determine, on the abscissa, theactual equivalence of micrograms of sequenceshomologous to the DNA on the filter in thecurve of Fig. 4. The result can be expresseddirectly in micrograms, units of contour length,or can be converted to the fraction of theplasmid DNA that is homologous to anotherplasmid. This calculation corrects for tailingand for incomplete hybridization on the basis ofthe assumption that these will be the same for

0 0.02 0.04 0.06DNA on the filter ,g

FIG. 4. Hybridization between plasmids showingpartial homology. The conditions and presentation ofthe data are the same as in Fig. 3. The curves arelabeled with the lengths of the plasmids used in OXRFunits. The first length gives the plasmid on the filter,and the second length gives the plasmid that is insolution. Also shown is a curve for the nonhomologous0.74- and 1.08-unit plasmids.

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the same DNA preparation in solution regard-less of whether the hybridizable sequences onthe filter are furnished by the same or adifferent DNA. If the hybridization indicatedby the corresponding curve on Fig. 4 is not closeto saturation, there will be an error resultingfrom a difference in concentration of any spe-cific hybridizable sequence on the filter in thecase of homologous hybridization as comparedwith self-hybridization. This difference arisesfrom the use of a constant microgram amount ofthe DNA on the filter that is of a differentmolecular complexity and contains a differentfraction of hybridizable sequences in the twocases. The problem should be minimal in thetwo homologies considered here because theyare so extensive and are limited to pairs of aboutthe same size. The effect can be allowed forwhen curves for self-hybridization of each plas-mid, such as those in Fig. 3, are available by amore detailed kinetic analysis. In the presentcase, the limited precision of the data does notjustify the more complex treatment.The results of calculations based on the data

for the smallest amount of DNA on the filter arepresented in Table 3. They have been calcu-lated in terms of the amount of DNA in OXRFunits that was found to be homologous in eachof the two separate experiments. This conver-sion is based on the assumption that the homol-ogous sequences are unique. The agreement isonly fair in the case of the 1.08-unit and theColEl plasmids, possibly because of the errorsmentioned above resulting from failure to reachsaturation and the approximate nature of thecorrection for retention. For the same reasonsthe data for the points with more DNA on thefilter yielded values that average 38% lower.The pairs of plasmids showing no homology

were also examined in this concentration range.The bottom curve of Fig. 4 is representative.

TABLE 3. Hybridization between plasmids showingpartial homologyd

Homologous length,Plasmid I Plasmid II 4XRF units

I b 2c 3d

1.08 unit ColEl 0.93 0.54 0.740.29 unit 0.35 unit 0.23 0.22 0.23

a Hybridization under conditions of excess DNA insolution (see Results).

b Calculated from experiments in which plasmid Iwas on the filter and plasmid II was in solution.

c Calculated from experiments in which plasmid IIwas on the filter and plasmid I was in solution.

d Calculated from measurements on electron micro-graphs of heteroduplexes.

The results are not given in more detail becausethey do not add anything to the conclusionsdrawn from Table 3.The extent of homology between the plasmids

in each of the pairs that showed extensivehybridization was also determined by measure-ment of the heteroduplexes formed betweentheir strands. Electron micrographs are shownin Fig. 5, and length measurements of thedouble- and single-stranded regions are given inTable 4. The designation of the regions to whichthese measurements refer is shown in Fig. 5Dfor the 1.08-unit, ColEl heteroduplex. The pos-sible summations of these lengths to make com-plete circular molecules is given in Table 4.When these summations are compared with thelengths of the 1.08-unit plasmid and ColEl, it ispossible to make an unambiguous choice of theassociation of the single-stranded regions withthe two plasmids forming the heteroduplex.This choice indicates that regions a + c + e + fare contributed by the 1.08-unit plasmid, andthat b + d + e + f are contributed by the ColElfactor. This conclusion has been incorporated inthe diagram in Fig. 5D. The loop in the 0.29-0.35-unit heteroduplex also appears to containnonhomologous single-stranded regions of dif-ferent length. The shorter single-strand is soshort that it is difficult to visualize directly. Inconcluding that it exists, we relied mainly onthe measurement of the double-stranded regionwhich was found to be 0.23 units in length(Table 4). This is significantly shorter than the0.29-unit length of the smallest plasmid. Thesummation of the double- and measurable sin-gle-stranded regions is 0.32 units or somewhatless than the expected 0.35 units (see Table 4).The two large plasmids could not be satisfac-

