JOURNAL OF BACTERIOLOGY, Feb. 1976, p. 635-642Copyright 0 1976 American Society for Microbiology

Vol. 125, No. 2Printed in U.S.A.

Isolation and Characterization of Escherichia coliChromosomal Mutants Affecting Plasmid Copy Number

DEAN E. CRESS' AND BRUCE C. KLINE2*

Department of Biochemistry, The University of Tennessee, Knoxville, Tennessee 37916

Received for publication 20 August 1975

We have isolated chromosomal mutants of an Escherichia coli K-12 strain thatmaintain higher levels of an F' plasmid. The mutants are designated as plasmidcopy number (pcn) mutants. They were detected by selecting for increasedlactose fermentation in bacteria deleted for the lac operon but harboring an

F'lacI,Ppro+ plasmid. When examined for the amount of F' plasmid deoxyribo-nucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show twoto seven times as much covalently closed, circular (CCC) DNA as does theparental strain. The increased plasmid level in one mutant strain (pcn-24) was

confirmed by DNA-DNA hybridization; however, this latter technique indicatedabout a twofold lower increase when compared with the increase measured forpcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F'DNA reflects a proportional increase in monomeric-size plasmid moleculesbecause oligomeric forms are not found. Also, in mutant pcn-24 the extra CCCplasmid copies do not seem to be randomly distributed throughout the cell'scytoplasm but appear complexed in situ with their host's folded chromosome. Inall pcn mutants examined to date, the classical sex factor F is maintained atnormal levels, whereas the viral plasmid PI CM is maintained at two to threetimes the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on

the chromosome and not on the plasmid. Finally, the absolute amount of CCC F'DNA detectable in lysates of the six different pcn mutants examined decreased50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNAoccurs when lysates of the parental F' strain are incubated at 37 C.

Biochemical requirements for the replicationof stringent plasmids are poorly understood.Both plasmid and chromosomal gene productsare involved (14). The diversity of plasmid typesand roles probably reflects a diversity of mech-anisms for the control of plasmid replication.The classical sex factor, F, appears to be themost chromosome-like of the stringent plasmidsin that F is a monomolecular replicon main-tained at a ratio of approximately one or twoplasmid copies per chromosome (2, 6, 11).Further, the macromolecular requirements for Freplication are similar but not identical (20, 21,28) to those for chromosome replication. Thesefindings suggest that F is a good model systemfor studying the control of stringent deoxy-ribonucleic acid (DNA) replication.We have chosen to study the control of F

replication through the isolation of mutantsaltered in that control. A number of tempera-

'Present address: Department of Biology, University ofUtah, Salt Lake City, Utah 84112.

'Present address: Department of Microbiology, MayoClinic, Rochester, Minn. 55901.

ture-sensitive F' segregation/replication-defec-tive mutants have been studied (10, 13, 15, 17,18) but have yielded little insight into thebiochemical control of replication. Thus, wesought to isolate mutants with control altered inan opposite manner-increased rounds of plas-mid replication per cell cycle. Such mutantshave been obtained for several R plasmid sys-tems (24, 26, 27) by assaying for increasedantibiotic resistance and for the Clo DF13plasmid by assaying for increased cloacin pro-duction (23). We have used the same logic inassaying for increased lac genes in bacteriaharboring an F'lacI,P pro+ plasmid. This ap-proach has been successful, and a number ofmutants have been isolated that harbor plas-mids at an increased level per chromosomalequivalent. This article describes the isolationand preliminary chracterization of these mu-tants.

