Mol Gen Genet (1981) 184:56-61

© Springer-Verlag 1981

Replication Control Functions of Plasmid R 1 Act as Inhibitors of Expression of a Gene Required for Replication

Janice Light 1 and Soren Molin z 1 Department of Genetics, University of Leeds, Leeds LS2 9JT, England 2 Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

Summary. Construction of translational fusions betwen the repA gene of plasmid R 1 (required for replication) and the laeZ gene has allowed a quantitative analysis of expression of this gene. It is suggested that the replication of R 1 is controlled by two replication control functions acting as inhibitors of repA expres- sion.

Introduction

Replication of many different bacterial plasmids has been shown to require most of the chromosomal replication functions (Broda 1979), but the plasmid molecules themselves carry genetic infor- mation for their own replication control (Nordstr6m et al. 1972; Veltkamp and Nijkamp 1976). Plasmids R 1, R6 and R 100 (in- compatibility group FII) are low copy number plasmids of enter- ic bacteria (one copy per chromosome equivalent on average at high growth rates) (Falkow 1975). The fact that the number of replication events per cell cycle is very low puts special de- mands on the control system, and determination of the molecular mechanisms involved is therefore of interest. For these plasmids the control genes have now been localized on the replicons (Mo- lin et al. 1979; Timmis et al. 1978; Taylor and Cohen 1979), and their gene products have been identified (Molin et al. 1981 ; Stougaard et al. 1981b). However, almost nothing is known about the way in which these factors interact with each other and with the replicon when they exert their regulatory effect.

Genetic analysis of the replication control system of plasmid R 1, including the characterisation of mutant plasmids with in- creased copy numbers and the cloning of wild type and mutant control genes, has led to the following conclusions: All genes required for replication and its control are located in the vicinity of the replication origin (basic replicon). Two independent, plas- mid-specific cytoplasmic factors, which act as inhibitors of repli- cation, have been identified. One of these products is a small, unstable RNA molecule (copA gene product). The other is a small, basic polypeptide (copB gene product). Kollek et al. (1978) showed that disruption of the DNA sequence between the cop genes and the replication origin prevents R 1 directed replication, and similar observations have been made for the other Inc FII plasmids R6-5 (Timmis et al. 1978) and R 100 (Miki et al. 1980). The suggestion for a positively acting element required for repli- cation of R 100 had previously been made by Yoshikawa (1974), who designated the gene repA.

We are investigating how genes involved in replication of R 1 are expressed in vivo using gene fusion techniques. Fusions

have been created such that promotion of transcription of the gene of interest directs the synthesis of an enzyme product which is easy to assay. The laeZ gene has been used extensively for such purposes (Casadaban and Cohen 1980). We have used vec- tors constructed by Casadaban in which the insertion of a DNA fragment resulting in a protein fusion is required for the expres- sion of /~-galactosidase from the lacZ gene (Casadaban et al. 1980).

Materials and Methods

Bacterial Strains and Plasmids. The Escherichia coli K-12 strain used in all experiments was CSH50, pro, lac, rpsL (Miller 1972). The plasmids used are listed in Table 1.

Growth Media and Measurements. The cells were grown in LB medium (Bertani 1951) or minimal medium A + B (Clark and Maaloe 1967), supplemented with 0.2% glucose, 1 lag/ml thia- mine and 20 gg/ml proline.

LA plates contain LB medium with 1.5% agar, McConkey- lactose plates were prepared as described by the manufacturer (Difco), and X-gal plates were prepared by adding 40 gg/ml 5-bromo-4-chloro-3-indolyl-/?-D-galactoside to A + B minimal medium supplemented as described above.

Antibiotics were dissolved in the plate media at the following standard concentrations: Ampicillin (50 gg/ml), Kanamycin (50 gg/ml), Chloramphenicol (50 gg/ml) and Tetracycline (10 ixg/ ml). Growth of cultures was measured as optical density at 450 nm (OD45o) using a Zeiss PMQ3 spectrophotometer.

