Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning

PLASMID

7, 163-179

(1982)

Genetic Studies in Copy Number

of F Plasmid Maintenance Control, Incompatability,

RALPH W. SEELKE, BRUCEC.

KLINE, JOHN D.

Genes Involved and Partitioning

TRAWICK,

ANDGRAHAM

D.

RITTS*

Department of Cell Biology, Section of Microbiology, Mayo Medical School, Mayo Foundation, Rochester, Minnesota 55905, and *University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota 55455 Received

April

6, 1981; revised

July 9, 1981

We have identified a 2.1 -kilobase (kb) region (44.1 to 46.19 kb) in F that is necessary and sufficient to form low copy number minireplicons. Within this region we have mapped (i) mutations (cop) inducing 4.4- to 28-fold increases in copy number and (ii) two separate regions that determine incompatability (incB and incC). The 2. I-kilobase region has also been shown by others to contain (i) an origin of replication, ori (ii) a locus (aos) necessary for sensitivity to the plasmid replication inhibitor, acridine orange, and (iii) nine, 19- to 22-base-pair direct repeat sequences organized in two clusters. In the present work we more accurately locate the aos locus and show that it, as well as ori, incB, incC, and some copmutations, map within or overlap the direct repeat regions. Analysis of other cop mutations indicates that they reduce or destroy the incompatability reaction associated with the 2.1 -kb region; however, these cop mutations do not map within the incB or in&2 determinants, A 2-fold copy number elevation and unstable plasmid maintenance also results from deletion of the 46.19- to 49.2-kb region. Results described here and elsewhere suggest that the instability of the deletion mutant reflects the loss of partitioning gene, a gene that is probably identical to an inc locus, incD, that had been identified in this region in prior work. Whether or not the incD locus has anything to do with the slight copy number elevation is unknown.

The normal F plasmid is 94.5 kb in length (Ohtsubo et al., 1974). After digestion of this plasmid with EcoRI restriction enzyme, two of the resulting fragments f5 (Lovett and Helinski, 1975; Timmis et al., 1975) and f7 (Lane and Gardner, 1979) can form mini-F plasmids. Comparison of the maintenance properties of f5- and f7-derived replicons shows that former but not the latter are typical of normal F (Lane, 1981). The purpose of this paper is to describe our genetic analysis of maintenance genes in f5-derived mini-F plasmids. Our results in conjunction with those of others have allowed us to approximate the f5 sequences essential for replicon formation and to identify an adjacent region encoding partitioning functions. In this paper we also identify and/ or map functions in the essential region that control copy number and incompatibility. Our results are consistent with the hypoth163

esis that control of F replication is negative and that incompatibility is related in part to this control mechanism. However, our results also show a somewhat complex relationship between the incompatibility determinants and replication. Further, our results suggest that yet another incompatibility determinant is closely related to the plasmid partitioning determinants, A preliminary report of this work has been presented (Kline et al., 1981). MATERIALS AND METHODS Bacteria and plasmids. The bacteria used in this study are: (i) CSHSO aruA(lacpro)thi str and (ii) BK342 which is essentially a recA derivative of CSHSO that also contains an F’lac plasmid. These strains have been described previously (Manis and Kline, 1978). The plasmids used in this work are described in Table 1. 0147-6

19X/82/0201

63-l 7$02.00/O

Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved

164

SEELKE

ET AL.

TABLE

1

PLASMIDS

Plasmid pMB9 pML31 pGA36 pBR322 psc101 pMF21 pMF45 pMF46 pBK50 pBK55 pBK63 pBK77 pBK80 pBK96 pBKl03 pBKl04 pBKlO5 pBKl06 pBKl07 pBKll0 pBKl13 pBKl18 pBKl38-2 pBK163 pBK207 pBK211 pBK213 pBK214 pBK215 pBK216 pBK217 pBK232

Description” a ColEl-like cloning vector, Tc’ Mini-FKm’ (see Fig. 1) pl SA-derived cloning vector, Cm’ Tc’ a ColEl-like cloning vector, Ap’ Tc’ Tc’ pML31 A[F40.8-43.1 kb]” pMF21 tl Tn3 46.19 kb, Ap’ Km’ (see Figs. 1 and 3) pMF45 A[EcoRI kun+ fragment], Ap’ Km” (see Fig. 3) pSClOl:pML31 [EcoRI /con+ fragment], Km’ Tc’ pBKSOzpML31 [ 40.8-43.1 kb], BornHI-mediated recombinant, Km’ Tc’ pMF45 cop44 pMF45 cop48 pMF45 cops0 pMF45 incD’ pMF21 D Tn3 45.99 kb A[F45.99-47.5 kb], Ap’ Km’ (see footnotes d and e, Table 2) pMF21 R Tn3 46.04 kb A[F46.04-46.19 kb], Ap’ Km’ (see footnotes d and e, Table 2) pMF21 St Tn3 45.99 kb, Ap’ Km’ (see footnotes d and e, Table 2) pMF21 t? Tn3 46.04 kb A[ F46.04-46.09 kb], Ap’ Km’ (see footnotes d and e, Table 2) pMF21 tl Tn3 45.99 kb A[F45.99-47.53 kb], Ap’ Km (see footnotes d and e, Table 2) pMF21 Q Tn3 46.04 kb, Ap’ Km’ (see footnotes d and e, Table 2) pMF21 51Tn3 46.04 kb A[F46.04-49.3 kb and 1600 bp of the EcoRI /can fragment], Ap’ Km’ (see footnotes d and e, Table 2) Mini-FAp derived from the smallest BarnHI fragment of pBKlO5 (see Fig. I) Mini-FAp derived from a KpnI digest of pBKlO5; note other spontaneous deletions have occurred since no KpnI sites are in this plasmid (see Fig. 1) pSClOl:pBKl03 [Tn3 1.5-4.5 kb/F47.5-49.3 kb/ EcoRI kun+ fragment], BumHI-mediated recombinant, Km’ Tc” (see Fig. 1) pBR322:pMF45 [44.1-45.88 kb], PstI-mediated recombinant, Ap” Tc’ (see Fig. 1) pMF45 cop21 I pMF45 cop21 3 pMF45 cop21 4 pMF45 cop215 pBKSO:pBK211 143.1-46.19 kb/Tn3 4.5-1.5 kb], BarnHI-mediated recombinant, Km’ Tc” (see Table 3) pSClOl:pBK211 [Tn3 1.5-0.0 kb/F46.19-49.3/EcoRI /can+ fragment], BamHI-mediated recombinant, Ap’ Km’ (see Table 3) pBR322:pMF45 [45.88-46.19 kb/Tn3 4.5-4.0 kb], PstI-mediated recombinant, Ap’ Tc’ (see Fig. 1)

Referen& Rodriquez et al. (1979) I-ovett and Helinski (1976) An and Friesen (1979) Bolivar et al. (1977) Cohen and Chang (1973) Manis and Kline (1977) Manis and Kline (1978) Manis and Kline (1978) Manis and Kline ( 1978) Manis and Kline (1978) Manis Manis Manis Kline Kline

and Kline (1978) and Kline (1978) and Kline (1978) (1979) and Palchaudhuri (1980)

Kline and Palchaudhuri

( 1980)

Kline and Palchaudhuri

( 1980)

Kline and Palchaudhuri

(1980)

Kline and Palchaudhuri

(1980)

Kline and Palchaudhuri

(1980)

Kline and Palchaudhuri

(1980)

Kline and Palchaudhuri

(1980)

Kline and Palchaudhuri

(1980)

GENETICS

OF MINI-F TABLE

Plasmid pBK233 pBK242 pBK257 pBK258 pBK261 pBK263 pBK264 pBK280 pBK299 pBK350 pBK374

PLASMID

165

MAINTENANCE

I-Continued.