MATERIALS AND METHODSBacterial strains. The genotypes and sources of

the Escherichia coli strains and plasmids used are635

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636 CRESS AND KLINE

shown in Table 1. Plasmids were transf'erred tovarious plasmid-f'ree recipients by conjugation withdonors listed in Table 1. Appropriate combinations oflactose fermentation and antibiotic resistance wereused for identification and counterselection, respec-tively. Plasmid P1 CM was transferred by transduc-tion (30).Media. Bacteria were assayed for lactose f'ermenta-

tion on EMB agar base (Difco) plates supplementedwith 1.0% filter-sterilized lactose. Bacterial conjuga-tion (9) and growth of Proteus mirabilis were carriedout in Penassay broth (Difco). E. coli CSH50 wascultured in M9 medium (16) supplemented with10-i M MgSO4, 0.2% glucose, 0.5% Casamino Acids, 5jg of thiamine per ml, and 250 gg of deoxyadenosineper ml. Thymine auxotrophs were supplemented withthymine (2 Hg/ml) or thymidine (4 jig/ml).Mutagenesis and isolation of mutants. Bacteria

harboring an F'lacI,P pro A+,B+ plasmid (hereafterdesignated F'lac pro+) in a host deleted for the lac-proregion ferment lactose poorly at 30 C. When culturedon EMB-lactose plates at 30 C the colonies appearwhite until after 24 to 30 h of incubation, at whichtime they turn pink. Mutants putatively relaxed incontrol of plasmid replication should have greaternumbers of lac genes and, therefore, be detectable asstronger lactose fermenters. To produce such mu-tants, bacteria were grown at 37 C to midexponentialphase in Penassay broth, N-methyl-N-nitro-N'-ni-trosoguanidine (NTG) was added to a final concentra-tion of 100 gg/ml, and the cultures were incubated at37 C for 30 min. After washing, aliquots of' bacteriawere subcultured overnight to allow genotypic segre-gation, diluted, plated on EMB-lactose agar at a levelof' 100 to 500 colonies/plate, and incubated at 30 C.After 18 to 24 h of' incubation, strong lactose ferment-ers (purple) could be readily distinguished. They werepurified by streaking into EMB-lactose and stored(25) at room temperature in Penassay agar. Only onemutant was selected from each subculture to ensurethe independent origin of each mutant.

Isolation of DNA. Crude lysates were preparedfrom midexponential phase cultures (4 x 108 cells/ml)continuously labeled with ["4C]thymine (0.1 MCi/ml;110 mCi/mmol) or [methyl-3H]thymidine (1 MCi/ml;

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25 Ci/mmol). The Brij-deoxycholate (DOC) lyticprocedure (5, 19) was used routinely. Where indi-cated, Brij-DOC lysates were treated with pancreaticribonuclease (RNase) at a final concentration of 50,gg/ml and/or treated with autodigested Pronase at afinal concentration of 1 mg/ml. In some experiments,Triton X-100 or sodium dodecyl sulfate was substi-tuted for Brij 58. The f'inal concentrations of thesubstituted detergents were the same as the finalconcentration of Brij 58. Alternately, Sarkosyl lysateswere prepared according to the procedure describedby Barzaral and Helinski (1), except that treatmentwith RNase and 37 C incubation were omitted.Dye-CsCl density gradient centrifugation and

sucrose density centrifugation. The techniques usedwere those previously described (19, 29).Diphenylamine assay. Total bacterial DNA was

determined by the diphenylamine procedure de-scribed by Friesen (12) with deoxyadenosine as astandard.

Hybridization. The amount of F'lac pro+ DNA as apercentage of total DNA was determined by DNA-DNA hybridization of isotopically labeled total E. coli(Flac pro+) DNA with excess total DNA from an F' P.mirabilis strain, designated Pml (F'lac+) (Table 1). AProteus strain harboring an F'lac+ plasmid of differ-ent origin than F'lac pro+ was employed because wewere unable to detect any conjugal transfer of F'lacpro+ into an F- Pml5 recipient, a derivative of Pml.The general protocol of Collins and Pritchard (6) wasfollowed for the hybridization reactions. The designsof the hybridization tests and controls are presentedin Table 2. Labeled DNA components were purifiedby two successive CsCl centrifugations of crude ly-sates treated with RNase and Pronase to insure 100'7recovery of DNA. Pml (F'lac+) DNA was purifiedaccording to the method Brenner et al. (3). Thereassociation reactions were carried out to a Cot value(4) of about 100 for the unlabeled component and of'about 0.04 for the labeled components. The amount ofannealed DNA was assayed with the single-strand-specific S1 nuclease from Aspergillus oryzae accordingto the procedure of' Crosa et al. (8). The correctivefactors required by the use of Si nuclease are pre-sented in Table 2. The enzyme was isolated according

