Moi Gen Genet (1984) 193:349 357 © Springer-Verlag 1984

Genetic Structure and Stability of a Copy Number Mutant of IncFI Group Plasmid ColV-K94 inEscherichia cob" K-12 Gopa Mitra and Sunil Palchaudhuri Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA

Summary. Mutants pWS10, pWSll and pWS12 were derived from an IncFI group plasmid ColV-K94 by the insertion of a transposon Tn903 (Kmr). These plasmids were all approximately 130 kb in length. The plasmid pWS12 resembled the wild type ColV-K94 in transmissibility, incompatibility and stable maintenance. Cells harboring pWSI 1 were poor conjugal donors but resistant to the same level of kanamycin as pWS12 containing hosts. In contrast, pWS10 conferred a higher resistance to kanamycin and exhibited reduced incompatibility properties in comparison with pWS12. The higher drug resistance associated with pWS10 appeared to be a consequence of an increase in its copy number and the generation of miniplasmids of varying sizes. Electron microscope analysis of the copy mutant pWS 10 revealed that Tn903 was located at a site adjacent to a region 32.6F to 35.3F. The latter region appears to be the primary replicon of ColV-K94 and is homologous with the secondary replicon of F. The insertional mutagenesis with Tn903 brought about an extensive DNA rearrangement including the duplication and translocation of the stems of two inverted repeat structures. The DNA alterations of pWS10 were distinguishable through comparison of its E c o R I digestion patterns with those of pWS11 and pWSI2.

Introduction Viruses and plasmids can replicate as autonomous replicons, physically separate from the chromosome. In steady state growth, plasmid DNA doubles at the same rate as chromosomal DNA. Over-replication occurs when the number of plasmid copies is increasing to its steady value. Transposons and insertion sequences forming a portion of the plasmid are not known to replicate autonomously but there are duplicate copies whenever these elements appear at new locations. This is a form of over-replication which, if unchecked, would result in a gradual increase in the abundance of transposable elements in the genome. Transposons (Tn) with insertion sequences (IS) at their termini have been frequently used as biological mutagens (Starlinger 1980) and are also capable of causing interference in the normal functioning of the host genome. The major outcome of the stringent control of plasmid replication is the maintenance of defined number of copies of the plasmid per chroOffprint requests to: S. Palchaudhuri

mosome equivalent. Copy number mutants have been isolated from the related members of the IncFII group plasmids R6, R100 and R1 (Morris et al. 1974, Uhlin and Nordstr6m 1975; Timmis et al. 1981). Analysis of these copy mutants has revealed the generation of miniplasmids in vivo (Goebel and Bonewald 1975; Mickel and Bauer 1976). Electron microscope heteroduplex analysis of the miniplasmids indicates an involvement of Tn-mediated deletions and transpositions for such generation of miniplasmids (Ohtsubo et al. 1978). As most of the copy number mutants have been shown to possess altered incompatibility properties (the inability of the two related plasmids to coexist stably in a bacterial host cell in absence of any selection pressure), a close functional relationship has been postulated between the incompatibility and copy number control genes (Molin and Nordstr6m 1980). Furthermore, the copy control genes are closely linked to the origin of replication. For IncFI group plasmids, copy number mutants have been isolated (Manis and Kline 1978) from mini-F (the 9 kb selfreplicating EcoRI fragment of F). Mini-F, like F, has stringently controlled replication, is stably maintained and is cured by acridine orange. Three loci termed incB (45.1-45.8F), incC (45.8-46.4F) and incD (47.6-49.4F) which are responsible for IncFI incompatibility have been mapped on mini-F (Kahn et al. 1979; Lane 1981) and mutations in the incB incC region are found to be associated with elevated copy number and altered incompatibility properties (Manis and Kline 1978). It has been proposed that a repressor coded by the incB incC region might be responsible for the regulation of copy number as well as incompatibility properties. In a more recent work, Tolun and Helinski (1981) have indicated the presence of a 22 base pair repeat sequence at the incC locus rather than gene expression in incC incompatibility. However, detailed studies of copy number mutants have not been done with IncFI group plasmids. The plasmid ColV-K94 has been shown to be related to other F-like plasmids on the basis of DNA sequence homology as studied by electron microscope-heteroduplex analysis (Palchaudhuri and Maas 1977; Sharp et al. 1973). In addition to the homology in transfer regions, homology in the incD region 47.6F-49.4F and incE region 32.6F35.4F, between F and ColV-K94 have been established (Lane 1981). Here we report the isolation and characterization of an incompatibility deficient, copy number mutant of plasmid ColV-K94 by mutagenic insertion of the kanamycin resistance transposon, Tn903. This plasmid mutant

