JOURNAL OF BACTERIOLOGY, Feb. 1981, p. 780-7870021-9193/81/020780-08$02.00/0

Vol. 145, No. 2

Transformation of Escherichia coli with PlasmidDeoxyribonucleic Acid: Calcium-Induced Binding ofDeoxyribonucleic Acid to Whole Cells and to Isolated

Membrane FractionsA. WESTON,t M. G. M. BROWN,: H. R. PERKINS, J. R. SAUNDERS, AND G. 0. HUMPHREYS*

Department ofMicrobiology, University of Liverpool, Liverpool, L69 3BX United Kingdom

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichiacoli at 0°C in the presence of divalent cations. During incubation at 42°C, 0.1 to1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-re-sistant DNA binding and the ability to produce transformants became saturatedwhen transformation mixtures contained 1 to 2 ug of plasmid NTP16 DNA andabout 5 x 10' viable cells. Under optimum conditions, between 1 and 2 moleculeequivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonucleaseresistant. Despite this, only 0.1 to 1% of viable cells became transformed bysaturating amounts of the plasmid. The results suggest that transport of DNAacross the inner membrane is a limiting step in transformation. After transfor-mation the bulk of labeled plasmid DNA remained associated with outer mem-branes. However, in vitro assays indicated that plasmid DNA would bind equallywell to preparations of inner or outer membranes provided divalent cations werepresent. Divalent cations promoted differing levels of binding to isolated innerand outer membranes in the order Ca2 >> Ba2+ > Sr2+ > Mg2+. This parallelstheir relative efficiencies in promoting transformation. Binding of plasmid DNAwas greatly reduced when outer membranes were treated with trypsin; thissuggests that protein components may be required for the binding or transport ofDNA (or both) during transformation.

Transformation of bacteria involves DNAbinding to the cell surface followed by uptakeacross the wall-membrane complex into the cy-toplasm. The binding and uptake processes havebeen most studied in Bacillus subtilis, Strepto-coccus pneumoniae, and Haemophilus influ-enzae, organisms which become naturally com-petent during growth (13). In contrast, Esche-richia coli cells can take up phage (4, 22), chro-mosomal (7, 25), or plasmid (6, 10) DNA, butonly in the presence of high concentrations ofCaCl2. The overall process is very inefficient,and at best only 1 to 2% of the cells survivingthe CaCl2 treatment are transformed (15, 26).Calcium ions promote DNA binding to the outercell surface (19), but only a small proportion(<1%) of this bound DNA can be converted toa DNase-resistant form in a subsequent uptakestep. It is not known whether this DNase-resist-ant material is taken solely into transformablecells, or whether all cells can take up the DNA

t Present address: Department of Microbiology, QueenMary College Industrial Research Ltd., London, El 4AAUnited Kingdom.

: Present address: Department of Genetics, University ofLeicester, Leicester, LEI 7RH United Kingdom.

but only a small proportion of these cells arecapable of establishing the plasmid as a replicon.Consequently, a direct analysis of the uptake oftransforming DNA molecules is difficult. All at-tempts to isolate the subpopulation of trans-formable cells have so far failed.

In this paper we have examined the factorswhich influence the binding of plasmid DNA towhole cells and to isolated inner and outer mem-brane fractions of E. coli. The results are dis-cussed in terms of their relevance to transportof DNA during transformation.

MATERIALS AND METHODSBacterial strains. E. coli K-12 C600 was obtained

from B. J. Bachmann (3). E. coli K-12 UB1139, metthy leu nalA (20), was used for the preparation of 3H-labeled plasmid DNA. E. coli K-12 W3805, galE,incorporated galactose predominantly into lipopoly-saccharide and was obtained from J. P. Beard.Preparation of plasmid DNA. Nonradioactive

DNA of the nonconjugative plasmid NTP16 (molecu-lar weight, 5.7 x 106), which codes for resistance toampicillin and kanamycin (2), was extracted fromstrain C600 (NTP16) by the method of Humphreys etal. (11).

