Multimerization and replication of plasmid pBP11

PLASMID

8,

126-140 (1982)

Multimerization FRIEDRICH Institut fiir

Medizinische

SCHMIDT, Mikrobiologie

and Replication ULRICH

VAN

TREECK,

und Immunologie

of Plasmid pBP1 1 AND

der Universittit

BERND

WIEDEMANN

Bonn. 5300 Bonn, West Germany

Received December 2, 1981 The plasmid pBPl1 has a size of 26,400 nucleotides and carries genes for resistance to ampicillin and sulfonamides but lacks Tra functions. By conjugational transfer it was derived from R-factor R 1767, which confers multiresistance in Salmonella typhimurium. A physical map was constructed by employing restriction enzyme analysis combined with fragment cloning and characterization of homoduplex molecules in the electron microscope. pBPl1 exhibits a symmetrical structure with unique and duplicated segments, the latter ones coding for resistance. Replication occurs in a unidirectional mode from three independent origins of replication. Multimeric forms of pBPl1 were produced with a constant frequency-a process which appeared to be reversible and recA dependent. Formation of pBPl1 and its multimerization by intra and intermolecular recombination is discussed.

We have isolated an R-factor from Saltyphimurium, RI 767, which is a self-transmissible plasmid and confers multiple resistance (Richmond and Wiedemann, 1974). RI767 was found to rearrange frequently its DNA sequence, involving amplification and dissociation of r determinants with insertion, deletion, inversion, and duplication of DNA (Wiedemann, 1981). We believe that the reassortment of RI767 can serve as a model for the evolution of plasmids. Although plasmid R1767 shows frequent reassortment of its genetic material and therefore should provide a convenient system for studying this reassortment, its instability and complexity creates some problems in experimental procedures. For this reason we have decided to study first the simpler plasmids which arise after transfer of R1767. It is also of interest that some of the smaller plasmids themselves show rearrangement so that we do not only get information about the structure of R 1767 but also some of the mechanisms which are involved in the rearrangement of R1767 DNA. In this paper we concentrate on pBPl1, a derivative of R1767. In first place, it has an unusual genome organization with its resistance determinants in large duplicated segments separated by two unique DNA semonella

0147-619X/82/050126-15$02.00/0 Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

126

quences, and second it promotes the formation of multimers. MATERIALS

AND METHODS

Bacterial strains, plasmids, and bacteriophages. The bacterial strains used in this

study are listed in Table 1. Plasmid Rl767 was originally isolated from a clinical strain of Salmonella typhimurium. Media. Growth media were previously described (Schmidt et al., 1976). Minimum inhibitory concentrations (MICs) were determined according to Ericson and Sherris (1971). Identification of @-lactamases. Analytical isoelectric focusing on polyacrylamide gels was used for characterization of plasmid mediated /3-lactamasewith crude extracts by the method of Matthew et al. (1975). Isolation of plasmid DNA. Plasmid DNAs were isolated according to Guerry et al. (1973) or to Clewell and Helinski (1969). Labeling and isolation of replicative intermediates of plasmid pBPl1 and the conditions of centrifugation were identical to those described by Eichenlaub et al. (1979). Digestion of DNA with restriction endom&eases. Restriction endonucleases were

purchased from Boehringer-Mannheim

or

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TABLE 1 BACTERIAL STRAINS AND PLASMIDS

Strains

urA lac thr leu thi tonA recA st’ thr leu lac gal Bl Pl’ sbcBl5 hsr4 hsm+ thy lac thr leu Tl’

Grinstedt et al. (1972) Achtman et al. (1971) G. Lebek, Bern, Switzerland R. Helmuth, Berlin, FRG H. Kneser, KiSln, FRG

R1767 ColEl pRI46 psc105 pBPl1 pVT5

Ap Su Tc Cm Sm Spe Co11Tra Cel+ Ap Su Sm Tra Km Tc Ap Su su

pVT6

su

Richmond and Wiedemann (1974) Clewell and Helinski (1969) van Embden et al. (1978) Nisen et 01. (1977) This paper This paper; EcoRI fragment B of pBPl1 inserted in ColEl This paper; EcoRI fragment C of pBPl1 inserted in ColEl

E. coli W3110 Nal’ E. E. E. E.

Origin or reference

Relevant characteristics

coli coli coli coli

C600 C600 Sm’