TABLE 1. Bacterial strains and plasmids

Strain Chromosome genotype Plasmid genotype Sourceand phenotyrpe

CSH41 (F'lacpro+)8CSH50CR34 (F)pcn (F'lac pro')

P. mirabilis Pml (F'lac+)

A(lac pro) galE thiara A (lac pro) strA thithr leu thi thy lacCSH50 plus pcn

lac thy nic

F'lacI,PproA +,B+F-

F'lacI,PproA+,B+

F'lac+Plcm clr 100 (P1 CM)

J. MillerJ. MillerD. HelinskiSingle-colony

isolates of'NTG -mutagenizedCSH50 (F'lacpro+)

L. S. BaronViral lysate from

J. Rosner

Parenthetic expressions indicate plasmids harbored by the indicated host.

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PLASMID COPY NUMBER MUTANTS 637

TABLE 2. Hybridization assays for F'lac pro+ DNA

AvgLabeled Unlabeled Avg input reasso- Avg (7.LabdUeAviciated hvbridi- Purposecomponentsa component counts/min" counts! zation

mine

'H-labeled CSH50 CSH50 (F'lacpro+) 25,464 21,241 83.4 Determine maximum(Flac pro+) amount of reassociation of

labeled DNA under theexperimental conditions

'H-labeled CSH50 Pml (F'lac+) 28,214 292 1.24 Determine chromosomal ho-mology between E. coliand P. mirabilis

3H-labeled CSH50 Pml (F'lac) 'H, 13,278; 14C, 558 5.87 Measure F'lac pro+ level in(F'lac pro+) + 3,352 36 1.08 CSH50 with internal con-"IC-labeled trol for background chro-CSH50 mosomal hybridization

'H-labeled pcn Pml (F'lac+) 'H, 12,426; 14C, 2,027 19.56 Measure F'lac pro+ level in(F'lac pro+)-24 + 3,197 27 1.21 pcn-24 with internal con-"4C-labeled trol for background chro-CSH50 mosomal hybridization

'H-labeled pcn Pml (F'Iac+) 'H, 12,868; 14C, 1,848 17.25 Measure ratio of F'lac pro+(F'lac pro+) -24 + 3,258 145 5.36 levels in pcn-24 and"4C-labeled CSH50 with internal con-CSH50 (Flac trol for variable recoverypro+) of plasmid during lysis

and purification; valuesare averages of two experi-ments

a In several cases the labeled DNA was copurified from a mixture of differentially labeled exponentialcultures that had been grown to identical cell densities.

b Each value represents the average of duplicate samples. The 'H values reported are within 2.5% precision,whereas 14C values are within 5% precision.cThe observed trichloroacetic acid-precipitable counts per minute were corrected for Si-resistant single-

stranded DNA and hydrolyzed double-stranded DNA by the following relation: Actual counts per minute =(observed counts per minute - [input counts per minute x fraction of single-stranded DNA resistant to S1])/(1- [fraction of single-stranded DNA resistant to Si] - [fraction of double-stranded DNA hydrolyzed by Si]).The percentage of double-stranded DNA hydrolyzed by Si nuclease was 6.3% as determined by separatehydrolysis of purified double-stranded DNA. The percentage of single-stranded DNA resistant to S1 was takenfrom the zero time denatured sample. The latter values ranged from 6.7 to 8.5%.

d % Hybridization = (corrected counts per minute of reassociated DNA as in footnote c)/(input counts perminute) x (100)/(83.4) x (100).

to the procedure of Sutton (33) and was a generousgift from David Vaughn.Folded chromosome isolation. Folded chromo-

somes were isolated from E. coli cultures as describedby Kline and Miller (22). The association betweenF'lac pro+ and the folded chromosome was assayed bysedimentation of folded chromosomes in the 4,000 x gsupernatant through a 20 to 31% neutral sucrose

density gradient onto a shelf containing CsCl (p =1.70 g/cm') and 35% sucrose. Under these conditions,the open circular and covalently closed circular(CCC) forms of the F'Iac pro+ molecules not associ-ated with the folded chromosome should appear asmaterial sedimenting at rates of 72 or 11OS, respec-tively.