Radioactively labeled NTP16 DNA was prepared

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from strain UB1139 (NTP16). The bacteria weregrown overnight in 5 ml of M9 minimal medium (1)enriched with 1 ug of thymine, 40 pg of methionine,and 40 pg of leucine per ml. The cells were diluted 10-fold with fresh medium and incubated at 37°C withaeration for 20 min. At this time 4 mCi of [methyl-3H]thymidine (specific activity 24 Ci/mmol; Radi-ochemical Centre, Amersham, United Kingdom) wasadded, and the cultures were incubated until theyreached an absorbance at 660 nm of 0.7. Chloram-phenicol was then added to a final concentration of 50jig/ml, and the cells were incubated for a further 2 hto allow amplification of the plasmid DNA.Transformation. Cells of E. coli K-12 C600 were

transformed under optimal conditions by the methodpreviously described (5, 10). Frequencies of transfor-mation are expressed as transformants per viable cellat the end of the expression period.

Isolation ofinner and outer membranes. Mem-branes were isolated by the method of Osborn et al.(17). Cells of E. coli K-12 C600 were grown overnightin nutrient broth at 37°C without shaking. The over-night culture (50 ml) was diluted with 1 liter of pre-warned nutrient broth and incubated at 370C withaeration until the cells reached an absorbance at 660nm of 0.2. The cells were then harvested by centrifu-gation at 00C, and all subsequent manipulations werecarried out at this temperature. The cells were sus-pended in 0.75 M sucrose-10 mM Tris-hydrochloride(pH 7.8) to a final absorbance at 600 nm of 10. Lyso-zyme was added to a final concentration of 100 pg/ml,and the mixture was incubated on ice for 2 min. Thesuspension was diluted twofold by the addition of 1.5mM EDTA-0.2 mM dithiothreitol (DTT) at a rate of1 ml/min. The suspension was then sonicated by usingan MSE sonicator in 30-s bursts at 40C for a total of2 min. The remaining intact cells and debris wereremoved by centrifugation (3,000 x g for 10 min in aSorvall RC-5 centrifuge). The membranes were pel-leted (40,000 x g for 90 min) and washed in 0.25 Msucrose-3 mM Tris-hydrochloride-1 mM EDTA-0.2mM DTT (pH 7.5). Inner and outer membranes wereseparated on a 25-ml linear sucrose gradient (25 to60%, wt/wt) by centrifugation at 40C (95,000 x g for17 h in an MSE swing-out rotor with three 25-mltubes). Inner and outer membrane fractions were har-vested by syringe, washed, and finally suspended in25% (wt/vol) sucrose-5mM EDTA-0.2 mM DTT (pH7.5). The protein concentration of membrane fractionswas determined by the method of Lowry et al. (14).The purity of outer membrane fractions was checkedby assaying succinate dehydrogenase activity (8) as aninner membrane marker. When D-[1-'4C]galactose wasadded to growing cells of a K-12 strain lacking galac-tose-4-epimerase, all of the incorporated label waslocated in the outer membrane fractions, confirmingthe lack of cross-contamination between the mem-branes (see below). Membrane fractions were storedat 40C for use in DNA-binding studies and were usedwithin 1 week of preparation.

Binding of 'H-plasmid DNA to whole ceils. E.coli K-12 C600 was grown overnight in nutrient brothat 370C. The culture was diluted 1:20 in fresh, pre-warmed broth, and incubation was continued for 90min. The cells were harvested by centrifugation,

washed once in 10 mM CaCI2 at 00C, and finallysuspended in 1/2o of the original culture volume of 75mM CaC12. This procedure is known to produce cellsof optimal transformation efficiency (5, 10). Variousamounts of 3H-labeled NTP16 DNA were added totransformation mixtures. The mixtures were kept at00C for 45 min and then transferred to 420C for 10min. The cells were washed three times by centrifu-gation in a Burkhard microfuge type 320 (14,000 x gfor 1 min) with 20% (wt/vol) sucrose-0.15 M NaCl.The total [3H]DNA bound to the cells was measuredby suspending the cells in distilled water, treating with50 mM Tris buffer (pH 8.0) and 50 ug of lysozyme perml and counting directly in aqueous Triton-toluenescintillant [300 ml Triton X-100, 100 ml distilled water,600 ml of toluene containing 6 g of 2(4'-tert-butyl-phenyl) -5(4"-biphenylyl) - 1,3,4 -oxadiazole (butyl-PBD; Koch-Light)].[3H]DNA bound to cells in a DNase-resistant form

was measured after the first wash by adding 20 plI ofbovine pancreatic DNase I (Sigma Chemical Co.; 2mg/ml in 10 mM MgCl2-0.2 M sodium acetate buffer,pH 5.0) and incubating at 00C for 5 min. After afurther wash the cells were lysed, and radioactivitywas assayed as described above.

