A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number

121-133

PLASMlD19,

(1988)

A Single Amino Acid Difference between Rep Proteins Affects Plasmid Copy Number BARBARA

Department

of Microbiology

J. FROEHLICH

and Immunology, Received

AND

JUNE

R.

of PI and P7

SCOTT

Emory University Health Sciences Center, Atlanta, Georgia 30322

February

8, 1988; revised

March

31, 1988

PI and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P 1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A Pl replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the Pl RepA protein (RepAl) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of Pl cop mutants drops to that of Pl cop+. However, when RepA is supplied, the copy number of P 1 cop and P 1 cop’ is higher than that when RepA 1 is supplied. This indicates that the single amino acid difference between the two versions of the o ,988 Academic RepA protein plays an important role in determining the plasmid copy number. Press, Inc.

Regulation of DNA replication can be studied most easily using plasmids, because such replicons are dispensable for cell viability. The mechanisms of replication control identified previously fall into several categories (reviewed by Scott, 1984). Some small plasmids, like ColEl, rely largely on plasmid-encoded negative regulators for replication control. Such plasmids do not encode any proteins required for their own replication. In contrast, large plasmids usually encode an essential replication protein, generally called Rep. In these large plasmids, the negative regulatory loop(s) usually involves indirect regulation of replication by control of the amount of available Rep protein. This is done at the transcriptional level (1) by repressor proteins in the FII incompatibility group plasmids (Rl , RlOO, etc.), (2) by plasmid-encoded RNA in the Staphylococcal plasmid pT 18 1, and (3) by the Rep protein itself for F and the incompatibility group Y plasmid P 1. In addition, several plasmids exSequence data from with the EMBL/GenBank sion No. 503308.

this article have been deposited Data Libraries under Acces-

hibit post-translational regulation of availability of Rep protein. In these cases (F, P 1, Rts 1, R6K, pSC 10 1, etc.), the Rep protein binds in an apparently nonfunctional manner to short (about 20 bp) iterated DNA sequences. This binding is thought to reduce the concentration of Rep protein available for functional binding at the replication origin region. The closely related plasmid prophages Pl and P7 are members of incompatibility group Y (Hedges et al., 1975) and, by heteroduplex analysis, P7 is 90% homologous to P 1 (Yun and Vapnek, 1977). Pl is maintained at the very low copy number of about 1 per chromosome in exponentially growing Escherichia coli K12 (Prentki et al., 1977; Scott et al., 1982). The minimal replicon of Pl (Fig. 1) contains an origin region and a gene (repA) encoding a protein (RepA) required for replication (Abeles et al., 1984; Austin et al., 1985; Chattoraj et al., 1985b). About half of the 245-bp region which functions as an origin consists of a set of five directly oriented inexact 19-bp repeats, called incC, located immediately 5’ to the repA gene

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FROEHLICH AND SCOTT

(Abeles et al., 1984). The purified RepA protein binds to these reiterated sequences in vitro (Abeles, 1986). This binding is probably necessaryboth for origin function (Austin et al., 1985) and for the negative autoregulatory activity of the Rep protein (Chattoraj et al., 1985a,b). As expected for a protein which is rate limiting for replication, an increase in the RepA concentration results in an increase in copy number (Pal and Chattoraj, 1987), at least at moderate RepA concentrations. At high concentrations, RepA appears to inhibit replication from a PI origin (Chattoraj et al., 1985b; Pal and Chattoraj, 1987) although experiments in vitro do not confirm this unexpected result (Wickner and Chattoraj, 1987). Directly 3’ to the repA gene is a region dispensable for replication but involved in copy number control (Chattoraj et al., 1984). This region, called incA, contains nine additional inexact copies of the Rep-binding repeats (Abeles et al., 1984). The incA repeats are thought to control copy number by binding the RepA protein so that it becomes unavailable for action at the origin (Chattoraj et al., 1984; Pal et al., 1986). To learn more about the regulation of replication of the Pl plasmid, we have studied three P 1 mutants (P 1 cop) which have a copy number at least five- to eightfold higher than that of wild-type Pl (Scott et al., 1982). The high-copy-number phenotype is caused, in each case,by a single base change in the repA gene (Baumstark et al., 1984) (Fig. 1). Severe plasmid-specific incompatibility prevents the use of two whole Pl plasmids in a complementation test (Cowan and Scott, 1981). Because incompatibility between nonidentical members of group Y (group-specific incompatibility) is less severe,we tested dominance of cop+ by performing complementation tests between Pl cop mutants and P7 wild type (Scott et al., 1982). One of these mutants, Pl copE38, which is temperature sensitive for high copy number, is complemented for copy number by P7 cop’ (i.e., a cell containing P7 cop+ and Pl copE38 has a low copy number of both plasmids). On the

other hand, the other two copy mutants tested (PI copN3 and N22) are not complemented by P7 cop+ (i.e., Pl copN3 or N22 retains its high copy number when present in a cell containing P7 cop’). This suggeststhat the replication region of P7 is similar enough to that of PI to reduce the copy number of one of the Pl mutants when present in truns and is consistent with the idea that the copE38 mutation is in a domain of the Rep protein different from that of the lesions of the other two copy mutants (Baumstark et al., 1984). To help learn more about plasmid replication, we have analyzed the similarities and differences between the replication regions of these two plasmids in further detail. The DNA sequence of the P7 replication region was determined and compared to that of Pl . We report here on the ability of wild-type Pl and P7 RepA proteins (RepAl and RepA7) to support replication from a PI origin and to complement the copy number of Pl cop mutant prophage. MATERIALS