In control experiments the same protocol was

followed except that cells from a differentially labeledF- culture were mixed with F'pcn cells. Then the cellmixture was lysed and analyzed for the release of F-cells of chromosome fragments similar in Svedbergunit value to F'lac pro+ DNA. None were found (datanot shown).

Alternately, folded chromosomes were isolated by amodification of the Worcel-Burgi procedure (34) inwhich Sarkosyl replaced Brij 58 as the lytic detergent.The crude lysate was incubated at 25 C, for 20 min,and the lysate was centrifuged through a 10 to 30%neutral sucrose density gradient. The material sedi-menting in a broad peak at 1,800S and the materialsedimenting at the top of the gradient were pooledseparately, and along with an aliquot of the crudelysate were treated with pancreatic RNase (100 lAg/

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638 CRESS AND KLINE

ml) and autodigested Pronase (1 mg/ml) for 10 min at37 C. Then the treated pools and crude lysate were

analyzed for percentage of CCC DNA by ethidiumbromide-cesium chloride centrifugation.

RESULTS

Isolation of mutants with increased plas-mid levels. Mutation to altered control ofstringent plasmid replication results in bacteriathat contain more than the usual one or twoplasmid molecules per replicating chromosome.A well-established approach to detecting suchmutants is to examine the bacterial populationfor increased expression of plasmid-linked genes(23, 24, 26, 27). In our application of thisapproach, we introduced an F'lacI,P pro+ plas-mid into a host deleted for the lac-pro region(Table 1). The i- mutation allows a constitutiveexpression of the lac operon, but the p- muta-tion seriously impedes the function of ribonu-cleic acid polymerase in transcribing the op-eron; the net effect is a weak fermentation oflactose. Thus, we reasoned that colonies thatstrongly ferment lactose would either be p+revertants or have increased copies of the lacI,Poperon. The latter could result from reiterationof the lacI,P operon in the same plasmid or fromincreased numbers of plasmids per cell. Withthis rationale as a basis for screening, we haveisolated 17 independently produced mutantsthat ferment lactose strongly after NTG muta-genesis. We have chosen to designate bacteriawith altered levels of plasmid DNA as pcnmutants.

Characterization of pcn mutants. (i) Iso-pycnic analysis. Cultures made from the 17isolates and the parental strain were examinedfor their amounts of CCC DNA by the dye-CsClequilibrium centrifugation technique. In 11 ofthe mutants, 3.0 to 8.0% of the DNA behaved as

CCC DNA (data not shown), whereas in theparental strain, the average amount of CCCDNA is 1.2% and has never been observed toexceed 2% (Table 3). Six isolates that showedthe greatest elevation were selected for furtherstudy. They are listed in Table 3.

Inspection of Table 3 shows that the recover-

ies of total DNA after centrifugation vary signif-icantly with lytic conditions and treatment ofcrude lysates. However, the percentage of CCCDNA in the parental strain is roughly invariant;in contrast, the percentage of CCC DNA in thepcn mutants varies over a great range of abso-lute values. These data suggest that pcn mu-

tants could erroneously appear to have elevatedlevels of plasmid DNA -because chromosomalDNA is preferentially lost during centrifuga-tion. However, when Sarkosyl-lysed cells were