Description”

Referen&

pBR322pBK211, construction analogous to pBK232, Ap” Tc’ (see Table 3) pBR322:pBK211, construction analogous to pBK207, Ap” Tc’ (see Table 3) pBR322:pBK216, [F43.1-46.19 kb/Tn3 4.5-1.5 kb], BarnHI-mediated recombinant, Ap’ Tc” (see Table 3) pBR322:pBK77 [F43.1-46.19 kb/Tn3 4.5-1.5 kb], BumHI-mediated recombinant, Ap’ Tc” (see Table 3) pMF45 A[Tn3 4.25-0.0 kb/F46.19-49.2 kb], BslEIImediated deletion, Ap’ Km’ (see Fig. 1) pBR322:pBK104 [F45.88-46.04 kb/Tn3 4.5-4.0 kb], PsrI-mediated recombinant, Ap’ Tc’ (see Table 3) pBR322:pBK105 [Tn3 4.0-4.5 kb/F45.99-47.3 kb], PstI-mediated recombinant, Ap’ Tc’ (see Table 3) pBK261 A[ F43.7-44.1 kb], PslI-mediated deletion, Ap’ Km’ (see Fig. 1) pMB9:pMF45 [Tn3 1.9-0.0 kb/F46.19-49.2 kb], BsrEII-mediated recombinant, Ap’ Tc’ pBK299 A[Tn3 0.6-0.0 kb/F46.19-47.3 kb], PstImediated deletion, Ap’ Tc’ (see Fig. 1) pGA36:pBK207 [45.0-45.35 kb BglII/SmaI fragment], recombinant made by using the .SmaI/BglII F DNA to replace a .SmuI/BgZII fragment excised from pGA36; Cm’

’ Plasmid nomenclature is according to the recommendation of Novick et al. (1976). The designation A[x-y kb] represents a deletion between the x and y kb coordinates. The designation B Tn3 45.99 kb represents Tn3 inserted at the 45.99-kb site on the F map. The notation pXy:pQD [x-v kb] represents a recombinant in which the vector pXy is recombined in vitro with the x-y kb fragment generated by the enzyme mentioned in the description from plasmid pQD. A map of Tn3 is given in Fig. 3. ‘All references are to this work unless specifically designated otherwise.

Isolation of copy number mutants. Copy number mutants were isolated from pMF45 (Table 1) after treatment with nitrosoguanidine (NTG) or ethyl methane sulfonate (EMS). Log phase cultures of BK343 (CSH50/pMF45) growing in AB3 medium (Difco Co.) at 37’ were adjusted to a cell density of 5 X lO’/ml and 2 pg NTG/ml final concentration was added. Cells were allowed to grow for 4 hr (about eight generations), then the culture was diluted IOOfold and 0.1 ml was spread on AB3 agar supplemented with 1000 pg ampicillin/ml and the plates were incubated for 18 hr at 37”. Between 100 and 500 small colonies (<2-mm diameter) and two to five large colonies (>3-mm diameter) per plate usually appeared after incubation. The large colo-

nies potentially harbored cop mutant plasmids. This was verified by purifying the plasmid, introducing it into a new CSHSO host by transformation, and then examining the growth of the transformant on AB3 plates containing 1000 pg of ampicillin/ml and by measuring the amount of covalently closed circular DNA (Table 2). Only one cop mutant was selected from each NTG- or EMStreated culture of BK343 to insure the isolation of independent copy number mutants. EMS-generated mutants were isolated by adding 10 ~1 of undiluted EMS to 1 ml of 5 X 10’ bacteria, culturing the bacteria for 2 hr at 37”, washing the cells once with AB3 medium, and then plating about 10’ bacteria on AB3 agar containing 1000 pg ampicillin/ ml. The plates were incubated at 37” over-

166

SEELKE

night and handled as described for NTGinduced mutants. Measurements

of plasmid

concentrations.

(i) Quantitative estimations of plasmid concentrations were done by the dye-CsCl technique (Radloff et al., 1967) using lysates made from 2 to 5 ml of bacteria labeled for three to five generations with [3H]thymidine. Bacteria were cultured overnight in M9 medium (Miller, 1972) supplemented with glucose, 0.2%, Difco casamino acids, 0.5%, deoxyadenosine, 250 pg/ml, and the appropriate antibiotic, then diluted into the same medium supplemented with [3H]thymidine (10 &i/ml) without antibiotic and grown for three to five generations. At this time the culture was harvested and the percentage of antibiotic-sensitive cells determined. Cultures with less than 95% were discarded. The Brij-desoxycholate procedure of Clewell and Helinski (1969) was used to lyse bacteria in preparation for isopycnic centrifugation; (ii) qualitative estimation of the Cop phenotype was done by culturing bacteria overnight in the same medium used for isotopic labeling. One milliliter of culture was lysed and treated as described by Weisblum et al. (1979) and the DNA in 50-100 ~1 of this lysate was subjected to electrophoresis through a 0.7 or a 1.6% agarose gel containing 0.04 M Tris, 0.2 M sodium acetate, 0.001 M EDTA, and 0.5 pg ethidium bromide. This technique is effective for plasmids up to 15 Mdal in mass; larger plasmids tend to comigrate with chromosomal fragments. Electrophoresis was done at 10 V/cm for 75 to 100 min or 1.25 V/cm overnight. Gels were visualized by ultraviolet transillumination. Copy mutants of the type described in Table 2 show a plasmid band easily visible to the naked eye whereas low copy plasmids such as pMF45 are invisible and can be detected only by fluorescence photography. Plasmid stability tests. Method 1. Bacteria were cloned on agar supplemented with the appropriate antibiotic (ampicillin 20 wg/ ml, kanamycin 40 pg/ml, or tetracycline 10 pg/ml), and a single colony was used to in-

ET AL.