Transfer of Plasmids to Bacteria. Plasmids were transferred by conjugation or transformation (Cohen et al. 1972).

Preparation of Plasmid DNA, Restriction Enzyme Treatment and Ligation of DNA Fragments. Plasmid DNA was prepared as described previously (Stougaard et al. 1981 a).

Digestion of DNA with restriction enzymes and ligation of DNA fragments using T4 polynucleotide ligase was as described by Molin et al. (1979).

Incompatibility. To test for the presence of the IncFII function on hybrid plasmids, plasmid Rldrd-19 or derivatives thereof were transferred by conjugation, and incompatibility investigated as described before (Molin et al. 1981).

Determination of Copy Numbers. Plasmid copy numbers were determined as levels of resistance to benzylpenicillin as described previously (Uhlin and Nordstr6m 1975). In some cases where

0026-8925/81/0184/0056/$01.20

Table 1. Plasmids

Plasmid Parent Resistance cop genotype Lac phenotype a Source/Reference replicon Phenotype

57

a) R1 derivatives R ldrd-19K-I R ldrd-19 Ap, Sin, Su, Cm copA, copB Beard and Connolly i975 pKN501 R ldrd-19 Ap eopA, eopB Molin et al. 1979 pKN501-1 pKN501 Ap copA Molin etal. 1981 pKN1562 Rldrd-19 Km copA, copB Molin et al. 1979

b) Cloning vehicles pBR322 pMB1 Ap, Tc Bolivar et al. 1977 pGA46 p15 Cm An and Friesen 1979 pMC874 p15 Km Lac- Casadaban et al. 1980 pMC1403 pBR322 Ap Lac- Casadaban and Cohen 1980 pOU491 pSC101 b Ap, Tc Meacock and Cohen 1979

c) cop hybrids pKN232 pBR322 Tc eopA, copB Molin etal. 1981 pOU423 pBR322 Tc copA Molin and Nordstr6m 1980 pOU16 pBR322 Tc, Ap copB E. Riise pOU494 pOU491 ° Tc, Ap, Km copA, copB This paper pOU393 pBR322 Tc copA- Molin and Nordstr6m 1980 pOU417 pBR322 Tc copA- Stougaard et al. 1981b pOU545 pGA46 Cm eopA Stougaard et al. 1981 b pOU15 pSC101 Tc copB E. Riise pOU30 pBR322 Tc, Ap copB- E. Riise

d) repA-lac fusion plasmids pJL99 pMC874 Km copA Lac + This paper pJL189 pOU491 Ap vopA Lac + This paper pJL127 pGA46 Cm eopA Lac (+~ This paper pJL128 pGA46 Cm copA Lac + This paper pJL111 pMCI403 Ap eopA, copB Lac (+~ This paper pJL133 pMCI403 Ap copA Lac + This paper

a The Lac phenotype is only indicated for plasmids that carry the structural lac genes. Lac + indicates a high level of expression detectable on McConkey-lactose plates, Lac (+~ indicates a low level of expression only detectable on X-gal plates, and Lac indicates no detectable expression

b Plasmid pOU491 is a Tn3 insertion derivative of plasmid pSC101repl03 which is temperature sensitive for replication as described by Meacock and Cohen (1979)

° Plasmid pOU494 consists of pOU491 and pKN1562 fused in the EeoR1 sites of the two plasmids. Resistance phenotypes indicate to which antibiotics the plasmids mediate resistance. The abbreviations used are: Ampicillin (Ap), sulfonamides (Su), chloramphenicol (Cm), kanamycin (Kin) and tetracycline (Tc). cop genotype refer to R1 copy number control genes (see text for details). The plasmids carrying only copB were constructed by E. Riise and P. Stougaard, and will be described elsewhere (Riise et al., in preparation). The genotypes copA- and copB- indicate that the respective genes are present but their functions inactivated by mutations

the plasmid did not mediate penicillin resistance the Tn3 transpo- son was inserted from a 2 phage carrying the transposon.