SK1592 CR34

Plasmid

New England Biolabs and used according to the manufacturer’s instructions. Gel electrophoresis. Restriction enzyme cleavage products were analysed on vertical slab gels as described recently (Van Treeck et al., 1981). Ligation of DNA fragments. Ligation of fragment and vector DNA was performed as described (Van Treeck et al., 1981). Ligation products were analysed electrophoretitally and used for transformation. Transformation of plasmid DNA. Plasmid transformation was carried out as described by Humphreys et al. ( 1979). The selective agar contained 20 rg ampicillin/ml or 500 pg sulfonamide/ml. Test of colicin production. Colonies grown for 12 hr at 37” on standard I agar were chloroform-treated, overlaid with a 2.5ml nutrient top agar containing lo8 cells/ml of the colicin-sensitive strain E. coli W3110, and further incubated. Inhibition of growth above and around a colony indicated colicin production. Test of colicin immunity. Standard I agar plates containing several colonies of the colicin-producing strain JC4 11 (ColE 1) were chloroform treated and overlaid with about

lo8 cells/ml of a single clone in nutrient top agar. Immune clones result in confluent growth. Electron microscopy. The techniques used for electron microscopy of native and selfannealed plasmid molecules were described by Davies et al. ( 1971) and by Van Treeck et al. ( 1981). For length calculations doubleand single-stranded DNA of phage 4X174 or fd was used as internal standard assuming a length of 5375 bp for $X174 (Sanger et al., 1977) and 6408 for f,, (Beck et al., 1978). The contour lengths of the DNA were measured with a Numonics digitizer. Isolation and analysis of Smal- and Sallcleaved replicative intermediates. E. coli

K12 CR34 thy- harboring pBP 11 was grown, labeled, and the plasmid replicative intermediates were isolated as described by Eichenlaub et al. (1979). Cells were harvested in the logarithmic phase of growth, resuspended in a medium lacking thymine, and incubated for 30 min. Cultures were shifted to 25”C, pulsed for different times (15 and 90 s) with [ ‘Hlthymidine followed by plunging it into a dry ice-ethanol bath and adding KCN to a final concentration of 25 mM to stop the incorporation. After thawing, cells

128

SCHMIDT,

VAN TREECK.

were lysed by the sodium dodecyl sulfatesalt method described by Guerry et al. (1973). Plasmid DNA was isolated by dyebuoyant densitiy centrifugation and material banding at the position between prelabeled ccc and open circular DNA was pooled, dialyzed, and used for digestion with restriction enzymes prior to analysis of replicative intermediates. DNA of these pools was cleaved with restriction endonuclease SmaI and Sal1 which create 1 and 6 fragments, respectively. Restriction fragments containing replication eyes and forks were photographed, measured, and fractional lengths of replicated and unreplicated segments were calculated. According to Delius et al. ( 1971), replicating molecules, displaying whiskers, were omitted from length measurements. Determination

of plasmid

copy number.

The number of copies of plasmid pBPl1 DNA per genome equivalent were estimated as previously described (Avramova et al., 1979). RESULTS Origin and Phenotypic Plasmid pBPl1

Markers

of

The plasmid pBPl1 was derived from exconjugants of a mating of Salmonella tyTABLE

AND

WIEDEMANN

21 R1767 with E. coli W3 110. Four exconjugants showed resistance to ampicillin (MIC, 250 pg/ml) and sulfonamides (MIC, 8000 pg/ml) and lack of transferability. By isoelectric focusing pBPl1 was shown to specify a /3-lactamase identical with the OXA- enzyme produced by plasmid R1818 (Matthew et al., 1975).

phimurium

Physical Mapping

of pBPl1

A map of the cleavage sites on pBP 11 was obtained by digestion of plasmid DNA with the restriction endonucleases SmaI, EcoRI, BumHI, PstI, BglII, SalI, and Hind111 (Table 2) which were used singly or in appropriate combinations of two enzymes followed by fragment analysis in ararose gel electrophoresis (Fig. 1). Some of the bands in the electrophoretic pattern proved to be more intense than others, indicating the existence of duplicated segments. To arrange these duplications in the physical map, hairpin loop structures of pBPl1 were examined in the electron microscope. They revealed characteristic snap-back structures which contain unique and repeated sequences,the latter ones duplicated in inverted orientation. The segments are organized symmetrically to each other as demonstrated in Fig. 2 (monomer). 2

