Isolation and Characterization of Escherichia coli Chromosomal ...

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

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

Isolation and Characterization of Escherichia coli Chromosomal 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 that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,Ppro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid PI CM is maintained at two to three times 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 decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.

Biochemical requirements for the replication ture-sensitive F' segregation/replication-defecof stringent plasmids are poorly understood. tive mutants have been studied (10, 13, 15, 17, Both plasmid and chromosomal gene products 18) but have yielded little insight into the are involved (14). The diversity of plasmid types biochemical control of replication. Thus, we and roles probably reflects a diversity of mech- sought to isolate mutants with control altered in anisms for the control of plasmid replication. an opposite manner-increased rounds of plasThe classical sex factor, F, appears to be the mid replication per cell cycle. Such mutants most chromosome-like of the stringent plasmids have been obtained for several R plasmid sysin that F is a monomolecular replicon main- tems (24, 26, 27) by assaying for increased tained at a ratio of approximately one or two antibiotic resistance and for the Clo DF13 plasmid copies per chromosome (2, 6, 11). plasmid by assaying for increased cloacin proFurther, the macromolecular requirements for F duction (23). We have used the same logic in replication are similar but not identical (20, 21, assaying for increased lac genes in bacteria 28) to those for chromosome replication. These harboring an F'lacI,P pro+ plasmid. This apfindings suggest that F is a good model system proach has been successful, and a number of for studying the control of stringent deoxy- mutants have been isolated that harbor plasmids at an increased level per chromosomal ribonucleic acid (DNA) replication. We have chosen to study the control of F equivalent. This article describes the isolation replication through the isolation of mutants and preliminary chracterization of these mualtered in that control. A number of tempera- tants. 'Present address: Department of Biology, University of MATERIALS AND METHODS Utah, Salt Lake City, Utah 84112. 'Present address: Department of Microbiology, Mayo Clinic, Rochester, Minn. 55901.

Bacterial strains. The genotypes and sources of the Escherichia coli strains and plasmids used are 635

636

J. BACTERIOL.

CRESS AND KLINE

shown in Table 1. Plasmids were transf'erred to various plasmid-f'ree recipients by conjugation with donors listed in Table 1. Appropriate combinations of lactose fermentation and antibiotic resistance were used for identification and counterselection, respectively. Plasmid P1 CM was transferred by transduction (30). Media. Bacteria were assayed for lactose f'ermentation on EMB agar base (Difco) plates supplemented with 1.0% filter-sterilized lactose. Bacterial conjugation (9) and growth of Proteus mirabilis were carried out in Penassay broth (Difco). E. coli CSH50 was cultured in M9 medium (16) supplemented with 10-i M MgSO4, 0.2% glucose, 0.5% Casamino Acids, 5 jg of thiamine per ml, and 250 gg of deoxyadenosine per ml. Thymine auxotrophs were supplemented with thymine (2 Hg/ml) or thymidine (4 jig/ml). Mutagenesis and isolation of mutants. Bacteria harboring an F'lacI,P pro A+,B+ plasmid (hereafter designated F'lac pro+) in a host deleted for the lac-pro region ferment lactose poorly at 30 C. When cultured on EMB-lactose plates at 30 C the colonies appear white until after 24 to 30 h of incubation, at which time they turn pink. Mutants putatively relaxed in control of plasmid replication should have greater numbers of lac genes and, therefore, be detectable as stronger lactose fermenters. To produce such mutants, bacteria were grown at 37 C to midexponential phase in Penassay broth, N-methyl-N-nitro-N'-nitrosoguanidine (NTG) was added to a final concentration of 100 gg/ml, and the cultures were incubated at 37 C for 30 min. After washing, aliquots of' bacteria were subcultured overnight to allow genotypic segregation, diluted, plated on EMB-lactose agar at a level of' 100 to 500 colonies/plate, and incubated at 30 C. After 18 to 24 h of' incubation, strong lactose fermenters (purple) could be readily distinguished. They were purified by streaking into EMB-lactose and stored (25) at room temperature in Penassay agar. Only one mutant was selected from each subculture to ensure the independent origin of each mutant. Isolation of DNA. Crude lysates were prepared from 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; TABLE 1. Bacterial Strain