Activities of 13-Galactosidase. Measurements of fl-galactosidase expressed from fusion plasmids were made essentially as de- scribed by Miller (1972). F rom an exponentially growing culture samples were taken for OD4so reading, and duplicate samples taken for activity measurements. Usually, 10 such samples were taken between 0D450 0.1-0.5, the activities plotted against OD~5o and expressed as OD420 per rain per OD450 per ml culture.

Chemicals. AI1 chemicals used were analysis grade. Restriction endonucleases were purchased from Boehringer

Mannheim GmbH, FRG. T4 polynucleotide ligase was obtained from Biolabs, Beverly, Ma., U.S.A.

Results

Construction of Fusions between the repA Gene and the lacZ Gene

We have constructed fusions between the repA gene and the lacZ gene by insertion of the Sau3A fragment carrying copA

(designated copA - Sau3A) in the BamH 1 site of plasmid pMC874 which carries the lac operon deleted of its promoter, translational start signal and the first 22 base pairs of the lacZ gene. Based on the nucleotide sequence (Rosen et al. 1980; Stougaard et al. 1981a), as shown in Fig. 1 such fusions should result in the synthesis of a polypeptide consisting of the 3 NH2-terminal ami- no acids of the putative repA protein followed by the/?-galactosi- dase protein (minus its first 7 amino acids). (It should be empha- sized that the assumption made here about the possibility of a translational fusion is based entirely on the nucleotide se- quence; no identification of the repA gene product has been published yet).

Transformants from a ligation mixture were screened for Lac ÷ phenotype on indicator plates and for incompatibility ex- pressed against R 1 derivatives. The presence of an R 1 incompati- bility function indicated that the Sau3A fragment carrying the copA gene and the first 3 codons of the repA gene was present on the hybrid plasmids. One plasmid conferring such a pheno- type, pJL99, was isolated and characterized. Restriction enzyme analysis showed that it consists of pMC874 carrying two contigu- ous Sau3A fragments from R1, one coding for the C-terminal

58

pKNI562

Expanded map of replication con{roE region

fi(3T GTfi ACTIGAT CTT EAE [[A [A[ TGA ETA ~AA fiT(3

repA

B2S3 B2P $3 S~ P 621 /.83 222 1

II II F 2 F I E

BASIC REPLICON

ori

P

CCG GAG 1 GAT CCE GT[ + GGC CT[ ETA G1GG CAG

[ zt I Yl pME8% II I , ;' '

SP H3B~ E

6GT GTfi ACTI fiAT [CC GTC CCA CA[ TfiA ETA ~[}G CAG

L--~Vo, t Thr Asp Pro Va[~l

pJL99 ff r~', sP

Fig. 1. Construction of a fusion between the repA gene and the lac operon. The map of pKN1562 shows the position of recognition sites for the enzymes PstI (P), BglII (B2), and Sau3A ($3), The numbers (1, 222, 483 and 621) refer to nucleotide numbers as previously pub- lished (Stougaard et al. 1981 a). Two copy number control genes (copA and copB), and the repA gene are indicated by the arrows which show the direction of transcription. The replication origin is shown as ori (Oertel et al. 1979). The nucleotide sequence around the Sau3A site (GATC) at position 222, and the predicted sequence of the NHz- terminal end of the repA gene product are indicated. Plasmid pMC874 carries the lac operon deleted of the first 7a/3 codons of the lacZ gene. Recognition sites for the enzymes SalI (S), PstI (P), HindIII (H3), BamH1 (B 0 and EcoR1 (E) are shown. The nucleotide sequence around the BamH1 site and the corresponding amino acid sequence of the NH2-terminal end of the deleted /~-galactosidase polypeptide are shown. Plasmid pJL99 was obtained by ligating Sau3A fragments from pKN1562 to BamH1 restricted pMC874 DNA. The hatched area indicates the insertion

end of the eopB polypeptide, and the other coding for the copA function and the NHz-terminal end of the repA polypeptide in the orientation shown in Fig. 1. The expression of/?-galactosi- dase from this plasmid shows that a fusion between the repA gene and the lac operon was established.

