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Preparation of plasmids from lambdoid phages and studies on their incompatibilities
PLASMID
1, 284-296 (1978)
Preparation
of Plasmids from Lambdoid Phages and Studies on their Incompatibilities KENICHI
Department
of Biochemistry,
MATSUBARA’ Kyushu University
AND YOKO OTSUJI School of Medicine,
Fukuoka,
812, Japan
Accepted January 27, 1978 Various lambdoid phages, including those carrying the immunity region of 434, 21. and 480, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm2ldv, and $8Odv, were similar to the already known plasmid Xdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids kdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.
phage. Bacterial receptor mutants and mucoid mutants are not selected, because a mixture of phages with different receptor specificities and a galdel host are used. With this technique, the genetic region of the genome that can be extracted as a plasmid and that functions in the autonomous replication of the plasmid has been determined (Matsubara, 1976; Berg, 1974). Here, the same technique is employed with other phages to see if the relationship between the phage and its plasmids can be generalized. It is shown that all the lambdoid phages tested, including imm434, imm2 1, and 480 [for descriptions of these phages, see Hershey (1971) and Fig. l), can give rise to plasmids similar to hdv. This paper describes the properties of these plasmids and their interactions in the cell. The latter problem seemed of particular interest, as this system provides a model for studying the mechanism of plasmid incompatibility [for reviews, see Novick (1969) and Clowes (1972)]. Our results show that incom’ Present address: Laboratory of Molecular Cepatibility is primarily determined by the netics, University of Osaka Medical School, Kita-ku, “immunity region” on the plasmid genome Osaka, 530, Japan.
The plasmid xdv is an autonomously replicating DNA fragment of the bacteriophage genome consisting essentially of only three genes (tof, 0, and P), one promoteroperator (pRoR) for transcription, and an origin (ori) for replication (Matsubara, 1976; Berg, 1974). It is only about one-tenth the size of the parental phage genome, and about sixty copies are found per carrier bacterial chromosome. Recently a technique was devised for preparing new hdv from A phage (Matsubara, 1974a). This technique makes use of the fact that Xdv plasmids arise in a population of cells infected with uv-irradiated nonlysogenizing A phage (viz., c1 or vir mutants) and that, even when the frequency of appearance of these plasmid-carriers is low, they can be selected by their ability to form colonies on plates seeded with the phage. The tolerance is specific, and the same cell is sensitive to infection by heteroimmune
0147-619X/78/0013-0284$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
284
PLASMIDS FROM LAMBDOID
I
Head
I
Tail
285
PHAGES
Recombination
Regulation
1 Lysis
1
A ircN-34
;un Q80 cm
rex cl
N PL OL
tof
cl1
0
P
0’
ori
PR OR
a
hdvl
n
Advo21
__ . a
b
c
. ..
FIG. 1. Genomes of bacteriophage A, imm434, imm21, 480, and some dv plasmids. Open bars show the segments originating from A. Hatched, cross-hatched, or dotted bars show regions of nonhomology. For symbols of genes, see Campbell (1971). The endpoints of Xdvl and AdvO21are adopted from Chow er al. (1974). Endpoints of imm434dv and imm2ldv have not been mapped precisely. Arrows indicate sites of cleavage by restriction endonuclease EcoRI (Thomas and Davis, 1975; Murray et al., 1977; Streeck and Hobom, 1975). Since locations of genes and EcoRI cleavage sites along the 980 genome have not been mapped precisely, the $80dv genome is not included. For explanation of a, b, and c, see text.
that carries an autorepressor gene (Matsubara, 1976)and a promoter-operator (DNA sequences recognized by the products of repressor gene tof, intermingled with the promoter sequence used in the initiation of transcription). MATERIALS AND METHODS Bacteria and plasmids. A derivative of Escherichia coli K12, km723 [recAl, his, str, (gal-att)de*], has been described pre-
viously (Matsubara, 1974a). Strain km960 is a thymine-requiring derivative of km723. The derivatives of hdv, hdvgall,, (Berg et al., 1974), hdvkan, (Berg, personal communication), and hdvamp, (Matsubara, to be published), carry, respectively, genes that code for fermentation of galactose, inactivation of kanamycin, and inactivation of ampicillin. The kanamycin-inactivating and ampicillin-inactivating genes are carried on transposons from the plasmids JR67 (Berg et al., 1975) and RM201 (Odakura et al. 1974), respectively. km960 carrying one of these plasmids was prepared by transformation (Mandel and Higa, 1970; Hashimoto and Matsubara, 1974).
Bacteriophages. The following bacteriophages were used. Phages imm434, imm21, 480, and the repressor-negative mutants, AcI,, and imm434c&, have been described previously (Matsubara, 1972a, b). The repressor-negative mutants, imm2 ICI, and @kI,, were prepared by mutagenesis of E. coli K12 recA lysogens with iV-nitro-N’nitrosoguanidine and selection of clear plaque-formers that appeared during subsequent incubation. For simplicity, these phages will be abbreviated as AC,imm434c, imm2lc, and $8Oc, respectively. The hybrid phages, hs0imm434c and hs0imm21c, were prepared by crossing imm434c or imm2lc with h*OimmA(Matsubara, 1974a). In these hybrid phages, the “left arm” that carries genes A, B, . . . J is from $180, whereas the “right arm” that carries genes att, int, c1, 0, P, and R is from imm434 or imm21. The h*imm+80 and h*imm~80gal+ were prepared by crossing phage $80~ with A and Apga15cIg5,,respectively, and selecting recombinants in which the left arm was from A and the right arm was from 480. The hm80imm~80cgal+was prepared by crossing hYmm$80cgal+ with a hybrid phage, h@‘80immAc18,,gal+ (Matsubara, unpublished
286
MATSUBARA
data), in which the left arm was from 480 and the right arm from A. For simplicity, a phage that shares the same immunity region as the plasmid will be referred to as homoimmune, while phages that do not share this region will be referred to as heteroimmune. For example, A is a homoimmune phage to the plasmid hdv, whereas it is heteroimmune to imm434dv. Media. The media used, PB, PBMM, PBB, PBB agar, EMBO, EMB galactose agar, and MacConkey galactose agar, have been described previously (Matsubara, 1972a, 1974a). Enzyme. The restriction endonuclease EcoRI was prepared according to the method of Yoshimori (1971). Assay of the rate of appearance of plasmid-minus segregants. Approximately 500 cells were spread on PBB agar to form colonies. They were then replica-plated onto EMBO agar coated with 10’ particles of homoimmune clear plaque-forming phage. After incubation for 16 h, the plasmid-carriers appeared as white- to pinkcolored prints on the replica plate, because they were tolerant to phage infection, whereas the segregants appeared dark red due to lysis. Segregation of Xdvgal, Advkan, or Advamp was assayed by observing colonies for their loss of the ability to ferment galactose or of resistance to the drugs. Extraction of plasmid DNA, sucrose gradient centrifugations, CsCl-ethidium bromide isopycnic centrifugations, and agarose gel electrophoreses. The methods used have been described previously (Matsubara et al., 1975).
AND OTSUJI
mixture containing nonirradiated phage at a multiplicity of infection of 0.1. Under these conditions, bacterial receptor mutants or mucoid mutants would not be recovered (Matsubara, 1974a). Furthermore, lysogens would not be formed because the phage could not lysogenize. Only carriers of a defective phage genome would survive and form colonies, due to their ability to interfere with growth of the homoimmune phage. Using 3 x lo8 cells infected with the irradiated phage mixture, I to 300 colonies were obtained. These colonies were crossstreaked, first against imm434c and then against imm2lc. About 79% of the colonies were tolerant to homoimmune imm434c, but were sensitive to the heteroimmune imm2lc, a behavior characteristic of imm434dv carriers (see below). Carriers of im2ldv were made similarly, except that a mixture of imm2lc and hmsoimm21c was used and the resulting colonies were cross-streaked over imm2lc first and then over AC. Four colonies each of imm434dv and imm2ldv carriers were chosen at random, purified, and subjected to marker rescue tests (Matsubara and Kaiser, 1968). All of these plasmids donated the genes O+ and P+, but not A+, J+, Nf, Q+, or R+. This pattern of rescue is identical to that of Adv (Matsubara and Kaiser, 1968), suggesting that these plasmids carry a segment of phage genome quite similar to that of the prototype Adv (see Fig. 1). Since there is a region of nonhomology between A and imm21 which extends from the left end of gene N to the right of the cI1 gene, the possibility that some of the imm2ldv’s might carry gene Nzl was not ruled out by these tests. RESULTS Sucrose gradient sedimentation analyses Preparation of imm434dv and imm2ldv and agarose gel electrophoreses showed Plasmids that all of the colonies tested carried plasmid A mixture of imm434c and hmsoimm434c DNA that could be isolated as the small, was irradiated with uv light to give a sur- covalently closed circular form (see below). vival of 10e2,and then adsorbed to bacterial strain km723 at a ratio of one phage per Preparation of @Odv’s Cells carrying plasmids derived from 480 bacterium. The infected cells were immediately plated on PBB agar with an identical were first sought by the technique described
PLASMIDS FROM LAMBDOID ABCDEFG
HIJKLMNO
Xdvgal+ Advl(D) hdvl(M)
XdvO21
FIG. 2. Analysis of dv plasmids by agarose gel electrophoresis. Plasmid DNAs in the covalently closed circular form where purified by CsCl-ethidium bromide density gradient centrifugations, as described previously (Matsubara ef al., 1975), and subjected to electrophoresis on a 0.9% agarose slab gel. (A) A mixture of hdvgal+, Advl dimer, Xdvl monomer, and XdvO21; (B) and (I) imm434dvCQ2; (C) and (J) imm434dvCQ7; (D) and (K) imm2ldvABS; (E) and (L) imm2ldvAB7; (F) and (M) @OdvDU9; (G) and (N) $lOdvDOl; (H) a mixture of hdvl and Adv021; (0) whole A phage DNA. (A)-(G) Samples without EcoRI treatment; (H)-(O) samples treated with EcoRI. Small fragments appearing in the @Odv digests are not shown in the picture.