AND METHODS

Media. LB broth, LB agar, and tryptone agar (Scott, 1974) were supplemented with 20 &ml ampicillin (Sigma Chemical Co.), 40 &ml chloramphenicol (Sigma), and 30 &ml kanamycin (Sigma) where indicated. EMBO plates were made as described by Gottesman and Yarmolinsky ( 1968). Bacterial strains. The bacterial strains are all derivatives of E. coli K 12. N 100 (Gottesman and Yarmolinsky, 1968) is recA sup+. CH734 (Coleman et al., 1980), which was used for plasmid DNA transformation (Cohen et al., 1972), is trpA36, ly.sA, ilvD130 argH xyl gly T6R. Phage strains and plasmids. Pl Cm was described by Kondo and Mitsuhashi (1964). P7 was described by Smith (1972). Copy mutants of PI Cm were constructed by crossing the Pl Ap Cm cop mutants (Scott et al., 1982) with appropriate Pl amber mutants. The WW phage mixture (Stemberg, 1976) contains 10’ plaque-forming units per

REPLICATION

REGION OF Pl AND P7

milliliter of each of the following: W30 which is X b2c and W248 which is X h80 del(att int). X-P1:5R contains the P 1 plasmid replication region cloned into a X vector (Sternberg, 1979; Sternberg and Austin, 198 I, 1983). X-Pl:SR repA is derived from X-Pl:SR and contains an amber mutation in the repA gene (Austin et al., 1985). Plasmid pALA is pBR322 containing a fragment of Pl DNA (bp 606 to 1569; Fig. 1) that includes the repA reading frame and about 1.5 incA repeats (Austin et al., 1985). This plasmid expressesthe RepA protein constitutively from unknown pBR322 sequences (Chattoraj et al., 1985b; Austin et al., 1985). Plasmid pMAC295 is a cointegrate of pBR322 with the Pl replication region (Capage and Scott, 1983). Plasmid pCM7 is pBR327 containing a promoterless chloramphenicol-resistance gene from pBR328 flanked by Hind111 sites (Close and Rodriguez, 1982). Plasmid pGP l-2 contains the P 15A origin, a kanamytin-resistance gene from pACYC 177, and the T7 RNA polymerase gene under the control of the temperature-sensitive X repressor, ~1857 (Tabor and Richardson, 1985). Plasmid pT7.6, which is similar to pT7.1 (Tabor and Richardson, 1985), is a derivative of pBR322 containing the polylinker region from pUC 13 downstream from a T7 RNA polymerase promoter (Tabor and Richardson, unpublished). The orientation of the Blactamase gene has been altered so that its promoter transcribes in the opposite direction from the T7 promoter. Construction of pEUP7. P7 plasmid DNA was digested with BamHI and HindIII. The 5.2-kb P7 fragment corresponding to the Pl plasmid replication region (Yun and Vapnek, 1977; Iida and Arber, 1979) was purified by electroelution from an 0.8% agarose gel followed by passageover an Elutip-d column (Schleicher & Schuell). The P7 fragment was ligated (BRL T4 DNA ligate) to pUC9 that had been digested with Hind111 and BamHI. Construction of pEU90.19. Plasmid pALA (Austin et al., 1985) was digested at a unique XmaIII site which lies in the

123

pBR322 DNA 10 bp from the pBR322 junction with the Pl incA region. Limited digestion of the ends of the linear DNA was carried out with Ba131 nuclease (IBI; slow) using the conditions recommended by the supplier. The ends of the DNA were repaired with T4 DNA polymerase (BRL) and the linear DNA was ligated (T4 DNA ligase; BRL) in the presence of a fivefold molar excess of BamHI linker (Pharmacia). The extent of deletion of PI DNA was determined by DNA sequence analysis (Sanger et al., 1977). The Pl DNA fragment extending from the BamHI linker to the PvuII site at bp 1379 in the repA gene (Fig. 1) was ligated to M 13mp8 digested with BamHI and HincII. The DNA sequence of the Ml 3 clones was determined by the Sanger dideoxy chain termination method (1977). The Pl DNA in this chimera extends from bp 606 to bp 1531 (Fig. 1). Construction of pEU90.197. Since the P7 RepA protein differs from that of Pl by a single amino acid (see below), a Pl DNA fragment containing this region was replaced in pEU90.19 by the corresponding P7 DNA fragment. Plasmids pEU90.19 and pEUP7 were digested at the unique AsuII and MluI sites (Fig. 1). The larger of the two fragments from pEU90.19 (missing bp 949- 1202 of the repA gene; Fig. 1) and a 253-bp fragment from pEUP7 (bp 949-1202; Fig. 1) were purified from agarosegels. These two fragments were ligated (T4 DNA ligase; BRL) and the replacement of the PI AsuII/MluI fragment by the P7 AsuII/MluI fragment was confirmed by DNA sequence analysis (see below). Construction of pEU100. A promoterless Hind111 fragment (bp 606 to 1856; Fig. 1) containing the complete reading frame of repA was isolated from pMAC295 (Capage and Scott, 1983) and ligated to pT7.6 digested with Hind111 (Tabor and Richardson, 1985, unpublished). The orientation of the P 1 insert was determined by restriction analysis, and a clone with the Pl fragment in the proper orientation for expression of repA from the T7 promoter was picked.