oculate antibiotic-free AB3 broth. This culture was maintained by serial transfer at 24hr intervals. The descendants were isolated by plating periodically onto AB3 agar without antibiotic and then 100 colonies from each plate were transferred to agar containing antibiotic to score for the loss of plasmid phenotype. Stable plasmid phenotypes were verified by showing the existence of autonomous plasmid DNA at the end of the examination period. Method 2. A storage culture of bacteria with plasmid was inoculated into AB3 medium supplemented with antibiotic and grown overnight. A high dilution of this culture was transferred to antibiotic-free AB3 medium and serially maintained in this medium as described above. Periodically the fraction of plasmid-free cells was measured as a function of cell growth by comparing cell titers in triplicate on AB3 plates with and without antibiotics. In both Methods 1 and 2 initial and final cell titers were determined for each serial transfer in order to calculate the number of elapsed generations. Recombinant DNA techniques. Restriction enzymes were used according to the suppliers recommendations. The production of in vitro recombinants or deletions or the cloning of various restriction fragments all proceeded by virtue of a common methodology that has been described (Manis and Kline, 1977). Without exception, recombinant or deletion plasmid were made by using restriction fragments purified by agarose gel electrophoresis. Also in every case, recombinant and deletion mutant plasmid structures were confirmed by restriction analysis of the purified plasmid DNA. Plasmid purification was done either by the dye-CsCl method or by the rapid procedure recently described by Klein et al. (1980). RESULTS Construction

of Mini-F

Plasmids

The majority of our research with mini-F involves the use of plasmids containing the

GENETICS pML3

I

R 40.3

pMF2

I

42

R

43

44

45

46

47

48

43.

R

kan - -------A

---

49

B

-_-_-_-_--e-r I

B 1

18

amp

7 I .5

45.930

amp

pBK138-2 -. 0

45.99 pBKl63

167

MAINTENANCE

WB 40.8

pl3Kl

PLASMID

B

B, 41

OF MINI-F

I I .4

2

J

___-__--_--

P pBK207

P

q pBR322

P pBK232

pBK261

pBK280

pBR322

c1

ds P

43.6

49

P

BS p8K350

-

+-

-m

44.1

p-

--

_

-

---

-J

.

46.19

Bs

________-

J

P

pMB9

FIG. 1. Maps of mini-F and mini-F chimeras. Map units for F are in kilobases and refer to the position on the 945kb, wild-type F plasmid as described by Ohtsubo er al. (1974). The symbols for restriction enzyme recognition sites are: (R) EcoRI; (B) BumHI; (P) PsrI; and (Bs) BsrEII. Table 1 contains complementary information regarding the plasmids in this figure. Discontinuities in the maps represent deletions. Restriction enzyme symbols shown at deletion sites represent “half sites.” For example, pMF21 has only one BumHI site formed by the junction of the 40.8- and 43.1-kb “half’-BumHI sites. The larger double bars represent the indicated cloning vectors while the narrower double bars represent pieces of Tn3. The coordinates for the Tn3 piece in pBK232 are 4.5-4.0 kb and in pBK350 are 2.0-0.6 kb; the latter represent BstEII/PstI termini. The orientation of the F sequences in pBK207 and 232 are as depicted. The orientation of the F sequences in pBK163 and pBK350 have not been determined.

ampicillin resistance transposon Tn3. The purpose of including Tn3 was to provide a resistance phenotype that is proportional to gene dosage (copy number) and to have a structural marker that was easily mapped. Our basic mini-F target has been pMF21 (Fig. 1). Previously, we mapped Tn3 insertions at three locations, 45.83, 46.35, and 46.45 kb (Manis and Kline, 1978; Kline and Palchaudhuri, 1980). These insertion sites were estimated by gel electrophoresis without knowledge of the appropriate nucleotide sequences and with rather large restriction fragments of bacteriophage X DNA as standards. Since this time appropriate sequence data have become available (Morutsu et al.,

1982) and more precise molecular length standards of pBR322 (Sutcliff, 1978) have been employed to remap the Tn3 insertion sites. The logic used for mapping and some typical electrophoresis results are presented in Fig. 2. The old (new) estimates of insertion sites are as follows: 45.83 (45.988 kb), pBK103, 105, 107, and 109; 46.35 (46.040 kb), pBK104, 106, 110, and 113; and 46.45 (46.194 kb), pMF45. By comparing pBR322 Hue11 fragment sizes determined by gel electrophoresis to their size determined by sequence data, we have determined empirically that the error in the gel method of analysis is about 10 base pairs. The smallest mini-F isolated by us is

168

SEELKE ET AL.

FIG. 2. Mapping Tn3 insertion sites in pMF21. The basic mapping strategy is to identify a restriction fragment consisting of F DNA and a terminal piece of Tn3, estimate its length in nucleotide base pairs, and then subtract from this length that portion of the DNA consisting of Tn3 sequences. The difference represents the length of F sequences which were then used to calculate the insertion site. A sample calculation is given for pBKlO5 (lane 2). Based on Psrl mapping data (Fig. 3 and Kline and Palchaudhuri) we were able to identify the 570-base pair PstI fragment as the F:Tn3 species. We also knew from this previous work that Tn3 in all our plasmids is inserted to the right of the 45.878kb PstI restriction site mapped by nucleotide sequence analysis (Morutsu et al., 198 I ). From the sequence data of Hefferon et al. (1979) we also know that the distance from the Tn3 terminus to the first PstI site is 460 base pairs. Hence, the amount of F DNA is 110 base pairs and the insertion is 45.878 kb plus 0.110 or 45.89kb. Results from three separate analyses are shown. In the first analysis pBKl03, 105, 107, and 109 were each digested with PsrI and electrophoresed against pBR322 HpoII fragments (lanes I, 3, and 5). The results with pBK103, 107, and 109 were the same as shown for pBKlO5. In the second and third experiments the plasmids pBK104, 106, 110, 113, and pMF45 were digested first with PsrI then with BstEI. This produces PsrIF:Tn3 BsrEII hybrid fragments. From sequence data, the BsrEII site is 92 base pairs from the Tn3 terminus. Hence, this value subtracted from the total fragments sizes of 254 base pairs for pBK104 (lane 4), 106, 110,

pBK138-2 (Fig. 1 and Kline and Palchaudhuri, 1980); it is a deletion mutant of pBK105. The F sequences in pBK138-2 were originally estimated as 44.0 to 45.83 kb. Remapping the Tn3 insertion site in pBK105 to 45.99 f .OlO kb indicates that the coordinates of pBK138-2 are 44.0 to 45.99 kb. The exact site of the leftward coordinate is unknown; however, it lies between the KpnI site at 43.9 kb and the PstI site at 44.1 kb (Kline and Palchaudhuri, 1980). Maintenance of pBK138-2 is stable in overnight cultures without selective pressure. However, this plasmid is a copy number mutant (Table 2) and is resistant to elimination by acridine orange. Both of these properties are typical of the parent plasmid pBK105 (Table 2 and Wechsler and Kline, 1980) but not typical of a normal F replicon. Insertion of Tn3 at 46.19 in pMF21 (pmF45, Fig. 3) does not cause a copy number change (Manis and Kline, 1980) nor does it induce acridine orange resistance (Wechsler and Kline, 1980). This implies that we might construct a more normal miniF from the 44. l- 46.19-kb sequences. To approximate this construction, we first deleted in vitro two small BstEII fragments from pMF45 to produce pBK26 1 (Fig. 1). pBK261 lacks most of Tn3 and the entire 46.19- to 49.20-kb region of F. The only coding information extraneous to the 44.1- to 46.19-kb region still in pBK26 1 determines synthesis of protein D (Fig. 7). Synthesis of protein D is prevented by making the 43.7to 44.1 -kb PstI deletion (Fig. 3 and Wehlmann and Eichenlaub, 1980). To accomplish this, pBK261 was digested in vitro with PstI and the two largest PstI fragments joined and 113 or 408 base pairs for pMF45 (lane 6) allows us to calculate the insertion sites from the 45.878 kb PsrI reference point as 46.04 and 46.19 kb, respectively. Electrophoresis was in an 8.0% gel made with an acrylamide/methylenebisacrylamide ratio of 29: 1 at 20 V/ cm. The buffer was 5 mM Tris, 50 mM borate, 1 mM EDTA, pH 8.3. After electrophoresis the DNA was stained with ethidium bromide (0.5 pg/ml) in the same buffer.