torily separated, and a mixture of them wasused in all experiments. Examination of thismixture did not reveal any heteroduplex forma-tion between strands of the two differentlengths. We conclude that the 5.7- and 7.2-unitplasmids have no homology. In any calculationsinvolving this mixture, the DNA was assumedto have a complexity equivalent to a contourlength of 12.9 units.The amount of double-stranded DNA in each

heteroduplex is entered in Table 3. Comparisonwith the results obtained by hybridization indi-cates that our method for quantitating hybridi-zation yields values that are in reasonableagreement with the direct heteroduplexmethod.Homology between plasmids and host

DNA. The DNA of S. dvsenteriae Y6R, treatedas described to remove contaminating plasmidDNA, was fixed to the membranes. The amount

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VOL. 116,1973 PLASMIDS OF S. DYSENTERIAE Y6R 171

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FI.5 eeoulxe ewe lsis (,B )Htrdpexsbtente10-ui lsi n hCoE ato.Adaga hwigtedobe ndsnl-trne egosad hi elto o htanso9htwolasmd DNs isshof inD .h.etr* eintngec einrfrt h lnt esrmnsgvniTabe. F,G)Heerdupexs eteenth 029 anth035uiplsdsTelagrmeceinGssingle-tranded0X1 74 On'eerdpe in ac pane clal1hwigesrne op

TABLE4.Lengthmeasurementsof double- and sigle-stranded regions of the two heteroduplexe

Heterouplex egiona Length ±4. SD SmainLength ±~SDHeteroduplex | Region9 | (tXRF units) Summation (LXRF units)

1.08-unit plasmid-ColEl a, SS 0.174 ± 0.023 a + c + e + f 1.13 ± 0.061b,SS 0.026 0.005 b+d+e+f 1.23X0.066c,SS 0.213±0.032 a+d+e+f 1.38±0.064d, SS 0.467 0.045 b + c + e + f 0.98 0.057e, DS 0.389 ± 0.039 1.08-unit plasmid 1.08 ± 0.041f, DS 0.353 ± 0.026 ColEl 1.19 ± 0.064

0.29 to 0.35-unit plasmid SS 0.088 ± 0.023 SS + DS 0.32 ± 0.028DS 0.231 ± 0.016 0.35 unit plasmid 0.35 ± 0.012

aThe letter designation refers to the diagram in Fig. 5C; SS indicates single-stranded, and DS indicatesdouble-stranded region.

° Lengths were measured on 22 to 35 heteroduplexes and on the same number of standard length molecules inthe same spreadings.

used varied from 5 to 20 Mg of which an average micrograms of homologous sequences and as theof 80% was retained. The results for the lower fraction of the plasmid genome that theseamount are given in Table 5. This amount is sequences represent. There is sufficient homolo-such, as shown by the results, that all hybridi- gous DNA in the host genome to account for 2.4zations fall on the part of the curve of Fig. 3 for genomes of the 1.08-unit plasmid and for 0.6the same plasmid size corresponding to 0.005,ug genomes of the ColEl factor, but only for aor less on the filter. They were therefore calcu- minor fraction of the DNA of the other plas-lated by the method used for the homologous mids. Control filters containing M. luteus DNAplasmids. The results are expressed both as or without DNA retained no radioactivity above

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PORTER, KOLODNER, AND WARNER

TABLE 5. Hybridization between the DNA of the host(S. dysenteriae Y6R) and the various plasmidsa

MicrogramsMicrograms of homologous

Plasmid bound per 4 DNA per Fraction'Ag of host microgramsDNAb of host

DNAc

5.7 + 7.2 unit 0.0023 0.0010 0.07e1.08 unit 0.0194 0.0032 2.40.74 unit 0.00156 0.00025 0.30.35 unit 0.00104 0.00011 0.20.29 unit 0.00074 0.00008 0.2ColEl 0.006 0.0009 0.6

aHost DNA (4 jAg; corrected for retention) was fixedto the filter in each case. The 0.2-jug 9H DNA insolution had the same specific activity as that given inTable 1.