124

FROEHLICH

Isolation of plasmid DNA. For restriction analysis, plasmid DNA was isolated by the alkaline extraction procedure of Bimboim and Doly (1979). For fragment purification and transformation, plasmid DNA was isolated by a modification of the alkaline extraction procedure (Maniatis et al., 1982). DNA purified by either technique was used for sequencing of supercoiled plasmid DNA. Complementation tests.The copy number of PI Cm was determined by the method of Shields et al. ( 1986). The plasmid-containing cells were grown with shaking in LB broth plus antibiotic to 2-4 X lo* cells/ml as determined by counting in a Petroff-Hauser chamber. Cold sodium azide was added to 0.02% to stop cell growth, the cells were harvested by centrifugation, and the pellet was resuspended to a concentration of about 2 X lo* cells/ml in M9 salts (42 mM Na2HP04, 22 InM KH2P04, 8.5 mM NaCl, 19 mM NH&l) containing 0.02% sodium azide. The cells were applied to nitrocellulose filters and lysed, and the DNA was immobilized as described by Shields et al. (1986). Probes for DNA-DNA hybridization were made by labeling linear DNA with [a-32P]dATP (Amersham; 3000 Ci/mmol) using the Klenow fragment of DNA polymerase (IBI) (Feinberg and Vogelstein 1983, 1984). At least 1 X 10’ cpm of denatured 32P-labeledprobe was used per filter. Filters were washed at 62°C in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, and 0.1% SDS. Washing at lower stringency resulted in a high nonspecific background. The filters were autoradiographed with Kodak X-Omat AR film using a DuPont Hi-Plus intensifying screen at -70°C for 4 to 48 h. The relative amount of probe hybridizing to each sample was measured by two-dimensional scanning of the autoradiogram using a Bio-Rad video densitometer 620. Similar results were obtained when each sample was cut out of the nitrocellulose filter and counted in a Beckman liquid scintillation counter. Antibody production. RepA production was induced as described by Tabor and Richardson (1985). CH734 containing both

AND

SCOTT

pGPl-2 and pEU 100 was grown with shaking at 30°C in LB broth plus drug to about 4 X lo* cells/ml as determined in a PetroffHauser chamber. The cells were shified for 30 min to 42°C to induce the T7 RNA polymerase and therefore RepA. Rifampicin was added to 100 &ml to prevent E. coli RNA polymerase-directed transcription. The cells were incubated for 2 h at 37°C with shaking, harvested by centrifugation, and washed with 10 IYIM MgS04, and the pellets were stored at -80°C. RepA purification. RepA was purified by a modification of the method of Abeles (1986). After the cells were lysed and the cell debris was pelleted as described, the protein solution was dialyzed overnight against RepA buffer (50 mM Tris, pH 7.5; 1 InM EDTA; 10% glycerol; 1 mM 2-mercaptoethanol) containing 0.25 M NaCl. During dialysis, most of the RepA protein formed a precipitate (Abeles, 1986). The protein was harvested by centrifugation at 17,000g at 4°C and the pellet was suspended in RepA buffer containing 0.5 M NaCl. MgC12was added to 10 mM and the protein solution was incubated with pancreatic DNase (to 1 pg/ml) for 10 min at room temperature. The protein solution was dialyzed against 3 changes of RepA buffer containing 0.25 M NaCl and then loaded onto a phosphocellulose column (Whatman P 11) which had been equilibrated with RepA buffer containing 0.25 M NaCl (Abeles, 1986). The RepA was eluted with a gradient of 0.25 to 1.15 M NaCl in RepA buffer and the fractions were assayed for RepA by SDS-polyacrylamide electrophoresis (Abeles, 1986; Laemmli, 1970). The fractions containing RepA were pooled and stored at -80°C. This procedure gave RepA which was at least 99% pure as determined on a silver-stained polyacrylamide gel. Preparation of antibody to RepA. A New Zealand White rabbit was injected intramuscularly with about 500 pg RepA homogenized in complete Freund’s adjuvant. After 4 weeks, a second intramuscular injection of the same material was given. Bleedings were at 7 weeks after the booster injection and the

