JOURNAL OF BACTERIOLOGY,

Vol. 172, No. 7

JUlY 1990, p. 3849-3858

0021-9193/90/073849-10$02.00/0

Copyright C) 1990, American Society for Microbiology

Replication of the Broad-Host-Range Plasmid RK2: Direct Measurement of Intracellular Concentrations of the Essential TrfA Replication Proteins and Their Effect on Plasmid Copy Number ROSS H. DURLANDt AND DONALD R. HELINSKI*

Center for Molecular Genetics and Department of Biology, University of California, San Diego, La Jolla, California 92093 Received 31 January 1990/Accepted 26 April 1990

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 0.6 ng of TrfA-32 and 1.8 + 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean + standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.

RK2 is a 60-kilobase self-transmissible plasmid of the IncPl incompatibility group (22, 25). As with other members of this group, RK2 has an unusually broad host range among gram-negative bacteria (9, 27, 28, 35, 36). Its copy number has been previously estimated as four to seven per chromosome equivalent in Escherichia coli (14). Plasmid replication has been shown to require two plasmid loci, the cis-acting origin of replication (oriV) and the trans-acting trfA gene whose product(s) is essential for replication initiation (13, 46, 51, 55, 56). The trfA gene has been shown to encode two protein products of 43 and 32 kilodaltons (designated TrfA43 and TrfA-32, respectively) as a result of independent translational initiation at two different start codons within the same open reading frame (23, 39, 44). At least one of these proteins is required for replication in all hosts tested (23, 35, 36, 39). Previous reports have shown that TrfA-32 is sufficient for plasmid replication in many hosts, including E. coli (23, 39). The role of TrfA-43 is unclear, although evidence indicates that it plays a critical role in RK2 replication or maintenance in Pseudomonas aeruginosa (11, 41). The importance of intracellular levels of the TrfA proteins in RK2 replication has been the focus of much study. Numerous lines of evidence show that TrfA synthesis is regulated at the transcriptional level and perhaps at the posttranscriptional level as well. The products of the RK2 korA (trfB) and korB genes are known to repress transcription from the trfA operon promoter (PtrfA) (4, 37, 40, 61), probably by binding to a pair of inverted repeats of similar sequence that overlap the promoter (43, 50). The RK2 kilD

(kilBI) determinant, which is associated with the promoter region and/or the first open reading frame of the trfA operon, may modulate the activity of korA and korB to prevent overrepression of PtrfA (37). The korA and korB genes also behave as replication control elements, especially when their dosages or expression levels are elevated. Under some circumstances they can interfere with replication of RK2 derivatives, presumably by repressing TrfA protein levels below the minimum required for initiation (37, 53). This can be overcome by expressing trfA constitutively from a heterologous promoter. In addition, Thomas and Hussain (54) have shown that plasmids consisting of oriV, trfA, korA, and korB have copy numbers similar to that of the intact RK2 plasmid, about 7.5 per chromosome equivalent, and that deletion of korB increases the copy number approximately 60% to 12 per chromosome. These observations have led to the proposal that RK2 replication is primarily controlled by maintaining the TrfA protein(s) in rate-limiting amounts. This is achieved at least in part through transcriptional repression by korA and korB (37, 53). Additional loci (kilD, incC, korE, and korF) may also be involved in this control (37, 52). However, the lack of an assay for the TrfA proteins has prevented a direct test of this hypothesis. In addition, it is clear that plasmid replication can be regulated in the absence of any transcriptional control of trfA. This was demonstrated by studies of plasmids that contain oriV and a constitutively transcribed trfA gene (35, 36, 54). If TrfA levels were strictly rate limiting, such plasmids would replicate autocatalytically as so-called runaway replicons (19). In fact, such plasmids replicate stably at defined copy numbers, despite the lack of any known controls on TrfA protein synthesis. This implies the

* Corresponding author. t Present address: Center for Biotechnology, Baylor College of

Medicine, The Woodlands, TX 77381.

3849

DURLAND AND HELINSKI

3850

J. BACTERIOL. TABLE 1. Strains and plasmids used

E. coli strain or

plasmid E. coli strain D1210 HB101

K38 Plasmid pAD9

Source or reference

Remarks

HB101 lacIq lacY+ F- leuB6 proA2 recAl3 thi-J ara-14 lacYl galK2 xyl-5 mtl-i rpsL20 hsdS20 rB mB HfrC (A)

-

supE44

33 6 32 A. Das (unpublished)

A. Greener (unpublished) A. Greener (unpublished) A. Greener (unpublished) A. Greener (unpublished) 49 Pharmacia This work This work

pSV16

pUC9 derivative with the E. coli rpoC transcriptional terminator inserted as an EcoRI fragment; Penr RSF1010 replicon containing the promoterless trfA gene; Tetr pAL100 with R6K pir* promoter inserted upstream of trfA; Tetr pAL100 with the Tn5 neo promoter inserted upstream of trfA; Tetr pAL100 with E. coli tac promoter inserted upstream of trfA; Tetr P1SA replicon; heat-inducible T7 RNA polymerase plasmid; Kanr pBR322 replicon; expression vector containing E. coli tac promoter; Penr pKK223-3 with cat inserted into bla; Camr pT7-5 with promoterless trfA gene inserted downstream of the T7+10 promoter; Penr Naturally occurring plasmid; Tetr Penr Kanr RK2 replicon; 700-bp HaeII-oriV; Penr Kanr, requires trfA in trans for

pT7-5

replication ColEl replicon; contains T74i10 promoter upstream of a polylinker; Penr