above. On plates seeded with a mixture of 480~ and h*imm+80c, 10 times more colonies were recovered from uv phage-infected cultures than were found in the above experiments with imm434 or imm2 1. Some (ca. 15%) of these colonies were tolerant to infection by homoimmune phage ($SOc), but were sensitive to heteroimmune phage (Xc). However, in marker rescue tests, all (50) colonies tested donated gene 19, which is equivalent to gene R in X (Sato, 1970). Furthermore, at least 10 of them carried extrachromosomal DNA which was indistinguishable in size from the whole 480 genome, although they did not produce any infectious particles upon prolonged incubations after uv irradiation. These bacteria apparently carried the entire 480 genome that had presumably undergone a mutation in the gene equivalent to iV in X (Signer, 1969; Kleckner and Signer, personal communication). It seems therefore that the
PHAGES
287
$80 genome is prone to produce such plasmids. Next, a phage mixture was made from two imm480c phages, each carrying the bacterial gal gene (h~80imm~80cgal+ and hximm$80cgal+), and added to galdel, recA cells. The infected cells were plated on MacConkey galactose agar. About 50% of the colonies showed the red Gal+ phenotype and were discarded as carriers of the whole phage genome. About 20% of the colonies were white to pink in color and were tolerant to homoimmune phage (480~)) but sensitive to AC. Phage-producing colonies were discarded, leaving 11 clones that were purified and examined for extrachromosomal DNA. Eight were found to carry a small circular DNA, viz., +80dv DNA, while the rest (3) carried DNA of the same size as the 480 genome. Appropriate superinfecting phages were not available, so marker rescue tests could not be done. Size, Oligomeric State, and Content of Plasmid Copies in a Cell
The phage-derived plasmids can be isolated as covalently closed circular DNA and have a molecular weight of 3 to 10 x 106. The electrophoretic patterns of some of these DNAs are shown in Fig. 2, and their molecular sizes in Table 1. Each of the plasmid DNAs was treated with EcoRI restriction endonuclease and run in sucrose gradients or in agarose gels. From cleavage maps of the lambdoid phage genomes, it was predicted that the hdv and imm2ldv DNAs would be cleaved once by EcoRI at a site within gene 0, yielding one unique fragment. In contrast, imm434dv DNA would be cleaved at two sites, one in gene c1 and one in gene 0, and would yield two fragments (Helling et al., 1974; Streeck and Hobom, 1975; Thomas and Davis, 1975; also see Fig. 1). Figure 2 shows that these predictions were verified: Different imm2ldv’s each produced one unique product; different imm434dv DNAs each produced two frag-
288
MATSUBARA
AND OTSUJI
TABLE I SIZE,~LIGOMERIC FORM, AND NUMBEROFCOPIESOFDIFFERENT dv PLASMIDS Molecular weight
Plasmid
Whole moleculen x 10-e (A)
hdvl dimer hdvl monomer AdvO21 imm434dvCQ2 imm434dvCQ7 imm2ldvABS immZldvAB7 imm2ldvABl4 480dvDU9 &OdvDOl
9.4 4.7 2.2 9.9 6.6 8.3 9.5 7.3 3.5 7.6
No. of copies per chromosome
EcoRI digest’ x 10-G (B)
2.8 + 2.4 +
3.3 + 3.5 +
4.7 4.7 2.2 0.83 = 0.83 = 4.2 3.1 3.3 0.39 = 0.39 =
3.6 3.2
3.7 3.9
Oligomeric form
CC DNA contentc
(C =A/B)
(D)
2 1 1 3 2 2 3 2 1 2
0.17 0.12 0.036 0.25 0.29 0.52 0.26 0.27 0.24 0.29
Whole moleculed W
66 95 60 90 156 226 101 137 245 136
Monomer units (E x Cl
132 95 60 270 312 452 303 274 245 272
a Molecular weight of closed circular DNA was estimated from its sedimentation velocity or mobility in agarose gels, using hdvgal+ (13.4 x lOa), hdvl dimer (9.4 x lOa), Advl monomer (4.7 x lOa), and Adv021 (2.2 x 106)as standards. b Molecular weights of linear duplex molecules were estimated from their sedimentation velocities or mobilities in agarose gels, using Advl, Adv021, and whole A phage DNA, all treated with EcoRI, as standards. imm434dv’s and 480dv’s each produce two fragments upon treatment with EcoRI. c Ratio of the amount of DNA in the closed circular (CC) form to the amount of chromosomal DNA, as assayed by CsCl-ethidium bromide isopycnic centrifugation (Matsubara et al., 1975). d D x 2.5 x 109/A, assuming that the molecular weight of E. coli chromosome is 2.5 x 109.
ments. One of the latter fragments is unique in size (MW, 0.83 x 106), suggesting that it is the fragment b in Fig. 1. The other fragment varies in size, depending on the plasmid species, and this suggests that it consists of regions a and c, which have joined together (see Fig. 1). Like imm434dv’s, +80dv DNAs gave rise to two fragments after&OR1 digestion, viz., they carry two EcoRI sites. The smaller fragment was unique in size (MW, 0.39 x 10s), whereas the large fragment was variable. One of these EcoRI sites must be at one end of fragment number six in the cleavage map of 480 (Helling et al., 1974), and the other is a new site, not previously reported, perhaps because of its proximity to the former site. No attempts were made to locate these sites on the $80dv genome. By comparing the sizes of intact, covalently closed circular molecules with the sum of the molecular weights of the EcoRIgenerated fragments, it was possible to as-
sess the oligomeric states of the intact molecules. Table 1 summarizes the results of these analyses. The molecular weight of the monomeric unit was found to fall within the range of 3 to 5 x 106. Many dv plasmids were found to be in the dimeric state, although some were in monomeric form and yet others were in the form of higher oligomers. The amount of plasmid DNA in the closed circular form relative to that of chromosomal DNA was measured by isopycnic centrifugations of cell lysates in CsClethidium bromide. The data obtained are also shown in Table 1. The number of plasmid copies per host chromosome was calculated assuming that the molecular weight of the latter is 2.5 x log. It can be seen that the various phage-derived plasmids and hdv occur in 50 to 200 copies per carrier cell. With each plasmid, the copy number is constant, and is not affected by the age of the cells. The carrier cells must possess some
PLASMIDS FROM LAMBDOID
289
PHAGES
TABLE 2 EFFICIENCYOF PLATING OF LAMBDOID PHAGESON BACETERIACARRYINGVARIOUSdv PLASMIDS Efficiency of plating” Bacteria km723 km723[Advl] km723[hdvBBl] km723[imm434dvCQ2] km723[imm434dvCQ7] km723[imm2ldvABS] km723[imm2ldvABl4] km960 km960[+80dvDU9] km960[+8OdvDO l] km960[imm2ldvABl4] [hdvl] km960[480dvDU9] [Advl]
imm434c
imm2lc
imm@Oc
1.0 -3 x IO-’ -3 x 10-1 0.10* 0.36 0.7 0.92 1.0 0.93 0.96
1.0 0.026 0.27b 5 x lo-3c 0.02’ 0.65 0.59 1.1 1.1 1.1
1.0 1.0 0.97 0.69 0.93 <2 x IO-’ <2 x 10-r 0.92 1.1 1.1
1.0 0.86 1.1 1.05 0.93 1.0 0.88 0.85 <5 x 10-1 <5 x 10-1
<5 x 10-7
0.03*
<2 x 10-T
0.84
<5 x lo-’
0.07b
1.1
<5 x 10-7
AC
a Bacteria grown overnight in PB were plated on PB agar with an appropriately diluted phage, and the efficiency of plating of the phage was scored relative to that on km723 or km960 (which do not carry plasmids) after overnight incubation at 37°C. The phages used are described under Materials and Methods. * These plaques were small and irregular in shape. c These plaques were extremely tiny. Plaques of normal size appeared with a frequency of less than 10-r.
mechanism for maintaining number.
a constant
Rate of Appearance of Plasmidless Segregants Cells carrying these plasmids were grown for 50 generations, then spread over PBB agar to determine the number of plasmidless segregants. It was observed that, among carriers of Adv, imm434dv, im2ldv, and $80dv, respectively, 0.7,0.5,0.4, and 0.7% of the population were segregants.