b Calculated from fraction of counts bound and thespecific activity. Average of three experiments, usingtwo different preparations of host DNA and three ofplasmid DNA. The standard deviations of the abovesamples are about +10%. Only in the cases of theplasmids showing very little homology to the host doesthe counting error enter significantly to the standarddeviation.

c Calculated from the curves in Fig. 3 as explained(see Results).

d Fraction of the plasmid DNA genome homologousto one genome of host DNA. The host genome wasarbitrarily assumed to have a molecular weight of 2.8x 10'.

eIn this case + is the fraction of the sum of thegenome size of the two plasmids in the mixture, sincethey are nonhomologous when tested for heteroduplexformation.

background levels. When amounts of S.dvsenteriae DNA greater than 5 jig were fixed tothe filter, the amount of hybridization in-creased. As would be expected from the natureof the curves in Fig. 3, this increase was lessthan proportional to the amount of DNA.Number of plasmids per cell. Although

there is some loss of plasmids during the pre-parative procedure, the recovery of the smalland intermediate plasmids is good enough toprovide an estimate of the number of each percell at the end of log phase growth. When onlyone alkali-denaturation step was employed, theyield of these two classes varied from 1 to 3% ofthe DNA of the cell. The sucrose gradientsemployed in separating these plasmids permitthe rough estimates that there was about 1.5times as much DNA from the intermediate asfrom the small class. In the intermediate classthere was about twice as much 1.08- as 0.74-unitplasmid DNA, and in the small class abouttwice as much 0.29- as 0.35-unit plasmid DNA.

Taken together, these figures yield the followingnumbers of each plasmid per host cell genome of2.8 x 10' dalton size: 6, 4, 6, and 15 of the 1.08-,0.74-, 0.35- and, 0.29-unit plasmids, respec-tively.The yield of the large plasmids was too poor

to estimate their number in the same way. Anaccurate determination was obtained from theequilibrium centrifugation in CsCl of a celllysate previously referred to. The amount ofDNA in the satellite peak (p = 1.700) was 5.6%of the total DNA in both peaks. This is equiva-lent to 3.5 copies of the DNA of each plasmidper genome of host, assuming that the samenumber of each of the two species is present.

DISCUSSIONThe plasmids of S. dysenteriae Y6R are

present in multiple copies per cell and, with oneexception, are not homologous to each othernor, again with one exception, are they homolo-gous to the DNA of the host cell. Extensivehomology was found between the two smallestplasmids (0.29 and 0.35 unit). Since no functionis known for either of these plasmids, no signifi-cance can be attached to the duplication of theirDNA in this way. A more interesting relationwas that found between the 1.08-unit plasmidand the ColE1 factor. The extensive homologybetween them, taken together with the im-munity of S. dysenteriae Y6R to the El colicin,and the fact that it does not produce an activecolicin, suggest that the 1.08-unit plasmid is adefective ColEl factor. S. dysenteriae Y6R wasfound to be sensitive to colicin E2, which sharesthe same membrane receptors as El, but differsin immunity properties (7). Although there issome uncertainty as to whether the receptors forcolicins El and E2 are actually identical (10),this can be interpreted to indicate that alteredmembrane receptors are not the basis for theresistance of S. dysenteriae Y6R to colicin El.Genes on the ColEl factor have recently been

mapped by Kingsbury and Helinski (personalcommunication) who found mutants that aredeficient in colicin production, but which retainimmunity. Also missing in the 1.08-unit plas-mid as compared with the ColEl factor is therelaxing factor phenomenon that has been ex-tensively investigated by Helinski and his co-workers (11). This could result either from theloss of the locus on the DNA to which therelaxing factor attaches or to the absence ofrelaxing factor formation in a different host. Itis clear from the nature of the heteroduplexesshown in Fig. 5 that the 1.08-unit plasmid couldnot have been formed from the ColEl factor by