S. Tabor and C. Richardson

pAL100 pAL102 pAL103 pAL104 pGP1-2 pKK223-3 pKK223-3CAT pRD113-34 RK2

pTJS65

pVR5 pVR5CAT

pVR5t1 pVR5t1CAT pVR5t2

pVR5t2CAT

pUC8 with 700-bp HaeII-RK2oriV inserted as a blunt fragment into the SmaI site; Penr pKK223-3 with the promoterless trfA gene inserted downstream of the tac promoter; Penr pVR5 with cat inserted into bla; Camr pVR5 with one copy of the EcoRI terminator fragment of pAD9 inserted between tac and trfA; Penr As with pVR5t1; Camr pVR5 with two copies of the EcoRI terminator fragment pf pAD9 inserted between tac and trfA; Penr As with pVR5t2; Camr

existence of at least one mechanism that can regulate RK2 replication independent of TrfA concentration. In this study, we report the preparation of a rabbit antiserum directed against TrfA-32 that reacts with both TrfA-32 and TrfA-43. The antiserum was used in immunoblotting (Western blotting) assays to measure intracellular levels of TrfA produced by RK2 and by other trfA-bearing plasmids. By altering the transcription level of a cloned trfA gene, we were able to vary the TrfA concentration by almost 200-fold. The copy numbers of RK2 and an RK2 origin plasmid under these conditions were determined by DNA hybridization. The results of these experiments and their implications for control of RK2 replication are presented and discussed in this paper. MATERIALS AND METHODS Strains and plasmids. The strains and plasmids used in this study are listed in Table 1. Plasmid pRD113-34 was constructed by cloning an EcoRI-to-PstI fragment containing the promoterless trfA gene into the phage T7 RNA polymerase expression vector pT7-5 (Fig. 1). The fragment carrying the trfA gene was derived from pCT88A34 by partial digestion with HaeII followed by PstI linkering at the HaeII site immediately downstream of the trfA gene. The resulting EcoRI-to-PstI fragment contains base pairs 408 through 1618 of the trfA gene (44) but lacks the trfA operon promoter and the promoter-proximal 13-kilodalton open reading frame. pSV16 (S. Valla, unpublished results) was constructed by

22 S. Valla (unpublished)

(unpublished) 36 V. Rosenthal (unpublished)

This work This work This work This work

This work

ligating together the EcoRI-to-BamHI oriV fragment of pTJS65, an EcoRI-to-BamHI fragment containing the pBR322 ,-lactamase gene (coordinates 3104 through 4363) (48), and a BamHI fragment containing the kan gene from pUC4K (pUC5) (60). pTJS65 is pUC8 (60) containing the 700-bp HaeII oriV fragment from RK2 cloned into the SmaI site of the polylinker. pAL100 (see Fig. 5) is an RSF1010 replicon containing the promoterless trfA fragment from pRD113-34 cloned downstream of a polylinker. pAL102, pAL103, and pAL104 contain a derivative of the R6K pir promoter (45), the TnS neo promoter (5), and the E. coli tac promoter (Ptac) (2), respectively, inserted into the polylinker upstream of trfA. These plasmids were constructed by A. Greener. Plasmid pVR5 is pKK223-3 (Pharmacia) with the EcoRI-to-PstI trfA fragment from pRD113-34 inserted downstream of Ptac (V. Rosenthal, unpublished results). Insertion of one copy of the E. coli rpoC transcriptional terminator from pAD9 into the EcoRI site of pVR5 in the native orientation (i.e., the same orientation relative to transcription as in rpoC) resulted in pVR5t1. Two tandem copies inserted in the reverse orientation resulted in pVR5t2. pKK223-3, pVR5, pVRSt,, and pVR5t2 were converted from penicillin resistance to chloramphenicol resistance as follows. A 773-base-pair (bp) TaqI fragment containing the promoterless cat gene was isolated from pUC8-CAT- (R. Durland, unpublished results) and treated with Klenow fragment to blunt the ends. This fragment was inserted into the ScaI site of each of the above four plasmids in the same

PLASMID RK2 TrfA REPLICATION PROTEINS

VOL. 172, 1990

T7 RNA polymerase

3851

cI857

P15A

origin kan

ColEI origin

trfA

ColEI origin Pst I

FIG. 1. Plasmids used to overproduce TrfA. pGP1-2 is a kanamycin-resistant P1SA replicon that contains the T7 RNA polymerase gene downstream of the A PL promoter and the thermosensitive A cI857 repressor gene downstream of the E. coli lac promoter (Plac). At 42°C the repressor is inactivated, allowing high levels of synthesis of the polymerase. pT7-S and pRD113-34 are ColEl derivatives that are compatible with pGP1-2 and carry resistance to penicillin (bla). pRD113-34 is derived from pT7-S by insertion of the promoterless trfA gene (shaded region) downstream of the T7(i10 promoter. T7 RNA polymerase protein synthesized by pGP1-2 recognizes T7410, leading to high levels of transcription of the downstream sequences in pRD113-34 and pT7-5.

relative orientation as the P-lactamase gene. The resulting plasmids were designated pKK223-3CAT, pVR5CAT, pVR5t1CAT, and pVR5t2CAT, respectively. Overproduction and partial purification of TrfA. Liquid cultures of E. coli K38 containing pGP1-2 and either pT7-5 or pRD113-34 were stored in 50% glycerol at -70°C, since prolonged storage at room temperature on agar plates resulted in the gradual loss of TrfA inducibility. Portions of frozen cultures were diluted into LB containing 50 ,ug of kanamycin and 250 jig of penicillin G per ml and shaken overnight at 30°C. The following morning, the cultures were diluted 1:125 into fresh medium and shaken at 30°C to an optical density at 590 nm (OD590) of 0.3 to 0.4. The cultures were shifted to 42°C for 30 to 60 min, rifampin was added to a final concentration to 100 ,g/ml, and the cultures were shaken for an additional 2 h at 37°C. The cells were harvested at 4°C by centrifugation for 5 min at 4,000 x g, washed once with 20 mM Tris chloride, pH 7.5-100 mM KCl-1 mM EDTA, and suspended in the same buffer containing 5 mM dithiothreitol and 40 pug of phenylmethylsulfonyl fluoride per ml at a final volume of 5 mlAiter of original culture. The cells were lysed by extensive sonication on ice; a temperature probe was used to ensure that the temperature remained below 15C. All subsequent steps were performed at 0 to 4°C. Insoluble debris and unlysed cells were pelleted for 30 min at 30,000 rpm in a Ti6O rotor. The recovered supernatants typically contained 10 to 15 mg of protein per ml as determined by the Bio-Rad Protein