Similar hybrids carrying only the single copA- Sau3A frag- ment (pJL108) have also been isolated, but they produce very low levels of /?-galactosidase only detectable on X-gal plates. Plasmid pJLl l l is another repA-lac fusion plasmid in which a translational fusion between the genes was obtained by inser- tion of a PvuII fragment from pKN1562 in the SmaI site of the promoter cloning plasmid pMC1403 (Fig. 2), Also in this case the nucleotide sequence of R100 indicated the possibility of obtaining a protein fusion. In pJLl l l more than 80% of the putative repA polypeptide is covalently combined with fl- galactosidase. Both the copA and the copB genes are present in pJLl l l , and in contrast to the relatively high level of lac

expression from plasmid pJL99 as observed on McConkey-lac- rose plates plasmid pJL111 results in much less synthesis, which can only be seen on X-gal plates. As described below this differ- ence in repA- lae expression is caused primarily by the difference in genotype of the two plasmids. The expression of/~-galactosi- dase from the plasmids pJL99 and pJL111 shows that the repA gene is both transcribed and translated.

Mapping of the repA Promoter

Although the copA- Sau3A fragment carries the promoter proxi- mal end of the repA gene, the very low level of/~-galactosidase expressed from the hybrid plasmid pJL108 carrying only this fragment on pMC874 compared to that from pJL99 indicates that the repA promoter is not present on this fragment. Also, this difference in lae expression shows that very little, if any, promoter activity from the vector plasmid is responsible for the lac expression from pJL99.

In order to map the repA promoter, different DNA fragments carrying the repA-lac fusion have been transferred to other vector plasmids. Thus, the HindIII-SalI fragment from pJL99 carrying the gene fusion was inserted into plasmid pOU491 re- sulting in plasmid pJL189. This plasmid expresses /~-galactosi- dase at a level comparable to that of pJL99. However, the PstI repA-lac fragment from pJL99 inserted into the PstI site of pGA46 gave rise to two types of plasmids depending on the orientation of the inserted fragment: One (pJL128) which is Lac + on McConkey-lactose plates, and another (pJL127) which is weakly Lac + on X-gal plates but Lac on McConkey-lactose plates. It was also found that insertions of various PstI fragments in the PstI site upstream from the repA-lae fusion in pJL99 result in reduction of lac expression. It therefore seems that the repA promoter is located in the lefthand cloned Sau3A frag- ment of pJL99 (i.e. R1, cf. Fig. 1) near the PstI site.

Regulated Expression of repA - lac

Since expression of the repA gene may be required for R 1 replica- tion and thus could represent a target for the replication control functions, we tested the effect of the presence of various plasmids carrying the replication control genes on repA-lac expression. The test assay consisted of transforming such hybrid plasmids to CSH50 harbouring pJL99 and plating on McConkey-lactose plates containing kanamycin to select for pJL99 and another antibiotic selecting for the incoming plasmid. The Lac phenotype was scored as Lac + or Lac- according to the colour of the colonies on the indicator plates. As shown in Table 2, all plas- raids carrying either the copA or the copB gene or both, irrespec- tive of gene dosage, were found to switch off repA-lae expres- sion from pJL99. In all cases the test plasmids used were compati- ble with pJL99 and did not affect its copy number. Using a plasmid vector that is temperature-sensitive for replication as carrier of the cop genes (pOU494) it was possible to show that this repression was reversible: Transfer of Lac- colonies from 30 o C to 42 ° C resulted in a reversion to Lac + phenotype from pJL99 as the hybrid plasmid was lost. In order to verify that the inhibition of repA- lac expression was copA and copB specif- ic, mutant hybrids which do not express any replication control function as judged from incompatibility and replication studies were also tested. Plasmid pKN393 carries the PstI F1 fragment from the copy mutant pKN104. Sequence analysis has shown that a single base substitution in the copA gene is responsible for the lack of copA activity in this mutant (Stougaard et al. 1981a). This plasmid does not repress repA-lac expression.