CSH41 (F'lacpro+)8 CSH50 CR34 (F) pcn (F'lac pro')

25 Ci/mmol). The Brij-deoxycholate (DOC) lytic procedure (5, 19) was used routinely. Where indicated, Brij-DOC lysates were treated with pancreatic ribonuclease (RNase) at a final concentration of 50 ,gg/ml and/or treated with autodigested Pronase at a final concentration of 1 mg/ml. In some experiments, Triton X-100 or sodium dodecyl sulfate was substituted for Brij 58. The f'inal concentrations of the substituted detergents were the same as the final concentration of Brij 58. Alternately, Sarkosyl lysates were prepared according to the procedure described by Barzaral and Helinski (1), except that treatment with RNase and 37 C incubation were omitted.

Dye-CsCl density gradient centrifugation and sucrose density centrifugation. The techniques used were those previously described (19, 29). Diphenylamine assay. Total bacterial DNA was determined by the diphenylamine procedure described by Friesen (12) with deoxyadenosine as a

standard. Hybridization. The amount of F'lac pro+ DNA as a percentage of total DNA was determined by DNADNA 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). A Proteus strain harboring an F'lac+ plasmid of different origin than F'lac pro+ was employed because we were unable to detect any conjugal transfer of F'lac pro+ into an F- Pml5 recipient, a derivative of Pml. The general protocol of Collins and Pritchard (6) was followed for the hybridization reactions. The designs of the hybridization tests and controls are presented in Table 2. Labeled DNA components were purified by two successive CsCl centrifugations of crude lysates treated with RNase and Pronase to insure 100'7 recovery of DNA. Pml (F'lac+) DNA was purified according to the method Brenner et al. (3). The reassociation 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 of annealed DNA was assayed with the single-strandspecific S1 nuclease from Aspergillus oryzae according to the procedure of' Crosa et al. (8). The corrective factors required by the use of Si nuclease are presented in Table 2. The enzyme was isolated according strains and plasmids genotype Plasmid and phenotyrpe

Chromosome genotype

Source

A(lac pro) galE thi

F'lacI,PproA +,B+

ara A (lac pro) strA thi

F-

thr leu thi thy lac CSH50 plus pcn

J. Miller J. Miller D. Helinski

F'lacI,PproA+,B+

Single-colony isolates of'

NTG -mutagenized CSH50 (F'lac pro+) P. mirabilis Pml (F'lac+)

lac thy nic

F'lac+ Plcm clr 100 (P1 CM)

Parenthetic expressions indicate plasmids harbored by the indicated host.

L. S. Baron Viral lysate from J. Rosner

PLASMID COPY NUMBER MUTANTS

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637

TABLE 2. Hybridization assays for F'lac pro+ DNA Avg reassoU e Labd Unlabeled Labeled AvgAv input iciated counts! counts/min" component componentsa mine

Avg (7.

hvbridization

'H-labeled CSH50 CSH50 (F'lacpro+) (Flac pro+)

25,464

21,241

83.4

'H-labeled CSH50 Pml (F'lac+)

28,214

292

1.24

3H-labeled CSH50 Pml (F'lac) (F'lac pro+) + "IC-labeled CSH50

'H, 13,278; 14C,

558 36

5.87 1.08

'H-labeled pcn Pml (F'lac+) (F'lac pro+)-24 + "4C-labeled CSH50

'H, 12,426; 14C, 3,197

2,027

19.56

27

1.21

'H-labeled pcn Pml (F'Iac+) (F'lac pro+) -24 + "4C-labeled CSH50 (Flac

'H, 12,868; 14C,

1,848 145

17.25 5.36

3,352

3,258

pro+)

Purpose

Determine maximum amount of reassociation of labeled DNA under the experimental conditions Determine chromosomal homology between E. coli and P. mirabilis Measure F'lac pro+ level in CSH50 with internal control for background chromosomal hybridization Measure F'lac pro+ level in pcn-24 with internal control for background chromosomal hybridization