350 Table l. Bacterial strains and plasmids

Strains

Plasmid

Chromosomal/plasmid genotype

Reference

JC41 t

ColV-K94

argG his leu metB lac str-309 R17s

D. Helinski

thi-I ilv arg met recA Sm~ thr-l, leuB6, thi-1, argE3 his-4, proA2, recA13, lacY galK2, mtl-1, xyl-5, ara-16 strA31, tsx-33, 2supE66 Same as AB2463 B1, thr leu B1 thr leu argG, metB, hisG, leuB6, reeA mtl-2, xyl-7, malA, gal6, lacY strA 104, tonA2, tsx-1, supE44 arg met leu his Sm ~ his, argG, metB, leu, thr, pro xyl, thy, lacY, strA, polAts214 B1, leu, metB, his, argG, lac B1 thr leu metE thy polAts polA12 thy rha lac strA

Palchaudhuri et al. (1976) Palchaudhuri et al. (1976)

AT753 AB2463

AB2463 C600 C600 r - m JC1553

KLF1 (F"thr +leu +) pML21 (ColE1-Km~)

JC1533 BK202

KLF15 (F'his +)

MA123 C600 r-m JGtll MM383

has been found to be of one size, generates miniplasmids and replicates stably. The site of insertion of Tn903 on the plasmid has been located by EM-heteroduplex analysis and the relationship of the insertion stimulated D N A sequence rearrangements to the mutant phenotypes of the plasmid has been analyzed. Materials and Methods

Bacterial strains and plasmids utilized in this work are listed in Table 1. The media used included TYE, M-9, LB medium, minimal medium (Palchaudhuri et al. 1976) supplemented with thymine and specific amino acids and Penassay base agar (1.5%). Kanamycin (Km) was added to the media at the concentration of 80 gg/ml. Beef-extract broth, pH 7.6 (Hirota 1960) was used for cultures and dilution in acridine orange experiments. For plasmid isolation, E. coli strains were grown in M-9 minimal medium with the following modifications: casamino acid (CAA), 3 mg/ml; thymine, 50 gg/ml; B1, 50 lag/ml. Biological and Genetic Techniques. Methods for the testing of phenotypes, conjugal transfer, acridine orange (AO) treatment and determination of sensitivity to phage R17 have been described previously (Palchaudhuri et al. 1976). When plasmid containing strains were used as recipients, they were converted into "phenocopy" by growing for 27 h to late stationary phase. When one of the mating pairs was a colicin producing strain, a filter-disc method of mating was used. Log phase cultures of the colicin producing strain were transferred onto a sterile filter disc, washed with 50 ml of L-broth and then mixed with recipient cells on the disc. The disc was asceptically placed on L-agar medium, incubated for i h at 37° C, resuspended in sterile saline and spread on selective plates. Isolation o f Mutants. The IncF1 group transmissible plasmid ColV-K94 lacks genetic markers suitable for genetic

D. Helinski S.N. Cohen A. Bachmann

B. Bachmann B Kline W. Maas S.N. Cohen B. Bachmann Monk and Kinross (1972)

selection and incompatibility testing. Genetic labeling of ColV with a kanamycin transposon, Tn903 was carried out. The plasmid mini-ColE1 carrying Tn903, referred to as pML21 (Lovett and Helinski 1976), was introduced by transformation into an E. coli K12 recA strain, AT753, already carrying ColV-K94. Purified transconjugants were tested for the stable coexistence of pML21 (Km r) and ColV (R17S). The resulting strain carrying both CoW and pML21 was used as a donor in mating with an E. coli K12 points recipient BK202. Donor and recipient cultures, grown to log phase in L-broth at 32° C and 37° C respectively, were mixed in a ratio of 1:5 and mating was carried out at 42 ° C for I h. Mating mixture was plated on selective media containing kanamycin (60 gg/ml) and lacking some of the metabolic requirements of the donor. The plates were incubated at 42 ° C. Since replication of the ColE1 plasmid (pML21) is polA1 dependent, it did not survive at 42 ° C in BK202. A few BK202 (Km r) transconjugants obtained following this mating presumably resulted from the transposition of Tn903 into ColV-K94. This insertion of Tn903 (Km r) into ColV was further confirmed by the 100% conjugal cotransfer of ColV and Km r character into another E. coli recA recipient AB2463, As a control experiment, BK202 was transformed with pure pML21 DNA and Km r transformants were selected at 32° C and 42 ° C on L-agar plates supplemented with thymine (20 lag/ml) and kanamycin (80 gg/ml). At 32° C, the number of transformants obtained was 2.5 x 10~ whereas no transformant appeared at 42 ° C. Other available polA mutant like MM383 and J G l l l (Monk and Kinross 1972) under identical conditions, produced a few Km r transformants even at 42 ° C and therefore were not used in this work. Furthermore, Km r transformants of BK202 obtained at 32 ° C were cured of plasmids pML21 (ColE1-Km r) following its growth at 42 ° C. Colicin V Assay. ColicinV was assayed according to the method described by Binns et al. (1979).