action was seen with A and imm434: hdv partially interferes with growth of the imm434 phage, and imm434dv interferes with that of the A phage. This type of weak interaction has been reported previously (Matsubara, 1972a; Berg and KellenbergerGujer, 1974). Compatibility between Two Different Plasmids
The addition of hdv DNA to Ca2+-treated E. coli cells transforms them to Adv-carriers (Hashimoto and Matsubara, 1974; Growth Interference of Superinfecting Berg et al., 1974). This technique was Phage employed to construct cells that carry two The plasmid carriers were tolerant to different plasmids. As shown in Table 3, the super-infection by homoimmune phage, viz., efficiency of transformation by Advl was the the phages from which the plasmid was de- same, regardless of whether or not the rerived (Matsubara, 1972a). However, the cipient cells were carriers of resident plasinteraction was specific, and these cells mid imm2ldv or 480dv. These transwere sensitive to heteroimmune phages. formants carried both the resident plasmid The efficiencies of plating of various phages and Advl, as judged from their tolerance on a set of different plasmid carriers are to phages A as well as imm21 and 480, shown in Table 2. An exceptional inter- and from the presence of two molecular
290
MATSUBARA
AND OTSUJI
TABLE 3 EFFICIENCYOF Adv TRANSFORMATIONOF CELLSTHAT~ARRY A RESIDENTPLASMID~ Efficiency of transformation (x 1O-s) Experiment Recipient bacteria
Donor DNA
km723 km723[imm21dvAB14] km960 km960[480dvDU9] km723 km723[imm434dvCQ2] km723[imm434dvCQ7] km723[Advl] km723[Adv021] km723[AdvBBl] km960 km96O[Advamp]
hdvl Advl Advl Advl Advgal+ Advgal+ Advgal+ Advgal+ Advgal+ AdvgaJ+ Advkan Advkan
Selection of transformants Tolerance to A
Selection of Gal+
Selection of KanR Selection of Kans, AMPa
1
2
3
560 430 190 180 132 105 42 24 36,34
480 58.5 228
650 770 750
4
740 370
a Donor DNAs in the closed circular form were purified by CsCl-ethidium bromide isopycnic centrifugation. Transformation was done using Ca*+-treated recipient bacteria as described previously (Hashimoto and Matsubara, 1974). The hdvgal+ transformants were scored as galactose-fermenting colonies on EMB galactose agar plates after incubation for 36 h at 37°C. For phenotypes of the Gal+ transformants, see text. The Advkan transformants were selected on PBB agar containing the indicated drugs (Kan = kanamycin, 20 pg/ml; Amp = ampicillin, 40 @ml). The efficiency of transformation was calculated as the number of transformants divided by the number of DNA molecules added.
species in extrachromosomal DNA preparations (Fig. 3). The components each exhibited the sedimentation velocity characteristic of the original plasmid, indicating that the two plasmids are carried independently, and not in a fused or “composite” form. The contents of plasmid DNAs in the double-carriers are additive (Table 4). From the double-carrier clones, each plasmid species was lost independently, as shown in Table 5. Clones that had lost both plasmids simultaneously appeared at a somewhat high frequency, but the difference was not significant. Based on these observations, we concluded that hdv and imm2ldv, and also hdv and $80dv, both pairs of which represent combinations derived from heteroimmune phages, are compatible, viz., these different species of plasmids can coexist independently in the same cell. Double-carriers of imm434dv and Xdv were constructed similarly, except that in this
case Advgal+ DNA was added to Gal- cells and Gal+ transformants were selected on EMB galactose agar. The use of hdvgal+ was necessary because carriers of imm434dv are partially tolerant to A phage infection, thus precluding the use of A for selection of Ad+ transformants. Table 3 shows that the efficiency of transformation was almost the same, whether or not the cells carried the resident plasmid imm434dv. However, the double-carriers in the combination were unstable, as will be shown below. Gal+ transformants of the control, plasmid-free recipient km723 produced black (Gal+) colonies, which rarely yielded Galsegregants, and contained extrachromosomal DNA indistinguishable from that of the original Advgal+ DNA (data not shown). On the other hand, galactose-fermenting transformants that had harbored the resident imm434dv were unstable. These colonies appeared brown (Gal’ phenotype) on EMB galactose plates because of the
PLASMIDS FROM LAMBDOID
A
Y’ C
d
IO FRACTION
NUMBER
FIG. 3. Analyses of extrachromosomal DNA in double dv-carriers by sucrose density gradient centrifugations. Double dv-carriers were prepared by transformation as described in Table 3, footnote a. The double-carriers harbor Xdvl and immZldvABl4 (A), Advl and @OdvDU9 (B), Xdvgal+ and imm434dvCQ7 (C). 3H-Labeled extrachromosomal DNAs in covalently closed circular form were prepared and sedimented at 37,000 rpm for 195 min at 20°C through 5-20% sucrose gradients containing 0.1 M Tris (pH 7.5), 1 M NaCl, and 0.01 M EDTA. Peaks were assigned from standards. These data are omitted for clarity. Sedimentations are from right, to left. Profiles similar to A or B were obtained when independently obtained double-carrier clones were tested. On the other hand, five independent clones carrying Xdvgal+ and imm434dv all exhibited different ratios in areas of the two plasmid peaks, although the peak positions were reproducible. This indicates that the Advgal+ and imm434dv coexist only in an unstable fashion.
high content of Gal- segregants. Upon restreaking, lo-40% of the cells in the brown colonies again produced Gal’ colonies and 60-90% produced Gal- segre-
291
PHAGES
gants. Even after several cycles of cell isolations, the Gal’ traits were maintained. Figure 3C shows a sedimentation profile of extrachromosomal, covalently closed, circular DNA prepared from such Gal’ cells. Two components are seen, corresponding to DNAs of Xdvgal+ and imm434dv. However, the ratio of the two DNAs varied, depending on the clones studied and also on the number of cell divisions. Because of the continuous segregation, no attempts were made to estimate the number of the plasmid copies per cell. Thus the heteroimmune hdv and imm434dv, which are closely similar, can be maintained as separate entities, but cannot coexist stably. When Xdvgal+ DNA was added to cells carrying a resident hdv, “galactose-fermenting” primary transformants appeared on EMB galactose plates at a reduced frequency (Table 3). These primary transformants had a small growth advantage over non-galactose-fermenting parental cells, and formed slightly larger colonies than the others, although they were neither black nor brown. The majority of cells in these colonies were Gal-, since, upon restreaking, Gal+ or Gal’ colonies were not detected (less than 0.1%). In a separate experiment, the galactose-fermenting primary transTABLE 4 CONTENTOFPLASMID DNA IN DOUBLE dV-CARRIER CELLS'
Bacteria km723[Advl] km723[imm21dvAB14] km723[imm2ldvAB14][Advl] km960[+8OdvDU9] km960[+80dvDU9][Advl]
Content of CC DNA (%) 12.5 25.3 37.9 20.2 38.1
o Plasmid-carrying cells were uniformly labeled with [3H]thymidine and lysed with lysozyme-Na-Sarkosynate. Then the crude lysates were subjected to isopycnic centrifugations to separate covalently closed circular plasmid DNA (CC DNA) from chromosomal DNA, as described previously (Matsubara et al., 1975). The values given are ratios of the amount of closed circular DNA to the amount of chromosomal DNA.
292
MATSUBARA
AND OTSUJI
TABLE 5 INDEPENDENTSEGREGATIONOFPLASMIDSFROMCELLSTHAT~ARRY Two DIFFERENT~V'S" Percentage of segregants that have lost: Bacteria km723[bdvl] km723[imm21dvAB14] km960[+8OdvDU9] km723[hdvl][imm2ldvAB14] km96O[hdvl][+8OdvDU9]
kdv 0.96 12.6 1.5
immfldv
480dv
Both dv’s
0.46 8.2 -
0.70 2.5
0.22 1.6
B One colony of plasmid-carrying cells was inoculated into 2 ml of PBB, aerated overnight at 37°C (the cells underwent about 30 cycles of doublings during this incubation), plated, and scored for plasmid-less segregants, as described under Materials and Methods.
formants were kept for 2 weeks on EMB galactose plates at 25°C to allow increased selection for the Gal+ cells before isolation. In this case, some black, stable Gal+ colonies appeared. All of them carried DNA indistinguishable from that of incoming hdvgal+, and no detectable amount of resident Xdv DNA was seen. These observations indicate that the two homoimmune Adv’s are incompatible. In the former case the resident plasmid must have prevented establishment of hdvgal+, while in the latter case the Advgal+ must have displaced the resident Adv plasmid. In order to confirm that two homoimmune Adv’s are incompatible, cells carrying two Adv’s with different drug-resistance markers (Advkan and Advamp) were constructed. Colonies that grew on a plate containing kanamycin and ampicillin were restreaked on drug-free plates, and colonies were tested for drug resistance. The results in Table 6 show that the kanamycin and ampicillin markers do not coexist stably: The double-carriers became single carriers of either resistance soon after elimination of the antibiotic selective pressure. We conclude, therefore, that homoimmune two Adv’s are incompatible.
ments that can replicate autonomously in a plasmid state. These fragments are about one-tenth of the parental phage genomes in size, and consist of those genes and structures that act in the process of replication. Berg and Kellenberger-Gujer (1974) observed a similar situation with the imm2 1 phage. Genetic and physical studies have shown that there is only one unique “replicator region” in the lambdoid phage genome, and this region can be extracted as Adv in the plasmid state (Matsubara and Kaiser, 1968; Davidson and Szybalski, 1971; Chow et al., 1974). No rearrangement of genes or mutational events seem to be required. There is a possibility that a similar process, viz., extraction of a simple plasmid replicon from a complex genome, may well be possible with other temperate phages. Recently, Hobom and Phillipsen (personal communication) showed that Adv-like plasmids can be generated from A phage DNA in vitro using genetic engineering techniques. Similar methods have been applied to the sex factor F and drug-resistant plasmids (Timmis et al., 1975; Lovett and Helinski, 1976). The reverse process, viz., construction of a complex phage genome from a simple DISCUSSION plasmid genome, has also been reported: In this paper we show that various tem- Matsubara and Kaiser (1968) described perate phages, including imm434, imm21, “new phages” whose entire “replicator and $80, as well as A, give rise to DNA frag- region” is derived from Adv and the re-
PLASMIDS FROM LAMBDOID
293
PHAGES
TABLE 6 INCOMPATIBILITY
Expt A
First growth with No drugs
BETWEEN
hdvkan
AND
Advamp”
Number of progeny colonies which are: Kana Amp* clone no.