GENETICS

OF MINI-F

PLASMID

TABLE COPY

2

NUMBERS OF VARIOUS MINI-F

PLASMIDS

Percentage ccc

Copy effect*

12.4 12.4 12.4 12.4

1.0 9.3 12.5 11.9

1.0 9.0 12.0 12.0

pBK26 1 pBK280

7.8 7.5

1.0 1.2

1.6 2.0

~BK104~ ~BK106~ pBK1 10d pBK1 13d

12.2 12.2 12.3 9.3

6.7* 7.6* 6.8* 5.6*

6.7 7.6 6.8 7.4

pBK103’ pBKlO5’ pBK107’

11.2 12.3 11.4

5.7* 6.0* 4.0*

6.3 6.0 4.4

pBK118 pBK138-2

3.3 2.6

1.7 1.4

6.9 6.6

12.4 12.4 12.4 12.4

13.9 27.9 13.4 12.4*

14.0 28.0 13.0 12.0

Plasmid pMF45 pBK63 pBK77 pBK80

pBK211 pBK213 pBK214 pBK215

Molecular weight

169

MAINTENANCE

Reference’ Manis Manis Manis Manis

and and and and

Kline Kline Kline Kline

(1978) (1978) (1978) (1978)

’ Each value is an average of two determinations in which the amount of covalently closed circular DNA(CCC) was measured by the dye-CsCl technique. The actual values deviated by no more than 10% from the reported value. Those values with an asterisk were measured only once. Values given are percentages: (cpm of CCC DNA + chromosomal DNA) X 100. b Copy effects are compared arbitrarily to the cop+ plasmid pMF45 which is given a relative value of 1.O. Actually though, 1% CCC DNA is equivalent to about 2.0 plasmids per chromosomal equivalent. ’ All values refer to results reported in this paper unless explicitly indicated otherwise. d pBK104, 106, I 10, and 113 each have Tn3 inserted,at coordinate 46.04 kb in the same orientation as in pMF45. In pMF45, Tn3 inserted at coordinate 46.19 kb (Kline and Palchaudhuri, 1980). ’ pBK103, 105, and 107 each have Tn3 inserted at coordinate 45.99 kb and in the same orientation; however, this orientation is opposite that in pMF45 (Kline and Palchaudhuri, 1980).

to form pBK280 (Fig. 1). pBK280 is equivalent to pBK261 deleted for the 43.7- to 44.1 -kb region. As shown in Table 2 both pBK261 and pBK280 have low copy numbers but are, nonetheless, elevated about twofold compared to pMF45. Because of the small plasmid sizes involved, this assay method does not reliably measure a twofold increase. Nevertheless, the dye-CsCl data are generally correct since copy numbers measured by single cell resistance (Nordstrom ef al.,

1980) to kanamycin were elevated about 1.5to 2.0-fold for both pBK261 and pBK280 hosts compared to a pMF45 host (data not shown). Both pBK261 and 280 were eliminated completely from their host by growth overnight in AB3 broth (pH 7.6) containing 100 pg of acridine orange per milliliter. The properties of pBK26 1 and 280 are consistent with the notion that low copy number plasmids, sensitive to acridine orange, can be formed from the 44. l- to 46.19-kb sequences.

170

SEELKE

SKS

ET AL.

K p,

40.8

43. I

! 1

4.5

Tn3

R

Ap 1 *

;

4

kan* ,-

3

2

I

0

FIG. 3. Structure and restriction map of PMF45. The restriction enzyme recognition sites are: (B) BumHI; (B2) BglII; (Bs) BsrEII; (C) Cl& (K) KpnI; (P) PsrI; (R) EcoRI; and (S) SmoI. The EcoRI /can+ fragment is 4.8 Mdal in mass and is insensitive to all the restriction enzymes shown except for KpnI and SmaI each of which cut it a few times. The sensitivity of this fragment to ClaI is unknown.

Identification and Mapping of inc+ Genes

The procedure used to detect and map inc genes consisted of cloning known PstI and BamHI restriction fragments from the miniF plasmid pMF45 and pBK103 (Fig. 3 and Table 1) into plasmid vectors compatible with F, introducing the recombinant plasmids into an Flat+ host by transformation, selecting for the recombinant phenotype on Maconkey lactose agar, and observing whether or not the Lac+ phenotype was lost (Manis and Kline, 1978). Maps of the Incf recombinants (pBK163, 207, 232, and 350) that were formed are shown in Fig. 1. This approach indicated that three different regions of F express an incC function: 44.1 to 45.88 kb (pBK207), 45.88 to 46.19 kb (pBK232), and 47.5 to 49.3 kb (pBK163 and 350; see also the derivatives of pBK211 in Table 3). The inc functions in these regions are designated, respectively, in&, incC, and incD (Kline and Lane, 1980). Upon completion of the above analysis, we found that the incB locus could be narrowed to the 45.0to 45.35-kb region (pBK374, Table 1). Production and Mapping of cop Mutations

Besides the finding that Tn3 inserted at either 45.99 or 46.04 kb, but not at 46.19 kb (pMF45), caused a cop mutation (Table 2), we previously had found that cop mutations can be induced in pMF45 by NTG (Manis and Kline, 1978). We knew from this latter work that the NTG mutations ~0~44,

48, and 50 mapped somewhere within the 43.1- to 46.19-kb region. Because NTG causes multiple mutation at a high frequency, we wanted to see if cop mutants could be isolated after a milder mutagenesis with EMS. Also, we wanted to know if the chemically induced cop mutations mapped between the Tn3-induced cop mutations or elsewhere. As shown in Table 2, EMS-induced mutations cop21 1, 213, 214, and 215 were isolated and they have copy numbers similar to those of the NTG-induced mutants. We have mapped the chemically induced cop mutations by making in vitro recombinants between cop+ and cop- variants of pMF45, pMF46, or pBK261. In Fig. 4 we show the structure of those recombinants that gave a Cop- phentoype. The reciprocal recombinants were also constructed and found to be Cop+. The Cop- phenotype in this study was defined as normal colony growth rate and size on agar containing 1.0 mg of either ampicillin or kanamycin per milliliter. The phenotype of several recombinants in both the Cop- and Cop+ classes was confirmed by direct examination of copy numbers using gel electrophoresis (data not shown). The only sequence common to all Cop- plasmids examined is the 45.35- to 45.88-kb region defined by SmaI and PstI sites as shown in Fig. 4. Therefore, of the cop mutations examined, cop48. 50, 211. 213, and 214, each must map in this region; cop44 and cop215 have been mapped to somewhere within the 45-O- to 46.19-kb re-

