Mol Gen Genet (1990) 220:320 324

© Springer-Verlag t990

Comparison of the CopB systems of plasmids R1 and ColV2-K94: A single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid ColV2-K94 Amit Banerjee, Peter C. Weber, and Sunil Palchaudhuri Department of Immunology and Microbiology, WSU School of Medicine, Detroit, MI 48201, USA

Summary, We have isolated a deletion mutation and a point mutation in the copB gene of the replication region Repl of the IncFI plasmid ColV2-K94. Subsequently, this copB gene with and without point mutation was cloned and se- quenced, and the point mutation was mapped in the coding region of copB with a change of one amino acid from argi- nine to serine. Furthermore, this copB mutant had an ap- proximately 10-fold increase in copy number. The CopB- phenotype of ColV2-K94 could be complemented in trans by the copB gene of coresident IncFII plasmids such as R1 and R538, but not R100, suggesting that ColV2-K94 and R1 or R538 contain the same copB allele.

Key words: copB - ColV2-K94 - Plasmid Point mutation - Allele

The RepA replicon has been extensively characterized in the IncFII plasmids R1, R100, and R6-5 (for recent reviews, see Scott 1984; Nordstrom et al. 1984). DNA synthesis in these plasmids initiates at the origin of replication oriR and requires a 33 kDa protein, the product of the repA gene. Expression of the repA gene, and therefore the initia- tion of DNA synthesis, is regulated at the translational and transcriptional levels by the products of copA and copB genes, respectively. A 91 bp untranslated RNA molecule encoded by the copA gene binds to complementary regions of repA transcripts and inhibits their translation into repA protein; this copA RNA also functions as the mediator of IncFII incompatibility. Regulation of replication is also effected by a protein, the product of the copB gene, which binds to the promotor of the repA gene and represses its transcription. Since the constitutively synthesized copB pro- tein is present at saturation levels, transcription from the repA promotor is normally completely repressed. Thus sup- plying additional copies of the copB gene on a high copy number vector has no effect on repA miniplasmid copy number, hence the copB gene is not involved in IncFIi in- compatibility.

A comparison of the eopB nucleotide sequences found in IncFII plasmids revealed a region of non-homology start-

Offprint requests to . S. Palchaudhuri

ing at codon 12 between copB of R1 and copB of R100 and R6-5 (Ryder et al. 1982). Nordstrom and Nordstrom (1985) have compared the copB functions of these plasmids and found that deletion of the copB gene resulted in a 3.5-fold increase in copy number of R100 and an 8-fold increase in copy number of R1; these deletion mutants could be complemented only by homologous and not heter- ologous copB protein. However, when the copB genes were left intact, R100 and R1 both had the same stringently controlled copy number. Thus, although there are two structurally different forms of the copB protein, they both serve an analogous function in the RepA replication system. These alternate forms of the copB gene are therefore alleles, and will be referred to in this work as copBI (the R1 allelic form) and copBII (the R100 and R6-5 allelic form).

Recently, we have identified a RepA-like replication sys- tem, Repl, in the IncFI plasmid ColV2-K94 or its Km r derivative pWS12 (Weber et al. 1984). This replicon has been found to be structurally and functionally homologous to the RepA replicon of R1, but differences in the nucleo- tide sequence of the copA gene have caused a loss of IncFII incompatibility in Repl (Weber and Palchaudhuri 1986). In the present work, we have identified the copB open read- ing frame (ORF) of Repl which may code for a protein essential for normal copy number control in pWS12. This gene appears to be the copBI allele, based on complementa- tion studies and sequence homology with copB of R1, an IncFII plasmid.

The bacterial strains and plasmids used in this work are listed in Table 1. The construction of the ColV2-K94 derivatives pWS12, pWS17, and pWS15, and the cloning of restriction fragments containing the Repl replicon have been described previously (Weber et al. 1984; Weber and Palchaudhuri 1986). For sequencing by the dideoxy meth- od, PstI generated fragments of pWS109 and pWS139 were cloned in M13mp19 bacteriophage using JM103 as host strain. The media employed included LB medium and M9 medium. Antibiotics were used in the following concentra- tions: ampicillin, 50 gg/ml; kanamycin, 100 gg/ml; tetracy- cline, 20 gg/ml; and chloramphenicol, 50 gg/ml.