Assay. The pRD113-34 and pT7-5 extracts were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining to visualize the induced TrfA protein bands. Glycerol was added to each cleared lysate to a final concentration of 10%. Extracts from 2 liters of cells (-100 mg of protein) were loaded onto a column (0.9 by 15 cm) containing about 5 ml (bed volume) of heparin-Sepharose CL-6B (Pharmacia) equilibrated with 20 mM Tris chloride, pH 7.5, 1 mM EDTA, 100 mM KCl, 10% glycerol, 5 mM dithiothreitol, and 40 ,ug of phenylmethylsulfonyl fluoride per ml. The column was washed until the A280 of the flowthrough was reduced to the background level. Elution was carried out with a linear 0.1 to 1.0 M gradient of KCI in the above buffer. The TrfA-containing fractions were identified by comparison to the equivalent control fractions from the pT7-5 lysate by using SDS-PAGE (Fig. 2A, lanes 3 and 4). The TrfA proteins coeluted at approximately 500 mM KCl, although TrfA-43 consistently eluted slightly earlier than TrfA-32. The peak TrfA fractions for the pRD113-34 lysate and the corresponding control fractions from the pT7-5 lysate were each pooled and dialyzed for 90 min against 50 volumes of elution buffer lacking KCl. The dialysates (containing 1 to 3 mg of total protein) were loaded onto a column (0.9 by 15 cm) containing 3 ml (bed volume) of CM Bio-Gel A (carboxymethyl agarose; Bio-Rad) equilibrated with elution buffer plus 50 mM KCl. The column was washed as de-

DURLAND AND HELINSKI

3852

J. BACTERIOL.

FIG. 2. Purification of the TrfA proteins. Coomassie blue staining (A) and Western blotting with anti-TrfA antiserum (B) of proteins separated in a 10%' SDS-polyacrylamide gel. Lanes 1 through 6 are from heat-induced lysates of E. coli K38 containing pGPl-2 and either pT7-5 (lanes 1, 3, and 5) or pRD113-34 (lanes 2, 4, and 6). Lanes: 1 and 2, cleared lysates; 3 and 4, proteins eluting from heparin-Sepharose between 450 and 550 mM KCI; 5 and 6, proteins eluting from carboxymethyl agarose between 150 and 250 mM KCI; 7, SDS-polyacrylamide gel-purified TrfA-32; 8, gel-purified TrfA-43.

kD, Kilodaltons.

scribed above and eluted with

linear 50 to 500 mM

a

KCI

gradient. TrfA fractions were identified by SDS-PAGE (Fig. 2A, lanes 5 and 6). The proteins coeluted at about 200 mM KCI. As before, TrfA-43 eluted slightly ahead of TrfA-32. At this point, the proteins were estimated to be 30 to 40% pure on the basis of Coomassie blue staining of SDS-polyacrylamide gels. Total recovery from 2 liters of cells was esti-

Rxg

mated to be 30 to 60

of TrfA-32 and 20 to 40

pLg

of

TrfA-43.

Preparation of anti-TrfA antisera. CM BioGel A-purified TrfA was electrophoresed in a 2-mm-thick 10% SDS-polyacrylamide gel and stained with Coomassie brilliant blue to visualize the proteins. The portion of the gel containing TrfA-32 (-50 rg) was excised and extensively destained. The gel slice was neutralized by soaking it in 100 mM Tris

chloride, pH 7.6, and crushed in water. Material equivalent mixed 1:1 with to approximately 25 pg of TrfA-32 was

Freund

complete adjuvant,

injected

into the

lymph

nodes

and

equal proportions

of each of

two

were

New Zealand

White rabbits. Rabbits were boosted subcutaneously at 1-month intervals with mixture (1:1) of TrfA-32 and incomwith crushed gel containplete adjuvant. The first boost a

was

ing TrfA-32

b

with

-15

as

g

described above.

of TrfA-32

that

Subsequent boosts

had

been

were

eluted from

SDS-polyacrylamide gel (see below). Rabbits

were

an

bled 7 to

days after each boost, and the presence of antibodies in serum that recognized TrfA-32 and TrfA-43 was assayed by using the Western blot procedure described below. of antiserum was used in all experiA single ments in this study.

10

each

preparation

All antisera exhibited reactivity with number of host in total E. coli lysates that interfered with the a

proteins

detection of

low levels

alleviated

follows. E. coli

90

to

as

Klett

units) was

of TrfA.

This

problem was partially

HB101 from 1 liter of cult'ure (80 harvested and suspended in Tris-

buffered saline (TBS) (20 mM Tris

chloride, pH 7.5,

150 mM

NaCi). The suspension was lysed by sonication and added to i

g

(wet weight)