GENETICS

OF MINI-F

PLASMID TABLE

STATUS OF

F DNA SO”rCe pBK21 pBK216 pBK2l pBK21 pBK2l pBK77 pBKIO4 pBKlO5

I

COP copz/ l” c0p.?ll

I I I

COp.?l/

cop48 coplO coplO

Cloned

region

43.1 to 46.19 43.1 to 46.19 44.1 to 45.88 45.88 to 46.19 46. I9 to 49.0 43.1 to46.19 45.88 to 46.04 45.99 to 47.3

171

MAINTENANCE

3

inc GENES IN cop MUTANTS“

inc gene(r) in cloned region EC BC B C D BC C C

Transformantb phenotypes Cloning vector psclol pBR322 pBR322 pBR322 psclol pBR322 pBR322 pBR322

Recombinant pBK No. 216 257 242 233 217 258 263 264

LaC+

LaC-

Observed Inc phenotype

839 II 0 0 IO 8 4690 ICOO

46 739 900 542 1480 578 I 3

1ncFootnote hlc+ InC+ hlc+ Footnote I!lC. Inc.

c

E

*This table should be read in conjunction with Fig. 5. b The heading “Tmnsfonnant Phenotypes” refers to transformation by the recombinant plasmid in the column immediately preceding the transformation data. For example, pBKZl6 and 257 produced 839 and 11 Lac+ antibiotic-resistant transformants. respectively. The incompatibility assay is based on the ability or inability of the recA(F’/~+) recipient host to retain the I..ac+ phenotype when transformants were plated on MscConkcy lactose agar containing an antibiotic that selected for the transforming DNA, e.g.. pBK216, 257, 242. etc. (Manis and Kline. 1978). ‘About 70% of the transformants four white colonies (Inc+); but about 30% of the colonies have markedly red centers. Sibling analysis of several such c&nits indicated that 5% of the siblings also pmduccd colonies with fed centers. For reamns discussed in the text, we consider this to represent a reduction in incompatibility (Inc*) compared to the same F sequcnas cloned fmm a cop* plasmid. “The control for this experiment has been publiihed by Manis and Kline (1978). They found that a 43.1~ to 46.19-kb region taken from the cop+ plssmid pMF45 and cloned into the pSClOI vector produced I Lx+ and 1328 Lx- transformants. A similar degree of incompatibility is seen when the same sequences are cloned into pBR322 (data not shown).

gion. To summarize, Cop- phenotypes can be induced by mutations within 45.35 to 45.88-kb region, by Tn3 insertion at 45.99 and 46.040 kb and by deletion of the 46.19to 49.2-kb region. Status of inc Genes in Mutants Cop- Phenotype

with a

To investigate the status of inc genes in plasmids originally isolated as copy number mutants, we cloned the appropriate regions into pBR322, pSC101, or a pSClO1 ‘Km’ derivative, pBK50. The data in Table 3 show that the chemically induced mutation cop21 1 did not cause inactivation of any inc+ function when each function was cloned individually. Essentially identical results were found with ~0~44, 48, 50, 213, 214, and 215 (data not shown). By contrast, the insertion of Tn3 into the 45.99- and 46.04-kb coordinates of pMF21 to form, respectively, pBK105 and 104, inactivated the incC gene (see pBK263 and 264 in Table 3) but not the incB+ and incD+ genes (data not shown). That the Tn3induced inactivation of incC was real and not a failure of the recombinant to express the incC gene is supported by the fact that

the PstI fragment from pBK104 was cloned in the same orientation as shown for the incP:pBR322 recombinant, pBK232 (Fig. 1). Furthermore, the incC PstI fragment (Tn3 4.0-4.5 kb/F 45.99-47.3 kb) from pBKIO5 also was cloned in the same orientation in pBR322. For reasons that are unknown, we have not found pBK263 and 264 plasmids with the F sequences cloned in the opposite orientation. Modijication of inc Gene Expression by Cloning Vectors and cop Mutations The fact that incB+ and inch* expressions when cloned individually into pBR322 were not inactivated by chemically induced cop mutations is not surprising given that these cop mutations do not map in the inc determinants (see Discussion and Fig, 7). However, these findings do not agree with our earlier observation that chemically induced ~0~44, 48, and 50 mutations do inactivate the Inc+ phenotype determined by the 43.1to 46.19-kb sequences from cop+ plasmids when these sequences are cloned in pBK50 (Manis and Kline, 1978). Using the analytical protocol outlined both in Fig. 5 and in

172

SEELKE General

Procedure

cop’

COD-

I -- A

A’

-\

/

A:B’or

n/:B

Sma

43.6

and

purify

recombine

-

43-fI 44 :

-

restrict 0’

determine

Bglll

-

Plasmids

I 6

I transformants

40.350.8

ET AL.

Bgln I

45I,

46 I,

Ska: c

I ; I

Pst

phenotype

47 48

49

:

I Pbt w 1 ;

44.1

45.3

46.4 45.88

FIG. 4. Mapping chemically induced cop mutations. The NTG mutations mapped are cop48(pBK77) and copSO(pBK80); the EMS mutations are cop211 (pBK21 l), cop213(pBK213), and cop214(pBK214). Recall that all cop mutants were produced from pMF45 (cop’). The Bg0I recombinants were made directly between BglII fragments of pMF45 and cop mutants. The SmaI recombinants were made between cop+and cop- plasmids that had first been deleted of the EcoRI kan+ fragment to form pMF46-like plasmids (see Table 1). These recombinants are all mini-FAp’ plasmids. To form the PstI recombinants, the cop+and cop-plasmids first were deleted of four of their seven PHI restriction sites, that is, they were converted to the pBK261-like structures. Next, the appropriate Ps?I fragments of cop+and cop-plasmids were recombined to give mini-F plasmids with only two PstI sites, that is, pBK280-like plasmids. The pBK280-like plasmids are all mini-FKm’ plasmids. The three types of recombinants shown above were formed from pBK211. Also, the reciprocal structures were formed from this plasmid. Only the structures depicted in this figure had a Cop- phenotype. The only sequence common to all three recombinants is the 45.35-kb SmaI to 45.88-kb PstI sequence. Based on the findings with pBK211, we mapped the copmutations in pBK77, 80, 213, and 214 by making just the SmaI and PstI recombinants.

