Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

PLASMID

5,277-291

(1981)

Complementation Analysis of Replication and Maintenance Functions of Broad Host Range Plasmids RK2 and RPI CHRISTOPHER Depurtment Department

M.

THOMAS

of Biology B-022, University of California, Sun Diego, La Jolla, California 92093, and of Genetics,’ University of Birmingham, P.O. Box 363, Birmingham BIS 2TT, England Received

October

14, 1980

It has previously been concluded that regions tentatively designated trf A and trf B, located at 16-18.7 and 54-56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, oriRK*, located at 12 kb on the genome, is also required. A segment of DNA containingori,,, can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans -acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trf A region alone can provide the trans -acting functions necessary for replication from oriRK2. Deletion of the trf B region in truns to an oriRK plasmid does not correlate with alteration in copy number or stability of the.ori aK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trf A and trf B regions as indicated by complementation analysis of three different mutants. The trJ’A and trf B regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori aKZplasmid to replicate at 30°C. When the source of trf A and trf B is a trf B mutant the ori aK2 plasmid is temperature stable but is temperature sensitive when the source is a trf A mutant. This confirms that only trf A is essential for initiation at and elongation from oriRK which is probably the primary event in RK2 replication and suggests that the trf B region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori,,,, trf A, and trfB.

Plasmid DNA replication has been studied as a means of understanding the basis for the autonomy of these interesting genetic units and as model systems for replication of any circular DNA molecule (for reviews see Thomas and Helinski, 1979; Kolter and Helinski, 1979). In general, plasmid replication depends both on host and plasmid genes. There is considerable evidence that many plasmids carry genes whose products are required to act either positively (Inuzuka and Helinski, 1978; Kolter et al., 1978) or negatively (Molin and Nordstrom, 1979; Shepard et al., 1979; Twigg and Sherratt, 1980) in their replication. It is thought that i Address to which reprint spondence should be sent.

requests

and corre-

277

by studying these products and the genes that encode them some understanding of plasmid replication and its control may be gained. For the plasmid RK2 it has been clearly demonstrated that replication from the origin of vegetative replication requires a positively acting factor encoded elsewhere on the plasmid (Figurski and Helinski, 1979; Thomas et al., 1980). The 56-kilobase pair (kb)* plasmid RK2 * Abbreviations used: kb, kilobase pair; LB medium, 10 g tryptone, 5 g yeast extract, 10 g NaCYliter; CFU, colony forming units; M9-CAA, 1 g NH&l, 1.5 g KH,PO,, 3.5 g Na,HPO,, 5 g Difco casamino acids, 2 g glucose, 5 mg thiamine/liter; TE buffer, 100 mM TrisHCl, pH 8.0, 20 mM EDTA; TNE buffer, 100 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 8.0; bp, base pair. 0147-619X/81/030277-15$02.00/0 Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

278

CHRISTOPHER

belongs to incompatibility group P whose members are generally able to transfer between, and be maintained stably in, a wide range of Gram-negative bacterial species (Datta and Hedges, 1972;Olsen and Shipley, 1973; Beringer, 1974; Cho et al., 1975). The replication properties of RK2 have been studied in Escherichia coli where three regions of the plasmid genome totaling 5.4 kb appear to be involved in replication (Thomas et al., 1979, 1980). One of the regions (ori& has been identified as the origin of unidirectional DNA replication (Meyer and Helinski, 1977). The other two regions (trfA and tr-B), each about 2 kb in size, together encode at least one transacting function required to allow replication from oriRK (Figurski and Helinski, 1979; Thomas et al., 1980). Deletion of part of the trf A region results in inactivation of the trans-acting replication function(s) necessary for oriRK activity indicating that the trf A region codes for one such function. A different deletion in a mini-RK2 replicon removes parts of regions encompassing both trf A and trfB. The resultant RK2 replicon pCT27 (Thomas, et al., 1980), functions poorly, being unstable, of low copy number, and inefficient in transformation. This defect can however be complemented in trans by the DNA segment encompassing the trfB region. It was therefore concluded that the trfB region specifies a tram -acting function involved in plasmid replication or maintenance. The site of action of this function is unclear, however, because the partially defective replicon carries oriRK and at least parts of both trf A and trf B regions (Thomas et al., 1980). It is possible therefore that the trf B function acts on a site in the trf A or trf B regions rather than at oriRK . It should be noted that the trfA and trfB regions are of considerable size and the exact location of the genes of interest is not known. Since initiation at and elongation from on.., is probably the primary event in RK2 replication we have studied further the plasmid genes required to provide the trans-

M. THOMAS

acting function(s) involved in this process. A major step forward has been the cloning of the trf A region without the trf B region allowing independent investigation of the two regions. We have tested these regions for the ability to allow replication in tram from oriRK and to complement mutant Pgroup plasmids temperature sensitive for replication or maintenance (Urlapova et al., 1979; Robinson et al., 1980; Thomas, unpublished). Construction of hybrid plasmids carrying trfA and trfB from mutant plasmids has allowed determination of whether oriRK replication, dependent on these functions, is temperature sensitive as a result of these mutations. Our results indicate that only trf A is essential for replication from ori,,,. However, temperaturesensitive defects in P-group plasmid replication or maintenance can arise in both trf A and trf B, although only the trf A mutation affects oriRK replication. It is concluded that the trf B region is involved in RK2 maintenance at a point other than initiation at and elongation from ori,,,. MATERIALS

AND METHODS

Bacterial strains, plasmids, and growth conditions. Escherichia coli strains used in this study were C600AtrpE5; C6OOthy-; C6OOAtrp ESrec A56; HB 101(AB266 leu pro lac gal str’ thi his ret A r; m,); KS02 (hsrhsm+, gal, supE, lac, met) (all from the

collection of D. R. Helinski); C2110 (polA1 his rha) (from M. Kahn); C2110 Ch3/86 (pol Al his rha trf A+ trf B+); CSH4 (ret A hug A 251 Tn5) (from R. Kolter). Plasmids

utilized in this study are listed in Table 1. Plasmids constructed during the course of this study are listed in Table 2. Media used were LB (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter) or MPCAA (1 g NH&l, 1.5 g KH,P04, 3.5 g N+HP04, 5 g Difco casamino acids, 2 g glucose, 5 mg thiamine per liter) for selection of trp E+. Then 1.5% (w/v) agar was added for solid medium. For selection of antibiotic resistance medium was supplemented with benzyl penicillin