REPLICATION

125

REGION OF PI AND P7

specificity of the antiserum was tested by Western blots (Towbin et al., 1979). The antiserum was purified by three adsorptions to CNBr-activated Sepharose 4B (Pharmacia) crosslinked to protein purified (as described above) from control cells lacking RepA (strain CH734 containing pT7.6 and pGPl-2). Quantitative Western blots. The cell extracts were prepared as described by Swack et al. (1987). Protein concentration was determined as described by Schaffner and Weissman ( 1973). Equal amounts of extracts were loaded into wells of an SDS-polyacrylamide gel and the proteins were separated by electrophoresis (Laemmli, 1970) in a Hoeffer Mighty Small slab gel unit at 10 mA per gel for 2.5 h. The polypeptides in the gel were electrotransferred onto a nitrocellulose filter (0.45 pm; Schleicher & Shuell) for 30 min at 2.5 mA/cm2 using the ABN Polyblot system. The nitrocellulose filters were incubated for 2 h in PBS-Tween (0.01 M sodium phosphate, pH 7.4; 0.15 M NaCl; 0.5% Tween 20) for 1 h in a 1 to 500 dilution of anti-RepA in PBS-Tween and then washed three times for 10 min in PBS-Tween. The filters were incubated 1 h in PBS-Tween containing 0.1 pCi/ml 12%labeled protein A (Amersham; 30 mCi/mg), washed three times for 10 min in PBS-Tween, air-dried, and then autoradiographed with Kodak X-Omat AR film. The relative amount of ‘251-protein A binding to each sample was measured by scanning the autoradiogram using a Bio-Rad video densitometer 620. Complementation for replication. NlOO carrying pEU90.19, pEU90.197, or no plasmid was grown to log phase in LB broth at 37°C infected with a m.o.i. of 5 X-Pl:SR or a rep mutant derivative of this phage. After a 15-min incubation at 32°C for adsorption, cells were plated with WW selector phage (see above) on EMBO medium and incubated at 30°C for 2 days. Lysogens form white colonies. DNA sequence analysis. DNA sequence was obtained from supercoiled plasmid DNA using a modification (Zagursky et al.,

1985) of the dideoxy chain termination method (Sanger et al., 1977). Supercoiled plasmid DNA which had been denatured by incubation for 5 min at room temperature in 0.4 N NaOH, was neutralized by adding sodium acetate (pH 4.6) to a concentration of 0.4 M. Oligonucleotide primers were end-labeled with “P ([X-32P]ATP; Amersham, 3000 Ci/mmol) using polynucleotide kinase (Pharmacia) and added to the DNA at a ratio of 3O:l (primer:plasmid DNA), and the whole mixture was ethanol precipitated. The dNTP/ddNTP ratio in a reaction containing 0.1 pmol plasmid DNA was 5:l (dNTP at 200 PM and ddNTP at 40 PM). Chain elongation was carried out by reverse transcriptase (Life Sciences) at a concentration of 0.1 unit/p1 at 42°C. RESULTS

Dlyerences in SequencebetweenPI and P7. DNA from Pl and P7 hybridizes in at least part of the inc-rep region (Yun and Vapnek, 1977; Meyer et al., 1986), but the restriction maps in this region of these plasmids are quite different (Iida and Arber, 1979). Both similarities and differences between PI and P7 in this region should help identify the features that are important for replication and its regulation. We determined the DNA sequence of the entire origin-repA-incA region from P7. In Fig. 1, the P7 sequence is compared to the sequence of the same region of Pl (Abeles et aI., 1984; Baumstark et al., 1984). In the origin region (bp 366-6 11, Fig. 1; Chattoraj et al., 1985b) there are four single base differences between PI and P7. Two of these differences are in the origin (incC) repeats (repeats 10 and 13; Fig. 1) and could affect RepA binding. The base substituted in repeat 10 lies within the region deleted from the PI mutant rep-l 1 (Austin et al., 1985), which cannot be complemented for replication and therefore defines a region containing sequences required for replication. The possible significance of the two other base differences between PI and P7 in this DNA

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FIG. 1. A comparison of the Pl and P7 replicons. The P7 replication region was sequenced as described under Materials and Methods. The sequence of the Pl replication region is from Abeles et al. (1984). A postulated dnuA box (bp 387-404) of five dam methylation sites (designated Me) are bracketed (Abeles et al., 1984). The -35 and -10 regions of the repA promoter (Chattoraj et al., 1985b) are labeled above the line. The 19-bp RepA-binding repeats of the origin (numbers 14-10) and in& (numbers 9-l) are indicated by arrows. The direction of the arrow shows the orientation of the repeat. The start codon (bp 664; designated Rep) and termination codon (bp 1521;designated stop) for the RepA protein are bracketed above the line. The positions of the repA mutants, repAl (Austin et al., 1985), and the cop mutans (Scott et al., 1982; Baumstark et al., 1984) are shown above the line. The DNA sequence differences between PI and P7 are marked with (*). A colon between basesofthe two sequencesindicates identity.