Copy numbers of cloned Repl regions in the poIA strain C2110 (where pBR322 vector replication is inhibited) were determined as follows. Plasmids were isolated from cultures grown for 14 h in M-9 medium using the rapid alkaline procedure of Birnboim and Doly (1979). The copy numbers ofcopB mutants (i.e., pWS139, pWS109A 1) were compared

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Table l. Bacterial strains and plasmids

Escherichia coli strain or plasmid

Genotype and phenotype Reference or source

Bacterial strains : AB2463 thi thr leuBproA argE Nordstrom et al. (1984)

his rpsL20 fecAl3 lacy HB101 hsdS20 recA13 ara-14 Nordstrom et al. (1984)

proA2 lueB6 lac Y1 galK2 rpsL20 SupE44

C2110 polA1 his rha Nordstrom et al. (1984)

Plasmids : R538 Cm r Sm r Su r Cooper (1971) R100 Cm r Sm r Su r Tc r Hg r Cooper (1971) R1 Apr Smr Su r Km r Cooper (1971) pER35 R1 RepA + PstI fragment Weber et al. (1984)

F2 (Tc r) pWS 12 ColV2-K94 : : Tn903 Weber and

(Km r) Palchaudhuri (1986) pWS17 pWSI2A 110.8V-68.6V Weber and

(Km r) Palchaudhuri (1986) pWS15 A high copy number Weber and

mutant of pWS17 Palchaudhuri (1986) pWSI 6 A deletion derivative of Weber and

the plasmid ColV2-K94 Palchaudhuri (1986) pWS139 H9 fragment ofpWSl5 This paper pWS109 Minimum Repl (H9) This paper

replicon of pWS17 cloned in pBR322

pWS109A1 pWS109 deleted of PstI This paper fragment P19 of H9 (Ap r)

Km, kanarnycin; Ap, ampicillin; Su, sulfonamide; Cm, chloram- phenicol; Tc, tetracycline; Sm, streptomycin; Hg, mercuric salts

to that of the wild-type Repl (i.e. pWS109) by densitometric scanning of Polaroid negatives o f these rapid alkaline isola- tion gels using a Helena Laboratories Quick Scan densit- ometer.

We cloned the Repl replicon on the self-replicating 42 kb EcoRI fragment El and the 4.0 kb HindIII fragment H9 of ColV2-K94 into a multicopy vector pBR322 termed pWS109. A comparison of the physical maps of Repl and RepA showed that both have homologous restriction sites (Fig. 1). The Pstl fragments ofplasmid pWS17 were blotted on to a nitrocellulose membrane and probed with the R100 RepA miniplasmid pRR933; the Repl-containing fragments P2, P18, and PI9 showed homology to RepA. The plasmid pWS109 could replicate in the polA strain C2110 and indi- cated complete recovery of the Repl replicon since the pBR322 replicon was not functional. Deletion of the 0.55 kb P19 from H9 (pWS109A 1) still yielded a functional Repl, but the copy number of this derivative was increased approximately 10-fold (Fig. 2). However, deletion o f both P18 and P19 from H9 abolished Repl function, indicating that the 0.55 kb P18 fragment was essential for replication.

In addition to this copB deletion mutant of Repl gener- ated in vitro (pWS109A 1), a copB point mutation was iden- tified in pWS15, a spontaneously derived high copy number mutant of pWS17. The cloned H9 fragment of pWSI5, termed pWS139, also had a high copy number in the polA host. This replication control mutation was not in the copA gene, as pWS139 exhibited normal Repl incompatibility, and the copA gene sequences of pWS109 and pWS139 were identical (Weber and Palchaudhuri 1986). The nucleotide sequence of the PstI fragments P18 and P19 of pWS109 indicated that Repl contained a cop BI gene (i.e. Rl- type allele) on the adjacent PstI fragments p18 and P19 (Fig. 1). This was supported by the behavior o f p W S l 0 9 A 1 : deletion of the PI9 fragment results in a 10-fold increase in copy number (Fig. 2), which is similar to that of copB mutants of R1 (Nordstrom and Nordst rom 1985). D N A sequence analysis of the P18 fragment of pWSI39 also demonstrated that the copB protein target region was unaltered, so that the copy number mutation of pWS15 had to reside in the promoter or coding region of the copB gene in the P19 fragment (Weber and Palchaudhuri 1986). Sequencing of P19 of pWS139 revealed a point mutation in the coding region of copB with subsequent alteration in an amino acid from arginine to serine at position 15 (Figs. 3 and 4). Out