Binding of 3H-labeled plasmid DNA to isolatedmembrane fractions. The method used to estimateDNA binding was based on that of Joenje et al. (12).Binding to outer membranes was measured in incu-bation mixtures (200 pi) containing 2.6 pg of 3H-labeledNTP16 DNA and outer membranes in a final concen-tration of 2.5% (wt/vol) sucrose-0.5 mM EDTA-0.02mM DTT (pH 7.5). The protein content ofmembranesadded was usually 40 to 50,ug. Various concentrationsof CaCl2 were added as described below. The bindingmixtures were incubated at 20°C for 10 min. Similarresults were obtained when the binding and centrifu-gation were performed at 0, 20, or 400C. Each mixturewas then layered onto a discontinuous sucrose gradient(2.7 ml of 15% [wt/vol] sucrose overlaid with 0.8 ml of5% [wt/vol] sucrose in 1.5 mM EDTA-0.2 mM DTT,pH 7.5) in a 4.2-ml polypropylene centrifuge tube andcentrifuged (155,000 x g) for 20 min at 200C in anMSE swing-out rotor with six 4.2-ml tubes. Aftercentrifugation the supernatant fluids were decanted,the inside walls of the tubes carefully wiped, and themembrane pellets were dissolved in 200 pl of 10% (wt/vol) sodium dodecyl sulfate. Duplicate samples (50 ul)were removed and counted directly in Triton-toluenescintillant.

Binding of [3H]DNA to inner membranes was car-ried out as for outer membranes. Binding mixturescontained 3.0,ug of 3H-labeled NTP16 DNA and innermembranes (40 to 50 pg of membrane protein) in afinal concentration of 0.5 mM CaC02.Trypsin treatment of outer membranes. Outer

membranes (138 pg of membrane protein) were mixedwith 50 p1 of trypsin solution (1 mg/ml) in a finalvolume of250 pl of 25% (wt/wt) sucrose-5mM EDTA-0.2 mM DTT (pH 7.5) and incubated at 370C for 1 h.CaC02 (final concentration, 75 mM) and 5.5 pug of 3H-labeled NTP16 DNA (specific activity, 7.7 x 104 cpm/pg) were then added, and the mixture was kept at 00Cfor 30 min. Controls were treated in the same mannerwithout the addition of trypsin. Binding mixtures were

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loaded on to 25 to 60% (wt/wt) sucrose gradients asdescribed above and centrifuged at 95,000 x g for 17h. Fractions were collected and counted directly inTriton-toluene scintillant.

RESULTSBinding and uptake of Plasmid DNA by

whole cell. The transformation process con-sists of an initial stage when cells are incubatedwith DNA at 0°C in the presence of CaW' ionsand a subsequent stage when the transformationmixture is subjected to a heat pulse at 42°C (6,7, 10). The DNA responsible for the productionof transformants only became resistant toDNase after the heat pulse (Fig. 1). Between 80and 100% of the DNA in a transformation mix-ture becomes cell associated (10). However, onlyabout 10% of this loosely bound DNA remained

101r

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Stage Stag1

Cold Heatadsorption pulse

30 1

20 1

l0

0 10 20 30 40 so 0

Time (min) before DNase addedFIG. 1. The efCt OfDNase onyelds oftransform-

ant. E. col K-12 CM0 was grown and renderdtransformable as dcribed in te text. Transforma-tion mixtures conaibed a sturating amount 63 pg ofDNA per 2 x 10 cetll) of NTP16 DNA; 20 i1 of asolution c 2 mg ofpanreatic DNase I permL 10 mM MsCI* and 50 mM sodium acetate (pH5.0) was added to each transormation mixture at thetimes shown. Mixtures were subsequently treated asin the normal tansformation protocoL Results areexpressed as apercentge of th tansfornation fre-quency (2.3 x ta s per viabk ceU)obtained with a contol mixture where DNase wasnot added.