pKN1562

+

pMC%03

Km R

CGG CAG ICTG ACG GCC GTC ~' GAC TGC

1 I\I'\'.'-'" 7.'-\\~ I s pl IP sp p

i I E P

/ ATT CCC / 55(5 GAT TAA fiSGi CCC CTA

<___ Oy As~

. A p R - ~ ~ " - z, y I S Smo ~;

59

/

CGG CAG/ GGG fiAT GCC fiTCi CCC £TA

pJL111 I Sma/ l I S ZSmo S S

I~B2 Fig. 2. Contruction of a fusion between the repA gene and the lac operon. The map of pKN1562 shows restriction enzyme sites for PstI (P), PvuII (P2), SalI (S), BglII (B2) and EcoR1 (E). The replication genes are indicated as in Fig. 1. The PvuII site in the 855 base pair repA gene is located 717 base pairs from the initiation codon of the gene, and the map shows the nucleotide and amino acid sequence derived from Rosen et al. (1980) around this site. Plasmid pMC1403 is analogous to pMC874 described in Fig. 1, carrying a deleted lac operon requiring protein fusions for expression of /~-galactosidase. However, this plasmid also has a SmaI site (Sma), useful for ligation of blunt-ended DNA fragments. The nucleotide and amino acid sequences around the SmaI site are shown. Plasmid pJL l l l was obtained by ligation of the PvuII fragment from pKN1562 (hatched region) into the Sinai site of pMC1403 screening for incompatibility against R1, and weak Lac + phenotype on X-gal indicator plates, The orientation and size of the inserted fragment was determined by restriction enzyme mapping analysis, and the indicated map shown to be correct

Table 2. Effects of cop gene products on repA lac expression from pJL99

Plasmid cop genes present added

Lac phenotype of cells carrying pJL99in presence of test plasmid on indicator plates

N o l l e a __

Rldrd-19K-I copA, copB pKN232 copA, copB pOU423 copA - pOU16 copB - pOU494 copA, copB 30 ° C - ; 42 ° C + b pOU393 copA- + pOU417 copA- + pOU30 copB - + pBR322 + pOU491 +(30 ° C)

PIasmid pJL99 itself carries the copA gene, but not the copB gene. b Plasmid pOU494 replicates under the control of pSC101 at 30 ° C,

but under R1 control at 42 ° C. Therefore, at 42°C pOU494 is sensitive to the presence of the copA function expressed from pJL99, is lost from the cells when selection pressure is dropped, and the lac genes are fully expressed

Another PstI F1 hybrid plasmid, pOU417, having a copA pro- moter muta t ion does not switch off repA- lac expression either.

Similarly, hybrid plasmids carrying the copB gene were un- able to inhibit repA-lac expression when the small BglII frag-

ment of the PstI F2 fragment was deleted. This deletion covers the copB promoter and more than half of the structural gene (Molin et al. 1981).

Mutat ions that cause loss of replication control activity in the copA and the copB genes thus simultaneously lead to loss of inhibi tory activity on repA- lac expression.

Plasmid pJL111 carries bo th the copA and the copB genes in addit ion to the repA- lac fusion. On the basis of the observa- tion made with pJL99 that addit ion of copB carrying plasmids greatly reduces repA-lac expression it is not surprising that only a low level of/~-galactosidase activity is found from this plasmid. F rom pJL111 a mutan t derivative was made (pJL133) by deleting the BglII fragment covering par t of the copB gene. This plasmid, which is genetically comparable to pJL199, ex- presses a high level of /%galactosidase activity detectable on McConkey-lactose plates.