KanR AmpR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
KanR Amps
KanS Ampa
KanS Amps
10
2 5
Total
9 0 0 0 9 0 3 8 8 0 2 7 8
1 0 0 0 20
20
54
65
No. of clones tested B
Kanamycin
90 86
12 8
78 78
0 0
C
Ampicillin
75 58
34 18
0 0
41 40
a Cells carrying Xdvkan and Xdvamp were constructed as described in Table 3, footnote a. In Expt A, 16 independent transformant clones were spread over drug-free plates. After overnight incubation (ca. 30 generations), 10 colonies from each clone were tested for the presence of the drug-resistant Adv’s by transfer with toothpicks onto two plates containing either kanamycin or ampicillin, and incubation at 37°C overnight. In Expt B, double-carrier clones of independent origins were transferred onto plates containing kanamycin, grown overnight, and then replica-plated onto two plates containing either of the two drugs. Experiment C was done similarly, except that the double-carrier clones were grown first on plates containing ampicillin.
maining regions from phage imm434. Such a phage appeared among the progeny of the imm434 phage that infected hdv-carrying cells. It was thought that the imm434 replicator region is replaced by that of the plasmid. Berg (1971) and Berg and Kellenberger-Gujer (1974) showed that such a new phage can emerge through formation of a tandem structure that contains two replicators, one from the heteroimmune phage and one from Adv. Thus, replicators in plasmids and viral genomes, both of which are major vehicles in infectious heredity, can be ex-
changed. The possibility that similar phenomena may occur in nature, at least in microbes, is intriguing. Thus, the genome of bacteriophage A can be regarded as a plasmid (hdv) genome that is connected to the control genes N and Q, and to other genes for morphogenesis and cell lysis (late genes). The genes N and Q flank the Adv genome; their products are positive regulators of transcription. Cell lysis and phage morphogenetic genes are located outside of these, and are under the control of the Q gene. Recent experiments
294
MATSUBARA
(Akaboshi, unpublished data) have shown that a variant A phage whose genome has two deletions, one of which covers the N gene and the other the Q gene, infects a cell and behaves indistinguishably from that of hdv plasmid. It was observed that about a hundred copies of the dv plasmids occur per host chromosome. For some unknown reason, except in a few instances, many of these dv plasmids were in a dimeric state. The implication of this phenomenon awaits further elucidation. As seen with the Xdvl-hdvgal and hdvkan-Advamp systems, two Adv plasmids are incompatible, although Adv replicates under a relaxed control (Rownd, 1969), and about a hundred copies occur per host chromosome. Cabello et al. (1976) and Kibe et al. (1976) demonstrated recently that the ColEI factor, which is similar to Adv in size and occurs in 20 to 30 copies per chromosome, also shows incompatibility. A similar observation has been made with an another non-self-transmissible small plasmid SSu (Smith et al., 1974). Thus, incompatibility is likely to be a general phenomenon that occurs between a pair of plasmids whose replication is controlled by the same regulatory mechanism. A theoretical consideration of incompatibility between two plasmids replicating under relaxed control will be dealt with separately (Ishii, Hashimoto-Gotoh, and Matsubara, manuscript in preparation). We have also shown that plasmids derived from different heteroimmune phages, such as Adv and @Odv or Adv and imm2ldv, are compatible. Since the genes and the other loci required for the replication of these plasmid genomes are known (Matsubara, 1976), we can determine which genetic element(s) is responsible for incompatibility. The compatible pair, hdv and imm2ldv, shares a segment that carries an origin for replication (ori) and also two initiator genes, 0 and P (see Fig. 1). It can be concluded, therefore, that these genes and loci are not
AND OTSUJI
involved in incompatibility. The genetic elements carried in the heterologous region are the genes rex, ~1, tof, ~11, and two promoter-operators (pRoR and p-e for transcriptions to the right and left of the genome, respectively) (see Fig. 1). Among these, genes rex, c1, and cI1 must be irrelevant in incompatibility, as these are dispensable and plasmids without them behave indistinguishably from plasmids that carry them. The role ofpre is not clear at present, but in phages its only known function is in the control of expression of irrelevant genes rex and c1. Therefore, as a working hypothesis, we propose that the remaining elements, viz., gene tof and the promoteroperator pRoR, are responsible for the determination of incompatibility. The pRoR controls expression of the plasmid operon, and the tof gene codes for a protein, the autorepressor, that negatively and specifically acts by binding at pRoR. The tofgene itself is under the pRoR control, and hence the tof-pRoR region constitutes a highly specific autorepression system (Matsubara, 1976; Berg, 1974). In contrast, the heteroimmune pair, imm434dv and Adv, exhibits weak incompatibility. This interaction could be due to cross-reaction between the A and 434 autorepression systems. Alternatively, the weak incompatibility could be ascribed to a hitherto unknown interaction involving a genetic site(s) located between the two right ends of the imm434 and imm21 immunity regions, although this region carries no known genetic function related to plasmid replication control. Further studies are necessary to elucidate these problems. The Adv plasmids interfere with growth of the superinfecting homoimmune phage, and divert its replication process into the plasmid state (Matsubara, 1972a, 1974b). The interference occurs only when the invading phage and plasmid genomes are homoimmune. Several lines of evidence have been presented which strongly suggest that the interference in phage growth is caused by the autorepressor established
PLASMIDS FROM LAMBDOID
by the resident plasmid to control its own replication (Matsubara, 1974b). Here again, weak interference was seen, as an exception to the rule, in hdv and imm434 phages or in imm434dv and A phages. In a clone carrying two plasmids, loss of one or the other of the plasmids occurred independently. Further examples of this must be examined, but this observation implies that the primary mechanism that leads to loss of the plasmid (viz., failure in the mechanism to perpetuate the plasmid) is again immunity specific, and is not caused by physiological effects of the host cell that block multiplication of the dv plasmids in general. ACKNOWLEDGMENTS The authors would like to thank Dr. Yasuyuki Takagi for his support and encouragement throughout this study, Midori Matsubara for her technical assistance, and Dr. D. Berg for sending us the Advgal+ and hdvkan plasmids. This work was aided in part by a grant from the Asahi Press and a grant from the Ministry of Education, Science and Culture, Japan.
REFERENCES BERG, D. E. (1971). Regulation in phage with duplications of the immunity region. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 667-678. Cold Spring Harbor Laboratory, New York. BERG, D. E. (1974). Genes of phage Lambda essential for hdv plasmids. Virology 62, 224-233. BERG, D. E., DAVIES, J., ALLET, B., AND ROCHAIX, J-D. (1975). Transposition of R factor genes to bacteriophage A. Proc. Nat. Acad. Sci. USA 72, 36283632. BERG, D. E., JACKSON, D. A., AND MERTZ, J. E. (1974). Isolation of a Adv plasmid carrying the bacterial gal operon. J. Virol. 14, 1063- 1069. BERG, D. E., AND KELLENBERGER-GUJER, G. (1974). N protein causes the Adv plasmid to inhibit heteroimmune phage imm434 growth and stimulate Adv replication. Virology 62, 234-241. CABELLO, F., TIMMIS, K., AND COHEN, S. N. (1976). Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature (London) 259, 285-290. CAMPBELL, A. (1971). Genetic structure. In “The
Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 13-44. Cold Spring Harbor Laboratory, New York. CHOW, L. T., DAVIDSON, N., AND BERG, D. (1974). Electron microscope study of the structure of Adv DNAs. J. Mol. Biol. 86, 69-89.
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295
CLOWES, R. (1972). Molecular structure of bacterial plasmids. Bacteriof. Rev. 36, 361-405. DAVIDSON, N., and SZYBALSKI, W. (1971). Physical
and chemical characteristics of lambda DNA. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 45-82. Cold Spring Harbor Laboratory, New York. HASHIMOTO, T., AND MATSUBARA, K. (1974). Transformation of Ca++-treated recA derivative of Escherichia co/i K12 by Adv DNA. Japan. J. Genet. 49, 97- 101. HELLING, R. B., GOODMAN, H. M., AND BOYER, H. W. (1974). Analysis of endonuclease R. EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14, 1235-1244. HERSHEY, A. D. (1971).
Lambda.” York.