351

Incompatibility Assay. In most cases, the test was performed by superinfecting a recA host carrying a resident plasmid with the plasmid to be tested. If the incoming and resident plasmids are incompatible, then selection for the establishment of the incoming plasmid should result in the loss of the resident plasmid. Incompatibility relationships between plasmids were determined as follows: twenty transconjugants selected for the markers borne by incoming plasmids were purified, grown to saturation in L-broth without antibiotic and spread on L-agar. Isolated colonies (100) from each plate were then replicated onto test media to determine the presence of the markers borne by incoming and resident plasmids. Incompatibility test was also carried out with transconjugants selected for the markers of both the resident and incoming plasmids. In that case, occasionally transposition of Tn903 (Km r) from pWS plasmids to the test plasmid or to the chromosome occurred under incompatibility pressure as was observed in a previous study (Palchaudhuri and Mitra •98•). Due to the absence of scorable marker on ColV-K94, the incompatibility test was accomplished only in one direction. Transconjugants were selected for the markers of incoming F's. In each mating with F', 20 pure transconjugants were grown in nonselective media and tested for colicin production indicative of the presence of ColV-K94.

Plasmid Copy Number Determinations. Copy number measurements of pWS10 and pWS12 were done by estimation of the amounts of radioactivity in covalently closed circular DNA when analyzed in dye-buoyant density gradients as described by Womble et al. (1977). This is a minimal estimate since with the D N A of this size a substantial amount of D N A may be nicked. Cells growing exponentially in 15 ml of M-9 minimal medium containing 250 gg deoxyadenosine per ml were labeled with 150 gl of [3H]-thymidine (Sp. activity ~ 6 Ci/mMol) by growing for 3 generations, lysed and centrifuged to equilibrium in a CsC1-EtBr density gradient in a fixed angle rotor 50 at 39,000 rpm for 40 h at 15° C. Both plasmid and chromosomal bands were well separated and visible in ultraviolet radiation and therefore that portion of the CsCl-gradient comprising of the visible bands was collected without cross-contamination. Six-drop fractions were collected from the bottom of the tube directly on 3 MM Whatman filter disc, washed and counted for the radioactivity as described previously (Palchaudhuri and Iyer •97•).

Preparation ofDNA. Plasmid DNAs (CoW or related plasraids) were prepared from 21 cultures grown to late log phase in modified M-9 medium following the protocol described previously (Palchaudhuri et al. 1976). The plasmid D N A in CsCl-ethidium bromide solution was extracted three times with CsCl-saturated isopropanol and dialyzed against the TEN buffer (10mM Tris-hydrochloride, 1 mM EDTA, 3 mM NaC1 pH 7.5). For identification of the plasmid D N A on agarose gels, the D N A fi'om lysates of transformants or transconjugants were prepared by a rapid lysis technique (Stougaard et al. 1979). Manipulation of Plasmid DNA. Endonuclease (EcoRI) digestion reactions were carried out for 1 h at 37 ° C in 20 gl volume containing approximately 0.5 gg D N A in the following incubation mixture: 100 mM Tris-HC1 (pH 7.5),

50 mM NaC1, 10 mM MgC12 and 2 units of EcolRI per gg/DNA. Fragments were analyzed by electrophoresis through vertical slab gels containing 0.7% w/v agarose following the method of So et al. (1978). Recombinant plasraids were constructed in vitro by ligation of endonuclease generated fragments with T4 ligase at a concentration of 1 to 2 U/ml at 4 ° C. D N A concentrations was 25 gg/ml. Transformation of E. coli with plasmid D N A was done as described by Cohen et al. (1972).