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simple deletion. Both of the nonhomologousregions must be classified as substitutions in theterminology of Davidson and Szybalski (3)because the single-strands in each loop are ofdifferent lengths. This also appears to be true ofthe heteroduplex between the two smallestplasmids.Another example of homology of ColEl with

plasmids was recently reported by Goebel andSchrempf (8). They detected hybridization be-tween ColEl DNA and minicircular DNA fromtwo new E. coli isolates and from E. coli 15. Inthese cases, no colicinogenic or immunity prop-erties were associated with the minicircularplasmids, and the homology was presumed toreflect common replicative functions.The detection of sequences equivalent to 2.4

copies of the 1.08-unit plasmid in the host DNAwas an unexpected finding because the relatedColEl is not known on genetic evidence to beintegrated with its host genome. This should bea reliable estimate because the amount ofhybridization found was within the range inwhich the self-hybridization curves (Fig. 3)should provide a satisfactory correction. It is notobvious from this relationship whether integra-tion and induction of this plasmid are regularlyassociated with its presence and replication.More information will be necessary to evaluatethe episomic properties of this DNA. ColElshowed homology with S. dysenteriae DNA tothe extent of 0.6 genome equivalents. In view ofits extensive homology with the 1.08-unit plas-mid, this observation and that of the 2.4 genomeequivalent homology of the 1.08-unit plasmidwith the host DNA can be reconciled only by theassumption that some of the latter homology isnot unique, but results from repeated sequencesof the nonhomologous regions of the 1.08 plas-mid. No information appears to be available onthe hybridization of ColEl with its host DNA.However, D. Kingsbury (personal communica-tion) has found more than 0.5 genome equiva-lents of ColEl to be hybridizable with the DNAof E. coli.The levels of hybridization of the nonhomolo-

gous plasmids with each other may result fromsmall residual cross-contamination. The similarresidual hybridization of plasmids with the hostDNA appears to be above the level that wecalculate could result from contamination. Inno case does it represent a large fraction of theplasmid genome. However, some evidencepoints to the possibility that this small degree ofhybridization could have specificity derivingfrom the relation of the plasmid to host possiblyas an episome, even though an entire genome

equivalent is not found except in the case of the1.08-unit plasmid. The levels of contaminationcalculated (see Results) were very conservativeinterpretations of the measurements, and allwere at least an order of magnitude below theresidual hybridization shown in Table 5. Hy-bridization with M. luteus DNA in place of thehost DNA and with OX174-RF DNA was un-detectable. This problem is discussed by Puga,Shleser, and Kohne (14) who found that about5% of 13S DNA is homologous to E. coli DNA,although no lysogenic relation is known for thisphage. Their approach is more precise than oursand was greatly facilitated by the availability ofthe phage DNA in the single-stranded form.The estimates of the degree of homology from

filter hybridization was shown in two cases to beconsistent with that determined from heterodu-plex formation, although a high degree of preci-sion cannot be claimed for the filter method.The conditions of hybridization used in thiswork have some advantages over other ap-proaches to DNA-DNA hybridization on filtersin that saturation is either achieved or thefailure to reach it is corrected for by theself-hybridization curves. Improvement couldbe made by treatment of the filter with asingle-strand cleaving nuclease such as thatfrom Neurospora (13, 15) and by employingDNA on the filter with a different label than onthat in solution to obtain an accurate measureof retention. Such refinements might justify thekinetic treatment of the results that can providea correction for failure to reach saturation dueto the complexity of the DNA.

ACKNOWLEDGMENTSWe thank Armand Stephanian and Fred Harwood for

expert assistance with the ultracentrifuge experiments.This investigation was supported by Public Health Service

grant CA-12627 and by grant P-564 from the AmericanCancer Society. R.K. was a predoctoral trainee on PublicHealth Service training grant GM-02063 from the NationalInstitute of General Medical Sciences.

LITERATURE CITED1. Britten, R. J., and D. E. Kohne. 1966. Nucleotide

sequence repetition in DNA. Carnegie Inst. Wash. YearB. 65:68-106.