“The Bacteriophage Cold Spring Harbor Laboratory, New
KELLENBERGER-GUJER, G. (1971). Recombination
in
bacteriophage Lambda II. The mechanism of general recombination. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 417-430. Cold Spring Harbor Laboratory, New York. KIBE, A., SHIMADA, K., AND TAKAGI, Y. (1976). Recombination and incompatibility of two differently marked colicin El factors. In “Proceedings, 1976 Molecular Biology Meeting of Japan, Kyoritsushuppan, Tokyo,” pp. 138-140. LOVETT, M. A., AND HELINSKI, D. R. (1976). Method for the isolation of the replication region of a bacterial replicon: Construction of a mini-F’km plasmid. J. Bacterial. 127, 982-987. MANDEL, M., AND HIGA, A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159- 162. MATSUBARA, K. M. (1972a). Interference in phage growth by a resident plasmid Adv. I. The mode of interference. Virology 50, 713-726. MATSUBARA, K. (1972b). Plasmid formation from bacteriophage A as a result of interference by resident plasmid Adv. Virology 47, 618-627. MATSUBARA, K. (1974a). Preparation of plasmid Adv from bacteriophage A: Role of promoter-operator in the plasmid formation. J. Viral. 13, 596-602. MATSUBARA, K. (1974b). Interference in bacteriophage growth by a resident plasmid Adv. II. Role of the promoter-operator. J. Virol. 13, 603-607. MATSUBARA, K. (1976). Genetic structure and regulation of plasmid Adv. J. Mol. Biol. 102, 427-439. MATSUBARA, K., AND KAISER, A. D. (1%8). Adv: An autonomously replicating DNA fragment. Cold Spring Harbor Symp. Quant. Biol. 33, 769-775. MATSUBARA, K., TAKAGI, Y., AND MUKAI, T. (1975). In vitro construction of Adv DNA in different
oligomeric forms and studies with their transforming activity. J. Virol. 16, 479-485. MURRAY, N. E., BRAMMAR, W. J., AND MURRAY, K. (1977). Lambdoid phages that simplify the recovery
296
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of in vitro recombinants. Mol. Gen. Genet. 150, E. S. (1974). Genetic and molecular characterization 53-61. of some non-transferring plasmids. Mol. Gen. Genet. 129, 229-242. NOVICK, R. P. (1969). Extrachromosomal inheritance STREECK, R. E., AND HOBOM, G. (1975). Mapping in bacteria. Bacterial. Rev. 33, 210-235. cleavage sites for restriction endonucleases in hdv ODAKURA, Y., HASHIMOTO, H., AND MITSUHASHI, plasmids. Eur. J. Biochem. 57, 595-606. S. (1974). R-factor mutant capable of specifying hypersynthesis of penicillinase. J. Bacterial. 120, THOMAS,R., AND DAVIS, R. W. (1975). Studies on the cleavage of bacteriophage Lambda DNA with EcoRI 1260-1267. restriction endonuclease. J. Mol. Biol. 91, 315-328. ROWND, R. (1969). The replication of a bacterial episome under relaxed control. J. Mol. Biol. 44, TIMMIS, K., CABELLO, F., AND COHEN, S. N. (1975). Cloning, isolation, and characterization of replica387-402. tion region of complex plasmid genomes. Proc. Nat. SATO, K. (1970). Genetic map of bacteriophage 480: Acad. Sci. USA 72, 2242-2246. Genes on the right arm. Virology 40, 1067-1069. YOSHIMORI,R. N. (1971). “A Genetic and BiochemiSIGNER, E. R. (1969). Plasmid formation: A new mode of lysogeny by phage A. Nature (London 223, cal Analysis of the Restriction and Modification of DNA by Resistance Transfer Factor.” Ph.D. 158-160. Thesis. University of California, San Francisco. SMITH, H. R., HUMPHREYS,G. O., AND ANDERSON,
1, 284-296 (1978)
Preparation
of Plasmids from Lambdoid Phages and Studies on their Incompatibilities KENICHI
Department
of Biochemistry,
MATSUBARA’ Kyushu University
AND YOKO OTSUJI School of Medicine,
Fukuoka,
812, Japan
Accepted January 27, 1978 Various lambdoid phages, including those carrying the immunity region of 434, 21. and 480, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm2ldv, and $8Odv, were similar to the already known plasmid Xdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids kdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.
phage. Bacterial receptor mutants and mucoid mutants are not selected, because a mixture of phages with different receptor specificities and a galdel host are used. With this technique, the genetic region of the genome that can be extracted as a plasmid and that functions in the autonomous replication of the plasmid has been determined (Matsubara, 1976; Berg, 1974). Here, the same technique is employed with other phages to see if the relationship between the phage and its plasmids can be generalized. It is shown that all the lambdoid phages tested, including imm434, imm2 1, and 480 [for descriptions of these phages, see Hershey (1971) and Fig. l), can give rise to plasmids similar to hdv. This paper describes the properties of these plasmids and their interactions in the cell. The latter problem seemed of particular interest, as this system provides a model for studying the mechanism of plasmid incompatibility [for reviews, see Novick (1969) and Clowes (1972)]. Our results show that incom’ Present address: Laboratory of Molecular Cepatibility is primarily determined by the netics, University of Osaka Medical School, Kita-ku, “immunity region” on the plasmid genome Osaka, 530, Japan.
The plasmid xdv is an autonomously replicating DNA fragment of the bacteriophage genome consisting essentially of only three genes (tof, 0, and P), one promoteroperator (pRoR) for transcription, and an origin (ori) for replication (Matsubara, 1976; Berg, 1974). It is only about one-tenth the size of the parental phage genome, and about sixty copies are found per carrier bacterial chromosome. Recently a technique was devised for preparing new hdv from A phage (Matsubara, 1974a). This technique makes use of the fact that Xdv plasmids arise in a population of cells infected with uv-irradiated nonlysogenizing A phage (viz., c1 or vir mutants) and that, even when the frequency of appearance of these plasmid-carriers is low, they can be selected by their ability to form colonies on plates seeded with the phage. The tolerance is specific, and the same cell is sensitive to infection by heteroimmune
0147-619X/78/0013-0284$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
284
PLASMIDS FROM LAMBDOID
I
Head
I
Tail
285
PHAGES
Recombination
Regulation
1 Lysis
1
A ircN-34
;un Q80 cm
rex cl
N PL OL
tof
cl1
0
P
0’
ori
PR OR
a
hdvl
n
Advo21
__ . a
b
c
. ..
FIG. 1. Genomes of bacteriophage A, imm434, imm21, 480, and some dv plasmids. Open bars show the segments originating from A. Hatched, cross-hatched, or dotted bars show regions of nonhomology. For symbols of genes, see Campbell (1971). The endpoints of Xdvl and AdvO21are adopted from Chow er al. (1974). Endpoints of imm434dv and imm2ldv have not been mapped precisely. Arrows indicate sites of cleavage by restriction endonuclease EcoRI (Thomas and Davis, 1975; Murray et al., 1977; Streeck and Hobom, 1975). Since locations of genes and EcoRI cleavage sites along the 980 genome have not been mapped precisely, the $80dv genome is not included. For explanation of a, b, and c, see text.
that carries an autorepressor gene (Matsubara, 1976)and a promoter-operator (DNA sequences recognized by the products of repressor gene tof, intermingled with the promoter sequence used in the initiation of transcription). MATERIALS AND METHODS Bacteria and plasmids. A derivative of Escherichia coli K12, km723 [recAl, his, str, (gal-att)de*], has been described pre-
viously (Matsubara, 1974a). Strain km960 is a thymine-requiring derivative of km723. The derivatives of hdv, hdvgall,, (Berg et al., 1974), hdvkan, (Berg, personal communication), and hdvamp, (Matsubara, to be published), carry, respectively, genes that code for fermentation of galactose, inactivation of kanamycin, and inactivation of ampicillin. The kanamycin-inactivating and ampicillin-inactivating genes are carried on transposons from the plasmids JR67 (Berg et al., 1975) and RM201 (Odakura et al. 1974), respectively. km960 carrying one of these plasmids was prepared by transformation (Mandel and Higa, 1970; Hashimoto and Matsubara, 1974).
Bacteriophages. The following bacteriophages were used. Phages imm434, imm21, 480, and the repressor-negative mutants, AcI,, and imm434c&, have been described previously (Matsubara, 1972a, b). The repressor-negative mutants, imm2 ICI, and @kI,, were prepared by mutagenesis of E. coli K12 recA lysogens with iV-nitro-N’nitrosoguanidine and selection of clear plaque-formers that appeared during subsequent incubation. For simplicity, these phages will be abbreviated as AC,imm434c, imm2lc, and $8Oc, respectively. The hybrid phages, hs0imm434c and hs0imm21c, were prepared by crossing imm434c or imm2lc with h*OimmA(Matsubara, 1974a). In these hybrid phages, the “left arm” that carries genes A, B, . . . J is from $180, whereas the “right arm” that carries genes att, int, c1, 0, P, and R is from imm434 or imm21. The h*imm+80 and h*imm~80gal+ were prepared by crossing phage $80~ with A and Apga15cIg5,,respectively, and selecting recombinants in which the left arm was from A and the right arm was from 480. The hm80imm~80cgal+was prepared by crossing hYmm$80cgal+ with a hybrid phage, h@‘80immAc18,,gal+ (Matsubara, unpublished
286
MATSUBARA
data), in which the left arm was from 480 and the right arm from A. For simplicity, a phage that shares the same immunity region as the plasmid will be referred to as homoimmune, while phages that do not share this region will be referred to as heteroimmune. For example, A is a homoimmune phage to the plasmid hdv, whereas it is heteroimmune to imm434dv. Media. The media used, PB, PBMM, PBB, PBB agar, EMBO, EMB galactose agar, and MacConkey galactose agar, have been described previously (Matsubara, 1972a, 1974a). Enzyme. The restriction endonuclease EcoRI was prepared according to the method of Yoshimori (1971). Assay of the rate of appearance of plasmid-minus segregants. Approximately 500 cells were spread on PBB agar to form colonies. They were then replica-plated onto EMBO agar coated with 10’ particles of homoimmune clear plaque-forming phage. After incubation for 16 h, the plasmid-carriers appeared as white- to pinkcolored prints on the replica plate, because they were tolerant to phage infection, whereas the segregants appeared dark red due to lysis. Segregation of Xdvgal, Advkan, or Advamp was assayed by observing colonies for their loss of the ability to ferment galactose or of resistance to the drugs. Extraction of plasmid DNA, sucrose gradient centrifugations, CsCl-ethidium bromide isopycnic centrifugations, and agarose gel electrophoreses. The methods used have been described previously (Matsubara et al., 1975).