of

powdered nitrocellulose. This mixture

was incubated overnight on a rotating wheel at room temperature. The nitrocellulose and bound proteins were recovered by centrifugation, and any remaining binding sites on the nitrocellulose were blocked by incubation for several hours at room temperature with TBS containing 3% bovine serum albumin (BSA). The supernatant was removed by centrifugation. One milliliter of antiserum was mixed with 49 ml of TBS containing 3% BSA. This solution was added to the nitrocellulose preparation and mixed overnight at room temperature to adsorb antibodies that recognize E. coli proteins. The powder was removed by centrifugation, and the supernatant was used for subsequent Western blots. This procedure greatly reduced the cross-reactivity of the antiserum with many of the host bands, especially those that were similar in molecular weight to TrfA. Preparation of TrfA standards for Western blots. Carboxymethyl agarose-purified TrfA was electrophoresed in a 1-mm-thick preparative 10% SDS-polyacrylamide gel in a single lane 10.5 cm wide. The estimated amount of TrfA loaded was 50 ,ug of TrfA-43 and 80 ,ug of TrfA-32. A sheet of nitrocellulose was carefully laid onto the gel to adsorb the proteins on the surface of the polyacrylamide, and reference notches were cut through the filter paper and the gel to aid in orientation. The adsorbed proteins on the filter were stained with India ink as described previously (21) to determine the location of the two TrfA proteins. Using the stained filter as a guide, the sections of the gel containing TrfA-32 and TrfA-43 were carefully excised. The intact fragments of polyacrylamide were soaked overnight in 50 mM NH4HCO3-0.05% SDS-1 mM EDTA at 37°C to elute the proteins. SDS was removed with Extracti-Gel D (Pierce Chemical Co.). The proteins were dialyzed against 10 mM NaPO4, pH 7.4-150 mM NaCl, lyophilized to dryness, and suspended in 0.5 ml of the above buffer. The concentration of the purified proteins was determined with the BCA assay (42) supplied by Pierce and by the amido black assay of Schaffner and Weissmann (34) with purified BSA as a standard. The BCA assay gave values of 37 ,ug/,l for TrfA-32 and 78 p.g/4dl for TrfA-43. The values obtained by the amido black method were 37 and 59 ,ug/,ul, respectively. The BCA values were used as references in determining TrfA levels throughout this study. Western blotting. Samples for Western analysis were prepared by determining the OD6. of the culture and harvesting measured volumes by centrifugation. Cell pellets were suspended in water and lysed with cracking buffer (final concentration, 62.5 mM Tris chloride, pH 6.8, 2% SDS, 10%

glycerol, 1% 3-mercaptoethanol, 0.01% bromphenol blue). Equivalent amounts of plasmid-free E. coli lysates were

mixed with known amounts of gel-purified TrfA-32 and TrfA-43 as standards. Samples and standards were heated to 100°C for 3 to 5 min and electrophoresed on 10% SDSpolyacrylamide gels as described previously (24). After electrophoresis, proteins were transferred to nitrocellulose (BA85, 0.45 jxm; Schleicher & Schuell, Inc.) overnight at 200 mA as described previously (59). Filters were washed with TBS and incubated at 37°C overnight with anti-TrfA antiserum (prepared as described above) diluted 1:1,000 in TBS containing 3% BSA and 0.03% NaN3. Filters were then washed three times for 5 to 10 min at room temperature with TBS and incubated at 37°C for 1 to 3 h with goat anti-rabbit immunoglobulin G (heavy and light chains) alkaline phosphatase conjugate (Bio-Rad; catalog no. 1706518) diluted 1:3,000 in TBS-BSA-NaN3. Blots were washed with TBS as before and developed in accordance with the recommendations of the manufacturer by using the reagents

VOL. 172, 1990

PLASMID RK2 TrfA REPLICATION PROTEINS

3853

supplied with the Bio-Rad Immun-Blot Assay Kit (catalog no. 170-6509).

RESULTS

Plasmid copy number determinations. Copy number was determined essentially by the procedure of Shields et al. (38) with the following modifications. All cultures were shaken at 37°C in LB with antibiotic selection for a minimum of five generations until the OD60 was 0.1 to 0.3. Portions of each culture were diluted and spread on LB with or without antibiotic to estimate the percentage of cells carrying the oriV-containing plasmid. In all experiments in this study this value was >95%. NaN3 was immediately added to 0.1%, and the cultures were chilled on ice. Culture volumes calculated to contain 0.1 and 0.5 OD6. units, respectively, were each transferred to separate 1.5-ml Eppendorf tubes and pelleted for 1 min at 4°C. After the supernatant was aspirated, pellets from the 0.5 OD600-unit samples were stored at -20°C for later determination of TrfA concentration by Western blot analysis. Pellets equivalent to 0.1 OD6. unit were suspended in 1.0 ml of lx M9 salts containing 0.1% NaN3 and kept on ice. A 0.1or 0.2-ml volume of each suspension was filtered onto nitrocellulose through a 96-well manifold (Schleicher & Schuell). Standards consisting of serial dilutions of purified pTJS65 DNA were prepared and bound to the membrane, also as described previously (38). After all samples had been loaded, the filters were treated with NaOH to lyse the cells, neutralized, and baked according to the published procedure. The 169-bp DraI-to-BamHI fragment of oriV isolated from pTJS65 was chosen as a probe for hybridization. This fragment contains part of the AT and GC-rich regions of oriV (bp 533 to 696) (46) but does not contain any of the repeated sequences. The probe was labeled by nick translation (30) and hybridized to filters as previously described (10). The labeled filters were autoradiographed with preflashed Kodak X-OMAT AR 50 or Ortho G 100 X-ray film at -70°C with an