Measure ratio of F'lac pro+ levels in pcn-24 and CSH50 with internal control for variable recovery of plasmid during lysis and purification; values are averages of two experiments

a In several cases the labeled DNA was copurified from a mixture of differentially labeled exponential cultures 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 singlestranded 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 separate hydrolysis of purified double-stranded DNA. The percentage of single-stranded DNA resistant to S1 was taken from 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 per minute) x (100)/(83.4) x (100).

to the procedure of Sutton (33) and was a generous

gift from David Vaughn. Folded chromosome isolation. Folded chromosomes were isolated from E. coli cultures as described by Kline and Miller (22). The association between F'lac pro+ and the folded chromosome was assayed by

sedimentation of folded chromosomes in the 4,000 x g supernatant 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 associated with the folded chromosome should appear as material sedimenting at rates of 72 or 11OS, respec-

tively. In control experiments the same

protocol

was

followed except that cells from a differentially labeled F- culture were mixed with F'pcn cells. Then the cell mixture was lysed and analyzed for the release of Fcells of chromosome fragments similar in Svedberg unit value to F'lac pro+ DNA. None were found (data not shown). Alternately, folded chromosomes were isolated by a modification of the Worcel-Burgi procedure (34) in which 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 sedimenting in a broad peak at 1,800S and the material sedimenting at the top of the gradient were pooled separately, and along with an aliquot of the crude lysate were treated with pancreatic RNase (100 lAg/

638

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CRESS AND KLINE

ml) and autodigested Pronase (1 mg/ml) for 10 min at 37 C. Then the treated pools and crude lysate were analyzed for percentage of CCC DNA by ethidium bromide-cesium chloride centrifugation. RESULTS Isolation of mutants with increased plasmid levels. Mutation to altered control of