TABLE 3. Dye-CsCl gradient analysis

Straina Lysate Avg ` Range AvggStraina treatmentb CCCC % CCC recovery

CSH50 None 1.2d 0.9-1.6 34CSH50 RNase + 1.2 1.0-1.6 >95

Pronase,37 C

pcn-24 None 9.0e 3.8-17.0 30pcn-24 RNase + 0.4 0.04-2.1 >95

Pronase,37 C

pcn-24 37 C 1.6 0.8-3.0 80pcn-18 None 4.0 2.0-4.9 38pcn-19 None 4.1 2.7-5.5 29pcn-21 None 6.6 4.0-7.6 35pcn-23A None 3.7 2.7-4.9 24pcn-23B None 4.2 3.2-6.0 32

a All strains contain the F'lac pro+ plasmid, 145 x106 daltons. This weight is equal to 5.8% of the weightof an E. coli chromosome.

'Pancreatic RNase was used at a final concentra-tion of 50 ug/ml, whereas final Pronase concentrationwas 1 mg/ml. Incubation time was 45 min.

cPercentage of CCC DNA is expressed as (totalcounts per minute in plasmid peak/total counts perminute in chromosomal peak) x 100%.

d Coefficient of variation of 11 separate determina-tions is 12.5%.

e Coefficient of variation of 14 separate determina-tions is 45%.

analyzed, we recovered up to 70% of the totalDNA and still found up to fivefold elevatedlevels of CCC DNA (data not shown). Theseresults indicate that the mutants do have ele-vated levels of CCC DNA but seriously questionthe quantitative reliability of the dye-CsClassay.

(ii) Enzymic analysis. To confirm our con-clusion about the mutants, we assayed thelevels of fl-galactosidase (25) in the mutant andparental strains as well as control strains ofknown lac genotype. The controls and parentalstrains gave expected and reproducible results,but the results with pcn mutants were notreproducible irrespective of the carbon source,temperature of incubation, and presence orabsence of enzyme inducer. We find the enzymelevels in any given pcn mutant can be higher,lower, or the same as the reasonably constantlevel of 200 U per ml (25) observed in theparental strain. We have no explanation forthis situation, but conclude that the enzymicresults are not of any value in confirmingplasmid levels determined by the dye-CsClprocedure.

(iii) DNA-DNA hybridization analysis.The technique of DNA-DNA hybridization per-

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PLASMID COPY NUMBER MUTANTS 639

mits a direct analysis of the plasmid level that isnot subject to the experimental vagaries ofisopycnic centrifugation or gene expression. Todo this analysis, we were forced to use anonisogenic F'lac+ plasmid in the Proteus hostbecause the F'lac pro+ plasmid does not fer-ment lactose well enough to be detected in thishost. Although this precludes an absolute deter-mination of the amount of F'lac pro+ DNA in E.coli, it does permit a valid comparison betweenE. coli pcn mutants and the parental strain.The results obtained (Table 2) indicate thatthere is about four times as much plasmid DNAin pcn-24 (18.3%) as in the parental bacteria(4.8%). Since plasmid DNAs purified from pcn-24 and the parental strain by dye-CsCl centrifu-gation both sediment on neutral gradients atthe same rates (Fig. 1), we conclude that thepcn-24 mutant has about four times as manyunit size plasmids per cell as the unmutatedparental strain. From the relative S values of72S observed for the open-circular form of F'lacpro+ plasmid, we calculate (32) that the mass ofthe unit size plasmid is 145 x 106 daltons.Note that the percentages of plasmid DNA

determined in Table 2 are quite precise,whereas those in Table 3 are relatively impre-cise, especially with lysates made from pcnmutants. We find with lysates from the pcn