associated after three washes with saline-sucrose(tightly bound DNA), and only about 1% wasDNase tant after th&eat pulse. The re-latioship betWeen the amount of [3H]DNAadded to tsformation mixtes and theamount bound to cell is ilustrated in Fig. 2.The capacityto bindplasmidDNA in a DNase-resistant form was saturated by 2 jig ofDNA pertransformation mixture, and the slope ofthe lineat nonsaturating DNA concentrations was ap-proximately 1. A similar curve was obtained fortransformants (Fig. 2 and reference 10), but inthis case saturation occurred at approximately0.8 ug of DNA per mixture. The curve for['H]DNA tightly bound to the celLs also had aslope ofapproximately 1, but saturation was notreached even at 10 jig of DNA per transforma-tion mixure.The concentration of CaCl2 present in trans-

formation mixtures had a considerable effect onthe quantity of 3H-labeled NTP16 DNA boundto intact cells of E. coli. The amount of DNAbound in a DNase-resistant form showed amarked increase with increasing Ca2+ concentra-tion up to a maximum at 100mM and thereafterdecreased dramatically (Fig. 3). Total DNAbinding was also dependent on Ca2+ concentra-tion (there being none in the absence of divalent

0

amount()dDNA in mixtureFiG. 2. Dose-response curves for 3H-labekd

NTP16 DNA bound to whole ceUs. Competent ceUs(approximately 10' per mixture) suspended in 75mMCaC12 were mixed with 3H-labekd NTP16DNA (spe-cific activity, &4 x 10 cpm/WJ in a total volume of0.5 mL Mixtus were incubated at 0°C for 45 minand subequently at 42°C for 10 min. Sampls uereremoved from the mixtures and treated as describedin the methods section: 0, total DNA binding; ,DNase-restan binding; U, transformantsper viablecell.

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cations), but it was efficient over a much widerrange of concentrations (20 to 100 mM) thanwas DNase-resistant binding (Fig. 3). TotalDNA binding and viability of cells were reducedsignificantly at concentrations of Ca2" above 100mM.

Isolation of 'H-labeled NTP16 DNA at-tached to inner and outer membranes aftertransformation. Competent cells of E. coli K-12 C600 were transformed with [3H]DNA andwashed to remove unbound DNA, and the innerand outer membranes were isolated by sucrosegradient centrifugation (17). The bulk of thelabeled DNA was attached to the outer mem-brane fraction with smaller amounts attached toinner membranes and as free DNA (Fig. 4).Little radioactivity was associated with themembranes when the cells were treated withDNase before membrane isolation, but most ofthis (>90%) gave a defined peak banding withinner membranes. DNA did not band at the

i.-

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u

(a

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Total DNA bound

we

position of inner or outer membranes in theabsence of membranes or Ca2" ions. Further-more, when the membranes were treated at anystage with concentrations ofEDTA above 5mM,all of the DNA was removed from membranematerial and sedimented in the position indi-cated for free DNA.DNA binding to isolated membrane frac-

tions. Experiments were carried out to deter-mine the conditions under which DNA wasbound to isolated membranes. Approximately1.0 ,ug of DNA was bound per 54 ,ug of outermembrane protein or 36 jig of inner membraneprotein. For subsequent experiments theamounts of membranes employed were adjustedso that they would bind approximately half ofthe [3H]DNA present. Maximum DNA bindingto outer and inner membranes, respectively, wasobtained at Ca2" concentrations of 5mM and 0.5to 1.0 mM (Fig. 5). All subsequent experimentswith inner membranes were carried out in 0.5mM CaCl2; at concentrations higher than 1 mMthe membranes aggregated and adhered to glass-ware.

All divalent cations tested facilitated bindingof labeled plasmid DNA to both inner and outermembranes, but calcium was the most effective(Table 1). No binding to either membrane was

0

400 F

300 [

0.

M

200

100 1

0

* 20 40 00 0 13 140

Concentraton of CaCI2(mM)FIG. 3. Effects of CaC12 concentration on binding

of 3H-labeled plasmid DNA to whole ceUs. E. colicells were prepared for transformation as describedin the text, except that after washing in 10mM CaC12the cells were suspended in the concentrations ofCaCl2 indicated. Transformation mixtures (0.5 ml)contained 3 pg of 3H-labeled NTP16 DNA (specificactivity, 8.4 x 104 cpm/pg) and approximately 109total cells of E. coli K-12 C600. Amounts of DNAbound were determined as described in the legend toFig. 2.