SUMMARYOF RESTRICTIONENZYME FRAGMENTSOF PLASMID pBPl1 Size (kilobases) Fragment

SmaI

A

26.4

B

C C D D’ E F F’ G G’

EcoRI 11.5 9.1 4.6

0.3

EamHI

Sal1

9.4 8.4 4.2 4.2 0.2 0.2

16.9 7.1 0.6 0.6

0.5 0.5

BglII 5.3 4.4 3.8 3.8 3.4

-

2.9 0.9 0.9 0.3 0.3

Hind111

PstI

13.6 8.6 1.9 1.9 0.25 0.25

A 14.9

i

B”

1.6 1.5 1.2

0.9 0.6 0.4 IO.3

C c’

2.3 2.3

o Digestion of pBP11 results in 10 fragments, the order of seven of them remains unclear. For practical use the segment covering these fragments is called fragment “B” (see also Fig. 1).

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FIG. 1. Circular restriction map of pBPl I monomeric form obtained by single and combined digestions with restriction endonucleases.Coordinates are given as kilobases relative to the single Smal site (clockwise). The repetitive region (6.8 kb) including the resistance genes is indicated with thick lines. Short inverted repeat sequencesin Hind111 fragment B are shown in black boxes, its orientation indicated by arrows (see also Fig. 1). The positions for the origins for replication are designated ori, and ori*, the direction of replication is indicated with an arrow. Restriction fragments located in the repetition are found twice and are distinguished by indices.

To align the inverted repeats to best fit the restriction map, snap-back structures of SmaI and EcoRI fragments of pBPl1 were examined electron microscopically. They demonstrated the position of the SmaI site outside of the inverted repeat sequence and one of the EcoRI sites inside of the duplication (Fig. 1). The average size of pBPl1 was found to be 26.4 + 0.7 kb. It was calculated by the sum of the DNA fragments and from the average length calculated from measurements of 36 snap-back molecules. To identify hairpin loop segments in the

duplicated DNA stretches, pBPl1 snapbacks were digested with single-strand endonuclease Sl. The remaining linear inverted repeats were denatured, renatured, and analyzed. All of the single-stranded fragments revealed two hairpin loop structures, formed by repeated sequences in inverted orientation (Fig. 2B). The larger stem has a size of 0.2 kb, the smaller one of 0.12 kb as calculated from the measurements of 20 palindromic structures. The position of these repeated sequences was localized as indicated in the physical map (Fig. 1) by

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SCHMIDT, VAN TREECK, AND WIEDEMANN

SMALL HAIRPIN

LOOP

FIG. 2. Electron micrographs of snap-backs observed from pBP I 1. Plasmid DNA was denatured and briefly reannealed as described in the text. (A) snap-backs of monomeric and multimeric forms of pBPl1. (B) Snap-back of inverted repeat segment of pBPl1 monomeric DNA. Monomeric snap-backs were digested with Sl to cut off the single-stranded loop segments 1 and 2 and were denatured and briefly renatured (Ohtsubo and Ohtsubo, 1976).

analysis of snap-back molecules of hybrid plasmids pVT5 and pVT6 (see below). Localization of Ampicillin and Sulfonamide Resistance Determinants on Plasmid pBPl1 To localize segments responsible for expression of drug resistance, EcoRI and BamHI fragments of pBPl1 were cloned by using ColEl and pSC 105 as cloning vehicles. Analysis of the recombinant plasmids thus obtained showed that BamHI fragment C carried the Ap and the Su gene while EcoRI fragments B and C carried the Su gene

alone. By these data the position of the Ap and Su genes could be localized inside a region of 4.07 kb belonging to the inverted repeat sequence as indicated in Fig. 1. Analysis of Diflerent Size Classes of pBPl1 Seven hundred eighty hairpin loop structures from one transformant clone were examined upon their size and conformation. Four different size classes could be found: monomers with a size of 26.4 kb, dimers with 53.5 kb, trimers with 81.2 kb, and tetramers with 103 kb. Figure 2 shows micrographs of

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OF PLASMID pBPl1

TABLE 3 DISTRIBUTION OF INVERTED REPEATS, COPY NUMBER, AND LENGTHS OF MONOMERIC AND MULTIMERIC FORMS OF pBPl1

Plasmid form Monomer Dimer A Dimer B Trimer Tetramer

Size (kb) 26.4 + 0.7 53.5 + 1.6 54.4 AZ1.3 81.2 + 2.2 103.0 2 3.5

Size of inverted repeats (kb)