REPLICATION

FUNCTIONS OF RK2 AND RPl

279

TABLE 1 PLASMIDS

Plasmid designation

Relevant characteristics

Molecular size (kb)

Source or reference

pRK353 pDS3 pMK2005 pRK248 pRK2501 pCT45 pMRS RPltsl2

R6K replicon; rrpE+ Pl5A replicon derived from pACYCl84; Cm’ ColE 1 replicon; rrpE+ RK2 replicon, Tcr Derivative of pRK248 Tc’ Km’ oriRK linked to Km’selective fragment Temperature-sensitive derivative of RPl Temperature-sensitive derivative of RPl

11 2.5 6.5 9.6 11 2.2 56 56

Kolter and Helinski (1978) David Stalker (unpublished) Mike Kahn (unpublished) Thomas et al., 1980 Kahn et al., 1979 Thomas ef al. (1981) Robinson et al. (1980) Urlapova et al. (1979)

(300 pg/ml) for Ap’; kanamycin (50 pg/ml) for Km’; tetracycline (20 puglml)for Tc’; and chloramphenicol (25 pg/ml) for Cm’. Plasmid DNA isolation. For rapid isolation of plasmid DNA a technique based on the Triton X-100 cleared lysate method was used. Overnight cultures grown in 30 ml of LB medium were harvested and resuspended in 1 ml of ice-cold 25% (w/v) sucrose, 50 mM Tris-HCI, pH 8.0, and 0.25 ml of lysozyme (10 mg/ml in the same sucrose buffer) was added. After 20 min on ice 0.25 ml 0.25~ EDTA, pH 8.0, was added and after a further 10 min on ice an equal volume (2 ml) of Triton X-100 solution (0.2% (w/v)), 50 mM Tris-HCl, pH 7.5, 62.5 mM EDTA was added very rapidly to ensure immediate mixing. After 5 min at room temperature the lysate was centrifuged at about 20,OOOgfor 15 min. The supematant was decanted, extracted with an equal volume of water-saturated phenol at neutral pH, and then the aqueous layer was extracted with diethyl-ether until all the phenol was removed. The DNA and RNA were precipitated by addition of an equal weight of isopropranol and storage at - 20°C for 30 min or more. The precipitate was pelleted, lyophilized, and resuspended in 100 ~1 of buffer (10 mM Tris-HCl, pH 7.5, 5 mM NaCl, 0.5 mM EDTA). For high copy number plasmids this yielded a DNA concentration of about 0.50 pg/pl. For purification of plasmid DNA using

cesium chloride/ethidium bromide gradients the method of Meyer et al. (1977) was used. Restriction endonuclease digestion, ligation, and bacterial transformation. Diges-

tion of plasmid DNA with restriction endonuclease was carried out with buffer conditions as recommended by the manufacturers (BarnHI, BgfII, HindIII, and Sst II were from Bethesda Research Laboratories (BRL); SafI was from Boehringer and Eco RI was prepared according to the method of Greene et al. (1978)). Restriction digests were analyzed by agarose gel electrophoresis (0.8- 1.1% (w/v) agarose depending on fragment size) using Tris/ borate/EDTA buffer (10.8 g Tris-base, 5.5 g boric acid, 0.925 g EDTA Na, per liter). For staining the DNA, the agarose and electrophoresis buffer contained ethidium bromide (0.2 pg/ml). Gels were photographed as described elsewhere (Kahn et al., 1979). DNA ligase was from BRL and recommended conditions were used. Bacterial transformation was carried out as described previously (Kahn et al., 1979). Isolation of plasmids carrying transposon Tn.5. The plasmid of interest was trans-

formed into strain CSH4. Transformants were purified and then 30 ml of LB medium was inoculated and incubated with shaking at 30°C until the culture had reached stationary phase. Plasmid DNA was extracted and supercoils were analyzed to determine whether the plasmid existed in a

280

CHRISTOPHER M. THOMAS TABLE 2 PROPERTIESOFDERIVEDPLASMIDSUSEDTOGENERATERESULTS IN THIS STUDY

Plasmid designation

Relevant RK2 functions

Structure and construction

Selective markers

Molecular size (kb)

TcrKmr

11

rrfA+ trf B+

trpE+

13.4

Derivative of pCT16 (Thomas et al., 1980)ColEl replicon

trf B+

trpE+

10.6

pCT141

pDS3 carrying Hind111 fragment derived from EcoRI hybrid of pRK2501 ahd pRK353

trfA+ trf B+

Cm’Tc’

10.7

pCT160

oriRK*linked to Cm’ P15A replicon from pACYC 184

orb

Cm’

pCT85

EglIUBamHI

oriRK , trfA+, trf B+

Tc’, trpB+

20.6

pCT85.3

SsrII deletion derivative of pCT85

trfA’

Tc’, trpE

13.1

pCT87

Hind111 fragment of pCT85.3 cloned in pDS3

trfA+

TcrCmr

10.2

pCT87:: Tn5#8b2

pCT87 with Tn5 inserted in the rrfA region

trfA+

TcrCmrKmr

16.0

pCT88

Hind111 deletion derivative of pCT87::TnS#Sb2

trfA+

Tc’Cm’Km”

9.6

pCT145

Hind111 hybrid of pRK2501ts3 and pDS3

oriRK , trfA+ trf B’”

Tc’Cm’

13.5

pCT145.1

EcoRI deletion derivative of pCT145

trfA+, trf B’”

Tc’Cm”

10.3

pCT153

BglIIIEamHI

or&., , trfAtS, trfB+

AprTcrKmr,

67.0

trfAts, trf B+

Tc’Km’Ap”,

pRK2501ts3

Temperature-sensitive derivative of pRK2501 from in vitro hydroxylamine mutagenesis

pCTlO1

Derivative of pCT14 (Thomas et al., 1980)ColEl replicon

pCT103

pCT153.1

hybrid of pRK248 and pRK353

hybrid of pMR5 and pRK353

EcoRI deletion derivative of pCT145

3.0

trpE+

54.0

trpE+

monomeric form, to avoid problems with the transposon inserting into only one of two tandem plasmid copies. If the plasmid DNA preparation was monomeric it was used to transform either C6OOAtrpE5 or C2110, selecting for either the plasmid marker alone or plasmid marker and kanamycin resistance. Comparison of the number of transformants under the two selective conditions gave an estimate of the proportion of plasmid molecules carrying Tn5 (this was generally about 1 in 104), Doubly resistant clones were streaked to single colonies on LB Kan,, plates a number of times to ensure segregation, through incompatibility, of any plasmid molecules not

carrying the transposon. DNA from as many as possible different CSH4 transformants carrying the original plasmid were used to increase the chance of isolating different insertions. Analysis of the location of Tn5 in the plasmid DNA isolated was carried out with Hind111 and Sal1 digests because Hind111 cuts Tn5 1.25 kb from each end and Sal1 cuts it approximately in the middle (Jorgensen ef al., 1979) thus allowing location of the site of insertion relative to both Hind111 and Sal1 sites in the plasmid DNA. Estimation of plasmid stability. Overnight cultures grown in LB medium with kanamycin (50 pg/ml) were diluted 104fold into fresh, nonselective medium and