a. Free InnerIr\ DNA membrane

nemb. \a I I

\o'- Ntop.!, .2.. w

10 15 20 25 30 35Fraction number

40 45

FIG. 4. Isolation of 3H-labeled plasmid DNA at-tached to membrane fractions. Bacteria (10' cells in2 ml of 75mM CaC4) were transformed with 10 pg of3H-labeled NTP16 DNA (specific activity, 8.4 x Idcpm/pg). The cels were washed inmediately after theheat-pulse step and lysed, and the membranes wereseparated by centrifugation on a 25 to 60% (wt/wt)sucrose gradient (95,00K) x g for 17 h) at 200C (17).Fractions were coUected by piercing the bottoms ofthe centrifuge tubes, and samples (180 I1) werecounted directly. The positions of outer membraneand free DNA and inner membrane (arrows) weredetermined in tubes centrifuged in parallel.

IN

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10-OF

x

E

c

E

z

0,'QCD

1-0

0-1

"I1

Concentrat on of CaCO2 (mM)FIG. 5. Effect ofCaC42 concentration on binding of

'H-labekdplamnidDNA to isolated membrane frac-tions. The amount of 'H-labekd NTP16 DNA boundto membrane fractions was measured as described in

the text. Binding mixtures contained 46 pg of outermembrane protein and 2.6 pg of 'H-labekd NTP16DNA (specific activity, 4.05 x 104 cpm/lg) or 42 pg ofinner membrane protein and 2.7 pg of 3H-labekdNTP16 DNA (specific activity, 3.9 x I04 cpm/g).CaCI2 was added to mixtures at the concentrationsindicated: 0, 3H-labeled NTP16DNA bound to outermembranes; *, 3H-labeled NTP16 DNA bound toinner membranes.

observed when divalent cations were omittedfrom the mixtures or when Na+ was substituted.Binding in the presence of high NaCl concentra-tions is apparently nonspecific since it also oc-curs with whole cells, but without inducingtransformation (10).

In all binding mixtures the concentration ofEDTA was 0.5 mM. This concentration wasnecessary for DNA binding to inner membranesregardless of the Ca2" concentration. Concentra-tions of EDTA above 1 mM inhibited DNAbinding to both inner and outer membranes.The reason for this rather strict requirement forEDTA to allow binding to isolated membranesis not yet clear.Pretreatment of outer membranes with tryp-

sin drastically reduced the amount of 3H-labeledNTP16 DNA bound (Fig. 6a). This reductionwas not the result of gross disruption in mem-brane structure because trypsin treatment didnot alter the buoyant density of the outer mem-branes (Fig. 6b).

DISCUSSIONTransformation of E. coli by DNA involves at

least three stages. First, DNA molecules are

TABLE 1. DNA binding to membrane fractionsConcen- Mem-

Divalent cation tration brane DNA bgunda(mM) fraction

None Inner 1.04Ca2+ 0.5 Inner 100.0Bae+ 0.5 Inner 27.3Sr2+ 0.5 laner 21.6Mg2+ 0.5 Inner 11.8Na+ 0.5 Inner 0.8

None Outer 1.3Ca2+ 5.0 Outer 100.0Ba2+ 5.0 Outer 22.0Sr+ 5.0 Outer 19.0Mg2+ 5.0 Outer 3.8Na+ 5.0 Outer 1.6

None Outer 1.6 (O)bCa2+ 75 Outer 100.0 (100)Ba2+ 75 Outer 45.0 (6.0)Sr2+ 75 Outer 42.7 (6.7)Mg2+ 75 Outer 14.4 (0.3)Na+ 75 Outer 5.0 (0)

aThe binding assays were carried out as describedin the text. Results are expressed relative to the valuesfor calcium (=100%).

b The numbers within parentheses refer to the rel-ative efficiency of divalent cations in inducing com-petence of E. coli K-12 C600 for transformation (10).