TABLE 3. Dye-CsCl gradient analysis

Straina

Lysate

Straina

treatmentb

CSH50 CSH50

None RNase + Pronase, 37 C None RNase + Pronase, 37 C 37 C

Range

Avg ` CCCC

% CCC

1.2d 1.2

0.9-1.6 1.0-1.6

Avgg

recovery 34 >95

9.0e 30 3.8-17.0 stringent plasmid replication results in bacteria pcn-24 0.4 0.04-2.1 >95 that contain more than the usual one or two pcn-24 plasmid molecules per replicating chromosome. A well-established approach to detecting such pcn-24 1.6 0.8-3.0 80 mutants is to examine the bacterial population pcn-18 None 38 2.0-4.9 4.0 for increased expression of plasmid-linked genes pcn-19 None 4.1 29 2.7-5.5 None 6.6 35 4.0-7.6 (23, 24, 26, 27). In our application of this pcn-21 24 3.7 2.7-4.9 approach, we introduced an F'lacI,P pro+ plas- pcn-23A None 4.2 3.2-6.0 32 mid into a host deleted for the lac-pro region pcn-23B None (Table 1). The i- mutation allows a constitutive a All strains contain the F'lac pro+ plasmid, 145 x expression of the lac operon, but the p- muta- 106 daltons. This weight is equal to 5.8% of the weight tion seriously impedes the function of ribonu- of an E. coli chromosome. cleic acid polymerase in transcribing the op'Pancreatic RNase was used at a final concentraeron; the net effect is a weak fermentation of tion of 50 ug/ml, whereas final Pronase concentration lactose. Thus, we reasoned that colonies that was 1 mg/ml. Incubation time was 45 min. cPercentage of CCC DNA is expressed as (total strongly ferment lactose would either be p+ revertants or have increased copies of the lacI,P counts per minute in plasmid peak/total counts per minute in chromosomal peak) x 100%. operon. The latter could result from reiteration d Coefficient of variation of 11 separate determinaof the lacI,P operon in the same plasmid or from tions is 12.5%. increased numbers of plasmids per cell. With e Coefficient of variation of 14 separate determinaa basis for we have as rationale this screening, tions is 45%. isolated 17 independently produced mutants that ferment lactose strongly after NTG mutagenesis. We have chosen to designate bacteria analyzed, we recovered up to 70% of the total with altered levels of plasmid DNA as pcn DNA and still found up to fivefold elevated levels of CCC DNA (data not shown). These mutants. Characterization of pcn mutants. (i) Iso- results indicate that the mutants do have elepycnic analysis. Cultures made from the 17 vated levels of CCC DNA but seriously question isolates and the parental strain were examined the quantitative reliability of the dye-CsCl for their amounts of CCC DNA by the dye-CsCl assay. (ii) Enzymic analysis. To confirm our conequilibrium centrifugation technique. In 11 of the mutants, 3.0 to 8.0% of the DNA behaved as clusion about the mutants, we assayed the CCC DNA (data not shown), whereas in the levels of fl-galactosidase (25) in the mutant and parental strain, the average amount of CCC parental strains as well as control strains of DNA is 1.2% and has never been observed to known lac genotype. The controls and parental exceed 2% (Table 3). Six isolates that showed strains gave expected and reproducible results, the greatest elevation were selected for further but the results with pcn mutants were not study. They are listed in Table 3. reproducible irrespective of the carbon source, Inspection of Table 3 shows that the recover- temperature of incubation, and presence or ies of total DNA after centrifugation vary signif- absence of enzyme inducer. We find the enzyme icantly with lytic conditions and treatment of levels in any given pcn mutant can be higher, crude lysates. However, the percentage of CCC lower, or the same as the reasonably constant DNA in the parental strain is roughly invariant; level of 200 U per ml (25) observed in the in contrast, the percentage of CCC DNA in the parental strain. We have no explanation for pcn mutants varies over a great range of abso- this situation, but conclude that the enzymic lute values. These data suggest that pcn mu- results are not of any value in confirming tants could erroneously appear to have elevated plasmid levels determined by the dye-CsCl levels of plasmid DNA -because chromosomal procedure. DNA is preferentially lost during centrifuga(iii) DNA-DNA hybridization analysis. tion. However, when Sarkosyl-lysed cells were The technique of DNA-DNA hybridization per-

PLASMID COPY NUMBER MUTANTS

VOL. 125, 1976

mits a direct analysis of the plasmid level that is not subject to the experimental vagaries of isopycnic centrifugation or gene expression. To do this analysis, we were forced to use a nonisogenic F'lac+ plasmid in the Proteus host because the F'lac pro+ plasmid does not ferment lactose well enough to be detected in this host. Although this precludes an absolute determination of the amount of F'lac pro+ DNA in E. coli, it does permit a valid comparison between E. coli pcn mutants and the parental strain. The results obtained (Table 2) indicate that there is about four times as much plasmid DNA in pcn-24 (18.3%) as in the parental bacteria (4.8%). Since plasmid DNAs purified from pcn24 and the parental strain by dye-CsCl centrifugation both sediment on neutral gradients at the same rates (Fig. 1), we conclude that the pcn-24 mutant has about four times as many unit size plasmids per cell as the unmutated parental strain. From the relative S values of 72S observed for the open-circular form of F'lac pro+ plasmid, we calculate (32) that the mass of the 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 imprecise, especially with lysates made from pcn mutants. We find with lysates from the pcn

0~~~~~~

53 S

X 10-

2

X

0~

20 10 FRACT ION FIG. 1. Neutral sucrose gradient centrifugation of purified F'lac pro+ DNA from pcn-24 and CSH50. DNA banding in the satellite position in cesium chloride-ethidium bromide gradients made from crude lysates was pooled and dialyzed to remove ethidium bromide. Portions of these two differentially labeled preparations were mixed and sedimented through a 5-ml, 20 to 31% neutral sucrose gradient containing 0.05 M sodium phosphate (pH 7.6), 0.005 M ethylenediaminetetraacetic acid, and 0.5 M NaCI. Centrifugation was for 55 min at 45,000 rpm in an SW 50.1 rotor at 15 C. Fractions of 10 drops were collected. Sedimentation is from right to left. Recovery of layered counts exceeded 90%. Symbols: 0, CSH50 (F'lac pro+); 0, pcn-24 (F'lac pro+).