The copy number control functions, which have previously been characterized as inhibitors of replication, are thus shown here to act as inhibitors of expression of the repA gene suggesting that the copy number of R 1 is determined by the level of this expression.

repA- lac Expression Reflects R1 Replication

We have performed a series of quanti tat ive comparat ive studies on the effects of addit ion of plasmids carrying the cop genes on plasmid replication and repA-lac expression (Table 3). Plas- mid p J L l l l which carries a repA-lac fusion as well as the

60

Table 3. Comparison of effects of cop gene products on replication of R 1 and on repA-lac expression

R 1 derivative Added plasmid

Plasmid cop genotype Plasmid cop genotype copy number

R 1 copy number

pKN501 copA, copB None pKNS01-1 copA None pKNS01-1 copA pOU545 copA pKNS01-1 cop A pOU15 copB

repA-lac fusion plasmid

pJL111 copA, copB None pJL 133 copA None pJL 133 copA pOU545 copA pJL 133 copA pOUl 5 copB

1 10

3-4 < 1 2 <1

Relative specific activity of fi-galactosidase from fusion plasmid

1 10

3-4 1 2 2 0.5-1

Plasmid copy numbers were determined as single cell resistance to penicillin as described in Materials and Methods, and are given by values relative to that of pKN501. Copy numbers below 1 indicate that a large fraction of the cells have lost the R 1 replicon. Activities of/~-galactosidase were measured as described in Materials and Methods, and the activity expressed from pJL111 was given the value 1

copA and the copB genes is genetically comparable to a wild-type R 1 replicon (e.g. pKN501), and the low activitiy of/~-galactosi- dase activity from pJLl l l may correspond to the low rate of replication of pKN501.

Deletion of the BglII fragment covering part of the copB gene results in a replication rate which is 8 10 fold increased (pKN501-1), and analogously the rate of repA-la¢ expression from pJL133 is increased approximately 10 fold compared to pJLl 11.

Addition of high copy number plasmids carrying the copB gene to cells harbouring wild-type R 1 plasmids has no effect on the replication frequency (copy number) of the R 1 replicon (Riise, manuscript in preparation). Likewise, addition of extra copies of the copB gene to cells carrying pJL111 does not signifi- cantly reduce repA-lac expression (not shown).

Introduction of plasmids carrying either copA or copB to cells harbouring pJL99 or pJL133, both of which are copB-, copA ÷, repA-lac reduces the levels of fl-galactosidase drasti- cally. In the examples in Table 3 cop-hybrid plasmids which have lower copy numbers than the repA-lac hybrid plasmid are used. The effect of the same cop-hybrid plasmids on the replication of the comparable R1 replicon pKN501-1 (copB-, copA +, repA +) is similarly strong: The penicillin resistance drops by more than a factor of 10, and most cells are found to have lost pKN501-1. There is thus a striking resemblance, qualitative as well as quantitative, between the pattern of regulation of plasmid replication and of repA expression.

D i s c u s s i o n

Construction of fusions between the repA gene of plasmid R 1 and the lacZ gene deleted of its NH2-terminal end has been the basis for an analysis of the interactions between the functions known to be important for controlled replication of the plasmid. The repA gene has been identified as a stretch of DNA near the replication origin which is required for replication of the IncFII plasmids (Kollek et al. 1978; Miki et al. 1980; Andres et al. 1979), and the nucleotide sequence analysis showed that a 33,000 dalton polypeptide could be encoded by this region (Rosen et al. 1980). Although the establishment of translational gene fusions described here show that a polypeptide is expressed from this region no direct evidence exists which identifies this protein with the repA encoded positive replication function. A high level of fl-galactosidase activity was only expressed from