adsorbed to the outside of the cell at 00C in thepresence of high concentrations of divalent cat-ions. This is followed by a heat pulse duringwhich the DNA becomes insensitive to DNase(6, 10, 19; this paper) and is presumably trans-ported to a site within the peripluns or insidethe inner membrane (or both). Finally, internal-ized DNA must be established either by forminga stable replicon itself or by recombining with aresident replicon. Experiments using plasmid,phage, or chromosomal DNA have demon-strated that much more DNA is bound to theoutside of the cells than is actually taken up (10,16, 19). Furthermore, under the conditions em-ployed in our experiments the capacity of E. colito bind DNA tightly on the outside of cells doesnot become saturated (Fig. 2). In contrast, theability to intemalize DNA, as judged by thelevel of DNase-resistant binding, and the pro-duction of transformants both showed satura-tion (Fig. 2). It is probable that a proportion ofthe DNase-resistant DNA is located in the per-iplasmic space. This might account for theslightly higher concentration of DNA requiredto saturate the DNase-resistant DNA binding(2 jg per mixture) when compared with thatrequired for saturation of the transformationfrequency (0.8 utg per mixture). Concentrationsof CaCl2 (75 to 100 mM) which give optimum

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a

Outr n-embrne Frev DNA

0

2 b

top

10 15 20 25 30 35 40 45Fraction number

FIG. 6. (a) Effect of trypsin treatnent on bindingof 3H-labeled plasmid DNA to outer membranes.Outer membranepreparations were treated with tryp-sin, mixed with 'H-Labeled NTP16 DNA, centigedthrough sucrose gradients (5 to 60%, wt/wt), frac-tionated, and assayed for radioactivity as describedin the text: 0, binding to typsin-treate$ outer mem-branes; *, binding to untreatedmembranes. (b) Effectof trypsin treatment on the buoyant density of outermembranes. Outer membranes labeled with j'4CJga-lactose were prepared from E. coli W38d6, galE,treated with trypsin, and analyzed on sucrose gra-dients as described for (a): 0, [14CJgalactose in tryp-sin-treated outer membranes; 0, [14CJgalactose inuntreated membranes.

transformation frequencies (10) also producedmaximal DNase-resistant binding (Fig. 3). Thissuggests that transport of DNA across the cell

envelope, rather than extemal binding, is a lim-iting step in producing transfonnants.When membranes were separated by the

method of Osbom et al. (17) immediately aftertransformation the bulk of bound DNA wasattached to the outer membrane fraction with asmall amount bound to the inner membranes(Fig. 4). This result is not surprising as DNAbinding to the outside of the cell is considerablymore efficient than uptake via the inner mem-brane into the cell. Treatment of intact heat-pulsed cells with DNase before separation of themembranes removed most of the bound DNA,

leaving only that which was attached to theinner membrane (see also reference 19). How-ever, isolated inner membranes were capable ofbinding amounts of DNA equivalent to thosebound by outer membranes. The DNA-bindingcapacity of inner membranes was saturated inthe presence of 0.5 to 1 mM CaCl2, and thecapacity of outer membranes was saturated in-the presence of 5 mM CaC12 (Fig. 5). This con-trasts with an optimum of 75 to 100 mM forboth DNase-resistant DNA binding to intactcells and the production oftransformants. Wholecells of E. coli are known to maintain low intra-cellular Ca2+ levels by active extrusion of thiscation (18, 21). Thus, high external concentra-tions of Ca2+ might be required to maintain therelatively low Ca2' concentration necessary topromote DNA binding to the inner membranesof intact cells. Furthermore, high external cal-cium ion levels might promote transport of Ca2+-DNA complexes down a Ca2+ concentration gra-dient into the cytoplasm as suggested by Grinius(9). Some DNA which is DNase resistant maybe bound in the periplasmic space, but we havenot investigated such binding in this study. Theprecise location of DNase-resistant DNA intransformed cells is under further study withDNA radioactively labeled to much higher spe-cific activity.Calcium or other divalent cations were abso-

lutely essential for DNA binding to inner andouter membranes and whole cells. The followingorder of effectiveness was obtained: Ca2+ > Ba2+> Sr2+ > Mg2+ (Table 1). The relative efficien-cies of these ions in promoting DNA binding areparalleled by their abilities to produce trans-formants (10).Very little plasmid DNA was bound to outer

membranes treated with trypsin (Fig. 6). Treat-ment of whole cells with trypsin or pronase inthe presence of Ca2+ also reduces the transfor-mation frequency (M. Brown, Ph.D. Thesis,University of LiverpooL 1980; and unpublishedobservations). Furthernor'e, pronase treatmentis known to reduce the ability of isolated cellenvelope preparations to inhibit transfection ofE. coli (23). These results suggest that integrityof the protein portion of the outer membrane isessential for DNA binding. van Alphen et al.(24) have described outer membrane particles inE. coli whose generation is stimulated by Ca2`and to a lesser extent by Mg2e ions. These par-ticles consist of aggregates of lipopolysaccharideand protein, and it has been proposed that theyfunction as aqueous pores (24). CaCl2 treatmentmay therefore either expose proteins which bindDNA or create pores for the binding or inwardpassage (or both) of DNA molecules.