REPLICATION

FUNCTIONS

grown at 37°C with shaking. When the culture had grown up to an E:&mnm of 1.00 (approx) it was diluted again about 105-fold. Samples were removed at (1) the start of growth, (2) after 2 h, (3) at the time of the second dilution, (4) 4 h later, and (5) after O/N incubation of the culture when it had been in stationary phase for a number of hours. After serial dilution the bacterial suspensions were plated on nonselective LB plates to estimate the number of CFU/per milliliter. The proportion carrying the oriRK plasmid (Km’) or the “helper” plasmids (Cml‘) was estimated by replica plating. The first 2 h of growth were taken to represent a lag phase. Between sample (2) and (4) was taken as exponential phase while between sample (4) and (5) represented continued exponential phase, late exponential phase, and stationary phase. Estimation of plasmid copy number. The plasmid of interest, pCT45, had to be separated from the helper plasmid on which it is dependent, so first total plasmid content was determined followed by agarose gel electrophoresis to separate the two plasmids and permit estimation of the ratio of the two plasmids. Total DNA was labeled in derivatives of C600thy - carrying the relevant plasmids using MPCAA medium supplemented with thymine (2 pg/ml) and [methyl3Hlthymine (10 $X/ml). Bacterial cultures, grown at 37°C with aeration and under selection for the resident plasmids by addition of kanamycin (50 &ml) were harvested at approximately 5 x lo* CFU/ml. Then 10 ml of culture was washed with TE buffer (100 mM Tris-HCI, pH 8.0, 20 mM EDTA) and resuspended in 0.8 ml TE buffer before addition of 100 ~1 of a predigested pronase solution (5 pg/ml) and 100 ~1 of 10% (w/v) sodium dodecyl sulfate solution. After incubation at 37°C for 30 min total ice-cold trichloroacetic acid (5%, w/v) precipitable radioactivity was determined. Analytical gradients were run after layering approximately 100,000 cpm of lysate onto 5 ml ethidium bromide (0.5 mg/ml), cesium chloride gradients (average density 1.55 g ml-‘). Preparative gradients with 0.5 ml of

OF RK2 AND RPl

281

the lysate were also run. The analytical gradients were centrifuged in an angle rotor at approximately 120,OOOgfor 40 h and then fractionated onto strips of Whatman No. 17 glass-fiber filter paper. The strips were immersed in 5% (w/v) ice-cold trichloracetic acid for 30 min, washed twice by immersion in ethanol, washed in acetone to assist drying, and the precipitated radioactivity in the plasmid and chromosome peaks was used to determine the total plasmid content. The preparative gradients, after similar centrifugation, were fractionated into tubes, after the chromosomal DNA had been removed out of the side of the tube using a hypodermic syringe. Aliquots of each sample were removed and the radioactivity was determined to check the position of the plasmid peak which was pooled, extracted with isopropanol to remove ethidium bromide, dialyzed against TNE buffer (100 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH S.O), and then precipitated with an equal weight of isopropanol after addition of 100 pg tRNA to aid precipitation. The pellet obtained was dried, resuspended in 100 ~1 Hind111 buffer, digested with restriction endonuclease HindIII, precipitated again, and resuspended in 15 ~1 of the same buffer before addition of 5 ~1 of g!ycerol/bromophenol blue/RNase mixture and electrophoresis through a 1% agarose gel. Bands representing the different plasmids were cut out of the gel and the radioactivity was determined by melting the gel in 1 ml of water, adding 10 ml of aquasol scintillation fluid (New England Nuclear), and counting the radioactivity in a scintillation counter. Plasmid copy number was estimated from plasmid size and the proportion of total cellular DNA that it represented. RESULTS

Deletion Analysis of Genetic Information Required in trans for Replication from oriRK Evidence for the involvement of two regions, trfA (16- 18.7 kb on the RK2 genome) and trfB (54-56 kb), in RK2

282

CHRISTOPHER M. THOMAS

R6K replicon

FIG. 1. Deletion of most of the trfB region using restriction endonuclease Sst II. pCT85 is one of two orientations of the hybrid formed by ligation of pRK353 digested withBum HI and pRK248 digested with BglII. pCT85 has four Sst II cleavage sites. Complete digestion with Sst II and ligation followed by transformation of C6OOAtrpES selecting for Tc’ and then screening for trp E- yielded a plasmid, pCT85.3 consisting of only the two fragments carrying the R6K replicon, Tc’, and trf A. A small amount of trj’J3 region (trfB*) also remains. The Hind111fragment carrying trf A and Tc’ was cloned in the single site of pDS3 by digestion of pCT85.3 and pDS3 DNAs with HindIII, ligating, and then selecting for Cm’ Tc’ after transformation of C600dtrpE5 with the DNA.

replication and/or maintenance in addition to the requirement for the replication origin, ori,,, (Thomas er al., 1980), has been described in the introduction. One or both of these regions provide function(s) necessary to allow replication from ori,,, (Figurski and Helinski, 1979; Thomas et al., 1980). The trfB region has been cloned without trfA and is insufficient to allow oriRK function, indicating a requirement for trf A. However,

the trf A region has not been cloned without ?rfB and therefore it has not been possible to decide whether the U-B region does or does not provide a trans-acting function required for oriRK function. Lack of suitable restriction sites in the reduced-size RK2 derivatives being utilized has hindered attempts to clone the trfA region alone. However, mapping of SstII sites in pRK248 (Fig. 1) made it clear that an SstII fragment contains all of