Overproduction and partial purification of TrfA. A twoplasmid system based on the properties of the phage T7 RNA polymerase was chosen to overproduce the TrfA proteins (Fig. 1). Plasmid pGP1-2 contains the T7 RNA polymerase gene cloned downstream of the A PL promoter. It also carries the temperature-sensitive X cI857 repressor gene, making transcription of the RNA polymerase gene heat inducible. The phage enzyme recognizes the T7410 promoter of pRD113-34, resulting in very high rates of transcription of the cloned trfA gene. Despite the reported efficiency of this and similar systems (47, 49), we observed that the maximum attainable level of TrfA was only 1 to 3% of total cell protein. This comparatively low level of protein synthesis is apparently specific to the trfA gene, since we obtained high levels of P-lactamase proteins using a related plasmid in which the bla gene was cotranscribed with trfA (data not shown). It is possible that the trfA message produced in this system is unstable or is poorly translated. A lysate of a culture containing pGP1-2 and pRD113-34 that was induced for TrfA synthesis was prepared as described in Materials and Methods. A control lysate was prepared from cells containing the vector plasmid pT7-5 (lacking trfA) instead of pRD113-34. Both lysates were subjected to the purification procedure described in Materials and Methods. At each step of the purification, equivalent fractions from the control and TrfA-containing lysates were electrophoresed side by side in 10% SDS-polyacrylamide gels. The TrfA proteins were identified by the presence of prominent bands of 43 and 32 kDa that were absent in the comparable control lanes. Figure 2A shows the protein profiles at different steps of the purification. The TrfA proteins were not readily detectable in cleared lysates in this experiment (compare lanes 1 and 2). However, after purification by heparin-Sepharose chromatography, the TrfA extract (lane 4) contained two prominent bands of approximately 43 and 32 kDa which were clearly absent from the control extract (lane 3). Further purification with carboxymethyl agarose (lanes 5 and 6) removed most of the largermolecular-weight contaminants, especially those that comigrated with the two TrfA proteins. Lanes 7 and 8 show SDS-polyacrylamide gel-purified preparations of TrfA-32 and TrfA-43, respectively, that were used as standards in later determinations of intracellular TrfA concentration (see below). Rabbit antisera directed against gel-purified TrfA-32 was prepared as described in Materials and Methods, and the presence of anti-TrfA antibodies was assayed by the Western blot procedure. The results of a Western blot performed on samples from various stages of TrfA purification are shown in Fig. 2B. The proteins were intentionally overloaded to emphasize the difference between lanes with and without TrfA. Except for one prominent host band of about 60 kDa present in the control cleared lysate (Fig. 2B, lane 1), only the lanes containing TrfA showed significant reactivity with the antiserum (Fig. 2B, lanes 2, 4, and 6). In each case, two major bands, corresponding to TrfA-43 and TrfA-32, can be seen. Additional less-intense bands of higher molecular weight were also present in the TrfA-containing lanes. Since they were not present in control lanes, these most likely represent multimeric forms of TrfA that were incompletely dissociated by the SDS gel system. These bands were also observed in the lanes containing gel-purified TrfA proteins (Fig. 2B, lanes 7 and 8). The presence of such bands in a

intensifying screen. Quantifying Western and copy number blots. Photographic negatives of Western blots and autoradiograms of copy number blots were scafined by using an LKB Ultroscan XL model 2222 laser densitometer. Peak areas were determined with a digitizing tablet (model 2210) and Sigma Scan (Jandel Scientific). The amounts of protein or DNA in the unknown samples were estimated by comparison with a standard curve prepared from the peak areas of the known samples. The concentrations of TrfA and oriV per mass of culture were calculated on the basis of the number of OD6. units in each sample. Values obtained by measuring two or more independent cultures differed by no more than 20%. Estimates of molecules per cell were calculated on the basis that an OD6. of 1.0 corresponds to 3.7 x 108 cells per ml. This value was determined empirically for E. coli HB101 growing logarithmically at 37°C in LB by using a Petroff-Hauser counting chamber. Other methods. Transformations (8), gel purification of restriction fragments (11), and CsCl purification of plasmid DNA (3) were done as described previously. SDS-PAGE was done by the method of Laemmli (24). Enzymes were obtained from New England BioLabs, Bethesda Research Laboratories, or Boehringer Mannheim Biochemicals and were used in accordance with the recommendations of the manufacturer. Materials used in obtaining anti-TrfA antisera were the generous gift of S. J. Singer. Fraction V BSA was purchased from Sigma Chemical Co.

3854

DURLAND AND HELINSKI a

TrfA-43

.

IrfA-32

'

b c Cl e t t

I

?

J. BACTERIOL. rI-n ri

ip

p

eV

FIG. 3. Western analysis of TrfA levels in E. coli HB101. All lanes contain 0.5 OD6. units of total cellular material from logarithmically growing cultures. Lanes a through e contain material from plasmid-free cells to which known amounts of the gel-purified TrfA-43 and TrfA-32 preparations (shown in Fig. 2A and B, lanes 7 and 8) have been added as standards. Lanes a through e contain 312.5, 62.5, 12.5, 2.5, and 0.5 ng of each TrfA protein, respectively. Lanes f and g contain material from duplicate plasmid-free cultures with no added TrfA. Note the presence of a host protein band that comigrates with TrfA-32. Lanes h and i contain material from duplicate cultures containing RK2, lanes j and k contain material from pAL100, lanes I and m contain material from pAL103, lanes n and o contain material from pAL102, and lanes p and q contain material from pAL104.

denaturing gel system is somewhat surprising and suggests that the TrfA proteins have a strong tendency to aggregate. Intracellular concentration of TrfA synthesized by RK2. Intracellular TrfA concentrations were measured by using quantitative Western blots of total lysates of E. coli HB101 containing various TrfA-producing plasmids. One such blot is shown in Fig. 3. Standards (lanes a through e) were prepared for each blot by mixing known quantities of each gel-purified TrfA protein with total lysates of plasmid-free cells. This eliminated errors in measurement due to possible interference by host proteins. The antiserum showed significant levels of reactivity against E. coli proteins (lanes f and g). Since these bands were not observed in cleared lysates (Fig. 2B, lane 1), it is likely that they are membrane proteins, and they may be surface antigens recognized by the rabbit serum. Several attempts to eliminate the reactivity against the host proteins were unsuccessful. A technique involving preadsorption of antiserum to total cell lysates bound to nitrocellulose (described in Materials and Methods) did significantly reduce the problem. However, it was still necessary to correct for the reactivity in plasmid-free cells when measuring TrfA-32 levels. Despite these difficulties, it was possible to detect and quantify both TrfA proteins in total lysates of HB101 containing RK2 (Fig. 3, lanes h and i). Western blots were performed on 10 independent cultures, and the total amount of each TrfA protein was estimated by laser densitometry and comparison with the standards. Under the conditions of these experiments, we estimate that there are 4.6 ± 0.6 ng of TrfA-32 and 1.8 ± 0.2 ng of TrfA-43 per OD6. unit of culture (mean ± standard deviation, n = 10). On the basis of determinations of the number of cells per OD6. unit, these values correspond to approximately 220 monomers of TrfA32 and 80 monomers of TrfA-43 per cell. RK2 plasmid copy number in these cultures was determined by using a dot blot hybridization procedure developed by Shields et al. (38) (Fig. 4). The technique is a modification of the colony blot procedure of Grunstein and Hogness (20) and allowed direct quantitation of the number of plasmids in a known amount of cell culture. Using the dot blot assay, we estimated that RK2 copy number is 5.6 x 109 ± 0.4 x 109 per OD6. unit, which corresponds to approximately 15 copies per cell. Effect of in vivo TrfA levels on the copy number of oriV. The