639

mutants that chromosomal DNA is preferentially trapped in the proteinaceous pellicle that forms on top of the CsCl density gradient during centrifugation (unpublished data). To obtain the data of Table 2, we eliminated this source of variation by pretreatment of the lysates with Pronase and RNase before centrifugation. As discussed next, these treatments are not possible when used in conjunction with the dye-CsCl assay. Other properties of pcn mutants. The enzymic treatments employed in the experiment described in Table 3 show that the pcn mutants are unusual in yet another respect. In lysates made from six mutants, but not lysates made from the wild-type parent, large losses of CCC DNA occur when the lysates are incubated at 37 C for 30 to 45 min. About a 95% loss occurs when Pronase and RNase are present during incubation, and about a 50 to 70% loss occurs if these enzymes are not present. Data for the mutant pcn-24 is shown in Table 3 and is typical for pcn-18, -19, -20, -23A, and -23B. The plasmid losses observed after incubation are absolute since the actual counts in CCC DNA peaks decrease. Thus, the losses are not merely relative due to the increased recoveries of linear DNA as alluded to above. Location and specificity of the pcn mutation. The pcn mutation maps on the chromosome in all 17 mutants examined. This conclusion is based on the strong fermentation of lactose after cured F-pcn mutants are reinfected with the unmutated parental F'Iac pro+ plasmid. Additionally, such remated F' bacteria derived from the pcn mutants listed in Table 3 also show about the same elevated levels of plasmid DNA as their original F'pcn parents as determined by dye-CsCl analysis (data not shown). Further, progeny derived from F'pcn x F-pcn+ crosses with the pcn mutants listed in Table 3 are weak lactose fermenters, i.e., pcn+. F'pcn-24, however, could not be tested this way because this host is not conjugally proficient even when it contains an F'lac pro+ plasmid that has never been exposed to a mutagen. The data in Table 4 show that the pcn mutants do have slightly elevated amounts of DNA. The extra DNA can be accounted for as plasmid DNA. We have converted the data on the total micrograms of DNA per cell of Table 4 into plasmid and replicating chromosome equivalents per cell with the assumptions listed in Table 4 and the relative amounts of these macromolecules as determined by our plasmid DNA-DNA hybridization data in Table 2. As we mentioned previously, the percentage of total DNA that is plasmid is not an absolute value

640

CRESS AND KLINE

J. BACTERIOL. TABLE 4. Plasmid copy num bers

GeneraGene Strain

Total

Chromosomal

DNA/cell time onal (min)

(ug/109)

(Dg/cl0)

Replicatinga chromo-

somes/

Plasmid (Ag/1O')

DNA/cell

Minimum plasmid

copies!/ cell

Minimum plasmid copies/ repli-

cating some

CSH50 (F'lacpro+) pcn-24 (F'lacpro+)

40 40

11.7 13.4

11.19 11.16

1.91 1.90

aThe molecular weight of the resting chromosome was taken to be 2.5 x

because the F'lac in Proteus is probably not identical. The error is not likely to be large, however, because the calculated number of F'lac pro+ plasmids per replicating chromosome in the unmutated parent is very close to the values reported by others (6) who used a similar experimental approach and analysis. Also, the theoretical amount of F' DNA expected in a cell lysate, based on the molecular weight of the plasmid, is 5.8%. This agrees well with the measured value (Table 2). The pcn mutation does affect the level of other plasmid systems as determined by dye-CsCl analysis; however, it does not affect all plasmid systems. Although the studies on which this statement is based are still in progress (Cress and Kline, unpublished data), we should like to mention some of the key findings to enhance a fuller appreciation of the Pcn phenotype. Firstly, in pcn mutants -18, -19, and -24, the classic sex factor F appears to be maintained at normal levels, whereas in pcn mutants -21, -23A, and -24, the viral plasmid P1 CM (30) is maintained at about a two- to threefold higher level. Moreover, the percentage of CCC DNA of the F+ plasmid in the pcn cell lysates is invariant even when the recovery of total CCC DNA in dye-CsCl gradients is low. Also, the CCC form of the F+ DNA is not lost when crude lysates of F+ pcn-24 are incubated at 37 C. In contrast, the CCC P1 CM plasmid form does decrease when crude lysates of pcn lysogens 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 autonomous 80S CCC F plasmid population of an F+ culture. cosediments with its host's 1,800 and 3,200S species of folded chromosomes on neutral sucrose gradients. These findings have led to the conclusion that nonintegrated F plasmid is complex to the host chromosome. The possibility that membrane serves as the linking moiety between plasmid and chromosome is suspect because the 1,800S species of chromosome has