REPLICATION

FUNCTIONS

the trfA region and only 150-200 bp of the rrfB region. A convenient way to delete the adjacent SstII fragment containing the rest of the trfB region was to utilize pCT85, one orientation of the hybrid plasmid formed by ligating pRK248 cut with BglII to pRK353 (a mini R6K trpE plasmid) cut with BumHI (Fig. 1). BgfII and BamHI produce identical four-base single-stranded cohesive ends but neither B&II nor BumHI sites are regenerated after the ligation. pCT85 consists of four SsrII fragments, three sites in pRK248 (one of which interrupts the Tc’ gene) and one in the trp region of pRK353. Digestion of pCT85 with SstII, ligation, and transformation of C600AtrpE5 with the DNA gave Tc’ transformants some of which were trpE-. Plasmid DNA from four such transformants were examined and one, pCT85.3, consisted of the two desired SstII fragments containing the R6K replicon, Tc’, and trfA (Fig. 1). This plasmid was transferred to two recA strains, C6OOAtrpE5 recA56 and HBlOl. It was found that an ori,,, plasmid (pCT45), which depends for replication on RK2 tram -acting replication functions, can be maintained in these strains. It was concluded that either the trfB region is not essential or that the remaining 150200 bp of the trfB region is sufficient to provide the trjJ3 function. Because of the lack of suitable sites to delete this remaining portion of the trfB region it was decided to attempt to insert transposon Tn5 into a nonessential part of the trf A region between the essential part of the trf A region and the remaining trf B region. Insertion of Tn5 which carries a gene conferring kanamycin resistance (Berg, 1977) was carried out as described under Materials and Methods. With pCT85.3 most insertions were found to be located in the R6K-derived DNA so the Hind111 fragment carrying Tc’, trf A, and trf B* was cloned into pDS3, a Cm’ Tc” derivative of pACYC184 (Chang and Cohen, 1978) to give plasmid pCT87 (Fig. 1). An apparently suitable insertion of Tn5 into pCT87 was obtained with the insertion located in the middle of the trfA region (Fig. 2) at the

283

OF RK2 AND RPl trfA;

Tll5 Km’

HindUI

I

digestion a

ligation

MA;

HtndM

FIG. 2. Use of TnS to clone the trfA regionin the of the rrfB region. Derivatives of pCT87 with Tn5 inserted were isolated as described under Materials and Methods. The point at which Tn5 is inserted was analyzed by Hind111 digestion and Sal1 digestion. One such isolate, pCT87::Tn5# 8b2, hadTn5 inserted as shown. Complete digestion with Hind111 followed by ligation and transformation of C6OOArrpE5 with the DNA, selecting for Cm’ Tc’ and screening for Km5 gave a plasmid, pCT88, which has only two Hind111 fragments, one is pDS3, the other carries Tcr, part of trf A (trfA*), and part of the inverted repeat of Tn5, but none of the trfB region.

absence of any

equivalent of 17.55 kb on the RK2 map, as estimated by separate Hind111 digestion and Sal1 digestion and analysis of the fragment pattern on agarose gels. This derivative still supported replication of an oriRK plasmid. This pCT87::Tn5 hybrid was digested with HindIII, ligated, and then C6OOAtrp E5 was transformed with the DNA. Selection for Cm’ Tc’ and screening for Km” yielded a plasmid consisting of the pDS3 replicon and a Hind111 fragment carrying Tc’, part of trf A, part of the Tn5 inverted repeat, but none of the trf B region. This plasmid,

284

CHRISTOPHER M. THOMAS TABLE 3

INSTABILITY AND LATE

OF pCT45 IN THE PRESENCE OF VARIOUS HELPER PLASMIDS DURING LAG, EXPONENTIAL, EXP~NENTIAL~~TATIONARY PHASE GROWTH IN LB AT 3PC WITH No PLASMID SELECTION

C6OOrhy(pCT141, pCT45) Number of generations 0 (Diluted from stationary phase) 3 7 12 25 (Sampled from stationary phase culture)

C6oothy (pCT88, pCT45)

C6OOrhy(pCT87, pCT45)

A”

B”

C”

A”

B*

CC

Aa

B”

CC

69 64 53 52

89 94 18 81

78 68 68 64

98 85 84 86

99 % 97 95

99 88 87 91

81 69 69 66

99 99 96 91

82 70 72 73

6

24

25

61

84

73

60

91

66

u Percentage of total population carrying pCT45 (Km’). b Percentage of total population carrying “helper” plasmid (Cmr). c Percentage of “helper” population carrying pCT45 (Km’Cm’).

pCT88, was found to retain the ability to support trans replication of oriRK*linked to a selective marker in both a ret+ and ret A56 strain. It was concluded that the necessary trans-acting function encoded by the rrfA region must be specified by the DNA between approximately 16 and 17.55 kb on the RK2 genome. Preliminary evidence indicates that the Tc’ trf A fragment from pCT88 linked to an oriRK fragment can function as an RK2 replicon (Thomas and Klauza, unpublished). Although the trf B region can be deleted without removing the ability to complement an oriRK plasmid it is conceivable that the trf B region provides functions normally involved in, but not essential for, maintenance of the oriRK plasmid. For this reason the stability and copy number of pCT45 replicating with various “helper” plasmids was investigated. In addition to using pCT88 and pCT87 as helper plasmids, both of which lack the complete trf B region, pCT141 which consists of pDS3 cloning vehicle and a Hind111 fragment containing the rrfA+ trf B+ Tc’ segment present in pRK248 was also used as a helper as a control for replication and maintenance in the presence of both trf A and trf B. pCT141 is the full helper plasmid most analogous to pCT88 and pCT87. Estimates of stability

(see Materials and Methods) indicated that pCT45 was least stable when pCT141 was providing the required truns-acting factors. The data from one experiment are shown in Table 3. After dilution of a stationary phase culture grown with kanamycin which selects for the oriRK, plasmid, pCT45, 99% of a C60&hy- (pCT87, pCT45) population that still carried the helper plasmid would form colonies carrying pCT45 whereas this figure was 82% with pCT88 and 78% with pCT 141. After growth in the absence of any selection these figures fell to 73% with pCT87, 66% with pCT88, and 25% with pCT141. It appears that with all three plasmids instability of pCT45 is most marked during lag phase or late exponential and stationary phase rather than exponential phase. With pCT141, pCT45 is unstable at all phases of growth but it may be important that pCT141 also shows marked instability during the experiment (only 25% of the final population carried pCT141 compared with 84 and 91% for pCT87 and pCT88, respectively). No definite conclusion can be drawn from these experiments although it does appear that deletion of the trfB region from the helper plasmid does not cause increased instability. The pattern of instability with pCT45 in the presence of pCT141, pCT87, and pCT88 where loss of the plasmid may be