Electron Microscope Homoduplexing and Heteroduplexing. Molecular lengths of duplex open circle plasmid D N A were obtained by length measurements of molecules spread by the aqueous basic protein film technique (Davis et al. 1971). Self-hybridized molecules and heteroduplexes were prepared from electron microscopy as described by Palchaudhuri and Maas (1977). The length of double stranded (or single stranded) ColV D N A was calculated from the ratio between ColV D N A and pRS5 DNA. pRS5 is a composite plasmid consisting of F-DNA, 32.6F-68.1F joined to pSC101 DNA of length 9.3 kb. The length of pRS5 D N A was assumed to be 44.8 kb (Palchaudhuri and Maas •977). Results

Characterization of ColV-Km r Derivatives Eight independent ColV-Km r transconjugants were tested for their incompatibility properties, resistance to kanamycin, self-transmissibility, R17 sensitivity and acridine orange curing. Two stable F' plasmids, K L F I (F'thr+leu+)and KLF15 (F'his +) were used as test plasmids and found to be as incompatible with seven ColV-Km r plasmids as with the parental ColV. One (ColV # 5) showed reduced incompatibility. Surprisingly, five of the seven of these Kmr-ColV derivatives were R17 R and showed very low frequency of transfer suggesting some alterations in transfer ability of the plasmid (Table 2). Table 2. Genetic characters of ColV-Kmr plasmids ColV-Kmr IncomKanamycin RI7 Transmissibilityc isolates/ patibility resistanceb sensi- (Frequency of BK202 index tivity recipients carrying donor plasmids per donor) ColV-K94 (wild type) 1 2 43 5 6 7 8

+

sensitive

S

2 x 10-1

+ + + + _a +

l ow l ow low low very h i g h l ow l ow l ow

R S R R S R R S

2 9 1 2 1.4 4 6 2

+

+

x x × x x x x x

10 7 10 . 2 10 -7 10 -8 10-1 10 - v 10 -7 10 -1

a Selectingfor IncFI plasmid KLF1 (F'thr+leu+) as incoming plasmid, ColV-Kmr isolates # 5 (pWS10) showed stable coexistence in 70% cases. The incompatibility index was measured by the method of Palchaudhuri et al. (1976) b "Very high" means survival of 100% cells at 400 lag kanamycin per ml and "low" means survival at 250 pg/ml or lower concentration of the drug c In transfer experiments, ColV-Kmr derivatives in BK202 strain were donors and JC1553 recA was the recipient

352 We selected ColV # 5, Colg # 3, and ColV # 8 designated as pWS10, pWS11, and pWS12 respectively as representative of classes of mutants obtained by the insertion of Tn903. Plasmid pWS12 behaved like wild type ColV with respect to incompatibility, transmissibility and R17 sensitivity, while pWS11 was R17 resistant and conjugation deficient. Plasmid pWS10 showed reduced incompatibility towards the test plasmid, and a higher resistance level to kanamycin compared to the properties in pWS12 and pWS/1, indicating a gene-dosage effect. We therefore decided to attempt a more quantitative analysis of these plasraids including an assay of the possibly higher copy number of pWS 10.

A

B

C

D

--ccc DNA, Colv. -Mini Plasmids ,-Chromosome

Copy Number Determination The relative amount of plasmid D N A in covalently closed circles (ccc) per chromosome of the host cell was determined for plasmids pWS/0 and pWS12 by agarose gel elctrophoresis and dye-cesium chloride density gradient centrifugation as described in Materials and Methods. As a preliminary study, plasmid-specific bands were identified on agarose gel after isolating the D N A from the isogenic recA strains of E. coli carrying plasmids, pWS10, pWSll, and pWS12 (control). Host cells were grown in modified M-9 medium to stationary phase and the plasmidenriched lysate was prepared by a modified lysis technique. Gel patterns showed a much higher yield for the pWS10 D N A in comparison to other K m r derivatives of ColV-K94 (Fig. 1), although all the strains were grown to same optical density. The yield of pWS10 D N A was measured by the band intensity supported the previous inference that the pWS10 was a copy mutant of ColV-K94. In the lysates of AB2463 (pWS10) several bands were seen moving much faster than pWS10 as indicated by arrows in Fig. 1. We designate these as "miniplasmids". In all cell lysates the chromosomal bands as marked in Figure 1 travelled a fixed distance and were confirmed by comparison with the chromosomal band isolated from a plasmid-less strain AB2463. In order to confirm that the observed differences between strains (Fig. 1) was not due to the variable recovery, these isolations were performed four times. Plasmid copy number was also estimated by ethidium bromide-cesium chloride density gradient centrifugation from the log phase cultures of AB2463, AB2463 (pWSI0) and AB2463 (pWSI2) labeled with 3H-thymidine as described in Materials and Methods. Results are shown in Table 3. The results show that the copy number of pWS10 is approximately 2.25 fold greater than that of pWS12 which exists a s / - 2 copies per cell.