Table 3, we found that the 43.1- to 46.19kb region from cop21 1, 214, and 215 is also Inc- when cloned in pBK50. For cop48.21 I, 213, and 214 the same fragment is grossly Inc+ when removed from the pBK50 recombinants and recloned into pBR322. However, there is a slight, but important, qualification to this last statement, which is given in the next paragraph. The strength of the incompatibility reaction with either pBR322 or pBK50 as the cloning vector for any individual or group inc+ sequences from cop+ plasmids is so

strong that even transient F’lac/vector:inc+ heteroplasmid cells cannot be isolated (Manis and Kline, 1978; Seelke, unpublished). Hence, the identification of pBK5O:F Inc(incB+incC+) reeombinants represents a very large loss of incompatability expression. When pBR322 replaced pBK50 as the also vector, a loss of incompatibility must have occurred since about 30% of the recipients transformed by pBK257 (pBR322:F cop-incB+incC) formed colonies with slightly red centers, a Lac’ phenotype. Sibling analysis of these Lac’ col-

GENETICS

OF MINI-F

PLASMID

B /amp*

R ----------I

l

43. I

kan+

R B

46.19 BamHl

B A

I

173

MAINTENANCE

pBK2 I I

pBK50

pBK2 I6

pBR322

pBK2 I I Pstl

44. I incC

incB , 45.8

+

pBR322

-

pBK242

+ pBR322 -pBK233 45.BG FIG. 5. Origins and history of various inc genes in recombinant plasmids. This figure represents a flow diagram for the experiments described in Table 3. The steps of the flow diagram that give rise to pBK216 and pBK257 were done not only with pBK211 but also with pBK80, 213, and 214 as starting material. The lower steps that give rise to pBK233 and 242 were done using pMF45 and all seven chemically induced cop mutants. tb

onies indicated that they were composed of 95% Lac- cells and 5% F’laclpBK257 heteroplasmid cells that in turn gave rise to colonies with Lac* phenotype. Comparable results were seen with plasmids analogous to pBK257 but containing different chemically induced cop mutations. We interpret this to mean two things: (i) the chemically induced cop mutations can reduce expression of the incompatability determined by the 43.1 to 46.19-kb region and (ii) the degree of reduction seen depends on the cloning vector. Stability

of Copy Number

Mutations

It was of basic interest to examine the stability of chemically and Tn3-induced copy number mutants. We found that three mutants, cop.50, 103, and 213, were unstably maintained. The segregation kinetics of each mutant is shown in Fig. 6. Several cop mu-

tants were stably maintained for at least 150 generations without selective pressure; these are ~0~44, 48, 104, 105, 107, 1 IO, 21 I, 214, and 215. Since the plasmid markers used to monitor stability are on transposons, we verified the plasmid’s presence or absence at the end of each experiment by gel electrophoresis. We also wished to know if the kinetic profiles shown in Fig. 6 were influenced by the ability of plasmid-free variants to double at a much faster rate than their plasmid-containing counterparts. We found by measuring optical densities that cells devoid of pBK80 (copJO), 105 (cop105), and 213 (~0~213) grew, within resolution, at the same rate as their parental types. However, the slight downward curvature in the loss of pBK80 and pBK2 13 indicates that plasmid-free variants of these hosts grow 1 to 3% faster than the nonvariants (Nordstrom et al.,

174

SEELKE

Generations

FIG. 6. Stabilities of select mini-F plasmids. The stability of copB50 (pBK80) was measured by Method 1 (0) and Method 2 (0). The stabilities of (0) pBKl03 and (m) pBK213 were measured by Method 1. The number of generations on the X axis refers to growth in the absence of selective pressure for the plasmid-determined phenotype. The loss of pBK213 is a reproducible event but the exact segregation profile is not reproducible. The basis for this is unknown.

1980) a fact that could not be detected by simply comparing growth rate of plasmidfree and plasmid-containing cultures. DISCUSSION

Results from several studies indicate that only sequences around the 44.1- to 46.19-kb region appear to be essential for formation of a low copy number replicon. Identification of the essential region is based on the findings that (i) deletions extending into it from either side are lethal (Wehlmann and Eichenlaub, 1980; Morutsu et al., 1981) (ii) insertions of foreign DNA at the 45.1-kb Bg/II or at the 45.88-kb Pstl site are lethal (Kahn et al., 1979; Kline and Palchaudhuri,

ET AL.

1980); (iii) deletions 40.8 to 43.1, 43.7 to 44.1, and 46.19 to 49.2kb are not lethal (Fig. I ), and (iv) mini-F can be easily formed containing only the F sequences 44.0 to 45.99 kb (pBK138-2) or 44.1 to 46.35 kb (pBAL16a) (Morutsu et al., 1982). The exact endpoints for the essential region cannot be identified unequivocally since the possibility exists that the non-F DNA present in each mini-F may make a contribution to replicon existence. Exceptions to our conclusions about the dimensions of the essential region have been found. In aggregate these exceptions indicate that just the 44.76- to 45.88-kb region can form a mini-F (Lane, 198 1; Eichenlaub et al., 1981; Kahn and Helinski, personal communication). However, more extensive results from a number of laboratories indicate that the larger 44.1- to 45.88-kb PstI fragment of F itself normally does not form a plasmid (Kahn et al., 1979; Kline and Palchaudhuri, 1980; Wehlmann and Eichenlaub, 1980; Morutsu et al., 1982) and that small deletions across the 44.1-kb terminus are lethal (Wehlmann and Eichenlaub, 1980; Morutsu et al., 1982). Hence, we feel that the smaller F plasmids represent mutants or exceptions likely arising because of some special property of the non-F DNA used as a phenotype marker. In one case, Eichenlaub et al. (198 1) reported that the 44. I- to 45.88kb PstI fragment could form a plasmid only if the 45.88 to 47.3-kb sequences were present on a separate plasmid. They concluded that gene A (Fig. 7) was necessary to complement replication of the 44.1- to 45.88-kb fragment. The existence of pBK138-2, pBK261, and pBAL16a makes their conclusion paradoxical. Several observations indicate that the 44.1- to 46.19-kb region is relevant to maintenance of larger F plasmids. (i) Cloned incB and incC determinants from this region disrupt stable maintenance of F’lac elements. (ii) Sensitivity to acridine orange, an inhibitor of F replication (Hohn and Korn, 1969) maps within this region. (iii) Many conditional replication mutants of F’gul plasmids

GENETICS

OF MINI-F

PLASMID

175

MAINTENANCE e incC

Is

I : hi.1

-.

:

-w

:

: : 44.5

:

:

ori . .. . -... -w-b-be : : ; : 45.0

Essential

-c

aos Jw

cop (inc?)

I I II I

1I: :

:‘:

:

L5.5

:

:

. ... .

+4-#-c : :A

:‘:

c

4y= /’

Region

.)‘

kb proteins

i

c

!