5 3 S0~~~~~~

0~X 10- 2 X

10 20

FRACT IONFIG. 1. Neutral sucrose gradient centrifugation of

purified F'lac pro+ DNA from pcn-24 and CSH50.DNA banding in the satellite position in cesiumchloride-ethidium bromide gradients made fromcrude lysates was pooled and dialyzed to removeethidium bromide. Portions of these two differentiallylabeled preparations were mixed and sedimentedthrough a 5-ml, 20 to 31% neutral sucrose gradientcontaining 0.05 M sodium phosphate (pH 7.6), 0.005M ethylenediaminetetraacetic acid, and 0.5 M NaCI.Centrifugation was for 55 min at 45,000 rpm in an SW50.1 rotor at 15 C. Fractions of 10 drops were col-lected. Sedimentation is from right to left. Recoveryof layered counts exceeded 90%. Symbols: 0, CSH50(F'lac pro+); 0, pcn-24 (F'lac pro+).

mutants that chromosomal DNA is preferen-tially trapped in the proteinaceous pellicle thatforms on top of the CsCl density gradient duringcentrifugation (unpublished data). To obtainthe data of Table 2, we eliminated this source ofvariation by pretreatment of the lysates withPronase and RNase before centrifugation. Asdiscussed next, these treatments are not possi-ble when used in conjunction with the dye-CsClassay.Other properties of pcn mutants. The en-

zymic treatments employed in the experimentdescribed in Table 3 show that the pcn mutantsare unusual in yet another respect. In lysatesmade from six mutants, but not lysates madefrom the wild-type parent, large losses of CCCDNA occur when the lysates are incubated at37 C for 30 to 45 min. About a 95% loss occurswhen Pronase and RNase are present duringincubation, and about a 50 to 70% loss occurs ifthese enzymes are not present. Data for themutant pcn-24 is shown in Table 3 and istypical for pcn-18, -19, -20, -23A, and -23B. Theplasmid losses observed after incubation areabsolute since the actual counts in CCC DNApeaks decrease. Thus, the losses are not merelyrelative due to the increased recoveries of linearDNA as alluded to above.Location and specificity of the pcn

mutation. The pcn mutation maps on thechromosome in all 17 mutants examined. Thisconclusion is based on the strong fermentationof lactose after cured F-pcn mutants are rein-fected with the unmutated parental F'Iacpro+ plasmid. Additionally, such remated F'bacteria derived from the pcn mutants listed inTable 3 also show about the same elevated levelsof plasmid DNA as their original F'pcn parentsas determined by dye-CsCl analysis (data notshown). Further, progeny derived from F'pcn xF-pcn+ crosses with the pcn mutants listed inTable 3 are weak lactose fermenters, i.e., pcn+.F'pcn-24, however, could not be tested this waybecause this host is not conjugally proficienteven when it contains an F'lac pro+ plasmidthat has never been exposed to a mutagen.The data in Table 4 show that the pcn

mutants do have slightly elevated amounts ofDNA. The extra DNA can be accounted for asplasmid DNA. We have converted the data onthe total micrograms of DNA per cell of Table 4into plasmid and replicating chromosomeequivalents per cell with the assumptions listedin Table 4 and the relative amounts of thesemacromolecules as determined by our plasmidDNA-DNA hybridization data in Table 2. As wementioned previously, the percentage of totalDNA that is plasmid is not an absolute value

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TABLE 4. Plasmid copy num bers

Minimum

Genera- Chromo- Repli- Minimum plasmidGene Total somal catinga Plasmid plasmid copies/Strain time DNA/cellonal chromo- DNA/cell copies!/ repli-

(min) (ug/109) (Dg/cl0) somes/ (Ag/1O') cell cating

some

CSH50 (F'lacpro+) 40 11.7 11.19 1.91 0.530 2.2 1.15pcn-24 (F'lacpro+) 40 13.4 11.16 1.90 2.242 9.3 4.90

aThe molecular weight of the resting chromosome was taken to be 2.5 x 10' (7).