FIG. 4. Dot blot analysis of the copy number of RK2 and pSV16. A total of 0.01 OD600 units of cellular material were prepared as described in the text and filtered onto nitrocellulose (rows A through D). For standards, twofold serial dilutions of purified oriV-containing pTJS65 DNA were also bound to the filter as described in the text. Lane 1 contains the controls for probe specificity. Rows A and B, E. coli HB101; C and D, HB101(pAL100). For lanes 2 through 6, rows A and B are duplicate spots from one culture and rows C and D are duplicate spots from an independent culture. Lane 2, rows A through D, HB101 (RK2); lane 3, rows A through D, HB101 (pAL100 pSV16); lane 4, rows A through D, HB101 (pAL103 pSV16); lane 5, rows A through D, HB101 (pAL102 pSV16); lane 6, rows A through D, HB101 (pAL104 pSV16). Standards (row E) were diluted by a factor of two between adjacent spots, with the darkest spot being equivalent to 2.5 x 108 molecules of plasmid. The results of a single experiment are shown, but the orientations of the spots have been altered from the original autoradiogram for clarity.

structures of plasmids used to measure the effect of different levels of TrfA on oriV copy number are shown in Fig. 5. Plasmid pSV16 contains only oriV and the resistance genes for kanamycin and penicillin. Replication of pSV16 is dependent on the synthesis of TrfA in trans. pAL100 is an RSF1010 replicon containing the promoterless trfA gene. Insertion of heterologous promoters of different strengths upstream of the trfA gene yielded the plasmids pAL102, pAL103, and pAL104, which specify substantially different levels of the TrfA proteins in vivo (Fig. 3, lanes j through q; Table 2). In some instances, there appear to be two TrfA-32 bands. The significance of this is unknown at present. The copy number of pSV16 in E. coli HB101 containing pAL100 or its derivatives was measured by the dot blot technique as shown in Fig. 4. Visual inspection of the blot indicated that the copy number of pSV16 did not change substantially in response to large variations in the amount of TrfA provided by the pAL100 plasmids. Densitometric analysis of the blots in Fig. 3 and 4 was used to quantitate the protein and DNA levels, respectively, in these cells. These data are presented in Table 2. For comparative purposes, the concentrations of TrfA-32 and TrfA-43 in each culture were combined and reported as total TrfA. Over a 20-fold range of total TrfA levels, there was no detectable change in the copy number of pSV16. In each case, the copy number of pSV16 is similar to that of RK2. Although pAL100 has no defined promoter upstream of trfA, it still produces about four times as much TrfA protein as plasmid RK2. This may be due to readthrough transcription from the pAL100 tet gene (Fig. 5). In order to measure the response of pSV16 to lower levels of TrfA, a different series of plasmids was constructed. These are based on the pBR322 expression vector pKK223-3, a penicillin resistance plasmid that carries the isopropyl-,-D-thiogalactopyranoside (IPTG)-