D:

I

iA; b

B;

-I

FIG. 7. A gene map of the f5 restriction fragment. Symbols are explained in the text. The ori at 42.6 kb was mapped by Eichenlaub et al. (1977). A second ori at 44.4 kb has been mapped by Figurski et al. (1978) but Eichenlaub et al. (1981) now report that this ori has been repositioned to coordinate 45.07 kb. The 19- to 22-base pair direct repeats discovered by Morutsu et al. (1981) and Tolun and Helinski (1981) are designated by arrows (-). The Tn3 insertions that induce cop mutations and resistance to acridine orange (aos-) are designated by (X). The proteins labeled A, B, C, and D and their approximate map locations arc from Wehlmann and Eichenlaub (1980).

have been localized to somewhere within the 43.9- to 46.9-kb region (Gardner et al., 1980). (iv) Originally Eichenlaub et al. (1977) indicated that only the 42.6-kb ori was used by mini-F pML3 1. A reexamination of this point shows that the 45.1 ori is also used (Eichenlaub, personal communication). (v) Replication control, identified by copy number mutations, maps in the region of 45.35 to 46.04 kb. Eichenlaub and Wehlmann (1980) and Eichenlaub et al. ( 1977) have made observations indicating that the ori at 42.6 kb and the C protein (Fig. 7) also are involved in normal F replication. In fact, amber mutations in the gene for protein C (gene C) cause a conditional replication-defective phenotype. For reasons that are not understood, deletion of the 40.8 to 43.1 BumHI fragment containing the 42.6-kb ori and gene C from the conditional mutants renders them replication proficient. One possibility is that protein C is multifunctional and is required for expression of ori 42.6 kb and represses expression of ori 45.1 kb. The latter is not likely, however, since we have found that

pBK138-2 and a plasmid with cloned gene C (pBK55) are stably comaintained for over 100 generations (i.e., are compatible). The genes, functions, and special structures found in the essential region are ori, inc, cop, acridine orange sensitivity (aos+), and nine small direct repeats of 19 to 22 base pairs (see Fig. 7). Morutsu et al. ( 198 1) and Tolun and Helinski ( 198 I), who independently discovered these sequences, have also found that these sequences cause incompatibility. Our results are consistent with this conclusion. Additionally, our results with Tn3 insertions imply that the incC set of repeats is involved in copy number control and sensitivity to acridine orange. From this it is logical to reason that acridine orange could inhibit F at the initiation step in replication. The apparent overlap of the direct repeats with most if not all of these functions is somewhat similar to the observation made with RK2 and R6K plasmids (Schafferman et al., 198 1). For ease of reference and discussion, we treat incB and incC loci as genes. Since incC, at least, evidently does not make a soluble

176

SEELKEETAL.

product (Tolun and Helinski, 1981), these genes may be operator- or promoter-like in their function. The summary map in Fig. 7 suggests that their primary functions apparently pertain to the replication process. On this basis it is reasonable to suspect that in& and incC cause incompatability by titrating essential replication components or blocking replication sites. In any event, we believe that incompatability should be viewed as an experimentally useful but secondary.consequence to the true functions of these genes. The number of cop genes in F is difficult to discern clearly. Each cop mutant induced by the same agent or technique has the same phenotype; each agent or technique induces a mutation in a unique map location; and also, each mutant type has some unique phenotypic differences. Chemically induced mutations result in the highest copy effect and show no loss of sensitivity to acridine orange. These cop mutations map outside the direct repeats shown to be the in& and incC determinants, yet the mutations apparently have a negative effect on expression of these incompatability determinants. Tn3induced cop mutations in contrast induce a rather uniform copy effect, resistance to acridine orange, and inactivate only the incC determinant. These differences suggest two cop genes are involved; however, it remains for future efforts to determine whether this is true. The 46.19- to 49.2-kb deletion gives a copy effect of only 1.5 to 2.0-fold. Several lines of evidence now indicate that this elevation is real. In addition to the dye-CsCl results (Table 2) and the single cell resistance data findings mentioned under Results, we have also found that making this deletion on a Tn3-induced cop mutant, pBK104 (Table 2), results in about another 2-fold increase in copy number above the Tn3-induced change (data not shown). Also, the frequency of plasmid-negative cells in single colonies harboring the plasmid pBK261 indicates a copy value of about 4. The basis for relating this type of observa-

tion to copy effects is discussed by Nordstrom et al. (1980). The 46.19- to 49.3-kb region encodes two proteins (A and B) according to Wehlmann and Eichenlaub ( 1980) (Fig. 7). As we shall see below, protein B maps in the region implicated in partitioning. This implies that protein A might be responsible for the slight effect on copy number; however, specific mutagenic analysis of protein A will be necessary to verify or disprove this implication. A different possibility is that the deletion could alter copy number control indirectly by fusion to a new promotor. Obviously, more information is needed to confirm or deny the existence of a cop gene in the 46.19to 49.3-kb region. The overall control of F replication is likely to be negative as the findings of Tsutsui and Matsubara ( 1981) indicate. Our observations that chemically induced cop mutations inactivate the Inc+ phenotype in F:pBKSO hybrids containing cop-incBincC sequences is superficially in agreement with the notion that the cop gene makes a repressor of replication that also determines incompatability (Uhlin and Nordstrom, 1975). The difficulty with our findings is that the cop mutations lie outside the incB and incC sequences. Further, these determinants when cloned into are incBi and in&+ pBR322 and assayed for their ability to eliminate an F’lac plasmid. In spite of this anomalous behavior, which might be explained by copy number differences between pBK50 and pBR322 vectors, we have recently found that the chemically induced cop mutations are recessive to the cop+ allele (Kline, Seelke, and Trawick, in preparation). Again, this is consistent with a negative model for replication control. In fact, if we were not aware of the incB and incC determinants, we would conclude from this finding that the 45.35- to 45.88-kb cop region was also an inc determinant. But whether this is the case or whether this cop region controls expression of incB and incC remains to be clarified. The other major process of stable plasmid

GENETICS

OF MINI-F

PLASMID

maintenance is partitioning. Ogura et al. (1980) have found that cloning the 47.3- to 49.3-kb PstI/EcoRI fragment from wildtype F into unstable, high copy number miniplasmids made from the Escherichia coli origin of replication (oriC) stabilized the miniplasmids. Likewise, Nordstrom (personal communication) has found that cloning a DNA fragment containing the 46.19to 49.3-kb sequences of F into partitioning defective (Par-) mutants of plasmid Rl gave Par+ recombinants. These results strongly imply that a par gene maps within the 47.3to 49.3-kb region, the region we and others (Lane, 198 1) have found to contain the incD locus. We have found that plasmids with a leaky incD mutation (pBK96; Kline, 1979) or with an incD deletion (pBK261 and pBK280, Fig. l), are lost at rates of about 0.5% per generation (unpublished data). This is the expected rate of loss for par mutants of this copy number value that are randomly distributed according to a binomial function (Nordstrom et al., 1980). Very possibly incD and par are equivalent genes. Replication or replication control and partitioning appear to be independent events at least for pSClO1 (Meacock and Cohen, 1980) for Rl (Nordstrom et al., 1980) and for NRl (Miki et al., 1980). The fact that the putative F par gene stabilizes maintenance of oriC and Rl plasmids, both of which are apparently unrelated to F replication genes, argues that the F par gene functions independently as well. Even par+ plasmids have a slight instability exemplified by a spontaneous loss of frequency of 10e2 and lop4 for F’plasmids. Hence, the instability associated with cop50, 103, and 213 mutations might merely be a manifestation of this since plasmid-free cells with slightly faster growth rate will eventually predominate in a culture (Nordstrom et al., 1980). However, this loss was not seen with all the other cop mutants examined. Possibly instead, these three mutants were not really lost but only their phenotypic determinants were spontaneously deleted. This