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The DNA-binding data presented in this pa-per support previous observations that transfor-mation is a very inefficient process in E. coli.Transformants only represent between 0.1 and1% of cells surviving the CaCl2 treatment (6, 10).It is not known whether all of the cells in apopulation of E. coli treated with CaC12 ormerely those fated to become transformants cantake up DNA molecules to sites protected fromthe action of deoxyribonuclease. Under the con-ditions used in our experiments transformationmixtures contained approximately 5 x 108 viablecells and 1011 molecules of NTP16 DNA (1 ,g)when the system was just saturating (see Fig. 2).Thus, mixtures contained approximately 200plasmid DNA molecules per viable cell or200,000 per transformant, assuming that 0.1% ofviable cells became transformed. Washing thecells removed much of this DNA, so that only10 to 20 molecules per viable cell or 10,000 to20,000 per transformant remained tightly bound.After treatment of cells with DNase only 1 to 2molecule equivalents per viable cell or 1,000 to2,000 per transformant remained attached. Itseems inherently unlikely that only transform-able cells would bind and internalize such largenumbers of DNA molecules. It is more likelythat all cells in the population can bind DNA,but that only a small minority are successful intransporting intact plasmid molecules to the in-terior of the cell where they might establishthemselves as replicons. There were sufficientplasmid molecule equivalents (1 to 2 per viablecell) bound in a DNase-resistant form to trans-form all of the viable cells (Fig. 2). However, anymolecules which had only partially traversed thecell envelope after the heat pulse would be dam-aged by DNase treatment. Therefore, measure-ment of DNase-resistant molecule equivalentswill certainly give an overestimate of potentialrunsformants. We have previously shown bysimultaneous transformation of E. coli withpairs of plasnids that even under saturatingconditions, the majority (probably >80%) oftransformable cells can only take up one DNAmolecule, whereas only 10 to 20% can take uptwo (26) and <1% can take up three (J. R.Saunders, unpublished observations) separateplasmid molecules. These findings taken to-gether indicate that the transition from exter-nally bound DNA molecule to established plas-mid replicon occurs with low probability.We conclude from the data presented that the

interaction of externally added DNA with intactcells and isolated membranes is highly ion spe-cific. However, even under optimal conditionsvery few cells in a population of E. coli areproductively transformed. It must be empha-sized that because such a small proportion of

cells are transformed, the results of binding ex-periments with isolated membranes from thetotal cell population may not directly reflectsteps in the transformation process. However,the parallels are intriguing. The reasons for thelow transfornation efficiency and the mecha-nisms of transmembrane transport of DNA areunder further study.

ACKNOWLEDGMENTSThe work described in this paper was supported by project

grants G976/457/C and G979/471/C to G.O.H. and J.R.S.from the Medical Research Council. M.G.M.B. was the recip-ient of a postgraduate scholarship from the Science ResearchCouncil.We are grateful for the excellent technical assistance of C.

Walker-Jones.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-lishers Inc., New York.

2. Anderson, E. S., E. J. Threlfall, J. Carr, M. Mc-Connell, and H. R. Smith. 1977. Clonal distributionof resistance plasmid-carrying SabnoneUa typhimu-rium, mainly in the Middle East. J. Hyg. 72:471-487.

3. Bachmann, B. J. 1972. Pedigrees of some mutant strainsof Escherichia coli. Bacteriol. Rev. 36:525-557.

4. Benzinger, R. 1978. Transfection of Enterobactericeaeand its applications. Microbiol. Rev. 42:194-236.

5. Brown, M. G. M., A. Weston, J. R. Saunders, and G.0. Humphreys. 1979. Transformation of Escherichiacoli C600 by plasmid DNA at different phases ofgrowth.FEMS Microbiol. Lett. 5:219-222.

6. Cohen, S. N., A. C. Y. Chang, and L Hsu. 1972. Non-chromosomal antibiotic resistance in bacteria: genetictransormation by R-factor DNA. Proc. Natl. Acad. Sci.U.S.A. 69:2110-2114.

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