AND OTSUJI
mixture containing nonirradiated phage at a multiplicity of infection of 0.1. Under these conditions, bacterial receptor mutants or mucoid mutants would not be recovered (Matsubara, 1974a). Furthermore, lysogens would not be formed because the phage could not lysogenize. Only carriers of a defective phage genome would survive and form colonies, due to their ability to interfere with growth of the homoimmune phage. Using 3 x lo8 cells infected with the irradiated phage mixture, I to 300 colonies were obtained. These colonies were crossstreaked, first against imm434c and then against imm2lc. About 79% of the colonies were tolerant to homoimmune imm434c, but were sensitive to the heteroimmune imm2lc, a behavior characteristic of imm434dv carriers (see below). Carriers of im2ldv were made similarly, except that a mixture of imm2lc and hmsoimm21c was used and the resulting colonies were cross-streaked over imm2lc first and then over AC. Four colonies each of imm434dv and imm2ldv carriers were chosen at random, purified, and subjected to marker rescue tests (Matsubara and Kaiser, 1968). All of these plasmids donated the genes O+ and P+, but not A+, J+, Nf, Q+, or R+. This pattern of rescue is identical to that of Adv (Matsubara and Kaiser, 1968), suggesting that these plasmids carry a segment of phage genome quite similar to that of the prototype Adv (see Fig. 1). Since there is a region of nonhomology between A and imm21 which extends from the left end of gene N to the right of the cI1 gene, the possibility that some of the imm2ldv’s might carry gene Nzl was not ruled out by these tests. RESULTS Sucrose gradient sedimentation analyses Preparation of imm434dv and imm2ldv and agarose gel electrophoreses showed Plasmids that all of the colonies tested carried plasmid A mixture of imm434c and hmsoimm434c DNA that could be isolated as the small, was irradiated with uv light to give a sur- covalently closed circular form (see below). vival of 10e2,and then adsorbed to bacterial strain km723 at a ratio of one phage per Preparation of @Odv’s Cells carrying plasmids derived from 480 bacterium. The infected cells were immediately plated on PBB agar with an identical were first sought by the technique described
PLASMIDS FROM LAMBDOID ABCDEFG
HIJKLMNO
Xdvgal+ Advl(D) hdvl(M)
XdvO21
FIG. 2. Analysis of dv plasmids by agarose gel electrophoresis. Plasmid DNAs in the covalently closed circular form where purified by CsCl-ethidium bromide density gradient centrifugations, as described previously (Matsubara ef al., 1975), and subjected to electrophoresis on a 0.9% agarose slab gel. (A) A mixture of hdvgal+, Advl dimer, Xdvl monomer, and XdvO21; (B) and (I) imm434dvCQ2; (C) and (J) imm434dvCQ7; (D) and (K) imm2ldvABS; (E) and (L) imm2ldvAB7; (F) and (M) @OdvDU9; (G) and (N) $lOdvDOl; (H) a mixture of hdvl and Adv021; (0) whole A phage DNA. (A)-(G) Samples without EcoRI treatment; (H)-(O) samples treated with EcoRI. Small fragments appearing in the @Odv digests are not shown in the picture.
above. On plates seeded with a mixture of 480~ and h*imm+80c, 10 times more colonies were recovered from uv phage-infected cultures than were found in the above experiments with imm434 or imm2 1. Some (ca. 15%) of these colonies were tolerant to infection by homoimmune phage ($SOc), but were sensitive to heteroimmune phage (Xc). However, in marker rescue tests, all (50) colonies tested donated gene 19, which is equivalent to gene R in X (Sato, 1970). Furthermore, at least 10 of them carried extrachromosomal DNA which was indistinguishable in size from the whole 480 genome, although they did not produce any infectious particles upon prolonged incubations after uv irradiation. These bacteria apparently carried the entire 480 genome that had presumably undergone a mutation in the gene equivalent to iV in X (Signer, 1969; Kleckner and Signer, personal communication). It seems therefore that the
PHAGES
287
$80 genome is prone to produce such plasmids. Next, a phage mixture was made from two imm480c phages, each carrying the bacterial gal gene (h~80imm~80cgal+ and hximm$80cgal+), and added to galdel, recA cells. The infected cells were plated on MacConkey galactose agar. About 50% of the colonies showed the red Gal+ phenotype and were discarded as carriers of the whole phage genome. About 20% of the colonies were white to pink in color and were tolerant to homoimmune phage (480~)) but sensitive to AC. Phage-producing colonies were discarded, leaving 11 clones that were purified and examined for extrachromosomal DNA. Eight were found to carry a small circular DNA, viz., +80dv DNA, while the rest (3) carried DNA of the same size as the 480 genome. Appropriate superinfecting phages were not available, so marker rescue tests could not be done. Size, Oligomeric State, and Content of Plasmid Copies in a Cell
The phage-derived plasmids can be isolated as covalently closed circular DNA and have a molecular weight of 3 to 10 x 106. The electrophoretic patterns of some of these DNAs are shown in Fig. 2, and their molecular sizes in Table 1. Each of the plasmid DNAs was treated with EcoRI restriction endonuclease and run in sucrose gradients or in agarose gels. From cleavage maps of the lambdoid phage genomes, it was predicted that the hdv and imm2ldv DNAs would be cleaved once by EcoRI at a site within gene 0, yielding one unique fragment. In contrast, imm434dv DNA would be cleaved at two sites, one in gene c1 and one in gene 0, and would yield two fragments (Helling et al., 1974; Streeck and Hobom, 1975; Thomas and Davis, 1975; also see Fig. 1). Figure 2 shows that these predictions were verified: Different imm2ldv’s each produced one unique product; different imm434dv DNAs each produced two frag-
288
MATSUBARA
AND OTSUJI
TABLE I SIZE,~LIGOMERIC FORM, AND NUMBEROFCOPIESOFDIFFERENT dv PLASMIDS Molecular weight
Plasmid
Whole moleculen x 10-e (A)
hdvl dimer hdvl monomer AdvO21 imm434dvCQ2 imm434dvCQ7 imm2ldvABS immZldvAB7 imm2ldvABl4 480dvDU9 &OdvDOl
9.4 4.7 2.2 9.9 6.6 8.3 9.5 7.3 3.5 7.6
No. of copies per chromosome
EcoRI digest’ x 10-G (B)
2.8 + 2.4 +
3.3 + 3.5 +
4.7 4.7 2.2 0.83 = 0.83 = 4.2 3.1 3.3 0.39 = 0.39 =
3.6 3.2
3.7 3.9
Oligomeric form
CC DNA contentc
(C =A/B)
(D)
2 1 1 3 2 2 3 2 1 2
0.17 0.12 0.036 0.25 0.29 0.52 0.26 0.27 0.24 0.29
Whole moleculed W
66 95 60 90 156 226 101 137 245 136
Monomer units (E x Cl
132 95 60 270 312 452 303 274 245 272
a Molecular weight of closed circular DNA was estimated from its sedimentation velocity or mobility in agarose gels, using hdvgal+ (13.4 x lOa), hdvl dimer (9.4 x lOa), Advl monomer (4.7 x lOa), and Adv021 (2.2 x 106)as standards. b Molecular weights of linear duplex molecules were estimated from their sedimentation velocities or mobilities in agarose gels, using Advl, Adv021, and whole A phage DNA, all treated with EcoRI, as standards. imm434dv’s and 480dv’s each produce two fragments upon treatment with EcoRI. c Ratio of the amount of DNA in the closed circular (CC) form to the amount of chromosomal DNA, as assayed by CsCl-ethidium bromide isopycnic centrifugation (Matsubara et al., 1975). d D x 2.5 x 109/A, assuming that the molecular weight of E. coli chromosome is 2.5 x 109.
ments. One of the latter fragments is unique in size (MW, 0.83 x 106), suggesting that it is the fragment b in Fig. 1. The other fragment varies in size, depending on the plasmid species, and this suggests that it consists of regions a and c, which have joined together (see Fig. 1). Like imm434dv’s, +80dv DNAs gave rise to two fragments after&OR1 digestion, viz., they carry two EcoRI sites. The smaller fragment was unique in size (MW, 0.39 x 10s), whereas the large fragment was variable. One of these EcoRI sites must be at one end of fragment number six in the cleavage map of 480 (Helling et al., 1974), and the other is a new site, not previously reported, perhaps because of its proximity to the former site. No attempts were made to locate these sites on the $80dv genome. By comparing the sizes of intact, covalently closed circular molecules with the sum of the molecular weights of the EcoRIgenerated fragments, it was possible to as-
sess the oligomeric states of the intact molecules. Table 1 summarizes the results of these analyses. The molecular weight of the monomeric unit was found to fall within the range of 3 to 5 x 106. Many dv plasmids were found to be in the dimeric state, although some were in monomeric form and yet others were in the form of higher oligomers. The amount of plasmid DNA in the closed circular form relative to that of chromosomal DNA was measured by isopycnic centrifugations of cell lysates in CsClethidium bromide. The data obtained are also shown in Table 1. The number of plasmid copies per host chromosome was calculated assuming that the molecular weight of the latter is 2.5 x log. It can be seen that the various phage-derived plasmids and hdv occur in 50 to 200 copies per carrier cell. With each plasmid, the copy number is constant, and is not affected by the age of the cells. The carrier cells must possess some
PLASMIDS FROM LAMBDOID
289
PHAGES
TABLE 2 EFFICIENCYOF PLATING OF LAMBDOID PHAGESON BACETERIACARRYINGVARIOUSdv PLASMIDS Efficiency of plating” Bacteria km723 km723[Advl] km723[hdvBBl] km723[imm434dvCQ2] km723[imm434dvCQ7] km723[imm2ldvABS] km723[imm2ldvABl4] km960 km960[+80dvDU9] km960[+8OdvDO l] km960[imm2ldvABl4] [hdvl] km960[480dvDU9] [Advl]
imm434c
imm2lc
imm@Oc
1.0 -3 x IO-’ -3 x 10-1 0.10* 0.36 0.7 0.92 1.0 0.93 0.96
1.0 0.026 0.27b 5 x lo-3c 0.02’ 0.65 0.59 1.1 1.1 1.1
1.0 1.0 0.97 0.69 0.93 <2 x IO-’ <2 x 10-r 0.92 1.1 1.1
1.0 0.86 1.1 1.05 0.93 1.0 0.88 0.85 <5 x 10-1 <5 x 10-1
<5 x 10-7
0.03*
<2 x 10-T
0.84
<5 x lo-’
0.07b
1.1
<5 x 10-7
AC
a Bacteria grown overnight in PB were plated on PB agar with an appropriately diluted phage, and the efficiency of plating of the phage was scored relative to that on km723 or km960 (which do not carry plasmids) after overnight incubation at 37°C. The phages used are described under Materials and Methods. * These plaques were small and irregular in shape. c These plaques were extremely tiny. Plaques of normal size appeared with a frequency of less than 10-r.