A copBcopA repA oriR I I " ~ - I l - -

B

97.1V H[n

Pst Pst V V

P6 1.1 kb

Pst Pst Pst V 7 V

F2 F1 0.55kb 0.55kb

98.6V 99.7V Pst Pst Pst v v v

P19 P18 0.55kb 0.5Bkb

V V V

V V V

I. ~7 V

Hae Hae Pst • • V

P4 1.6kb

Hae Pst • v R1

E 1.6kb

Tng03 [3.2 kbl

101.1Vq') Hoe H o e • • Hin II

P2 2.8kb

R100

i pWS109

I pWS109A1

I pWS109A2

102.6V Pst

pWS17

rep inc + ÷

÷ ÷

Fig. 1. A Genetic (top) and restriction (bottom) maps of the RepA replicons in the IncFII plasmids R100 and R1 (based on Nordstrom and Nordstrom 1985). B Restriction map of the Repl replicon in pWS17 or pWSI09, oriented with respect to the RepA maps in A. All restriction fragments shown were cloned in pBR322. Restriction sites shown are: PstI (Pst), HindIII (Hin), HaeII (Hae), and EcoRI (Eco)

"~8x

'~lx ,~10x

~ o o

322

AAAAc

Fig. 2. Plasmid copy number determinations of cloned Repl repli- cons. Rapid alkaline plasmid isolations were performed on C2110 (polA) strains carrying pWS109 (wild-type Repl from pWS17), pWS109A 1 (in vitro-generated copB mutant of Repl from pWS17), and pWS 139 (copB mutant of Repl from pW S 15). Increase in copy number was estimated by densitometric scanning of gel bands, using chromosomal bands as reference peaks. By this method, pWS139 and pWS109AI were found to have 8- and 10-fold in- creases in plasmid copy number, respectively, compared to pWS109

of 63 amino acids o f ColV2-K94 CopB protein, one amino acid differs from the copB protein of R1 plasmid. The codon GTT at the amino acid posi t ion 48 has changed to ATT (Fig. 4) and consequently the ColV2-K94 CopB protein contains isoleucine instead of valine in R1. The main differ-

G T

T A C G T A C G

pWS 109 pWS 139

AA AAA ~ G

T

Fig. 3. Sequences at the point mutation of pWS139 eopB gene com- pared to those of the same region ofpWS109

ence is observed at the init iat ion codon of RI : the absence of the A T G of R1 in our copB gene sequence may result in a smaller protein of molecular weight of 7443 dalton. The copB protein of R1 plasmid is 10500 dal ton in which the init iat ion codon is assumed to be 23 amino a d d s up- stream of the ColV2-K94 CopB init iat ion codon (Stougaard et al. 1981).

As copB mutat ions in RepA can be complemented by the cloned copB gene in trans to restore normal copy

Fig. 4. Nucleotide sequences of the copB gene in ColV2-K94. PstI fragments Pt8 and 19 were cloned in M13 mp19 vector and four clones each from pWSI09 and pWS139 were sequenced. Listed above the ColV2-K94 CopB sequence are differences found in the corresponding sequence for the R1 CopB (Stougard et al. 1981). The mutation of pWS139 at base 202 is marked by an asterisk. This change of C to A has brought about a change in the corresponding amino acid from argine to serine. The base at position 91, which is an A instead of a G in R1, starts the translation of the R1 CopB protein with a molecular weight of 10.5 kDa. Assignment of the protein coding region is based on the Pustell sequence analysis programs (International Biotechnologies)

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Table 2. Cross-reactivity of CopB proteins in RepA-like systems