0.530 2.242

2.2 9.3

1.15

4.90

10' (7).

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 the pcn-24 mutant by the same experimental approach (22). However, since incubation at 25 C to generate membrane-released folded chromosomes might destroy a significant amount of the F' plasmids (Table 3), we analyzed lysates made at 0 C for the presence of free 110 and 72S plasmid DNA. To do this, we simply lysed the bacteria and pelleted the folded chromosomes onto a CsCl cushion located at the bottom of a 20 to 31% sucrose gradient while sedimenting the 110S material to the center of the gradient. The results in Fig. 2 show that the amount of free 110 and 72S DNA in pcn-24 is less than 0.5% of the total DNA in the gradient. The simplest interpretation of these results is that all the plasmid copies in pcn-24 are associated with the folded chromosome. Subsequent to these results we have found that membranereleased folded chromosomes can be isolated from pcn-24 without loss of plasmid DNA by lysis with Sarkosyl in 1 M NaCl at 25 C for 20 min (see above). When purified, 1,800S folded chromosomes from pcn-24 were tested by dye-CsCl analysis, and 5.3% of the DNA banded in the CCC peak. Analysis of DNA sedimenting in the 0 to 300S region of the preparative sucrose gradients used to purify the 1,800S folded chromosomes showed that less than 5.3% of this DNA is in the CCC form. These results indicate no enrichment for free 110S CCC plasmid DNA in lysates of pcn-24 and substantiate the results of Fig. 2. We conclude all the F' plasmid copies in pcn-24 are complexed to folded chromosomes. DISCUSSION Our results indicate that an F' plasmid can be stably maintained within a range of copy levels, not just the stringent state of one plasmid per chromosomal equivalent. This conclusion is based primarily on the increased levels of plasmid DNA in pcn mutants and the absence of

VOL. 125, 1976

PLASMID COPY NUMBER MUTANTS

641

tants that contain plasmids capable of being

0~ ~ ~

~

~~

2

I

10

20

FRACTION FIG. 2. Free F'lac pro+ DNA from pcn-24 in cell lysates containing membrane-released folded chromosomes. Cells were lysed with Brij-DOC as described in the text, and the supernatant from 4,000 x g centrifugation was mixed with differentially labeled F'lac pro+ DNA purified by dye-cesium chloride centrifugation. The mixture was sedimented through a 20 to 31% neutral sucrose density gradient of the same composition as in Fig. 1, except that the gradient was prepared over a 0.5-mI shelf of 35% sucrose containing cesium chloride (p 1.71). Centrifugation was for 55 min 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-24 =

lysate. any plasmid DNA in one mutant that is larger than unit size parental plasmid. Our genetic studies show that only chromosomally determined pcn mutants have been isolated. We do not know the map position of these mutations, or even whether only one chromosomal mutation gives rise to the Pcn phenotype. Since we have used nitrosoguanidine to induce pcn-type mutations, our isolates may well be multiply mutated. Plasmids in lysates made from pcn mutants lose most of their CCC DNA form via an unknown mechanism or agent that is activated by heat (Table 3). Since our original observations were made, we have found that stored pcn mutants give rise upon subculture and subsequent lysis to lysates that have much less plasmid-destroying activity. Nonetheless, the elevated levels of plasmid in these subcultures do not appear reduced. We cannot explain these observations at present. Further, we point out that the plasmid-destroying agent is uniquely observable only in lysates made from pcn mu-