mechanism for maintaining number.
a constant
Rate of Appearance of Plasmidless Segregants Cells carrying these plasmids were grown for 50 generations, then spread over PBB agar to determine the number of plasmidless segregants. It was observed that, among carriers of Adv, imm434dv, im2ldv, and $80dv, respectively, 0.7,0.5,0.4, and 0.7% of the population were segregants.
action was seen with A and imm434: hdv partially interferes with growth of the imm434 phage, and imm434dv interferes with that of the A phage. This type of weak interaction has been reported previously (Matsubara, 1972a; Berg and KellenbergerGujer, 1974). Compatibility between Two Different Plasmids
The addition of hdv DNA to Ca2+-treated E. coli cells transforms them to Adv-carriers (Hashimoto and Matsubara, 1974; Growth Interference of Superinfecting Berg et al., 1974). This technique was Phage employed to construct cells that carry two The plasmid carriers were tolerant to different plasmids. As shown in Table 3, the super-infection by homoimmune phage, viz., efficiency of transformation by Advl was the the phages from which the plasmid was de- same, regardless of whether or not the rerived (Matsubara, 1972a). However, the cipient cells were carriers of resident plasinteraction was specific, and these cells mid imm2ldv or 480dv. These transwere sensitive to heteroimmune phages. formants carried both the resident plasmid The efficiencies of plating of various phages and Advl, as judged from their tolerance on a set of different plasmid carriers are to phages A as well as imm21 and 480, shown in Table 2. An exceptional inter- and from the presence of two molecular
290
MATSUBARA
AND OTSUJI
TABLE 3 EFFICIENCYOF Adv TRANSFORMATIONOF CELLSTHAT~ARRY A RESIDENTPLASMID~ Efficiency of transformation (x 1O-s) Experiment Recipient bacteria
Donor DNA
km723 km723[imm21dvAB14] km960 km960[480dvDU9] km723 km723[imm434dvCQ2] km723[imm434dvCQ7] km723[Advl] km723[Adv021] km723[AdvBBl] km960 km96O[Advamp]
hdvl Advl Advl Advl Advgal+ Advgal+ Advgal+ Advgal+ Advgal+ AdvgaJ+ Advkan Advkan
Selection of transformants Tolerance to A
Selection of Gal+
Selection of KanR Selection of Kans, AMPa
1
2
3
560 430 190 180 132 105 42 24 36,34
480 58.5 228
650 770 750
4
740 370
a Donor DNAs in the closed circular form were purified by CsCl-ethidium bromide isopycnic centrifugation. Transformation was done using Ca*+-treated recipient bacteria as described previously (Hashimoto and Matsubara, 1974). The hdvgal+ transformants were scored as galactose-fermenting colonies on EMB galactose agar plates after incubation for 36 h at 37°C. For phenotypes of the Gal+ transformants, see text. The Advkan transformants were selected on PBB agar containing the indicated drugs (Kan = kanamycin, 20 pg/ml; Amp = ampicillin, 40 @ml). The efficiency of transformation was calculated as the number of transformants divided by the number of DNA molecules added.
species in extrachromosomal DNA preparations (Fig. 3). The components each exhibited the sedimentation velocity characteristic of the original plasmid, indicating that the two plasmids are carried independently, and not in a fused or “composite” form. The contents of plasmid DNAs in the double-carriers are additive (Table 4). From the double-carrier clones, each plasmid species was lost independently, as shown in Table 5. Clones that had lost both plasmids simultaneously appeared at a somewhat high frequency, but the difference was not significant. Based on these observations, we concluded that hdv and imm2ldv, and also hdv and $80dv, both pairs of which represent combinations derived from heteroimmune phages, are compatible, viz., these different species of plasmids can coexist independently in the same cell. Double-carriers of imm434dv and Xdv were constructed similarly, except that in this
case Advgal+ DNA was added to Gal- cells and Gal+ transformants were selected on EMB galactose agar. The use of hdvgal+ was necessary because carriers of imm434dv are partially tolerant to A phage infection, thus precluding the use of A for selection of Ad+ transformants. Table 3 shows that the efficiency of transformation was almost the same, whether or not the cells carried the resident plasmid imm434dv. However, the double-carriers in the combination were unstable, as will be shown below. Gal+ transformants of the control, plasmid-free recipient km723 produced black (Gal+) colonies, which rarely yielded Galsegregants, and contained extrachromosomal DNA indistinguishable from that of the original Advgal+ DNA (data not shown). On the other hand, galactose-fermenting transformants that had harbored the resident imm434dv were unstable. These colonies appeared brown (Gal’ phenotype) on EMB galactose plates because of the
PLASMIDS FROM LAMBDOID
A
Y’ C
d
IO FRACTION
NUMBER
FIG. 3. Analyses of extrachromosomal DNA in double dv-carriers by sucrose density gradient centrifugations. Double dv-carriers were prepared by transformation as described in Table 3, footnote a. The double-carriers harbor Xdvl and immZldvABl4 (A), Advl and @OdvDU9 (B), Xdvgal+ and imm434dvCQ7 (C). 3H-Labeled extrachromosomal DNAs in covalently closed circular form were prepared and sedimented at 37,000 rpm for 195 min at 20°C through 5-20% sucrose gradients containing 0.1 M Tris (pH 7.5), 1 M NaCl, and 0.01 M EDTA. Peaks were assigned from standards. These data are omitted for clarity. Sedimentations are from right, to left. Profiles similar to A or B were obtained when independently obtained double-carrier clones were tested. On the other hand, five independent clones carrying Xdvgal+ and imm434dv all exhibited different ratios in areas of the two plasmid peaks, although the peak positions were reproducible. This indicates that the Advgal+ and imm434dv coexist only in an unstable fashion.
high content of Gal- segregants. Upon restreaking, lo-40% of the cells in the brown colonies again produced Gal’ colonies and 60-90% produced Gal- segre-
291
PHAGES
gants. Even after several cycles of cell isolations, the Gal’ traits were maintained. Figure 3C shows a sedimentation profile of extrachromosomal, covalently closed, circular DNA prepared from such Gal’ cells. Two components are seen, corresponding to DNAs of Xdvgal+ and imm434dv. However, the ratio of the two DNAs varied, depending on the clones studied and also on the number of cell divisions. Because of the continuous segregation, no attempts were made to estimate the number of the plasmid copies per cell. Thus the heteroimmune hdv and imm434dv, which are closely similar, can be maintained as separate entities, but cannot coexist stably. When Xdvgal+ DNA was added to cells carrying a resident hdv, “galactose-fermenting” primary transformants appeared on EMB galactose plates at a reduced frequency (Table 3). These primary transformants had a small growth advantage over non-galactose-fermenting parental cells, and formed slightly larger colonies than the others, although they were neither black nor brown. The majority of cells in these colonies were Gal-, since, upon restreaking, Gal+ or Gal’ colonies were not detected (less than 0.1%). In a separate experiment, the galactose-fermenting primary transTABLE 4 CONTENTOFPLASMID DNA IN DOUBLE dV-CARRIER CELLS'
Bacteria km723[Advl] km723[imm21dvAB14] km723[imm2ldvAB14][Advl] km960[+8OdvDU9] km960[+80dvDU9][Advl]
Content of CC DNA (%) 12.5 25.3 37.9 20.2 38.1
o Plasmid-carrying cells were uniformly labeled with [3H]thymidine and lysed with lysozyme-Na-Sarkosynate. Then the crude lysates were subjected to isopycnic centrifugations to separate covalently closed circular plasmid DNA (CC DNA) from chromosomal DNA, as described previously (Matsubara et al., 1975). The values given are ratios of the amount of closed circular DNA to the amount of chromosomal DNA.
292
MATSUBARA
AND OTSUJI
TABLE 5 INDEPENDENTSEGREGATIONOFPLASMIDSFROMCELLSTHAT~ARRY Two DIFFERENT~V'S" Percentage of segregants that have lost: Bacteria km723[bdvl] km723[imm21dvAB14] km960[+8OdvDU9] km723[hdvl][imm2ldvAB14] km96O[hdvl][+8OdvDU9]
kdv 0.96 12.6 1.5
immfldv
480dv
Both dv’s
0.46 8.2 -
0.70 2.5
0.22 1.6
B One colony of plasmid-carrying cells was inoculated into 2 ml of PBB, aerated overnight at 37°C (the cells underwent about 30 cycles of doublings during this incubation), plated, and scored for plasmid-less segregants, as described under Materials and Methods.
formants were kept for 2 weeks on EMB galactose plates at 25°C to allow increased selection for the Gal+ cells before isolation. In this case, some black, stable Gal+ colonies appeared. All of them carried DNA indistinguishable from that of incoming hdvgal+, and no detectable amount of resident Xdv DNA was seen. These observations indicate that the two homoimmune Adv’s are incompatible. In the former case the resident plasmid must have prevented establishment of hdvgal+, while in the latter case the Advgal+ must have displaced the resident Adv plasmid. In order to confirm that two homoimmune Adv’s are incompatible, cells carrying two Adv’s with different drug-resistance markers (Advkan and Advamp) were constructed. Colonies that grew on a plate containing kanamycin and ampicillin were restreaked on drug-free plates, and colonies were tested for drug resistance. The results in Table 6 show that the kanamycin and ampicillin markers do not coexist stably: The double-carriers became single carriers of either resistance soon after elimination of the antibiotic selective pressure. We conclude, therefore, that homoimmune two Adv’s are incompatible.
ments that can replicate autonomously in a plasmid state. These fragments are about one-tenth of the parental phage genomes in size, and consist of those genes and structures that act in the process of replication. Berg and Kellenberger-Gujer (1974) observed a similar situation with the imm2 1 phage. Genetic and physical studies have shown that there is only one unique “replicator region” in the lambdoid phage genome, and this region can be extracted as Adv in the plasmid state (Matsubara and Kaiser, 1968; Davidson and Szybalski, 1971; Chow et al., 1974). No rearrangement of genes or mutational events seem to be required. There is a possibility that a similar process, viz., extraction of a simple plasmid replicon from a complex genome, may well be possible with other temperate phages. Recently, Hobom and Phillipsen (personal communication) showed that Adv-like plasmids can be generated from A phage DNA in vitro using genetic engineering techniques. Similar methods have been applied to the sex factor F and drug-resistant plasmids (Timmis et al., 1975; Lovett and Helinski, 1976). The reverse process, viz., construction of a complex phage genome from a simple DISCUSSION plasmid genome, has also been reported: In this paper we show that various tem- Matsubara and Kaiser (1968) described perate phages, including imm434, imm21, “new phages” whose entire “replicator and $80, as well as A, give rise to DNA frag- region” is derived from Adv and the re-
PLASMIDS FROM LAMBDOID
293
PHAGES
TABLE 6 INCOMPATIBILITY
Expt A
First growth with No drugs
BETWEEN
hdvkan
AND
Advamp”
Number of progeny colonies which are: Kana Amp* clone no.