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128

FROEHLICH

segment is unclear, since they are in regions of the DNA sequence with no recognizable features. In the repA gene, there are 13 differences between Pl and P7 (Fig. 1). However, only 1 of the 13 base differences leads to a changed amino acid, converting a polar residue (Asn in Pl) to a basic one (Lys in P7). All of the other differences between these repA genes occur in the third base of a codon and do not alter the encoded amino acid. Three other single base differences occur between the origin repeats and the repA gene in apparently nontranslated DNA that contains no features identified as important for replication. In the incA region (bp 1524-l 808, Fig. 1), there are 14 single base differences between PI and P7. Nine differences in sequence between the plasmids occur in the DNA between the repeats and are therefore not likely to be important for RepA protein binding. The five other differences are in the incA repeats (repeats 1, 2, 8, and 9; Fig. 1). There are no insertions or deletions in P7 compared with Pl, showing that all spacing is conserved. Even though there is 50% divergence between the PI and P7 DNA sequences in the region between the last two incA repeats, the spacing between these two incA repeats is completely conserved. The P7 sequence 3’ to the last incA repeat (repeat 1) is totally unrelated to the PI sequence in this region. This result agreeswith the finding of Ludtke and Austin ( 1987) that there is no homology between Pl and P7 in this region which contains the partition locus (Austin et al., 1982).

AND

SCOTT

sured. Because of deletions in the X portion, this phage can lysogenize only if the Pl replicon it contains is functional (Sternberg, 1976). Complementations of two missense repA mutants (Austin, unpublished) and one amber mutant (repA 103; Austin et al., 1985) by RepAl (supplied by pEU90.19) and RepA (supplied by pEU90.197) were compared in a recA host. All three X-Pl:SR repA mutants lysogenized both N 100 (pEU90.19) and NlOO (pEU90.197) as efficiently as did X-PI :5R repA+, but no lysogens of the repA mutant phage were detectable in NlOO that did not contain PI DNA (data not shown). Therefore, RepA appears to be functionally equivalent to RepA 1 for P 1 plasmid replication. Complementation of Pl cop Mutants for Copy Number

Previously, we found that Pl copE38 is complemented by P7 cop+, but that PI copN3 and copN22 are not (Scott et al., 1982). It is impossible to perform complementation tests using two whole P 1 plasmids because of strong incompatibility (Cowan and Scott, 1981). We therefore constructed chimeric plasmids (see Materials and Methods) containing only the repA open reading frame from P 1 or P7 (pEU90.19 and pEU90.197, respectively) and completely lacking all DNA repeat regions that determine incompatibility. The copy number of PI cop mutant plasmids was determined in the presence of pEU90.19 or pEU90.197. Supplying either the RepAl protein of Pl or the RepA protein of P7 in trans reduces the copy number of P 1 copN3 to the level of P 1 Complementation between Pl and P7 cop+ (Table 1). This indicates that RepAl for Replication and RepA are dominant to this mutant, as If the RepA proteins of Pl and P7 are very well as to PI copE38 (Scott et al., 1982). This similar, as indicated by the DNA sequence, is probably also true for Pl copN22, but, RepA might be able to complement a Pl since the copy number in the complementarepA mutant for replication. To test this, ly- tion test of this plasmid with pEU90.197 is sogeny by X-P1:5R which contains a com- not very different from that of Pl copN22 plete P 1 minireplicon (Sternberg, 1979; alone, the experimental error makes the conSternberg and Austin, 1981, 1983) was mea- clusion uncertain for this mutant.

REPLICATION

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REGION OF Pl AND P7

TABLE 1 COMPLEMENTATION OF Pl copMUTANTS FOR COPY NUMBER’

Strain

Relative copy no.’

N 100 (P 1 Cm cop’) NlOO (Pl copN22) NlOO (Pl Cm copN3) N 100 (pEU90.19) (P 1 Cm cop’) NlOO (pEU90.19) (Pl Cm copN22) NlOO (pEU90.19) (Pl Cm copN3) NlOO (pEU90.197) (Pl Cm cop+) NlOO (pEU90.197) (Pl Cm copN22) NlOO (pEU90.197) (PI Cm copN3)

1 7.3 + 1.7 14.1 + 5.1 1.1 + 0.50 1.4 Y!I0.21 1.2 iI 0.30 4.9 AZ2.0 5.0 f 0.85 4.5 +- 1.2

a Log-phase cells were analyzed for copy number of the PI prophages by the method of Shields et al. (1986; see Materials and Methods). A 1.5-kb Hind111fragment from plasmid pCM7 containing the chloramphenicolresistancegene (Close and Rodriguez, 1982) was used as a PI Cm specific probe. Under the conditions used, this probe hybridized only to DNA from cells containing the Pl Cm prophage. Duplications of the Tn9 in PI Cm, which occur spontaneously at a relatively high frequency (Meyer and Iida, 1979), would be seen as an increase in copy number since the radioactive probe used in the DNA hybridizations is homologous to the chloramphenicol-resistance gene of Tn9. By restriction fragment analysis, we have ruled out such duplications in the Pl Cm plasmids used for these experiments (Froehlich, unpublished). bThe copy numbers were calculated relative to N 100 (Pl Cm cop’). Each number is the average of at least three separate measurements. For each experiment, quadruplicate samples of at least three dilutions were applied to the filter for each strain.