Fig. 1. Electrophoresis of DNA from cleared lysates in 0.7% agarose gel: (A) AB2463, (B) AB2463 (pWS12), (C) AB2463 (pWSI 1), (D) AB2463 (pWS/0). Arrow indicates the position of miniplasmids and chromosomes Table 3. Copy number measurement of pWSl0 and pWSl2 by Dye-cesium chloride density gradient centrifugation Plasmid

Percentage of plasmid-DNA in ccc f o r m a

Copy number b

pWS12 pWSI0

3.05 (2) 6.21 (3)

0.89 2.01

a Percentage of plasmid is calculated from dye-CsC1 gradients as (counts per minute in covalently closed DNA) x 100 + (counts per minute in linear DNA) b Copy number per chromosomal equivalent = percentage of plasmid + 100 x (chromosomal mass/plasmid mass). Values given are the averages of the number of determinations given in parenthesis

A

F

Mini P[asmids

B

C

D

-Chromosome

\

Transformation orE. coli K-12 by a Copy Mutant and Its Miniplasmids In order to confirm that the mutation in the pWS t 0 plasmid is responsible for the production of miniplasmids, the pure 130 kb molecule and mixture of miniplasmids were separately used to transform a suitable recipient, C600 r - m - . The transformation with the 130 kb pWS10 molecule resulted in the reappearance of miniplasmids as determined by agarose gel electrophoresis (Fig. 2, Lane A). Miniplasmids were not seen in the lysates prepared from log phase cultures ofAB2463 (pWSI0) (Fig. 2, Lane B). Only plasmid of 40 kb size was recovered from all the K m r transformants

Fig. 2. Electrophoresis of DNA from cleared lysates in 0.7% agarose gel: (A) pWS10 DNA and miniplasmids from Km r transformant obtained by transformation of C600 r - m - with pure pWS10 DNA, (B) pWS10 DNA from log phase culture of AB2463 (pWSt0), (C) AB2463 (KLFI), (D) Miniplasmids (40 kb) from Kmr transformant obtained by the transformation of C600 r - m with DNA consisting of a mixture of miniplasmids (8 kb-40 kb) only

353 obtained with the miniplasmid D N A (8-40 kb) and a representative result is shown in Fig. 2, Lane D. Because of the selection for kanamycin resistant transformants, miniplasmids which do not carry the Km r could not be detected in this study.

Further Studies of Incompatibility The incompatibility behavior of the kanamycin resistant CoW plasmid derivatives, pWS10 and pWS12 was examined by testing with two different F' plasmids KLF1 (F'thr+leu +) and KLF15 (F'his+). A donor recA strain carrying either KLF1 or K L F I 5 was mated with a recA strain carrying the pWS plasmid in "phenocopy" after which the markers of the incoming F' plasmid were selected. The results are shown in Table 4. In the case of pWS12, the transconjugants were found to be carrying the incoming F' plasmid only and the resident pWS plasmid was eliminated. However, when pWSI0 was the resident plasmid in phenocopied cells, the transconjugants were also selected for the markers of both the resident and incoming plasmids. The transconjugants were grown to log phase in L-broth and the examination by agarose gel electrophoresis revealed the presence of both K L F I and pWS10 D N A molecules. The test for the incompatibility was repeated in opposite direction using AB2463 recA (pWS plasmids) as the donor strains and recA strains carrying either KLF15 or KLF1 in "phenocopy" as the recipients. Selection was made for the kanamycin resistance genes on pWS plasmids. For pWS12 (control) no detectable change in incompatibility with KLF15 or KLF1 was observed but for pWSI0, reduced incompatibility was observed against F' (Table 4).

Table 4. Quantitative incompatibility test of plasmids pWS10 and pWSt2 ~ Incoming plasmid