elevated by the putative pcn gene products. Biochemical characterization of the plasmiddestroying agent is the subject of a future report (D. Cress and B. Kline, manuscript in preparation). Previous work by Kline and Miller (22) has demonstrated that the F plasmid, which is under stringent control of replication, is complexed in some noninte,grative fashion in the folded chromosomes of F+ bacterial hosts. This finding made it reasonable to expect that the Pcn phenotype might be an expression of mutants that are able to maintain plasmids in both complexed and noncomplexed states or in just the latter state. The results described in Fig. 2 show that all F' plasmids in the pcn-24 mutants are complexed to their host's folded chromosomes. We suspect the multiple-plasmids complex at just one chromosomal locus because F-pcn-24 progeny arise from pcn-24 (F'lac pro+) parents at the same or greater rates than F- progeny arise from isogenic F'pcn+ parents. In any event, the results of Fig. 2 indicate a deregulation of the plasmid copy number does not occur because the pcn-24 mutant can maintain its plasmids in the noncomplexed state. The ability to maintain multiple copies of homologous plasmid is very much analogous to an incompatibility breakdown. Normally, the incompatibility (inc) function of the host plasmid prevents stable inheritance of superinfecting, homologous plasmids. However, should the incompatibility function of the host plasmid be mutated and permit superinfection and stable maintenance of the incoming and resident plasmids, then in effect the inc mutation is pcn-like. In a somewhat comparable but not identical situation, San Blas et al. (31) have actually mutated bacterial host genes and successfully selected for a loss of plasmid incompatibility between an F'gal+ and F'his+ pair. In effect, they generated a pcn-like chromosomal mutation. Whether or not there is any biochemical or genetic similarity between their inc mutant and our pcn mutants is unknown. Finally, in closing, we point out that careful comparison of the results of Tables 2 and 3 will show that the dye-CsCl analytical technique is a poor quantitative tool. Nonetheless, it can be used in a screening procedure if one understands its limitations and does not relie on it for a definitive answer to plasmid levels in lysates of putative mutants. Clearly, of all the physical assays (including alkaline sucrose zonal centrifugation), only DNA-DNA hybridization assays

642

CRESS AND KLINE

do not seriously underestimate the true amount of plasmid DNA present in an extract. ACKNOWLEDGMENTS This work was supported by a University of' Tennessee predoctoral fellowship awarded to D. E. Cress and by Public Health Service research grant GM 18608 from the National Insititute of General Medical Sciences awarded to B. Kline.

LITERATURE CITED 1. Bazaral, M., and D. R. Helinski. 1968. Circular DNA forms of colicinogenic factors El, E2. E3 from Esche-

richia coli. J. Mol. Biol. 36:185-194. 2. Bazaral, M., and D. R. Helinski. 1970. Replication of a bacterial plasmid and an episome in Escherichia coli.

Biochemistry 9:399-406. 3. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. J. Citarella, and S. Falkow. 1969. Polynucleotide sequence relationships among members of Enterobacteriaceae. J. Bacteriol. 98:637-650. 4. Britten, R. J., and D. E. Kohne. 1966. Nucleotide sequence repetition in DNA. Carnegie Inst. Washington Yearb. 65:78-106. 5. Clewell, D., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166. 6. Collins, J., and R. H. Pritchard. 1973. Relationship between chromosome replication and F'lac episome replication in Escherichia coli. J. Mol. Biol. 78:143-155. 7. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of' Escherichia coli B/r. J. Mol. Biol. 31:519-540. 8. Crosa, J. H., D. J. Brenner, and S. Falkow. 1973. Use of single-strand specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexes. J. Bacteriol. 115:904-911. 9. Curtiss, R., III. L. G. Caro, D. P. Allison, and D. R. Stallions. 1969. Early stages of conjugation in Escherichia coli. J. Bacteriol. 100:1091-1104. 10. Cuzin, F.. and F. Jacob. 1967. Mutations de l'episome F d'Escherichia coli K-12. II. Mutants a replication thermosensible. Ann. Inst. Pasteur Paris 112:397-418. 11. Frame, R., and J. 0. Bishop. 1971. The number of sex-factors per chromosome in Escherichia coli. Biochem. J. 121:93-103. 12. Friesen, J. D. 1968. Measurement of DNA synthesis in bacterial cells, p. 625-640. In L. Grossman and K. Moldave (ed.), Methods in enzymology. vol. 12. part B. Academic Press Inc., New York. 13. Hathaway, B. G., and P. L. Bergquist. 1973. Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12. Mol. Gen. Genet. 127:297-306. 14. Helinski, D. R. 197:3. Plasmid determined resistance to antibiotics: molecular properties of' R-factors. Annu. Rev. Microbiol. 27:437-470. 15. Jacob, F.. S. Brenner, and F. Cuzin. 1963. On the regulation of DNA synthesis in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 16. Kahn, P., and D. R. Helinski. 1964. Relationship between colicinogenic factors El and V and an F factor in Escherichia coli. J. Bacteriol. 88:157:3-1579. 17. Kingsburgy, D. T., and D. R. Helinski. 197:3. Temperature-sensitive mutants for the replication of plasmids