Copy numberb

6.8 20.4 2 x 6.8 34.0 45.0

17-196 l-26 -0.002’ O.l-0.3* 0.04-0.08’

a The lengths are expressed as units of kilobases (kb). * Values are expressed as the number of copies of plasmid DNA per genome equivalent. ’ Values are derived from the numbers of monomer and multimer plasmid species as determined out of 30,000 intact pBPl1 snap-backs by using the electron microscope. Sizes were easy to distinguish by inspection. These estimates were confirmed by selective photography and molecule measurement.

typical snap-back structures of monomeric and multimeric species of pBP Il. To determine the overall content of each form per cell, the copy number of plasmid DNA per genome equivalent was estimated as described under Materials and Methods. DNA from buoyant density gradients was subjected to sucrose gradient centrifugation resulting in three peaks, which were used for calculation of the number of copies per chromosomal equivalent of each single size class. Thus monomers were found to be present with 18 copies, dimers with one copy and trimers with 0.2 copies per chromosomal equivalent (Table 3). The copy numbers of other forms could only be estimated indirectly by comparing the numbers of monomeric and multimeric plasmid speciesout of several EM quantitations. As mentioned above four independent clones were isolated from a single mating experiment of R factor R1767, bearing resistance to ampicillin and sulfonamides and lack of transferability. According to restriction analysis and characterization of snap-back structures, plasmid DNA, isolated from the four exconjugants, was found to be identical with pBP 11. One thousand snap-back structures from each of the clones were examined. Within the range of calculation error their size was found to be in accordance with the corre-

sponding forms of pBPl1 and the ratio of different size classes remains constant in these clones: About 90% resembled monomers, less than 10% dimers, 1% trimers, and 0.4% tetramers of pBPl1. This finding implies that multimers of pBPl1 may have arisen by intermolecular recombination between single monomers analogous to the multimer formation of ColEl DNA (Potter and Dressler, 1978). This process was found to be recA dependent for pBP 11: Monomeric and dimeric DNA of pBPl1 was separated electrophoretically, extracted from the bands, purified and used to transform E. coli SK1 595 and E. coli CBOOrecA. All of the resulting recA clones investigated harbored only the plasmid form originally used for transformation while E. coli SK1 595 which is not deficient in its recombination system always harbors the original set of monomeric and multimeric forms of pBPl1 when transformed with purified monomeric and dimeric plasmid DNA. Thus recA-dependent production of dimeric circles must result from dimerization of pBPl1 in tandem. This could be demonstrated by analysis of the genome organization of pBP 11 dimers (data not shown): (i) Restriction patterns of monomeric and dimeric DNA were found to be identical. (ii) By using the small palindromic se-

132

SCHMIDT, VAN TREECK, AND WIEDEMANN

quence, flanking one end of the monomer’s duplication distal to Su, as reference point, snap-back preparations of purified dimeric DNA were screened for defect structures. Their analysis revealed the expected genome organization for pBPl1 dimers as schematized in Fig. 4. Hairpin loop structures of this form must lead to two configurations which have been demonstrated. (iii) Final proof for the sequence organization of pBPl1 dimeric circles resulted from underwound loop structures (Broker et al., 1977) found in snap-back preparations,

showing that the DNA sequencesof the loop segments in pBPl1 dimers are homologous. Intramolecular Recombination Diferent Forms of pBPl1

within

pBP 11 exhibits large duplicated segments which should serve as substrates for homologous recombination leading to inversions within the replicon. These plasmids should only differ with respect to the relative orientation of the two segments located between the inverted repeat sequencesand are

MO

MONOMER heteroduple.

FIG. 3. Heteroduplex between two monomeric circles of different types. The region of homology was found to be 20.1 kb and equals the size of two inverted repeats and one loop segment. In some hairpin loop structures of pBPl1, loop segment 1 was found to exhibit a small snap-back (arrows). This palindromic structure was identified near one end of the single-strand region in the heteroduplex molecule and indicates that loop 1 has been inverted in one of the strands.