KanR AmpR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
KanR Amps
KanS Ampa
KanS Amps
10
2 5
Total
9 0 0 0 9 0 3 8 8 0 2 7 8
1 0 0 0 20
20
54
65
No. of clones tested B
Kanamycin
90 86
12 8
78 78
0 0
C
Ampicillin
75 58
34 18
0 0
41 40
a Cells carrying Xdvkan and Xdvamp were constructed as described in Table 3, footnote a. In Expt A, 16 independent transformant clones were spread over drug-free plates. After overnight incubation (ca. 30 generations), 10 colonies from each clone were tested for the presence of the drug-resistant Adv’s by transfer with toothpicks onto two plates containing either kanamycin or ampicillin, and incubation at 37°C overnight. In Expt B, double-carrier clones of independent origins were transferred onto plates containing kanamycin, grown overnight, and then replica-plated onto two plates containing either of the two drugs. Experiment C was done similarly, except that the double-carrier clones were grown first on plates containing ampicillin.
maining regions from phage imm434. Such a phage appeared among the progeny of the imm434 phage that infected hdv-carrying cells. It was thought that the imm434 replicator region is replaced by that of the plasmid. Berg (1971) and Berg and Kellenberger-Gujer (1974) showed that such a new phage can emerge through formation of a tandem structure that contains two replicators, one from the heteroimmune phage and one from Adv. Thus, replicators in plasmids and viral genomes, both of which are major vehicles in infectious heredity, can be ex-
changed. The possibility that similar phenomena may occur in nature, at least in microbes, is intriguing. Thus, the genome of bacteriophage A can be regarded as a plasmid (hdv) genome that is connected to the control genes N and Q, and to other genes for morphogenesis and cell lysis (late genes). The genes N and Q flank the Adv genome; their products are positive regulators of transcription. Cell lysis and phage morphogenetic genes are located outside of these, and are under the control of the Q gene. Recent experiments
294
MATSUBARA
(Akaboshi, unpublished data) have shown that a variant A phage whose genome has two deletions, one of which covers the N gene and the other the Q gene, infects a cell and behaves indistinguishably from that of hdv plasmid. It was observed that about a hundred copies of the dv plasmids occur per host chromosome. For some unknown reason, except in a few instances, many of these dv plasmids were in a dimeric state. The implication of this phenomenon awaits further elucidation. As seen with the Xdvl-hdvgal and hdvkan-Advamp systems, two Adv plasmids are incompatible, although Adv replicates under a relaxed control (Rownd, 1969), and about a hundred copies occur per host chromosome. Cabello et al. (1976) and Kibe et al. (1976) demonstrated recently that the ColEI factor, which is similar to Adv in size and occurs in 20 to 30 copies per chromosome, also shows incompatibility. A similar observation has been made with an another non-self-transmissible small plasmid SSu (Smith et al., 1974). Thus, incompatibility is likely to be a general phenomenon that occurs between a pair of plasmids whose replication is controlled by the same regulatory mechanism. A theoretical consideration of incompatibility between two plasmids replicating under relaxed control will be dealt with separately (Ishii, Hashimoto-Gotoh, and Matsubara, manuscript in preparation). We have also shown that plasmids derived from different heteroimmune phages, such as Adv and @Odv or Adv and imm2ldv, are compatible. Since the genes and the other loci required for the replication of these plasmid genomes are known (Matsubara, 1976), we can determine which genetic element(s) is responsible for incompatibility. The compatible pair, hdv and imm2ldv, shares a segment that carries an origin for replication (ori) and also two initiator genes, 0 and P (see Fig. 1). It can be concluded, therefore, that these genes and loci are not
AND OTSUJI
involved in incompatibility. The genetic elements carried in the heterologous region are the genes rex, ~1, tof, ~11, and two promoter-operators (pRoR and p-e for transcriptions to the right and left of the genome, respectively) (see Fig. 1). Among these, genes rex, c1, and cI1 must be irrelevant in incompatibility, as these are dispensable and plasmids without them behave indistinguishably from plasmids that carry them. The role ofpre is not clear at present, but in phages its only known function is in the control of expression of irrelevant genes rex and c1. Therefore, as a working hypothesis, we propose that the remaining elements, viz., gene tof and the promoteroperator pRoR, are responsible for the determination of incompatibility. The pRoR controls expression of the plasmid operon, and the tof gene codes for a protein, the autorepressor, that negatively and specifically acts by binding at pRoR. The tofgene itself is under the pRoR control, and hence the tof-pRoR region constitutes a highly specific autorepression system (Matsubara, 1976; Berg, 1974). In contrast, the heteroimmune pair, imm434dv and Adv, exhibits weak incompatibility. This interaction could be due to cross-reaction between the A and 434 autorepression systems. Alternatively, the weak incompatibility could be ascribed to a hitherto unknown interaction involving a genetic site(s) located between the two right ends of the imm434 and imm21 immunity regions, although this region carries no known genetic function related to plasmid replication control. Further studies are necessary to elucidate these problems. The Adv plasmids interfere with growth of the superinfecting homoimmune phage, and divert its replication process into the plasmid state (Matsubara, 1972a, 1974b). The interference occurs only when the invading phage and plasmid genomes are homoimmune. Several lines of evidence have been presented which strongly suggest that the interference in phage growth is caused by the autorepressor established
PLASMIDS FROM LAMBDOID
by the resident plasmid to control its own replication (Matsubara, 1974b). Here again, weak interference was seen, as an exception to the rule, in hdv and imm434 phages or in imm434dv and A phages. In a clone carrying two plasmids, loss of one or the other of the plasmids occurred independently. Further examples of this must be examined, but this observation implies that the primary mechanism that leads to loss of the plasmid (viz., failure in the mechanism to perpetuate the plasmid) is again immunity specific, and is not caused by physiological effects of the host cell that block multiplication of the dv plasmids in general. ACKNOWLEDGMENTS The authors would like to thank Dr. Yasuyuki Takagi for his support and encouragement throughout this study, Midori Matsubara for her technical assistance, and Dr. D. Berg for sending us the Advgal+ and hdvkan plasmids. This work was aided in part by a grant from the Asahi Press and a grant from the Ministry of Education, Science and Culture, Japan.
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CLOWES, R. (1972). Molecular structure of bacterial plasmids. Bacteriof. Rev. 36, 361-405. DAVIDSON, N., and SZYBALSKI, W. (1971). Physical
and chemical characteristics of lambda DNA. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 45-82. Cold Spring Harbor Laboratory, New York. HASHIMOTO, T., AND MATSUBARA, K. (1974). Transformation of Ca++-treated recA derivative of Escherichia co/i K12 by Adv DNA. Japan. J. Genet. 49, 97- 101. HELLING, R. B., GOODMAN, H. M., AND BOYER, H. W. (1974). Analysis of endonuclease R. EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14, 1235-1244. HERSHEY, A. D. (1971).
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of in vitro recombinants. Mol. Gen. Genet. 150, E. S. (1974). Genetic and molecular characterization 53-61. of some non-transferring plasmids. Mol. Gen. Genet. 129, 229-242. NOVICK, R. P. (1969). Extrachromosomal inheritance STREECK, R. E., AND HOBOM, G. (1975). Mapping in bacteria. Bacterial. Rev. 33, 210-235. cleavage sites for restriction endonucleases in hdv ODAKURA, Y., HASHIMOTO, H., AND MITSUHASHI, plasmids. Eur. J. Biochem. 57, 595-606. S. (1974). R-factor mutant capable of specifying hypersynthesis of penicillinase. J. Bacterial. 120, THOMAS,R., AND DAVIS, R. W. (1975). Studies on the cleavage of bacteriophage Lambda DNA with EcoRI 1260-1267. restriction endonuclease. J. Mol. Biol. 91, 315-328. ROWND, R. (1969). The replication of a bacterial episome under relaxed control. J. Mol. Biol. 44, TIMMIS, K., CABELLO, F., AND COHEN, S. N. (1975). Cloning, isolation, and characterization of replica387-402. tion region of complex plasmid genomes. Proc. Nat. SATO, K. (1970). Genetic map of bacteriophage 480: Acad. Sci. USA 72, 2242-2246. Genes on the right arm. Virology 40, 1067-1069. YOSHIMORI,R. N. (1971). “A Genetic and BiochemiSIGNER, E. R. (1969). Plasmid formation: A new mode of lysogeny by phage A. Nature (London 223, cal Analysis of the Restriction and Modification of DNA by Resistance Transfer Factor.” Ph.D. 158-160. Thesis. University of California, San Francisco. SMITH, H. R., HUMPHREYS,G. O., AND ANDERSON,