difference (Fig. 1). Although it is unlikely, it seemed possible that the amount of RepA produced from pEU90.19 might differ from that produced from pEU90.197, either because these base changes alter the message stability or because of possibly unfavorable codon usage. Densitometric analyses of Western blots (see Materials and Methods) indicated that the concentration of RepA in cells containing pEU90.19 was the same as that in cells containing pEU90.197 (Table 2). Thus, the increased copy number seen for all PI plasmids in the presence of pEU90.197 is due to differences in the properties and not in the concentrations of RepA 1 and RepA7. DISCUSSION

Sequence comparisons of the replication regions of Pl and P7 indicate that there are 33 base differences out of a total of 1443 bases (2.3%). This sequence divergence between the two plasmids is similar to that reported for their cl genes(2.1%; F. A. Osbom, S. Ashkar, S. R. Stovall, and B. R. Baumstark, submitted). The locations of the repA gene, the inc repeats, and other DNA sites thought to be important for replication are completely TABLE 2 THE RELATIVE AUOUNT OF RepA MADE BY pEU90.19 AND pEU90.197’

RepA Leads to a Higher Copy Number Than Does RepAI A striking and unexpected result shown in Table 1 is that RepA allows all three PI plasmids, including both cop mutants and cop+, to reach a higher copy number than does RepAl. In these experiments, Rep protein is produced in trans from a plasmid lacking incompatibility repeats and is apparently expressed constitutively from a promoter created during the fusion of the repA gene to the vector. The chimera providing RepA differs from that providing RepAl only in six single bases in the middle of the rep.4gene which lead to only one amino acid

Expt 1 2 3 4 5

w RepAh% extractb (pEU90.19) 107 + 71 k 96 k 70 + 142 +

9.2% 7.5% 8.0% 30% 4.0%

Relative amount of RepA ng RwA/rg extractb (pEU90.19/ (pEU90.197) pEU90.197) 159 + 86 k 76 + 96 k 150 j,

30% 10% 15% 14% 15%

1.5 1.2 0.8 1.3 1.1

a Log-phase cells were lysed and RepA concentration was measured on Western blots (Swack et al., 1987; see Materials and Methods). b The amount of RepA in the extracts was calculated relative to a series of dilutions of purified RepA protein on the same gel.

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identical in Pl and P7. This would be expected if the RepA proteins of PI and P7 bind to the DNA repeatsin a way dependent on their location on the helix, and suggests that the spacing is critical for function. In addition to conservation of spacing,P7 and PI also show sequenceidentity in the sites predicted to be involved in binding DnaA protein and in methylation, both of which are thought to be important for PI replication (Hansen and Yarmolinsky, 1986; Abelesand Austin, 1987). Although there is only 50% homology of the P7 and Pl sequencebetween the last two incA repeats, the repeat itself is conserved. This suggeststhat variation in this last repeat would be detrimental to function, either because a total of nine repeats is required or. becausethe last one in particular is important. Becauserepeatsas short as 19 bp or even shorter serve as “hot spots” for deletion or duplication of the intervening DNA (Farabaugh et al., 1978; Edlund and Normark, 198l), P7 and PI might have been expected to have a different number of inc repeats. The conservation of the number and spacing of incA repeatsas well as origin repeatssuggeststhat their presenceprovides a selective advantageto the plasmid. In fact, when two repeatswere deletedfrom the origin region of Pl (Austin et al., 1985),the resulting plasmid did not replicate. Most of the basesin the incA repeatsthat differ between P7 and Pl are in the most external part of the repeated sequence.Becausethese are the most divergent from the consensuseven within Pl, these differences are probably of little importance. The conservation of sequencebetween Pl and P7 in the central portion of all the incA repeatsis consistent with the expectation that this region, which is responsiblefor Y group-wide incompatibility (Austin et al., 1982) is functionally identical for both plasmids. On the other hand, there are single base differences between P7 and Pl in the middle of two of the incC repeats(repeats 13 and 10; Fig. 1). Either these basesmust be unimportant for