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133

The pulse labeled DNA was detected exhibiting a broad density distribution between the positions of supercoiled and open circular DNA fractions in CsCl-ethidium bromide gradients. Fractions in this region were collected and used as DNA molecules representing replicating forms as has been reported for ColEl, pSClO1, R6K, RSFlOlO, and RSF1030 (Lovett et al., 1974, 1975; Cabello et al., 1976; Crosa et al., 1975; de Graff et al., 1978; Conrad et al., 1979). Electron microscope studies on this intermediate fraction unequivocally identified replicative intermediates showing “eyes” and “forks.” To find the number of initiation sites and the mode of replication for pBP 11, DNA from these fractions was subjected to restriction analysis by cleavage with SmaI and SalI, the first of which is found to cut pBP 11 once, while the second one cleaves six times (see also physical map, Fig. 1). Linear molecules with internal replication loops were found to be of the same size-e.g., 26.1 + 0.58 kb for SmaI-cleaved intermediatesas the contour length of the corresponding SmaI or Sal1 fragments of unreplicating pBPl1 DNA spread on the same grid. Digestion of replicating molecules generated two classes of structures which can be observed in the electron microscope: Molecules with an internal loop of replicating DNA between the two linear ends and molecules consisting of a linear DNA segment bordered by one or two y-shaped branches of different length depending on the stage of replication and the kind of fragment (Fig. 5). For a plasmid with unidirectional replication from a fixed origin, the distance from the fork, which remains at the origin, to the enzyme cleavage site must always be the same. As shown in Fig. 6, all molecules of SmaI-cleaved intermediates appeared to replicate in one direction. The same holds true for SalI-generated fragments (Fig. 7). Mode of Replication of pBPl1 Evaluation of the different short unrepliA thymine-requiring mutant, E. coli CR34 cated DNA branches and transfer of the carrying pBPl1, was pulse labeled with corresponding replication points to the phys[ 3H]thymidine and lysed as described in de- ical map led to the disclosure of three lotail under Materials and Methods. cations of origin of replication in pBPl1.

thought to have arisen by reciprocal exchange of strands between the inverted repeats. Two micrometers yeast DNA is found to exhibit a related principle of genome organization as pBPl1; it contains unique DNA segments and large inverted repeats (Hollenberg et al., 1976). Occurring with a stoichiometry of 1:1 two types of monomeric circles of 2-pm yeast plasmids have been observed, carrying one of their unique sequences in inverted orientation to the other (Guerineau et al., 1976). To search for two types of monomeric molecules, pBPl1 was denatured and renatured for varying times, to allow reannealing of complementary strands. Samples were subjected to analysis of structures in the electron microscope. Only two structures out of 10,000 molecules have been detected, which could have been identified as heteroduplex monomers, presenting inhomology in loop segment 1 (Fig. 3). The rate of inversion cannot be estimated from these data. Therefore the finding only indicates a low frequency of inversion. Analogous results have been found from inversion formation in dimeric circles. Dimers with an altered genome organization due to reciprocal exchange between the inverted repeats should be easily detectable in their snap-back structure. pBP 11 dimeric form B shows the expected configuration (Fig. 2). Its sequence organization was deduced from analysis of defective hairpin-loop molecules and is schematized in Fig. 4. Four out of 3000 dimeric hairpin loop structures have been found to represent dimer form B (Table 3). This remarkably low frequency of inversion formation in dimeric circles indicates that inter- and intramolecular recombination in pBPl1 might follow two pathways.

134

SCHMIDT,

VAN

TREECK,

AND

WIEDEMANN

* a r” n

a z a I-

MULTIMERIZATION

AND

REPLICATION

OF PLASMID

pBPl I

FIG. 5. Electron micrographs of pBPl1 replicative intermediates cleaved with restriction endonucleases SmaI (A, B) and Sal1 (C, D). Replicating pBPl1 DNA molecules were prepared as described in the text. Structures arranged in the order of increasing extent of replicating (A-D). 1#~X174RF11 was used as internal contour length reference. (A) represents a SmaI fragment, showing two simultaneous replication sites, located in the repetitive sequence (ori,). (B) shows a SmaI fragment at more advanced stage of replication, while (C) and (D) represent molecules of Sal1 fragment A. Growing forks are indicated by arrows.

136

SCHMIDT, VAN TREECK, AND WIEDEMANN Smr

I

SMA

1

-

r t

I t 0.2

1.0

0.0

0.6

0.4

LENGTH

RELATIVE

FIG. 6. Line diagrams representing the location of the replicating portion (heavy lines) in the molecules of pBPl1 which were treated with restriction endonuclease SmnI. All of the replicative intermediates were normalized to a scale of 1.0. The data reveal three classesof molecules for which one branch point may be aligned on a vertical line. Classes one and two are located in the duplication and named ori, as shown in the map of pBPl1 at top (the extension of the duplicated segment of pBPl1 is indicated in black boxes). The third class may be associated with or&. The location of the other branch point varies for all molecules. The common branch point for each class is given in the text.