J. BACTERIOL. in Escherichia coli. I. Isolation and specificity of' host and plasmid mutations. Genetics 74:1-16. 18. Kingsbury, D. T., D. G. Sleckmann, and D. R. Helinski. 1973. Temperature-sensitive mutants for the replication of' plasmids in Escherichia coli. II. Properties of' host and plasmid mutations. Genetics 74:17-31. 19. Kline, B. C., and D. R. Helinski. 1971. F, sex factor of' Escherichia coli size and purification in the form of' a strand-specific relaxation complex of supercoiled deoxyribonucleic acid and protein. Biochemistry 10:4975-4980. 20. Kline, B. C. 1973. Role of DNA transcription in the initiation of' Escherichia coli sex factor (f) DNA replication. Biochem. Biophys. Res. Commun. 50:280-287. 21. Kline, B. C. 1974. Mechanism and biosynthetic requirements for F plasmid replication in Escherichia coli. Biochemistry 13:134-146. 22. Kline, B. C., and J. R. Miller. 1975. Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physicochemical approach to studying the unit of segregation. J. Bacteriol. 121:165-172. 23. Kool, A. J., and J. J. Nijkamp. 1974. Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid CIO DF13. J. Bacteriol. 120:569-578. 24. Macrina, F. L., G. G. Weatherly, and R. Curtiss III. 1974. R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K12. J. Bacteriol. 120:1387-1400. 25. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory of Quantitative Biology, Cold Spring Harbor, N.Y. 26. Morris, C. G., H. Hashimoto, S. Mickel, and R. Round. 1974. Round of replication mutant of a drug resistance factor. J. Bacteriol. 118:855-866. 27. Nordstrom, K., L. Ingram, and A. Landback. 1972. Mutations in R factors of Escherichia coli causing an increased number of R factor copies per chromosome. J. Bacteriol. 110:562-569. 28. Pritchard, R. H., M. G. Chandler, and J. Collins. 1975. Independence of F replication and chromosome replication in Escherichia coli. Mol. Gen. Genet. 138:143-155. 29. Radloff, R., Bauer, W., and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed-circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. 30. Rosner, J. L. 1972. Formation, induction, and curing of bacteriophage PI lysogens. Virology 48:679-689. 31. San Blas, F., R. Thompson, and P. Broda. 1974. An Escherichia coli K12 mutant apparently carrying two autonomous F-prime factors. Mol. Gen. Genet. 130: 153-16:3. 32. Sharp, P. A., M.-T. Hsu, E. Ohtsubo, and N. Davidson. 1972. Electron nmicroscope heteroduplex studies of' sequence relations among plasmids of Escherichia coli. I. Structure of' F-prime factors. J. Mol. Biol. 71:471-497. 33. Sutton, W. D. 1971. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240:522-531. 34. Worcel, A., and E. Burgi. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 71:127-147. 35. Worcel, A., and E. Burgi. 1974. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:91-105.