MAINTENANCE

177

explanation is unlikely because spontaneously generated, antibiotic-sensitive cells were found to be completely devoid of plasmid DNA (data not shown). An obvious explanation is that cop+ and par+ gene functions are not independent in F but as yet there is no other firm support for this hypothesis although the Cop- phenotype resulting from deletions of the 46.19- to 49.2kb region represents a potential connection. Finally, since cop50 and 213 are chemically induced mutants, a second cryptic mutation may exist in them and be reponsible for the instability. In closing we can summarize as follows: a region of F, 44.1 to about 46.19 kb, is essential and sufficient for replicon formation. Nevertheless, genes in the 40.8- to 43. lkb region also participate in replication when present but of themselves do not constitute a replicon. The components identified in the essential region are ori, cop, inc, aos, and nine direct repeats. The loci of the direct repeats overlap each of these functions. A relationship apparently exists between cop and two inc determinants but the details are unclear; however, accumulating evidence clearly indicates negative control of F replication. Partitioning function(s) (par) map outside of the essential region between 47.3 and 49.2 kb. They are likely identical to a third inc determinant that had been identified previously. Loss of the 46.19- to 49.19kb region which contains the par determinant leads to a slightly unstable phenotype apparently caused by random distribution of the plasmids at cell division. Available data indicate that the par+ gene(s) can function with plasmids other than F, indicating that they can work independently of a specific replicon type. ACKNOWLEDGMENTS This research was supported in part by a Public Health Service research grant to Bruce C. Kline from the National Institutes of Health (GM25602) and in part by the Mayo Foundation. We also gratefully acknowledge the expert technical assistance of Becky

178

SEELKE

Schmidt Arendt, Tim McLean, Ross Aleff, and Diane Nelson. We thank K. Nordstrom, R. Eichenlaub, and S. Levy for allowing us to cite their unpublished observations.

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ET AL. binant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3, 88-91. KLINE, B. (1979). Incompatability between Flat, R386 and FpSClOl recombinant plasmids: The specificity of F incompatability genes. Plasmid 2, 437-445. KLINE, B., AND LANE, D. (1980). A proposed system for nomenclature for incompatability genes of the Esckerickia coli sex factor, plasmid F. Plasmid 4, 231-232. KLINE, B., AND PALCHAUDHURI, S. (1980). Genetic studies of F plasmid maintenance genes. Plasmid 4, 281-291. KLINE, B., SEELKE, R., ANDTRAWICK, J. (1981). Replication and incompatibility functions in mini-F plasmids. In “Molecular Biology, Pathogenicity, and Ecology of Bacterial Plasmids” (S. B. Levy, R. L. Clowes, and E. L. Koenig, eds.), pp. 317-326. Plenum, New York. LANE, H. E. 0. (198 1). Replication and incompatability of F and plasmids in the IncFI group. Plusmid 5, 100-126. LANE, D., AND GARDNER, R. C. (1979). Second EcoRI fragment of F capable of self-replication. J. Bucreriol. 139,141-151. LOVETT, M. A.., ANDHELINSKI, D. R. (1976). Method for the isolation of the replication region of a bacterial replicon: Construction of a mini-F’km plasmid. J. Bucteriol. 127, 982-987. MANIS, J. J., AND KLINE, B. C. (1977). Restriction endonuclease mapping and mutagenesis of the F sex factor replication region. Mol. Gen. Gent. 152, 175182. MANIS, J. J., AND KLINE, B. C. (1978). F plasmid incompatability and copy number genes: Their map locations and interactions. Plasmid 1, 492-507. MEACOCK, P. A., AND COHEN, S. N. (1980). Partitioning of a bacterial plasmid during cell division: A cis acting locus that accomplishes stable plasmid inheritance. Cell 20, 529-542. MIKI, R., EASTON, A. M., AND ROWND, R. H. (1980). Cloning of replication incompatability and stability functions of R plasmid NRl. J. Bucteriol. 141, 8799. MILLER, J. H. (1972). “Experiments in Molecular Genetics,” p. 13-23. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MORUTSU, T., MATSUBARA, K., SUGISAKI, H., AND TAKANAMI, M. (1981). Nine unique repeating sequences appear in a region of the mini-F plasmid genome essential for replication and incompatability. Gene 15, 257-271. NORDSTROM, K., MOLIN, S., AND AAGAARD-HANSEN, H. (1980). Partitioning of plasmid Rl in Esckerickia coli. 1. Kinetics of loss of plasmid derivatives deleted for the par region. Plasmid 4, 215-227. NOVICK, R. P., CLOWES, R. C., COHEN, S. N., CURTISS, R., III, DATA, N., AND FALKOW, S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Bucteriol. Rev. 40, 168-189.

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OGURA, T., MIKI, T., AND HIROGA, S. (1980). Copy number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: Evidence for a control region of replication. Proc. Nat. Acad.

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OHTSUBO, E., DEONIER, R., LEE, H. J., ANDDAVIDSON, N. (1974). Electron microscopic heteroduplex studies of sequence relations among plasmids of Escherichia colj. IV. The F sequences in F14. J. Mol. Biol. 89, 565-584.

RADLOFF, T., BAUER, W., AND VINOGRAD, J. (1967). A dye-buoyant density method for detection and isolation of closed circular duplex DNA: The closed circular DNA in HeL cells. Proc. Nut. Acad. Sci. USA 57, 1514-1520. RODRIGUEZ, R. L., BOLIVAR, F., GOODMAN, H. M., BOYER, H. W., AND BETLACH, M. (1976). Construction and characterization of cloning vehicles. In “ICN-UCLA Symposium on Molecular and Cell Biology,” Vol. 5, “Molecular Mechanisms in the Control of Gene Expression” (D. P. Nierlich, W. J. Rutter, and F. Fox, eds.), p. 471-478. Academic Press, New York. SHAFFERMAN, A., STALKER, D. M., TOLUN, A., KOLTER, R., AND HELINSKI, D. R. (1981). Structurefunction relationships in essential regions for plasmid replication. In “Molecular Biology, Pathogenicity, and Ecology of Bacterial Plasmids” (S. B. Levy, R. C. Clowes, and E. L. Koenig, eds.), pp. 259-270. Plenum, New York.

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SUTCLIFF, G. (1978). pBR322 restriction map derived from the DNA sequence: Accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acid Res. 5,2721-2728.

TIMMIS, K., CABELLO, F., AND COHEN, S. (1975). Cloning, isolation and characterization of replication regions of complex plasmid genomes. Proc. Nut. Acad.

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