Under the test conditions replication occurs unidirectionally for each single origin in pBP 11. Two origins were localized at posi-

tions between the two resistance genes, 6.0 + 0.6 and 7.6 + 1.0 kb apart from the SmaI cutting site. They have to be homologous in SAL I

SAL1

SALI

t

0.2

RELATIVE

0.4

0.6

0.0

1.0

LENGTH

FIG. 7. Line diagrams of replicating pBPl1 as in Fig. 6 except that digestion was with endonuclease SalI. Due to a comparison of length calculations of Sal1 fragments with and without replication forks or eyes, S&-cleaved y- or eye-shaped replicative intermediates were correlated with fragment A and B, as indicated by the simplified map at top. Sal1 cleaved replicative intermediates in which ori, has been unequivocally identified, have not been isolated.

MULTIMERIZATION

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pBPl1

137

minants Ap and Su in large duplicated segments (6.8 kb) and can start its replication from three different locations (ori) unidirectionally. R6K (Crosa et al., 1975) and NRl (Perlman and Rownd, 1976) have been reported to have two sites at which initiation of replication occurs. In the case of NRl it appears that the bacterial host and the exact experimental conditions can influence whether one or more origins are observed (Perlman and Rownd, 1976). Under the assay conditions used for the isolation of replicative intermediates of pBPl1, the homologous origins located in the duplication (ori,) are expressedwith approximately equal frequency. It remains unclear, in which direction replication proceeds after the two replication forks meet each other. pBPl1 plasmid DNA exists in various discrete size classes. The data demonstrate that larger entities like dimers, trimers, and tetramers are circular multimers of the smallest size of pBPl1 DNA (Table 3). Several analogous results have been obtained for the multimerization of DNA from 4X174 and S13 (Thompson et al., 1975), and various plasmid DNAs like pMB9 (Bedbrook and Ausubel, 1976) and ColEl (Potter and Dressler, 1978). Potter and Dressler have isolated recombination intermediates (Chiforms) and characterized the mechanism leading to plasmid multimers by the aid of these structures (Potter and Dressler, 1978). They postulated that circular molecules undergo recombination via a Holliday intermediate, i.e., the process is recA dependent. This could be shown for pBPl1 too. Transformation with purified monomer and dimer DNA demonstrates that multimer formation does not occur in ret- hosts. Monomers and dimers propagate themselves stably in recA cells but undergo inter- and intramolecular recombination in ret+ background leading to a similar pattern of discrete size classes as found in wild-type cells. Thus multimer formation is reversible in ret+ hosts. Multimers of pBPl1 most probably arise by a DISCUSSION single reciprocal recombination process ocpBPl1 demonstrates an unusual genome curring at regions of homology between plasorganization. It carries its resistance deter- mid molecules rather than by a replication

their DNA sequence since they were localized at the same position in the duplicated segment in pBP 11. Therefore we have termed these origins of replication with the same symbol, ori,, as indicated in the physical map (Fig. 1). As the SmaI cut is located asymmetrically in the loop, fragments isolated by SmaI digestion deliver two classes of molecules, each starting their replication within one of the two duplications. As deduced from the number of replicating intermediates (Fig. 6), oril in each of the duplications is used with the same probability. As Sal1 delivers fragments with the same distance to the origin of replication, located in either of the duplicated segments, one cannot distinguish which one of these origins is expressed. The actual presence of two distinct replication initiation sites was confirmed by finding a unique class of molecules illustrated in Fig. 6, containing two clearly defined replication loops. They comprised about 5% of the identified replicating intermediates. The actual mode of replication of this minority remains unclear but will be discussed below. Furthermore a few molecules could be observed with a fragment size identical with pBPl1 dimers initiating their replication at ori,. They have not been listed in the plot of replicating intermediates. Fourteen percent of the molecules under investigation expressed a totally different origin of replication named orit which could be located 10.7 + 0.6 kb apart from the SmaI cut. Replicating molecules, exhibiting ori2 led to the conclusion that initiation of replication starts from ori, unidirectionally too. Our data locate or& within loop segment 2. Due to the lack of Sal1 fragments replicating from or&., we cannot distinguish which of the possible two sites in loop 2 are used as or&. Thus only one of the sites is arbitrarily designated ori, in the physical map (Fig. 1).