because the F'lac in Proteus is probably notidentical. The error is not likely to be large,however, because the calculated number of F'lacpro+ plasmids per replicating chromosome inthe unmutated parent is very close to the valuesreported by others (6) who used a similarexperimental approach and analysis. Also, thetheoretical amount of F' DNA expected in a celllysate, based on the molecular weight of theplasmid, is 5.8%. This agrees well with themeasured value (Table 2).The pcn mutation does affect the level of

other plasmid systems as determined bydye-CsCl analysis; however, it does not affectall plasmid systems. Although the studies onwhich this statement is based are still in prog-ress (Cress and Kline, unpublished data), weshould like to mention some of the key findingsto enhance a fuller appreciation of the Pcnphenotype. Firstly, in pcn mutants -18, -19,and -24, the classic sex factor F appears to bemaintained at normal levels, whereas in pcnmutants -21, -23A, and -24, the viral plasmidP1 CM (30) is maintained at about a two- tothreefold higher level. Moreover, the percentageof CCC DNA of the F+ plasmid in the pcn celllysates is invariant even when the recovery oftotal CCC DNA in dye-CsCl gradients is low.Also, the CCC form of the F+ DNA is not lostwhen crude lysates of F+ pcn-24 are incubatedat 37 C. In contrast, the CCC P1 CM plasmidform does decrease when crude lysates of pcnlysogens are incubated at 37 C.Physical location of the F' plasmids in the

pcn-24 mutant. Recently Kline and Miller (22)demonstrated that the otherwise autonomous80S CCC F plasmid population of an F+ culture.cosediments with its host's 1,800 and 3,200Sspecies of folded chromosomes on neutral su-crose gradients. These findings have led to theconclusion that nonintegrated F plasmid iscomplex to the host chromosome. The possibil-ity that membrane serves as the linking moietybetween plasmid and chromosome is suspectbecause the 1,800S species of chromosome has

been shown to be free of membrane (35).In light of this behavior with F+ plasmids, we

sought to locate the F'lac pro+ plasmids in thepcn-24 mutant by the same experimental ap-proach (22). However, since incubation at 25 Cto generate membrane-released folded chromo-somes might destroy a significant amount of theF' plasmids (Table 3), we analyzed lysatesmade at 0 C for the presence of free 110 and 72Splasmid DNA. To do this, we simply lysed thebacteria and pelleted the folded chromosomesonto a CsCl cushion located at the bottom of a20 to 31% sucrose gradient while sedimentingthe 110S material to the center of the gradient.The results in Fig. 2 show that the amount offree 110 and 72S DNA in pcn-24 is less than0.5% of the total DNA in the gradient. Thesimplest interpretation of these results is thatall the plasmid copies in pcn-24 are associatedwith the folded chromosome. Subsequent tothese results we have found that membrane-released folded chromosomes can be isolatedfrom pcn-24 without loss of plasmid DNA bylysis with Sarkosyl in 1 M NaCl at 25 C for 20min (see above). When purified, 1,800S foldedchromosomes from pcn-24 were tested bydye-CsCl analysis, and 5.3% of the DNA bandedin the CCC peak. Analysis of DNA sedimentingin the 0 to 300S region of the preparative sucrosegradients used to purify the 1,800S foldedchromosomes showed that less than 5.3% of thisDNA is in the CCC form. These results indicateno enrichment for free 110S CCC plasmid DNAin lysates of pcn-24 and substantiate the resultsof Fig. 2. We conclude all the F' plasmid copiesin pcn-24 are complexed to folded chromo-somes.

DISCUSSIONOur results indicate that an F' plasmid can be

stably maintained within a range of copy levels,not just the stringent state of one plasmid perchromosomal equivalent. This conclusion isbased primarily on the increased levels of plas-mid DNA in pcn mutants and the absence of

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I 2

0~~~ ~ ~~

10 20

FRACTIONFIG. 2. Free F'lac pro+ DNA from pcn-24 in cell

lysates containing membrane-released folded chromo-somes. Cells were lysed with Brij-DOC as described inthe text, and the supernatant from 4,000 x g centrifu-gation was mixed with differentially labeled F'lacpro+ DNA purified by dye-cesium chloride centrifuga-tion. The mixture was sedimented through a 20 to 31%neutral sucrose density gradient of the same composi-tion as in Fig. 1, except that the gradient wasprepared over a 0.5-mI shelf of35% sucrose containingcesium chloride (p = 1.71). Centrifugation was for 55min at 45,000 rpm in a SW 50.1 rotor at 15 C.Recovery of counts greater than 95%. Symbols: (0)"C-labeled F'lac pro+ marker, (0) IH-labeled pcn-24lysate.