REPLICATION

285

FUNCTIONS OF RK2 AND RPI

particularly associated with stationary phase is interesting. Estimates of copy number (Table 4) indicated that pCT45 was maintained at a similar level in the presence of pCT87 and pCT141. Under the conditions used for labeling (exponential phase in the presence of kanamycin) there was no necessity to correct for the insignificant proportion of the population of bacteria that could not form colonies on LB kan plates. This level was approximately 10 copies per chromosome equivalent, rather lower than previous estimates of 16 copies per chromosome equivalent for pCT45 (Thomas et al., 1981) but close to the lower end (11.5 copies per chromosome equivalent) of the range from which that average figure was obtained. Estimates for the copy number of pCT45 in the presence of pCT88 were about 5, i.e., about half of the level of that in the presence of pCT87 and pCT141. It was concluded that deletion of the majority of the trfB region does not affect pCT45 copy number. Deleting the rest of the trfB region and nearly half of the trfA region may cause a significant decrease in copy number but whether this is due to complete loss of trfB or the possibility that the TnS-generated deletion extends into the trf A region close to the function involved in replication from orzRK2is not known at present. Complementation Analysis of Temperature-Sensitive Defects in Plasmid Replication or Maintenance

Two methods were utilized to determine whether specific cloned segments of RK2 could complement temperature-sensitive defects of two mutants of RPl, a plasmid indistinguishable from RK2 (Burkhardt et al., 1979), and one mutant of pRK2501, a mini-RK2 replicon. The strain used was C600AtrpES recA56 which minimizes the chance that recombination between replicons is responsible for suppression of the defect and allows trpE+ to be used as a selective marker. For the most commonly used method, plasmid DNA was prepared

TABLE 4 COPY NUMBER FOR PLASMIDSCARRYING ALL OR PART OF THE rrfA AND trf B REGIONSAND FOR pCT45 REPLICATING IN THEIR PRESENCE

ESTIMATED

Strain C6OOthy- (pCT141, pCT45) C6OOthy- (pCT87, pCT45) C6OOthy- (pCT88, pCT45)

Copy number of “helper” plasmid

Copy number pCT45

18

10.5

20

9.5

17

5.0

for the plasmids to be tested for their ability to trans-complement. Strains of C6OOAtrpE5 recA56 carrying the temperature-sensitive plasmids were made competent after growth at 30°C and then aliquots were transformed with DNA of various plasmids. Selection for the incoming and the resident plasmids at 42°C should result in transformants only if complementation occurs. To control for normal reversion frequency a plasmid that carried no RK2 DNA was used as the incoming plasmid. Since only those cells that have taken up plasmid DNA can possibly grow the resulting background is obviously much lower than if the equivalent number of competent cells were plated at 42°C with selection only for the resident plasmid. The alternative method to test for trans-complementation was to use purified temperaturesensitive plasmid DNA and to transform this into C6OOAtrpES recA56 strains carrying the various plasmids to be tested for their ability to truns-complement. These methods have advantages over the construction of strains carrying both plasmids at 30°C and then testing for growth at 42°C under selection for the temperature-sensitive plasmid. For example, when double plasmid strains are constructed at 30°C and if complementation by the hybrid does occur there is no longer an easy way to check that the particular clone being picked for testing at 42°C carries a temperature-sensitive plasmid rather than a revertant. Thus a large number of clones need to be tested

286

CHRISTOPHER M. THOMAS TABLE 5 T~IWZS-COMPLEMENTATION OFTEMPERATURE-SENSITIVEP-GROUPPLASMIDS BYCLONED SEGMENTSOFRK~IN CfXOAtrpE5recA56

Transforming plasmid pMK2005 pCTlO1 pCT103 pDS3 pCT141 pCT88 pCT87

Ratio of the number of transformants selecting for incoming and resident plasmids at 42°C to that at 30°C

Region carried by transforming plasmid

pMR5”

RPl ts 12”

-

0.02

0.02

0.004

trfA + trfB

rrfA + rrfs

0.75 0.026
1.25 0.026
trfA trfA + trfs*

1.0

1.0

1.0

1.0

1.20 1.0
trfi

pRK2501 ts 3”

a Resident plasmid. b Transformations selecting for both resident and incoming plasmid at 30°C gave very small colonies.

individually to make certain that the majority are not revertants whereas by testing for complementation at the same time as introducing the second plasmid many clones can be tested very simply on a single plate. The plasmids used in the complementation tests carry either both the trfA and trfB regions, only one of the two regions, or neither. Plasmids pCTlO1 and pCT103 are trpE+ derivatives of pCT14 and pCT16 (Thomas et al., 1980) which carry, respectively, trf A and trf B, or only trfB. They were constructed by inserting a Hind111 trpE fragment from pRK353 into the Hind111 site in the Km’ of pCT14 and pCT16. These trp E+ Km” derivatives were used because all three temperature-sensitive plasmids carry Km”. pMK2005 was chosen as a control since it is a trp Et Km” derivative of pMK20, the parent ColEl cloning vehicle for pCT14 and pCT16. The test plasmids (pDS3, pCT141, pCT87, and pCT88) were all Cm’, being based on the pDS3 cloning vehicle, and are described earlier in this paper, or in Tables 1and 2. The results of tests using these plasmids to transform C600Atrp ES ret A56 carrying the temperature-sensitive plasmids are shown in Table 5. The degree of complementation is estimated by comparing the number of transformants selecting for both incoming and resident plasmid at 42” and at 30°C. In

theory an alternative method would have been to compare the number of transformants at 42°C with selection for both incoming and resident temperature-sensitive plasmids or just the incoming plasmid. This method would depend on the assumption that at the nonpermissive temperature the resident plasmid is just diluted out of the population if no complementation occurs. However, in C6OOAtrpES ret A56 carrying these temperature-sensitive plasmids incubation at 42°C seems to inhibit growth even with no selection for the temperaturesensitive plasmid. This was not observed for C6OOAtrp ES. Thus in C600 Atrp E5 ret A56 if no complementation occurs at 42°C no reliable estimate of the number of transformants for the incoming plasmid can be obtained. It was for this reason that the total number of transformants was determined at 30°C and the number which could complement the temperature-sensitive defect was determined at 42°C. The results indicated that pMR5 and RPlts12 are complemented by the trfA region but not the trfB region while pRK2501ts3 is complemented by the trfB but not the trfA region (Table 5). Cloning of trfA and trfB Regions from Temperature-Sensitive Mutants It would be nice to know whether the temperature-sensitive defects in these mu-