Resident plasmid

KLFI KLF15 pWSI2 pWS10 pWSI2 pWS10 KLF15 KLFt 5 KLFI KLF1 KLF1 KLF15

% Colonies Carrying

KLF15 KLFI KLFI5 KLFI 5 KLF1 KLF1 pWS12 pWSI 0 pWSI2 pWS10 ColV-K94 ColV-K94

Incoming plasmid

Resident plasmid

96 90 98 92 98 96 98 98 99 91 99 99

4 8 0 60 2 48 2 96 3 70 0 2

For incompatibility testing recA host strains (donors and recipients) were always used and the transconjugants were selected for the markers of the incoming plasmids. In each case, 20 pure transconjugants were tested for the incompatibility as described in Materials and Methods

a

A

B

C

D

Acridine Orange Sensitivity In contrast to the results of others ,(Kahn and Helinski 1964) we found ColV-K94 and its Km r derivatives were not curable by acridine orange (AO). An E. coli K-12 recA + strain (MA123) carrying KLF1 or KLF15 showed 95%-100% curing of these F's at subinhibitory concentrations of the drug (final conc. 35 gg/ml). An isogenic CoWK94 strain was not curable under identical conditions and a variety of different conditions of AO treatment. Usually only 1%-2% of these cells showed a loss of ColV-K94 in AO medium (pH 7.6). However, repeated dilutions of stationary phase cultures of JC411 (ColV-K94) to 104 per ml and their subsequent growth to saturation resulted in a higher percentage of CoW-less cells even in the absence of AO.

1

2\ 3~

~567 -8910-

Analysis of Restriction Enzyme (EcoRl) Digestion Pattern of ColV-K94

Fig. 3. EcoRI restriction patterns of ColV-K94 and its Kmr derivatives in 0.7% agarose gel: (A) pWSlt DNA, (B) pWSI2 DNA, (C) ColV-K94 DNA and (D) pWS10 DNA. In all cases eight bands (bands 2 and 3 are overlapped) occupy identical positions except in D, where bands I and 4 have been shifted. Several faint bands which seem to have originated from miniplasmids present in the pWS10 DNA preparation are also seen in Lane D

As shown above, the phenotype of pWS12 was found to be essentially similar to that of ColV-K94, in spite of its Tn903 insertion. Figure 3 shows that the EcoRI restriction patterns of pWS12 are also indistinguishable from those of original ColV. The presence of the Tn903 transposon in pWS12 is, nevertheless, clear from the 100% co-transfer of Km r and ColV-K94. EM analysis also confirmed that purified pWS12 D N A showed the expected lollipop structure, 1 kb stem flanking a 1 kb loop, upon its self hybridization (next section). EcoRI digests of ColV-K94, pWS10 and pWS12 contained 12 fragments ranging from 0.5 kb to 31 kb of which 10 bands are in Fig. 3. The sum of their

molecular lengths was approximately 128 kb, in close agreement with estimate of 130 k b + 3 kb obtained from the pWS12 by electron microscopy. In the case of the copy mutant pWS10, ColV-K94 fragments I and 4 were replaced by larger fragments of between 18.5 kb and 31 kb (Fig. 3). As is noted below, we believe this is a consequence of incorporation of 3 kb transposon, Tn903 within the region of ColV-K94 encompassed by fragments I and 4. Several indistinct bands which seemed to have originated from the EcoRI digestion of "miniplasmids" were also observed (Fig. 3, Lane D).

354

Xl

X2

1.4 ~X~

b[••0.7

X3 36 pWS 10

<]

A

-

/F

58F4

,co, v

A

A

5 5 ~ n9 0 ~ 1 0 . 7 3

Tronsfer genes 1.4 OX~.,~Tn 903

X2

1 1 2 3 ( 22,6.

12

6

12

1.9

275

pWS 12 (Col V- K94: :-In 9o3)

46.8E ~ \'WW qv~ 0.7 1.8 0.7 4.8 0.7 X3

59.1

[4_ X1 ~.2

1.4

( t1.2×,

30

1.2

6

1.2 10.7 , qVV¢,

"~

pWS10

60

T

/

pWS 10 36

B

c1~07

16

0.7 1.2 1.8 0.7 4.8 ~.,)-Tn 903

~~,-~Tn

1.L X1 C

pWS 10

903

30

3.5

Fig. 4A-C. Schematic representation of Co1V-K94/F heteroduplex (A), lengths, location and orientations of inverted repeats in single strands of pWSI2 (B) and pWS10 (C). The copies of the stem 1.2 kb are designated as a, b, d, g and the stems 0.7 kb are labeled as c, e, f, h respectively in order to specify the interactions which resulted three different structures during self hybridization as shown in Fig. 5. For the comparison, the identical letter designations have been used in both Fig. 4C and 5A, 5B and 5C

Fig. $A-C. Schematic representation of three structures resulting from the possible interactions occurring between duplicated regions of 1.2 kb DNA sequence as well as between duplicated 0.7 kb DNA sequence as shown in Fig. 4 B. All these structures have been verified by electron microscopy. One photomicrograph confirming one of these structures (A) is shown in Fig. 6