any plasmid DNA in one mutant that is largerthan unit size parental plasmid. Our geneticstudies show that only chromosomally deter-mined pcn mutants have been isolated. We donot know the map position of these mutations,or even whether only one chromosomal mutationgives rise to the Pcn phenotype. Since we haveused nitrosoguanidine to induce pcn-type muta-tions, our isolates may well be multiply mu-tated.

Plasmids in lysates made from pcn mutantslose most of their CCC DNA form via anunknown mechanism or agent that is activatedby heat (Table 3). Since our original observa-tions were made, we have found that stored pcnmutants give rise upon subculture and subse-quent lysis to lysates that have much lessplasmid-destroying activity. Nonetheless, theelevated levels of plasmid in these subculturesdo not appear reduced. We cannot explain theseobservations at present. Further, we point outthat the plasmid-destroying agent is uniquelyobservable only in lysates made from pcn mu-

PLASMID COPY NUMBER MUTANTS 641

tants that contain plasmids capable of beingelevated by the putative pcn gene products.Biochemical characterization of the plasmid-destroying agent is the subject of a future report(D. Cress and B. Kline, manuscript in prepara-tion).

Previous work by Kline and Miller (22) hasdemonstrated that the F plasmid, which isunder stringent control of replication, is com-plexed in some noninte,grative fashion in thefolded chromosomes of F+ bacterial hosts. Thisfinding made it reasonable to expect that thePcn phenotype might be an expression of mu-tants that are able to maintain plasmids in bothcomplexed and noncomplexed states or in justthe latter state. The results described in Fig. 2show that all F' plasmids in the pcn-24 mutantsare complexed to their host's folded chromo-somes. We suspect the multiple-plasmids com-plex at just one chromosomal locus becauseF-pcn-24 progeny arise from pcn-24 (F'lacpro+) parents at the same or greater ratesthan F- progeny arise from isogenic F'pcn+parents. In any event, the results of Fig. 2indicate a deregulation of the plasmid copynumber does not occur because the pcn-24mutant can maintain its plasmids in the non-complexed state.The ability to maintain multiple copies of

homologous plasmid is very much analogous toan incompatibility breakdown. Normally, theincompatibility (inc) function of the host plas-mid prevents stable inheritance of superinfect-ing, homologous plasmids. However, should theincompatibility function of the host plasmid bemutated and permit superinfection and stablemaintenance of the incoming and residentplasmids, then in effect the inc mutation ispcn-like. In a somewhat comparable but notidentical situation, San Blas et al. (31) haveactually mutated bacterial host genes and suc-cessfully selected for a loss of plasmid incom-patibility between an F'gal+ and F'his+ pair. Ineffect, they generated a pcn-like chromosomalmutation. Whether or not there is any biochem-ical or genetic similarity between their incmutant and our pcn mutants is unknown.

Finally, in closing, we point out that carefulcomparison of the results of Tables 2 and 3 willshow that the dye-CsCl analytical technique is apoor quantitative tool. Nonetheless, it can beused in a screening procedure if one under-stands its limitations and does not relie on it fora definitive answer to plasmid levels in lysatesof putative mutants. Clearly, of all the physicalassays (including alkaline sucrose zonal centrif-ugation), only DNA-DNA hybridization assays

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642 CRESS AND KLINE

do not seriously underestimate the true amountof plasmid DNA present in an extract.

ACKNOWLEDGMENTSThis work was supported by a University of' Tennessee

predoctoral fellowship awarded to D. E. Cress and by PublicHealth Service research grant GM 18608 from the NationalInsititute of General Medical Sciences awarded to B. Kline.

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