138

SCHMIDT,

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mechanism. These conclusions are supported by the following observation: In some preparations of pBP 11 so-called “figure-eight” structures have been observed, exhibiting short single-stranded arms at the region of contact. Figure-eight molecules represent recombination intermediates and show two genomes covalently held together at a region of contact (Potter and Dressler, 1978). As demonstrated by analysis of restriction fragments and snap-back structures, dimers are tandem repetitions of the monomer. Based on this evidence the genome organization of pBP 11 monomeric and multimeric forms are depicted in Fig. 4. Formation of multimers other than dimers are expected via the same mechanism of intermolecular recombination, e.g., trimers and tetramers might have arisen by a single reciprocal exchange between a monomer and a dimer or between two dimers, respectively. Similarly to the plasmid and phage replicons mentioned above, pBP 11 possesseslarge segments in duplicated form-structures which might be capable of undergoing intramolecular recombination. A few monomeric, dimeric, and multimeric circles of pBPl1 have been detected, which exhibit segments with inverted orientation. By these observations the rate at which inversions are formed in pBPl1 appears to be much lower than the rate of pBP 11 multimerization. The frequency of inter and intramolecular recombination is dependent on the mediating enzyme system provided by the host. Multimer formation of ColEl plasmids was shown to occur at high frequency in Proteus mirabilis but to a much lesser extent in E. co/i (Goebel and Helinski, 1968). The same holds true for the frequency of inversion. Guerineau et al. (1976) report a stoichiometry of 1:1 for the presence of different 2-pm monomeric circles, whereas this inversion does not occur when the yeast plasmid is cloned in E. co/i (Hartley and Donelson, 1980). In our experiments loop segments tend to invert with a frequency below 10e3. No inversion loop structures have been observed in recA hosts. The striking difference between multimer formation and inversion

AND

WIEDEMANN

formation is not yet understood. Both processesare recA dependent, but probably intramolecular recombination might be directed by additional functions. As reported recently an analogous observation has been found for chromosome rearrangements. While duplications are rather common in bacteria, inversions are extremely rare (Roth and Schmid, 1981). In addition low rates of pBPl1 inversion and its recA dependence indicates that inversion in pBPl1 is not catalyzed by a specific DNA “invertase” as has been reported by inversion of the C region in Pl DNA (Kutsukake and Iino, 1980) and the G-loop in Mu DNA (Kamp et al., 1979). Little is known of the formation of pBPl1 replicons from R1767. As derived by transfer of R1767 to ret+ recipients, several replicons have been isolated, carrying Ap Su resistance. Only pBPl1 could be shown to carry its resistance determinants in two copies (unpublished observation). Furthermore hairpin loop structures of R1767 do not reveal inverted repeats as large as found in pBPl1. In a single transfer of R1767 DNA all four exconjugants tested were found to yield the replicon pBPl1, indicating that production of duplications in pBPl1 is site specific. Structures including duplicated segments like pBPl1 have been demonstrated in procaryotic as well as in eucaryotic DNA as has been described for X dv mutants (Chow et al., 1974), for the arg genesin E. co/i (Charlier et al., 1979), for 2-pm yeast DNA (Guerineau et al., 1979), and for DNA isolated from chloroplast rRNA genes (Bedbrook et al., 1977). The mechanisms involved in formation of structures with large inverted repeats still remains obscure. Probably certain recognition sequencesbordering the duplication might be involved as hot spots of recombination as has been reported for the small palindromic sequencein pBP 11 (Schmidt et al., 1980). In further experiments we try to find a derivative of R1767 which might form deletion replicons like pBPl1. This will help to analyze the mechanism involved.

MULTIMERIZATION

AND REPLICATION

ACKNOWLEDGMENTS We thank J. Kratz for helpful discussionsand critical reading of the manuscript and A. K. Rottenbacher for excellent technical assistance. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to B. Wiedemann.

REFERENCES M., WILLETTS, N., AND CLARK, A. J. ( 1971). Beginning a genetic analysis of conjugational transfer determined by the F-factor in Escherichia coli by isolation and characterization of transfer defective mutants. J. Bocreriol. 106, 529-538.

ACHTMAN,

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