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SCOTT

RepA binding or the RepA proteins of the cognate plasmids must be altered in a compensatory manner to allow binding. Within the RepA reading frame, there are 13 single base differences between Pl and P7, but only 1 of them results in an amino acid difference (Fig. 1). In agreement with the expected similarity of the RepA and RepAl proteins, we found that RepA protein is able to complement both nonsense and missensePl repA mutants for replication. This indicates that the proteins of both plasmids are functionally very similar in the positive function of RepA in the origin region. We have investigatedthe effectsof RepA 1 and RepA on Pl copy mutants (Scott et al., 1982). These mutants each differ from the wild type by a single basechangein the repA coding sequencewhich alters an amino acid in RepA (Baumstark et al., 1984). (For copN3, the copy number shown in Table 1 may differ from that previously reported (Scott et al., 1982) becausea different host strain was used.) There are three obvious explanations for the high-copy-number phenotype: (1) more effectivebinding of the mutant protein (RepA 1cop) to the origin repeats, (2) greater availability of the mutant protein for binding to the origin caused by reduced binding to incA repeats, or (3) reduced effectivenessat repressingrepA transcription. The copy number of Pl cop is reduced to the level of PI cop+ when either RepAl or RepA is supplied in tram (Table l), again attesting to the similarity between these two proteins. If explanation (1) above is correct for the effect of the cop mutations (the wildtype RepA protein initiates replication less efficiently than does RepA 1cop), complementation of the high-copy phenotype might result from displacement of the RepAlcop protein from the incC region of PI by both of the wild-type RepA proteins (RepAl and RepA7), thus reducing the copy number. Explanation (2) for the copy mutants (RepAl cop binds less well to incA repeats than does RepAl) does not predict reduction of

REPLICATION

REGION OF PI AND P7

copy number when wild-type RepA is sup plied unless (1) is also true. If (3) above is correct (RepA 1cop is less effective at repressing repA transcription than RepAl), then supplying RepAl or RepA should lead to decreased repA transcription, less RepA for initiation, and thus a lower copy number. PI copN22 and copN3 mutants are not complemented by a whole P7 cop+ plasmid (Scott et al., 1982); however, at least copN3 is complemented for copy number when the RepA protein of P7 is provided in tram from a chimeric plasmid. Since there is almost twofold more RepA protein made from the promoter used in the chimera than from the wild-type Pl plasmid (Swack et al., 1987), it is possible that this difference in RepA concentration accounts for the difference in complementation results. Altematively, the incA repeats of P7 (present in the whole plasmid but not in the chimera) might bind so much of the RepA protein that there is not enough available for replication of the Pl cop mutants. Although P 1 cop+, Pl copN22, and Pl copN3 all have the same copy number when complemented with either RepA 1 or RepA7, there are about five times as many copies when RepA is provided (from pEU90.197) than when RepAl is supplied (from pEU90.19) (Table 1). Densitometric tracings of Western blots indicated that both chimerit plasmids produce the same amount of RepA (Table 2). Therefore, it appears that the single amino acid difference between the RepA protein and the RepAl protein has a major effect on activity. Either RepA is more effective at initiating replication from a P 1 origin than is the cognate protein of P 1 or RepA binds less well than RepAl to the PI incA repeats, so more RepA is available for initiation. Thus, although RepA is similar enough to RepAl to complement a Pl repA mutant plasmid for replication, RepA results in a higher copy number than does RepAl. Where Pl RepA has a polar asparagine, P7 RepA has a basic Iysine. Both the side chains and the charges of these amino acids are dif-

131

ferent, so they are likely to affect binding to the DNA. The effect may be direct if this position on the protein is a contact point with the DNA or indirect if it changes the conformation of the protein. We have also found that six independently isolated intragenie suppressors of the high-copy phenotype of P 1 copN22 result from three different alterations in a single codon in the RepA protein (Scott and Froehlich, in preparation). In light of the identification of specific amino acids that appear to be involved in DNA binding, a more detailed molecular study of the interaction of the RepA proteins with their DNA target sites should be revealing. ACKNOWLEDGMENTS This work was supported by Public Health Service Grant AI 17696.We thank A. Groff, S. Guritz, K. Smith, and S. Mackay for assistancein performing these experiments. We are very grateful to Stuart Austin for the X-Pl:SR phages, to Dhruba Chattomj for pALA69, and to Stanley Tabor and Charles Richardson for pT7.6 and pGPl-2.