Characterization of pWS12 and pWSlO by Electron Microscopy

A sample of circular D N A isolated from AB2463 (colVK94), AB2463 (pWS12) or AB2463 (pWS10) was denatured and self renatured. There is no difference between the self hybridized molecule o f ColV-K94 and its K m r derivative, p W S I 2 in terms of number, arrangement and order o f these transposon like structures except the plasmid pWS12 contains the transposon Tn903 (Kmr). Figure 4B represents a drawing of the molecule pWS12 with all three locations o f structures x 1, x 2 and x a. The transposon Tn903 (1 kb loop and i kb stem) is located at a site 1.9 kb away from the xl structure. F o r the purpose of comparison, we have included the final results of the structures of copy m u t a n t p W S I 0 in Fig. 4C. The stem-loop structure of x 1 is maintained b u t the number, location and orientation o f the 1.2 kb inverted repeat sequence o f x 2 and 0.7 kb inverted repeat sequence of x 3 are rearranged. The structures which can arise from such duplications and altered orientations o f the stems have been visualized by electron microscopy. These results are shown in Fig. 5. These diagrams (Fig. 5) were actually the self-hybridized molecules o f pWS10 and were analyzed by EM. One representative micrograph is shown in Fig. 6. In

As shown by previous investigators (20, 25) the self hybridized molecule of ColV-K94 contains three stem-loop structures. In order to indicate the extent of h o m o l o g y o f ColVK94 with F molecule and the coordinates of the stem-loop structures with reference to those h o m o l o g o u s regions, the ColV,I K 9 4 / F heteroduplex molecule is redrawn from previous works (20, 25) and presented in Fig. 4A. The stemloop structure x~ consisting o f a 1.2 kb stem and the 1.46 kb loop is seen at 94.5F corresponding to 98.6 V position on ColV; then the second stem loop structure x 2 consists o f a 1.2 kb stem and 5.6 kb loop and is located 24.6 kb away from the xx structure. The third structure x 3 is 27.0 kb clockwise from the x 2 structure. Thus, the x~ and x 2 structures have stems o f equal lengths. The third structure x 3 forms a 7.6 kb loop and another structure, referred to as x 3 forms a 4.6 kb loop but each structure seen has an identical stalk size of 0.7 kb. W e think this means that the D N A sequence forming stalk is repeated three times with intervening distances 2.3 kb and 4.6 kb respectively (Fig. 4B).

355

Fig. 6. A self-hybridized molecule of

pWS10. Lengths of the inverted repeats are indicated. The bar represents 1 kb fact, Fig. 5A is drawn on the basis of this micrograph. It shows a stem-loop structure of xl, the transposon Tn903 and two double stranded stems (0.7 kb and 1.2 kb). In the final structure as presented in Fig. 4C if the interactions occurring between b and g pair of 1.2 kb repeat sequences and between c and f copies of 0.7 kb repeat sequences, one will visualize the structure as presented in Fig. 5. The copies of 1.2 kb sequence are designated as a, b, d, g and the copies of 0.7 kb sequences as c, e, f, h, respectively in both figures 4C and 5. Finally, the pWS10 was hybridized with a cloned fragment of F, 32.6F-68.1F. In the heteroduplex molecule there are two regions of homology separated by a substitution loop. These homologous regions, 32.6F35.4F and 46.8F-68.1F resemble our earlier patterns observed in the heteroduplexes formed between F and ColVK94 (Palchaudhuri and Maas 1977). Thus the position of Tn903 was found to be I kb away from the 35.4F end of the 32.6F-35.4F region.

Cloning of Replication Region of pWSlO By comparison with the published works of copy mutant plasmids belonging to both IncFI group and IncFII group we assume that in pWSI0 the transposon Tn903 has also inserted into a copy control gene and is linked to the origin of replication. The plasmid pWS10 was, therefore, cleaved with EcoRI, treated with ligase and then used directly to transform the strain AB2463 recA to kanamycin resistance.