REPLICATION

287

FUNCTIONS OF RK2 AND RPl

tants are due to alteration in the functions required to allow replication from oriRK2. A way to test this is to clone the trf A and trfB regions and use them to allow replication of a physically separate oriRK plasmid which may or may not now be temperature sensitive. Such experiments have been carried out for pRK2501ts3 and pMR5. The scheme to construct a derivative of pRK2501ts3 carrying trfA and trfB but not oriRK is shown in Fig. 3. The cloning vehicle used was pDS3 (Table 1). One of the problems with pRK2501 ts3 was its relatively high reversion frequency so that retention of the defect needed to be checked at each stage. A hybrid between pRK2501ts3 and pDS3 was constructed by Hind111 digestion, ligation, and transformation into a polA strain (C2110) selecting for both plasmid markers (pDS3 is pof A+ dependent). Temperature-sensitive clones were selected and transferred to a pol A+ strain (K802) which was restriction deficient but will modify the DNA and would therefore not reduce transformation efficiency with DNA from C2110, an E. co/i C strain. Plasmid DNA was prepared and the orientation of the Hind111 fragments was determined. Temperature sensitivity of the plasmids on transfer back to C2110 was also tested. Eco RI digestion followed by transformation selecting for Tc’ and screening for Cm” gave plasmids (pCT145.1) that had lost oriRKP. pCT45, which consists of oriRK linked to a Km’ selective fragment, was then transformed into this strain which was made competent after growth at 30°C. Equally high numbers of transformants were obtained whether the selective plates were incubated at 30 or 42°C. The relative number of transformants was not affected by using C600Atrp ESrec A56 instead of C6OOAtrpES ret+, indicating that host recombination was not resulting in suppression of the defect by recombination between the two plasmids. Four transformants obtained at 30°C were tested for loss of pCT45 after growth in the absence of selection at 42°C. No significant increase in the rate of loss compared to 30°C was found. To check that the defect was still

TC’

5011

PDS3

IIf*

2 5Kb

EcoRI

Cm’

pRK250ltr3 Pl5A

1lKb HindP Km’

0

0 Hind

repkon

m

trt B

O”RK2

EcoRI

Hind= dagcrtion

Ilgation

Y 5.31 I lc’ HlndD, PISA I*pllCO” Cm’

trfA pCTlLS

ECORI

13.5Kb

HlndDl 0

trt B

‘=“RK2 EcoRI EcoRl

dIgestion

B ligotkon

1 trtil TC’ SC!,, pCTlLS.1 10.3Kb Hl”dL!I 0 P15A repllco”

trfs EC&l

FIG. 3. Cloning of trfA and trfB from pRK250lts3. pRK250lts3 and pDS3 were joined at their single Hind111 sites by Hind111 digestion, ligation, and transformation of a polA1 strain (C2110) selecting for Tc’ Cm’. Plasmid DNA extracted from temperaturesensitive clones was transformed into apolA+ restriction negative strain (K802), and pCT145 was selected as a plasmid with the orientation of the Hind111 fragments as shown and was still temperature-sensitive on transfer back to C2110. Digestion of‘pCTl45 DNA with EcoRI followed by transformation of C600AtrpE5 selecting for Tc’ and screening for Cm% yielded pCTl45.1.

present in these plasmids, pCTl45.1 was digested with Eco RI, ligated with Eco RIdigested pCT7 (a Km’ ColEl derivative carrying ori&, and the DNA was transformed into apol Al strain (C2110) at 30°C. Since the vehicles in both pCT145.1 and pCT7 are poZA+ dependent, Km’Tc’resistant transformants must carry hybrid plasmids with an RK2 replicon. About 60% of these transformants were found to be temperature sensitive. Since revertants to temperature stability appear to have considerable selective advantage over

288

CHRISTOPHER M. THOMAS

defect in pRK2501ts3 probably does not result in temperature sensitivity of factors positively required in tram for replication from oriRK*. A slightly different scheme was adopted to clone trfA and trfB from pMR5 (Fig. 4). A mini R6K rrp+ plasmid, pRK353, cut with BamHI was inserted into the BglII site of pMR5 by ligation and transformation. One orientation of this hybrid plasmid, pCT153, enabled an EcoRI fragment to be deleted, Km’ removing Ap’ and or-i,,, and leaving behind a trpE+ Tc’ Kmr trfA+ and trfB+ plasmid, pCT 153.1. Because of the selective markers present in pCT153.1 (Km’ Tc’ rrpE) the ori,,, Km* plasmid (pCT45) could not be used and so it was most convenient to EcoRI d!gestm B llgatm transfer pCT153.1 into a @Al strain (C2110) and then to use pCT160 (Table 2) as a Cm’ ori,,, plasmid. The P15A replicon in pCT160 ispofA+ dependent so that in C2110 pCT160replication will be dependentupon rep lication from oriRK*. When C21lO(pCT153.1) was grown at 30°C and transformed with pCT160 DNA, Cm’ transformants were obtained with selection at 30°C but not at FIG. 4. Cloning of trfA and rrf B from pMR5. pMR5 42°C whereas C2110 Ch3/86, which carried DNA digested with BglII and pRK353 DNA digested with BamHI were mixed and ligated, and GOOAtrpES trf A and tr-B functions from a nontemperature-sensitive RK2 replicon gave equal was transformed with the DNA selecting for Tc’, Ap’, Km’, and trpE+. Clones were analyzed and a hybrid numbers of Cm’ transformants at both 30 plasmid, pCTl53, carrying pRK353 inserted relative to and 42°C. When single Cm’ transformants pMRS as shown was chosen. Digestion of pCTl53 with obtained at 30°C were grown at 30 and 42°C EcoRI followed by transformation of C6OOArrpE5 in liquid medium without selection, pCT160 selecting Tc’, Km’, trpE+, and screening for loss of Ap’ was found to be stable in C2 110 Ch3/86 at gave plasmids like pCTlS3.1, with a single EcoRI site. Such plasmids have lost ~ri~,~ but retain trf A and trfB both 30 and 42°C and stable in C2110 and depend on the R6K replicon for replication. (pCT153.1) at 30°C but not at 40°C (Fig. 5). The helper plasmid pCT153.1 was found to pRK250 1ts3 in transformation, enrichment be stable at 42°C. In C2110 (pCT153.1) at for revertants in the construction between 42°C after an initial lag, possibly due to both pCT145.1 and pCT7 is likely to have a period of continued replication at 42°C occurred. Therefore the presence of a (Robinson et al., 1980) and dilution of majority of temperature-sensitive mutants pCT160 to approximately one copy per cell, after the construction was taken to indicate the rate of decrease in the proportion of the that a high proportion of the pCT 145.I DNA growing population that carried pCT160 is preparation consisted of mutant DNA. If rapid. However, the absolute number of this was the case then the lack of any Cm’ colonies continues to rise slowly increased rate of loss of pCT45 at 42°C with throughout the experiment rather than pCT145.1 as helper indicates that the reaching a plateau, possibly indicating temperature-sensitive defect does not result leakiness in the defect. In Fig. 5 the dashed in pCT45 instability and therefore that the line indicates the rate of loss expected for a pRK353 1lKb