PLASMID RK2 TrfA REPLICATION PROTEINS

VOL. 172, 1990

3855

rpoC trA

term. \

_

~~~~~~~~~~~I

_ ,

pVR5t2 CAT pVR5t 1 CAT pVR5CAT

, #I

P tac

trfA tet cat

oriV

RSF1I1O origin

pBR322 origin

kan

bla

FIG. 5. Structures of plasmids used in this study to determine the effect of TrfA level on plasmid copy number. Plasmid pAL100 is an RSF1010 replicon containing the promoterless trfA gene (shaded region). Plasmids pAL103, pAL102, and pAL104 were derived from pAL100 by insertion of the indicated promoter fragments upstream of the trfA gene. pKK223-3CAT is pKK223-3 with a chloramphenicol resistance gene (cat) inserted in the bla gene. pVRSCAT is pKK223-3CAT with the trfA gene inserted downstream of Ptac. pVRSt1CAT and pVR5t2CAT have one or two E. coli rpoC transcriptional terminators (0), inserted between Ptac and trfA. Plasmid pSV16 is an RK2 origin plasmid carrying a 700-bp HaeII fragment from oriV. Replication of pSV16 is dependent on trfA provided in trans.

inducible tac promoter (2) upstream of a polylinker. Because both pSV16 and RK2 also encode penicillin resistance, pKK223-3 was converted to chloramphenicol resistance by inserting the cat gene into the bla gene. The resulting plasmid, designated pKK223-3CAT, is shown in Fig. 5. Insertion of the trfA gene downstream of the tac promoter in pKK223-3CAT generated pVR5CAT. Expression of the trfA gene in this plasmid was regulated by maintaining it in the lacIq host D1210 (HB101 laclq lacY+) and adding different amounts of IPTG to exponentially growing cultures. Even in the absence of IPTG, however, this plasmid produces relatively high levels of TrfA (compared with RK2), presumably because of inefficient repression of Ptac. Interestingly, addition of sufficient IPTG to fully induce the tac promoter in pVR5CAT prevents growth of the host cell. This appears to be a direct effect of TrfA on the cell, since induction of the vector alone (pKK223-3CAT) does not influence growth (data not shown). In order to reduce the TrfA levels, one or two transcriptional terminators were inserted between Ptac and trfA (pVR5t1CAT and pVR5t2CAT, respectively). E. coli D1210 TABLE 2. Copy number of pSV16 supported by pAL100, pAL103, pAL102, and pAL104 TrfA source

pAL100 pAL103 pAL102 pAL104 a

Values

Total TrfA unit)

(ng/OD6w 28 54 280 530 are means +

No. of copies of pSV16a

(1u)/

Molecules

OD60

unit

4.9 5.1 4.9 4.8

0.4 0.3 0.6 0.7

standard deviations from two trials.

containing pVR5CAT, pVR5t1CAT, or pVR5t2CAT was transformed with pSV16. TrfA synthesis was induced with different concentrations of IPTG, and the protein levels and plasmid copy number were determined (Table 3). In the absence of IPTG, pVR5t2CAT synthesized TrfA at levels comparable to those of RK2. Under these conditions, pSV16 copy number was reduced to about half its normal value. Increasing TrfA concentration about twofold resulted in a copy number comparable to the copy number observed in the experiments presented in Table 2. Further increases in TrfA had no additional effect on copy number. These results, combined with the data from Table 2, demonstrate that pSV16 copy number is insensitive to TrfA concentration over at least a 50-fold range. A significant effect on pSV16 copy number was only observed when the TrfA concentration was reduced to approximately the level synthesized by RK2. TABLE 3. Copy number of pSV16 supported by the

pKK223-3CAT derivatives TrfA

IPG

source

(onM)n (ng/OD6w unit) 4LM)

Total TrfA

No. of copies of pSV16a

(OlO Molecules/cell ~ ~ ~Molecules D~unt

pVR5t2CAT

0 20 200

5 10 70

2.8 ± 0.7 5.5 0.2 5.6 0.7

7.5 ± 2 15 1 15 2

pVR5t1CAT

0 20 200

14 20 190

5.6 ± 0.5 5.8 0.2 5.5 0.2

15 ± 1 16 1 15 1

120

4.6 ± 0.5

12 ± 1

Molecules/cell

13 14 13 13

±

1 1 2 2

pVRSCAT

0

a Values are means

+

standard deviations from two trials.

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DURLAND AND HELINSKI

J. BACTERIOL. 10

TABLE 4. Copy number of RK2 in the presence of excess TrfA proteins Source of additional TrfA

pKK223-3CAT

pVRSt2CAT

pVRSt1CAT

pVRSCAT

IPTG concn

(p.M)

TtlTf

(ng/OD6w unit)

0 10 50 250

5.8 7.0 6.4 7.4

0 10 50 250

7.8 11 48 52

0 10 50 250

12 24 75 180

0 10 50 250

190 370 1100

9

8

No. of copies of RK2' Molecules (109)/ unit Mlclscl 5.4 ± 1.0 14 ± 3 5.6 ± 0.2 15 ± 1 5.9 ± 0.2 16 ± 1 5.4 ± 0.3 14 ± 1

D0D0

Molecules/ce

6.7 7.3 7.6 7.0

± 0.7 ± 1.1 ± 0.6

18 20 20 19

6.6 7.5 7.2 7.2

± 0.3 ± 0.5 ± 0.7 ± 0.4

18 ± 1 20 ± 1 19 2 19 1

± 0.3

7.6 ± 0.5 7.4 ± 0.4 7.6 ± 0.3

20 20 20

± 1 ±2

±3 ±2

1 1 1

b

a Values are means ± standard deviations from two experiments. b This strain was not viable under these conditions, presumably due to excess TrfA protein(s).

Effect of excess TrfA proteins on the copy number of RK2. The above data indicate that there is little dependency of oriV initiation frequency on TrfA concentration over a wide range of values. To test whether intact RK2 responds to TrfA concentration in a similar manner, we measured RK2 copy number in the presence of different amounts of TrfA supplied in trans by the pVR5CAT derivatives, with pKK223-3CAT serving as a control. Western and copy number blots were used to measure protein and DNA levels, respectively, and the results are presented in Table 4. As expected, induction of the tac promoter in pKK223-3CAT had no effect on RK2 copy number or on TrfA levels. In this experiment, the copy number of RK2 in the presence of pKK223-3CAT was 5.4 x 109 + 0.2 x 109 molecules per OD6. unit, corresponding to 15 copies per cell. The total TrfA concentration specified by RK2 was 6.4 + 0.8 ng per OD6. unit. Increasing the total TrfA level to 15 to 20 ng per OD6. unit, using the pVR5 derivatives, increased the RK2 copy number about 30% to an estimated 7.4 x 10' molecules per ODwo unit (approximately 20 copies per cell). Further increases in TrfA concentration, up to approximately 1,100 ng per OD6. unit (170-fold above normal), had no additional effect on RK2 copy number. Thus, both pSV16 and RK2 showed similar responses to TrfA levels in vivo. These results and the results from Tables 2 and 3 for pSV16 are presented graphically in Fig. 6. DISCUSSION Quantitative analyses of TrfA protein and RK2 plasmid levels indicate that an average single cell of an exponentially growing E. coli HB101 culture contains approximately 220 monomers of TrfA-32, 80 monomers of TrfA-43, and 15 copies of plasmid RK2. Since logarithmically growing E. coli normally contains multiple chromosomes per cell, our estimate of 15 copies per cell in E. coli HB101 is consistent with the earlier estimates of four to seven copies per chromosome equivalent (14). As in the case of other plasmids, such as

0

7

copy

nutnber ( 109molecules

0

0

0

00

6

0 0 0

*

5

S 0

t

4

per OD600)

0

*

0

t 2 1

I 2

5

10

20

50

100

200

500

1000

2000

total TrfA (ng per OD600)