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CHATTORAJ, D. K., CORDES, K., AND ABELES, A. (1984). Plasmid PI replication: Negative control by repeated DNA sequences. Proc. Natl. Acad. Sci. USA 81,6456-6460. CHATTORAJ, D. K., PAL, S. K., SWACK, J. A., MASON, R. J., AND ABELES, A. L. (1985a). An autoregulatory protein is required for PI plasmid replication. In “Sequence Specificity in Transcription” (R. Calendar and L. Gold, Eds.), pp. 271-280. A. R. Liss, New York. CHATTORAJ, D. K., SNYDER, K. M., AND ABELES, A. L. (1985b). Pl plasmid replication: Multiple functions of RepA protein at the origin. Proc. Natl. Acad. Sci. USA 82,2588-2592. CLOSE, T. J., AND RODRIGUEZ, R. L. (1982). Construction and characterization of the chloramphenicol-resistance gene cartridge: A new approach to the transcriptional mapping of extrachromosomal elements. Gene 20,305-3 16. COHEN, S. N., CHANG, A.-C. Y., AND Su, L. H. (1972). Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69,2 110-2 114. COLEMAN, R. D., DUNST, R. W., AND HILL, C. W. (1980). A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene. Mol. Gen. Genet. 177,213-272. COWAN, J. A., AND SCOTT, J. R. (198 1). Incompatibility among group Y plasmids. Plasmid 6,202-22 1. EDLUND, T., AND NORMARK, S. (198 1). Recombination between short DNA homologies causes tandem duplication. Nature (London) 292,269-27 1. FARABAUGH, P. J., SCHMEISSER, U., HOFER, M., AND MILLER, J. H. (1978). Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the IacI gene of Escherichia coli. J. Mol. Biol. 126, 847-85 1. FEINBERG, A., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132,6-13. FEINBERG, A., AND VOGELSTEIN, B. (1984). Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. B&hem. 137,266-267. GOTTESMAN, M. E., AND YARMOLINSKY, M. B. (1968). Integration negative mutants of bacteriophage lambda. J. Mol. Biol. 31,487-505. HANSEN, E. B., AND YARMOLINSKY, M. B. (1986). Host participation in plasmid maintenance: Dependence upon dnaA of replicons derived from Pl and F. Proc. Natl. Acad. Sci. USA 83,4423-4427. HEDGES, R. W., JACOB, A. E., BARTH, P. T., AND GRINTER, N. J. (1975). Compatibility properties of PI and & AMP prophages. Mol. Gen. Genet. 141, 263-267. IIDA, S., AND ARBER, W. (1979). Multiple physical differences in genome structure of functionally related bacteriophage Pl and P7. Mol. Gen. Genet. 173, 249-26 1.

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KONDO, E., AND MITSUHASHI, S. (1964). Drug resistance of enteric bacteria. IV. Active transducing bacteriophage PlCm produced by recombination of R factor with bacteriophage Pl. J. Bacterial. 88, 1266-1276. LAEMMLI, U. K. (1970). Cleavage of structured proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LUDTKE, D. N., AND AUSTIN, S. J. (1987). The plasmid-maintenance functions of the P7 prophage. Plasmid 18, 93-98. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, S. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring, NY. MEYER, J., AND IIDA, S. (1979). Amplification of chloramphenicol resistance transposons carried by phage PlCm in Escherichia coli. Mol. Gen. Genet. 176, 209-219. MEYER, J., STALHAMMAR-CARLEMALM, M., STREIFF, M., IIDA, S., AND ARBER, W. (1986). Sequence relations among the IncY Plasmid p 15B, PI, and P7 prophages. Plasmid 16,8 l-89. PAL, S. K., AND CHATTORAJ, D. K. (1987). RepA protein is rate limiting for PI plasmid replication. UCLA Symp. Mol. Cell. Biol. 47,441-450. PAL, S. K., MASON, R. J., AND CHATTORAJ, D. K. (1986). Pl plasmid replication: Role of initiator titration in copy number control. J. Mol. Biol. 192, 275-285. PRENTKI, P., CHANDLER, M., AND CARO, L. (1977). Replication of the prophage Pl during the cell cycle of Escherichia coli. Mol. Gen. Genet. 152,7 l-76. SANGER, F., NICKLEN, S., AND COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. SCHAFFNER, W., AND WEISSMAN, C. (1973). A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56, 502-5 14. SCOTT, J. R. (1974). A turbid plaque-forming mutant of phage Pl that cannot lysogenize Escherichia coli. Virology 62, 344-349. SCOTT, J. R. (1984). Regulation of plasmid replication. Microbial. Rev. 48, l-23. SCOTT, J. R., KROPF, M. M., PADOLSKY, L., GOODSPEED, J. K., DAVIS, R., AND VAPNEK, D. (1982). Mutants of the plasmid prophage Pl with elevated copy number: Isolation and characterization. J. Bacteriol. 150, 1329-1339. SHIELDS, M. S., KLINE, B. C., AND TAM, J. E. (1986). A rapid method for the quantitative measurement of gene dosage: Mini-F plasmid concentration as a function of cell growth rate. J. Microbial. Methods 6, 33-46. SMITH, H. W. (1972). Ampicillin resistance in Escherichia coli by phage infection. Nature New Biol. 238, 205-206. STERNBERG, N. (1976). A genetic analysis of bacteriophage lambda head assembly. Virology 71,568-582.

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some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.

WATKINS,C. A., AND SCOTT,J. R. (198 1). Characterization of bacteriophage D6. Virology 110,302-317. WICKNER,S. H., AND CHATTORAJ,D. K. (1987). Replication of mini PI plasmid DNA in vitro requires two Initiation proteins: The products of Pl repA and E. coli dnaA. Proc. Natl. Acad. Sci. USA 84,3668-3672. YUN, T., AND VAPNEK, D. (1977). Electron microscopic analysis of bacteriophage PI, phage PlCm, and phage P7: Determination of genome sizes, sequence homology, and location of antibiotic resistance determinants. Virology 77, 376-385. ZAGURSKY,R. J., BAUMEISTER,K., LOMAX, N., AND BERMAN,M. L. (1985). Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA. Gene Anal. Tech. 2,89-94. Communicated by Barry Polisky