trfAflmr

1

REPLICATION

289

FUNCTIONS OF RK2 AND RPI

nonleaky defect in replication. It was concluded that pMR5 does carry altered replication functions such that replication from OriRKPis temperature sensitive.

Culturr

2 L 6

6

growth (generations) 10 12

14 16 16 20

DISCUSSION

The results described indicate that while only one region (trf A) of RK2 is required to provide trans-acting functions necessary for replication from oriRK a second region (trfB) can affect replication or maintenance if present in the replicon. This result is fully consistent with previous results (Thomas et al., 1980) where the evidence that trfB provided a trans-acting replication or maintenance factor was from the ability of the trfB region to complement the partly defective RK2 replicon (pCT27) as discussed in the introduction rather than from a direct demonstration of the need for this region to provide trans-acting factor(s) necessary for replication from oriRKP. It may also explain the apparent ability to delete this region from RP4, a P-group plasmid indistinguishable from RK2 (Burkhardt er al., 1979), reported recently (Barth, 1979). The evidence presented indicates that deletion of all but 150-200 bp of the trfB region does not affect stability or copy number of an oriRK plasmid. In pCT88 where all of the trfB region has been deleted by means of a Tn5 insertion into the trfA region there is evidence that the copy number of an oriRK plasmid dependent on truns-acting functions provided by pCT88 may be lower. This could be due either to deletion of some nonessential replication function or to an effect on expression of the essential function that pCT88 still provides. For example, it has recently been demonstrated that Tn5 insertion can lead to lowlevel transcription from the ends of the transposon (Berg et al., 1980). If Tn5 had inserted into the normal promoter or between it and the structural gene then a reduced expression of an essential function could lead to the observed effect. Therefore, while the coordinates 16- 17.55kb define the DNA segment that must carry the structural

I

FIG. 5. Kinetics of loss of an on’,,, plasmid from a strain carrying rrfA and trfB from pMR5. Single colonies of C211OCh3/86(pCT160) and C2110(pCT153.1, pCT160) were inoculated into 10 ml LB medium and grown for 30 min at 30°C before the cultures were divided and half transferred to a 42°C water bath. Serial dilution and plating on selective or nonselective LB agar was used to determine the total number of bacteria, the total number of bacteria carrying rrf A and trfB, and the proportion carrying the oriRK plasmid, pCT160. C2110Ch3/86 (pCT160): (0) 30°C (0) 42°C; C2110 (pCT153-1, pCT160): (A) 30°C (A) 42°C. The dashed line indicates the slope expected for segregation of a nonreplicating plasmid initially present at one copy per bacterium after four generations.

gene for the essential trfA function it may not carry all the information normally involved in expression of that gene. How is it possible to rationalize the

290

CHRISTOPHER M. THOMAS

positive requirement for a trfB function? This region cloned in a ColEl plasmid appears to inhibit replication of the hybrid plasmid after chloramphenicol treatment (Figurski el al., 1979). It may be that in the trfB region there is a potential block to RK2 replication that can only be overcome by a trans -acting function, which is also specified by the trfB region. The observation that one class of revertant of pRK2501ts3 carried an approximately 200-bp deletion in the trfB region (C. Thomas, unpublished) is consistent with this hypothesis, since it can be postulated that the deletion removes the potential block to replication. On the other hand the trfA and trfB regions from pRK2501ts3 cloned into a PlSA replicon (pCT145.1) can be replicated at 42°C indicating that this replication block must not be a completely general phenomenon. The role of the trfB region in the replication and maintenance of RK2 replicons remains to be determined. The fact that pMR5 appears to be temperature sensitive in an RKZspecific replication function required for replication from oriRKSadds strength to the idea that there may be a protein encoded by the trfA region that acts in initiation of replication of RK2 plasmid DNA. The characteristics of segregation of an oriRK plasmid replicating at 42°C in the presence of the pMR5 trfA and trfB regions (Fig. 5) are different from those reported for segregation of pMR5 at 42°C (Robinson et al., 1980). However, the difference in the apparent residual replication rate may simply reflect the exact experimental conditions used. It remains to be determined in what way the temperaturesensitive function acts to allow replication from oriRKZ. While temperature-sensitive mutants of many different plasmids have been isolated the ability to dissect physically the RK2 replicon has allowed the in viva demonstration that a trans-acting function is affected by the mutation. Only in the case of R6K (Kolter et al., 1978; Inuzuka and Helinski, 1978) and certain staphylococcal plasmids (Wyman and Novick, 1974; Iordanescu,

1979) has a similar use been made of temperature-sensitive plasmid mutants. In conclusion, it appears that the replication and maintenance of RK2 may be a more complex process than imagined previously, consisting both of a machinery to initiate replication from or& and other steps as yet ill defined but which may include replication blocks that need truns-acting proteins to overcome them. ACKNOWLEDGMENTS I would like to thank Dr. P. M. Bennett and Dr. A. Sakanyan for the temperature-sensitive mutants derived from RPl, and Dr. D. R. Helinski for providing the facilities for part of this research. Financial support to D.R.H. was through Public Health Service Grant Al07194 from the National Institute of Allergy and Infectious Disease and NSF Grant PCM-04635 and through a University of Birmingham Faculty Research Grant to C.M.T. The following colleagues have been helpful in preparation of the manuscript, D. R. Helinski, T. Schmidthauser, D. Stalker, and A. Moir.