Transformants that were K m r were found to contain a single plasmid (pWS14) consisting of the Tn903 (Km r) and the ColV sequence of length 16 kb including the 32.6F35.4F region and the x 3 structure. The plasmid pWSI4 was completely stable. The incompatibility behavior of this plasmid with the incoming F' plasmid KLF1 was similar to that of its parent, pWSI0. However, the plasmid pWS14 was not highly stable upon its transfer into a polA strain BK202. The plasmid D N A isolated from the stationary phase cultures of AB2463 (pWS14) contained another smaller plasmid, pWS14-1 (8.0kb__0.5 kb). The self-hybridization of pWS14-1 and its analysis by EM revealed the presence of Tng03 and the ColV D N A (5.5 kb) including the 32.6F-35.4F region. Surprisingly, miniplasmid pWS14 and its deletion derivative pWS14-1 carry a common region of 3.8 kb encompassing the secondary replicon of F, 32.6F-35.4F. In addition to this, the insertion of Tn903 (km r) at a site 1.0 kb counterclockwise from 35.4F end induced the duplication and translocation of 0.7 kb and 1.2 kb stem sequences and in turn generated several miniplasmids.

Discussion

The plasmid pWSI0, obtained by the insertion of Tn903 (Km r) into ColV-K94, shows an increased level of kanamycin resistance, increased copy number, altered incompatibil-

356 ity behavior and generation of miniplasmids. In contrast to pWS10, the plasmid pWS12, genetically resembles the original ColV-K94 except for insertion of Tn903. Heteroduplex analysis shows sequence homology between the F-derivative pRS5 and ColV-K94 in two different areas, 32.6F-35.4F and 46.8F-94.4F (Palchaudhuri and Mass 1977). N o homology in the primary replicon of F (42.5F-46.7F) and ColV-K94 has been observed. More recently, 32.6F-35.4F has been characterized as secondary replication region of F by Lane and Gardner (1979). Besides being highly unstable, the replication of this secondary replicon is dependent on D N A polymerase I activity and maintenance is not sensitive to acridine orange. By EM-heteroduplex analysis, insertion of Tn903 in pWS10 has been found to be at a site closely linked to the region 32.6F35.4F of ColV-K94. Moreover, the replication of ColV-K94 also seems to be partially polA dependent as judged by the unstable maintenance of pWS14 in BK202 strain at 4 2 ° C and its maintenance is not affected by acridine orange. It has been observed that for both the incFI and incFII group plasmids, the genes controlling the copy number phenotype were located in the vicinity of the replication origin (Lane 1981 ; Molin et al. 1979). Similarly, Manis and Kline (1978) found that the insertion of Tn3 into primary replicon of F resulted in a five-fold increase in copy number. Hence, it is likely that the elevated copy number o f pWS10 is due to the insertion of Tn903 at a site adjacent to its replication genes. As has been previously noted, ColV-K94 contains a region, 46.8F-49.4F, c o m m o n to plasmid of I n c F I group (Palchaudhuri and Maas 1977). Recently the presence of incB incC regions in ColV-K94 has been suggested by hybridization of the 43.9-46.9 kb segment of mini-F with restriction fragments of the ColV plasmid (Lane 1981). Since only a partial homology in this 3 kb segment also can give rise to hybridization bands, it appears that in addition to incD, ColV-K94 may contain the part of incC region. F r o m the results of self-hybridization analysis of pWS10, it appears that the 1.2 kb stem of transposon like structure x 2 has been acting as an independent ' I S ' element analogous to the IS10 sequence of T n l 0 element (Kopecko et al. 1976). An additional copy of the inverted repeat (1.2 kb) has been inserted into a gene between 46.8F and the 0.7 kb stem of another transposon like structure x 3 (Fig. 4C). The exact mechanism of cross-over between the 0.7 kb stem segment and the 1.2 kb stem segment is at present unknown and could account for the inactivation of incC, and hence, the elevated copy number and altered incompatibility behavior of pWSl0. It has been known that acridine orange selectively inhibits F replication (Hirota 1960) and Wechsler and Kline (1980) find that incC and acridine orange sensitivity loci overlap. F r o m our results it appears that neither ColV-K94 nor its K m r derivatives are curable by acridine orange. This may be due to the fact that the primary replicon of ColVK94 (which is secondary for F) is not sensitive to acridine orange or that the part of incC which is present in C o W is not sufficient and functional F replication origin is essential for AO curing (Palchaudhuri and Mitra 1981). This work suggests two other important mechanisms controlling the stability of large plasmids: (a) the product ( R N A or protein) of the cop gene checks the over-replication of IS elements and consequently (b) the D N A rearrangements

including duplication, translocation and deletion of Tn elements generate miniplasmids. Acknowledgements. We thank Dr. Helinski and B. Kline for the

strains JC411 (ColV-K94) and BK202. We thank U. Syrowik and N. Kar for the technical assistance. This work was supported by a grant from the American Cancer Society (# NP333). References

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Communicated by G.A. O ' D o n o v a n

Received September 7, 1982 / June 27, 1983