FIG. 6. oriV copy number versus intracellular TrfA concentration. The data were taken from Tables 2 through 4. A portion of the curve representing pSV16 copy number is dashed to indicate that there are insufficient datum points in that region for accurate plotting; the indicated shape of that portion of the curve is speculative. Symbols: 0, RK2; *, pSV16.

R6K (15, 17, 18), F (31, 57, 58), and P1 (1, 7), one or both TrfA proteins bind to the 17-bp direct repeats in the RK2 oriV region. This is supported by the results of experiments using purified and partially purified TrfA proteins (R. Durland, Ph.D. thesis, University of California, San Diego, La Jolla, Calif., 1988; [29]). It is known that TrfA-32 alone can support RK2 replication (23, 39), but the role of TrfA-43 in RK2 replication is uncertain. Experiments in which the start codon of TrfA-32 was changed from ATG (met) to CTG (leu), abolishing TrfA-32 synthesis, suggest that TrfA-43 alone is also capable of supporting replication in E. coli (F. Fang, unpublished observations). Assuming that TrfA-43 and TrfA-32 are functionally equivalent, a growing cell contains about 300 TrfA monomers. The native form of TrfA in solution is not known, but three other plasmid replicons, R6K (16), F (26), and P1 (1), specify initiator proteins that are dimeric. If we assume that TrfA binds as a dimer to each of the nine 17-bp repeats in the RK2 origin region, then there are about 150 active TrfA molecules and 135 binding sites per cell. Thus, normal TrfA levels appear to be relatively low, which is consistent with the proposal that TrfA level is rate limiting for RK2 replication. Our experiments on the effects of various TrfA levels on RK2 replication indicate that the copy number of a minimal origin plasmid (pSV16) and of intact plasmid RK2 show relatively small changes in response to increasing concentrations of TrfA. For RK2, approximately doubling the normal TrfA level results in a 30% increase in the amount of plasmid DNA. Further increases in TrfA to levels that appear to be the maximum tolerated by the cell have no additional effect on the copy number. A similar response is seen with the RK2 origin plasmid pSV16. This suggests that the observed copy control mechanisms operating in pSV16 are likely to be the same as those that regulate RK2 itself. The copy number of pSV16, however, is somewhat lower than that of RK2 at any given concentration of TrfA (Fig. 6). The oriV sequences present in pSV16 include eight of the nine 17-bp direct repeats found in RK2. Previous work suggested that deletion of the single repeat found upstream of oriV results in elevated copy numbers relative to RK2, especially under conditions of excess TrfA (53, 54). Despite the fact that this

3857

VOL. 172, 1990

PLASMID RK2 TrfA REPLICATION PROTEINS

region is missing in pSV16, we did not observe a similar high copy number relative to RK2. Models proposing that TrfA is rate limiting for RK2 replication predict that plasmid copy number should respond positively to increases in TrfA concentration. We found this to be true only over a limited range of plasmid copy number and TrfA protein concentration, since the maximum observed increase in intact RK2 copy number was about 30%. Thus, the data indicate that TrfA protein is not strictly rate limiting for RK2 replication. Although the TrfA concentration does influence copy number, as evidenced by the observed 30% increase for RK2, one or more additional controls must exist that limit replication independent of TrfA level. The fact that these controls continue to operate even in a minimal replicon system suggests that they are a function of the properties of oriV, TrfA protein, or both. Alterations in the oriV region can result in two- to fourfold increases in copy number (53, 54, 56), implying that oriV itself is involved in the negative regulation of replication. In the accompanying paper (12), we describe genetic evidence that suggests that a critical regulatory element also resides in the TrfA protein. It is interesting that the overall response of pSV16 to variations in TrfA concentration appears to be similar to that of RK2 itself. This suggests that despite the complex controls on trfA transcription that are present in intact RK2 (including korA and korB), the basic elements regulating replication initiation of RK2 in E. coli may be inherent in the oriV region and the TrfA protein(s).

gene of Rhizobium meliloti is oxygen regulated. J. Bacteriol.

ACKNOWLEDGMENTS We thank P. Tooker and M. Filutowicz for advice and assistance during protein purification. We are grateful to S. J. Singer, M. Adams, and G. Anders for technical advice and assistance in preparing anti-TrfA antibodies. We also thank S. Valla, A. Greener, and V. Rosenthal for plasmid constructs and helpful suggestions. This work was supported by Public Health Service grant AI-07194 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Abeles, A. L. 1986. P1 plasmid replication. Purification and DNA binding activity of the replication protein RepA. J. Biol. Chem. 261:3548-3555. 2. Amann, E., J. Brosius, and M. Ptashne. 1983. Vectors bearing a

hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167-178. 3. Bazaral, M., and D. R. Helinski. 1970. Replication of a bacterial plasmid and an episome in Escherichia coli. Biochemistry 9:399-406. 4. Bechhofer, D. H., J. A. Kornacki, W. Firshein, and D. H. Figurski. 1986. Gene control in broad host range plasmid RK2:

expression, polypeptide product, and multiple regulatory functions of korB. Proc. Natl. Acad. Sci. USA 83:394-398. 5. Beck, E., G. Ludwig, E. A. Auerswald, B. Reiss, and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19: 327-336. 6. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472. 7. Chattoraj, D. K., K. M. Snyder, and A. L. Abeles. 1985. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc. Natl. Acad. Sci. USA 82:2588-2592. 8. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:2110-2114. 9.

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227:680-685. 25. Lanka, E., R. Lurz, and J. P. Furste. 1983. Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid 10:303-307. 26. Masson, L., and D. S. Ray. 1988. Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product. Nucleic Acids Res. 16:413-424. 27. Olsen, R. H., and P. Shipley. 1973. Host range of the Pseudomonas aeruginosa R factor R1822. J. Bacteriol. 113:772-780. 28. O'Neill, E. A., C. Berlinberg, and R. A. Bender. 1983. Activity

of plasmid replicons in Caulobacter crescentus: RP4 and ColEl. Genetics 103:593-604. 29. Pinkney, M., R. Diaz, E. Lanka, and C. M. Thomas. 1988. Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III. J. Mol. Biol. 203:927-938. 30. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 31. Rokeach, L. A., L. Sogaard-Andersen, and S. Molin. 1985. Two functions of the E protein are key elements in the plasmid F replication control system. J. Bacteriol. 164:1262-1270.

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