REFERENCES P. T. (1979). RP4and R3OOBas wide host-range plasmid cloning vehicles. In “Plasmids of Medical, Environmental and Commercial Importance” (K. N. Timmis, and A. Puhler, eds.), pp. 399-410. Elsevied North Holland, Amsterdam/New York. BERG, D. E. (1977). Insertion and excision of the transposable kanamycin resistance determinant Tn5. Ztl “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 205-212. Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y. BERG, D. E., WEISS, A., AND CROSSLAND, L. (1980). Polarity of Tn5 insertion mutations in Escherichiu coli. J. Bacterial. 142, 439-446. BERINGER, J. E. (1974). R factor transfer inZ?hizobium leguminosarum. J. Gen. Microbial. 84, 188-198. BURKHARDT, H.-J., RIESS, G., AND PLJHLER, A. (1979). Relationship of group Pl plasmids revealed by heteroduplex experiments: RPl, RP4, R68 and RK2 are identical. J. Gen. Microbial. 114, 341-348. CHANG, A. C. Y., AND COHEN, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the PlSA cryptic mini plasmid. J. Bacreriol. 134, 1141-1156. BARTH,

CHO, J. J., PANOPOULOS,

N. J., AND SCHROTH,

M. N.

(1975). Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwina species. J. Bacterial. 122, 192-198. DATTA, N., AND HEDGES, R. W. (1972). Host range of R-factors. J. Gen. Microbial. 70, 453-460.

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FUNCTIONS

D., AND HELINSKI, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent upon a plasmid function provided in trans. Proc. Nat. Acad. Sci. USA 76, 1648-1652. FIGURSKI, D., MEYER, R., AND HELINSKI, D. R. (1979). Suppression of ColEl replication properties by the incP-1 plasmid RK2 in hybrid plasmids constructed in vitro. J. Mol. Biol. 133, 295-318. GREENE, P. J., HEYNECKER, H. L., BOLIVAR, F., RODRIGUEZ,R. L., BETLACH,M. C., COVARRUBIAS, A. A., BACKMAN, K., RUSSEL,D. J., TAIT, R., AND BOYER, H. W. (1978). A general method for the purification of restriction enzymes. Nucleic Acids Res. 5, 2373-2380. INUZUKA, M., AND HELINSKI, D. R. (1978). Requirement of a plasmid-encoded protein for replication in vitro ofplasmid R6K. Proc. Nat. Acad. Sci. USA 75,

FIGURSKI,

5381-5385.

IORDANESCU,S. (1979). Incompatibility-deficient derivatives of a small staphylococcal plasmid. Plasmid 2, 207-215.

JORGENSEN, R. A., ROTHSTEIN,S. J., AND REZNIKOFF, W. S. (1979). A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177, 65-72. KAHN, M., KOLTER, R., THOMAS, C., FIGURSKI, D., MEYER, R., REMAUT, E., AND HELINSKI, D. R. (1979). Plasmid cloning vehicles derived from plasmids ColEl, F, R6K and RK2. In “Methods in Enzymology” (R. Wu, ed.) Vol. 68, pp. 268-280. Academic Press, New York. KOLTER, R., AND HELINSKI, D. R. (1978). Construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid 1, 571-580. R., INUZUKA, M., AND HELINSKI, D. R. (1978). Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15, 1199- 1208. KOLTER, R., AND HELINSKI, D. R. (1979). Regulation of initiation of DNA replication. Annu. Rev. Genet.

KOLTER,

13, 355-391.

MEYER, R., AND HELINSKI, D. R. (1977). Unidirectional replication of the P-group plasmid RK2. Biochim. Biophys. Acta 487, 109- 113. MEYER, R., FIGURSKI, D., AND HELINSKI, D. (1977). Physical and genetic studies with restriction endo-

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291

nucleases on the broad host-range plasmid RK2. Mol. Cert. Genet. 152, 129-135. MOLIN, S., AND NORDSTROM,K. (1979). Control of plasmid RI replication: functions involved in replication, copy number control, incompatibility and switch-off of replication. J. Bacterial. 141, 111-126. OLSEN,R. H., AND SHIPLEY,P. (1973). Host range and properties of the Pseudomonas aeruginosa R factor R1822. J. Bacterial. 113, 772-780. ROBINSON,M. K., BENNETT, P. M., FALKOW, S., AND DODD, H. M. (1980). Isolation of a temperaturesensitive derivative of RPl. Plasmid 3, 343-349. SHEPARD,H. M., GELFAND, 0. H., AND POLISKY, B. (1979). Analysis of a recessive plasmid copy number mutant: evidence for negative control of ColEl replication. Cell 18, 267-275. THOMAS,C. M., AND HELINSKI, D. R. (1979). Plasmid DNA replication. In “Plasmids of Medical, Environmental and Commercial Importance” (K. N. Timmis and A. Piibler, eds.), pp. 29-46. Elsevier/North Holland, Amsterdam/New York. THOMAS, C. M., STALKER, D., GUINEY, D., AND HELINSKI, D. R. (1979). Essential regions for the replication and conjugal transfer of the broad host range plasmid RK2. In “Plasmids of Medical, Environmental and Commercial Importance” (K. N. Timmis and A. Ptihler, eds.), pp. 375-385. Elsevier/ North Holland, Amsterdam/New York. THOMAS, C. M., MEYER, R., AND HELINSKI, D. R. (1980). Regions of broad host range plasmid RK2 essential for replication and maintenance J. Bacterial. 141, 213-222.

THOMAS, C. M., STALKER, D. M., AND HELINSKI, D. R. (1981). Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol. Gen. Genet. 181, l-7. TWIGG, A. J., AND SHERRATT,D. (1980). Transcomplementable copy-number mutants of plasmid ColEl. Nature (London) 283, 216-218. URLAPOVA,S. V., MYAKININ, V. B., AND STEPANOV, A. I. (1979). Temperature-sensitive mutant of the plasmid RPl. Russ. Genet. (Genetika) 15(3), 433-443.

WYMAN, L., AND NOVICK, R. P. (1974). Studies on plasmid replication IV. Complementation of replication-defective mutants by an incompatibility-deficient plasmid. Mol. Gen. Genet. 135, 149-161.