2. Clewell, D. B., and D. R. Helinski. 1970. Properties of asupercoiled deoxyribonucleic acid-protein relaxationcomplex and strand specificity of the relaxation event.Biochemistry 9:4428-4440.

3. Davidson, N., and W. Szybalski. 1971. Physical andchemical characteristics of lambda DNA, p. 45-82. InA. D. Hershey (ed.), The bacteriophage lambda. ColdSpring Harbor Laboratory, Cold Spring Harbor, NewYork.

4. Davis, R. W., M. Simon, and N. Davidson. 1971. Electronmicroscope heteroduplex methods for mapping regionsof base sequence homology in nucleic acids, p. 413-428.

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In L. Grossman and K. Moldave (ed), Methods inenzymology, vol. 21, part D. Academic Press Inc., NewYork.

5. Denhardt, D. T. 1966. A membrane-filter technique forthe detection of complementary DNA. Biochem. Bio-phys. Res. Commun. 23:641-646.

6. Domingo, E., C. S. Gordon, and R. C. Warner. 1972.Azotobacter phages: properties of phages A12, A21,A31, A41 and their constituent DNAs. Virology49:439-452.

7. Fredericq, P. 1957. Colicins. Annu. Rev. Microbiol.11:7-22.

8. Goebel, W., and H. Schrempf. 1972. Isolation of minicir-cular deoxyribonucleic acids from wild strains of Esch-erichia coli and their relation to other bacterial plas-mids. J. Bacteriol. 111:696-704.

9. Habich, A., C. Weissmann, M. Libonati, and R. C.Warner. 1966. Isolation of a fraction of Bacillusmegaterium DNA enriched in "minus" sequences. J.Mol. Biol. 21:255-264.

10. Hamon, Y., and Y. Per6n. 1966. Nouvelle classificationdes colicines appartenant au groupe E. Zentrabl.riol. Parasitenk. Abt. I. Orig. 200:375-379.

11. Helinski, D. R., and D. B. Clewell. 1971. Circular DNA.Bakteriol. Parasitenk. Abt. I. Orig. 200:375-379.

12. Lee, C. S., and N. Davidson. 1970. Physiochemicalstudies on the minicircular DNA in Escherichia coli 15.Biochim. Biophys. Acta 204:285-295.

13. Linn, S., and I. R. Lehman. 1965. An endonuclease fromNeurospora crassa specific for polynucleotides lackingan ordered structure. J. Biol. Chem. 240:1287-1293.

14. Puga, A., R. Shleser, and D. E. Kohne. 1970. Homologybetween the genomes of bacteriophage S13 and Esche-richia coli. Virology 43:507-510.

15. Rabin, E. Z., B. Preiss, and M. J. Fraser. 1971. A nucleasefrom Neurospora crassa conidia specific for single-stranded nucleic acids. Prep. Biochem. 1:283-307.

16. Rush, M. G., C. N. Gordon, and R. C. Warner. 1969.Circular deoxyribonucleic acid from Shigelladvsenteriae Y6R. J. Bacteriol. 100:803-808.

17. Rush, M. G., and R. C. Warner. 1970. Alkali denaturationof covalently closed circular duplex deoxyribonucleicacid. J. Biol. Chem. 245:2704-2708.

18. Stacey, K. A., and E. Simson. 1965. Improved method forthe isolation of thymine-requiring mutants of Esche-richia coli. J. Bacteriol. 90:554-555.

19. Studier, F. W. 1965. Sedimentation studies of the sizeand shape of DNA. J. Mol. Biol. 11:373-390.

20. Vinograd, J., J. Lebowitz, R. Radloff, R. Watson, and P.Laipis. 1965. The twisted circular form of polyoma viralDNA. Proc. Nat. Acad. Sci. U.S.A. 53:1104-1111.

21. Warnaar, S. O., and J. A. Cohen, 1966. A quantitativeassay for DNA-DNA hybrids using membrane filters.Biochem. Biophys. Res. Commun. 24:554-558.

22. Wetmur, J. G., and N. Davidson. 1968. Kinetics ofrenaturation of DNA. J. Mol. Biol. 31:349-370.

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