repA-lac fusion hybrids having the region near the PstI site, which separates the F1 and F2 fragments. This indicates that the repA promoter is approximately 300 base pairs upstream from the start of the structural gene and, as seen in Fig. 1, the spacing sequence contains the copA gene which is presumably transcribed in a direction opposite to that of repA (Stougaard et al. 1981b). However, as revealed by the nucleotide sequence (Stougaard et al. 1981a), there is also the possibility that a 61 amino acid polypeptide (7K) is encoded by this region overlap- ping the copA gene but transcribed in the same direction as repA. So far no evidence for the existence of this small polypep- tide is available, nor has any function been assigned to it, but the fact that the repA promoter is located upstream to the 7K gene indicates that it is transcribed, and possibly translated, in a fashion similar to that of repA. Supporting evidence for the position of the repA promoter comes from an analysis of in vitro transcripts from the analogous region of the related plasmid R100 (Rosen et al. 1981). The nucleotide sequence of R1 (Stougaard et al. 1981a) indicates two putative promoters (Pribnow boxes) at positions 535 and 495 (cf. Fig. 1). It is not known which of these is important for promoting repA transcrip- tion in R 1.

The two replication control genes, copA and copB, code for functions which have previously been shown to operate as inhibi- tors of replication (Molin and Nordstr6m 1980; Molin et al. 1981). The observation made here, that these functions also in- hibit repA expression, makes it tempting to conclude that plas- mid replication control is exerted at the level of repA expression, and that the target site(s) for these two functions are located between the copB and the repA genes. However, at present we cannot exclude that other sites exist in the plasmid on which these elements may also act.

The inhibitory effect on repA-lacZ expression of addition of extra copies of the copA gene shows that this function acts independently of copB which is absent in some of the hybrids. The presence of the copA gene in all fusion plasmids described here means that one cannot conclude from the presented data whether the copB function operates independently of the copA function, but recent experiments indicate that it does (Light and Molin, in preparation).

Measurements of copy numbers of a copB-, copA +, repA + plasmid and levels of /Lgalactosidase activity from a copB-, copA +, repA-lacZ fusion plasmid showed that relatively small increases in eopA gene dosage resulted in an apparently disporo-

61

port ional reduction in bo th the rate of replication of the R1 replicon and of repA-lac expression from the fusion plasmid. The sensitivity of R 1 replication, and of repA expression, to the dosage of the copA gene suggests tha t the action of the copA gene product may be similar to that proposed by Pri tchard et al. (1969) in the inhibi tor dilution model. Addi t ion of the copB gene also greatly reduced both replication and repA expres- sion. However, the inhibit ion is similarly strong with low and high copy number plasmids (not shown), which may indicate tha t the copB gene is autoregulated. Alternatively, the cellular concentrat ion of the copB gene product may be so high that addi t ional expression from extra gene copies does not significant- ly increase the level of inhibition. Al though no published data exist which directly show how the cop genes are expressed, there are indications tha t the copA activity is amplified when cloned on multicopy plasmids (Molin and Nords t r6m 1980; Andres et ai. 1979; Taylor and Cohen 1979), and we have recently ob- tained evidence using copB - lacZ gene fusions tha t also the copB gene is expressed in propor t ion to gene dosage (Light and Molin, in preparation).

In conclusion the similarity in the response of replication and repA-lac expression to the presence of the hybrid plasmids used in the experiments described here strongly suggests that replication of plasmid R1 is controlled by the action of the copA and copB gene products on the expression of the repA gene. This control may be exerted either on the level of expression of a positively acting and limiting polypeptide product of the repA gene, or on the level of t ranscript ion through this gene to the origin, or both. At present we cannot distinguish between these possibilities. This is a first step towards the unders tanding of the interactions between the replication control functions of a low copy number plasmid, and we are now using this system to analyse previously isolated copy mutants and to isolate new control mutants . Such analyses should lead to identification and characterizat ion of the impor tan t sites in control of replication.

Acknowledgements. The technical assistance of Eva Heyn Olsen is gratefully acknowledged. We thank our colleagues from our depart- ments for many helpful discussions, and E. Riise and P. Stougaard for making unpublished plasmids available. The project was supported by grants from the Danish Medical Research Council and NATO (Grant no. RG170.80).

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Communica ted by J. Schell

Received June 15 / September 9, 1981