Plasmid Relevant % loss of resident phenotypes plasmids

pWS15 (Km r) pWS16 high copy (Km r) mutant

pBR322 Tc r Ap r 0 0 pER35 RepA + and copB + func- 90* 2

tions of RI cloned in BR322 (Tc r)

pWS109 RepA1 +, copA + copB + 88 0 functions of ColV-K94 in pBR322

RI00 RepA +, eopA + copB + Tc r 0 1 R538 RepA +, copA + copB + Cm r 89 1

* pER35 complements the copB protein deficiency in pWS15 and causes reduction of its copy number to that of pWS17. The low copy number plasmid pWS17 lacks partition locus and is highly unstable, but its high copy number mutant pWS 15 is stable. Stabili- ty of plasmid inheritance was assessed by measuring the loss of antibiotic resistance characters of the recipient plasmid. The host strain for pWS16 was recA E. coli K12 derivative AB2463. The donor strain in all cases was the same, C2110, whenever the mating was involved

number (Molin et al. 1981), the copy number of the Co1V2- K94 derivative copB mutan t pWS15 was sensitive to coresi- dent I ncF I I plasmids that shared the same copB allele, such as RI or its clone pER35 or R538. Thus the IncFI I plasmid R1 (copBI allele), R100 (copBII allele), and R538 (unknown copB allele) were conjugated into HB101 (pWSI5) ; their effect on PWS15 copy number was measured by direct visu- al ization in rapid alkaline isolat ions and the loss of the resident plasmid was determined by kanamycin sensitivity, as pWS15 carries the kanamycin resistance determinant Tn903. The copy number of pWS15 (pWS17 CopB*) was reduced to that of pWS17 (CopB +) in the presence of R1 clone pER35 and R538, but not R100. The plasmid pWS17 (low copy number) cannot be stably mainta ined since it lacks the par t i t ion loci of ColV-K94. Table 2 shows that the presence of the wild-type CopB plasmid pER35 reduces the copy number of pWS15 and thus eliminates the plasmid pWS15. This effect was copB- and not copA-mediated, as the copA repressor o f RepA and Repl do not cross-react (Weber and Palchaudhur i 1986). This confirmed that ColV2-K94 contains the copBI allele, and R1 but not R100 could complement the CopB protein muta t ion of pWS15. The fact that R538 had the same effect on p W S I 5 copy number as R1 indicated that R538 also contains the copBI allele. These results were suppor ted by the observat ion that the kanamycin resistance level of pWS15 was reduced to that o f pWS17 in the presence of a coresident pER35 or R538 plasmid, and that the kanamycin resistance level of pWS15 was unaffected by a coresident R100 plasmid (Ta- ble 2). Unl ike ampicillin, the kanamycin resistance levels and copy numbers are not strictly p ropor t iona l (Nords t rom et al. 1980; Weber 1986), but there was a significant differ- ence in resistance levels of AB2463 (pWS15) and AB2463 (pWS17).

This work is a cont inuat ion of our character izat ion of Repl, a RepA-l ike repl icat ion system in the IncFI plasmid ColV2-K94. In these studies, we have isolated mutants of

the copB gene to show that the O R F of Repl replicon which is analogous to the CopB protein of RepA but smaller in size (?), is essential for the stringent regulat ion of plasmid copy number. In the copB mutant , the amino acid arginine had changed to serine at posi t ion 15 and such a change showed a different hydropa thy profile of the CopB protein as its amino-terminus (not shown). This raises the question why a poin t muta t ion in CopB protein failed to interact with its target D N A sequence at the RepA promoter . Based on complementa t ion studies with cross-reacting CopB pro- teins of IncFI I plasmids, it appears that ColV2-K94 and interestingly R538, both contain the copBI allele present in the R1 RepA, as opposed to the copBII allele present in the R100 RepA. The presence of identical copB alleles on ColV2-K94 and R538 are further evidence that ColV2- K94 obtained its Repl replicon by recombinat ion with this R plasmid. Genera t ion of a ColV2-K94-1ike chimeric plas- mid by in vitro recombinat ion between an F-l ike ColV fac- tor and R538 has been described previously (Cooper 1971). Such a recombinat ion event which could have produced the present ColV2-K94 probab ly occurred in the middle of the transfer (tra) operon; studies on the tra operons of F-l ike plasmids (Willetts and Skurray 1980) revealed that the alleles of the ColV2-K94 transfer genes traA, traM, traJ, andfinP situated proximal ly to the p romote r are ident- ical to those of F ; the alleles of the ColV2-K94 transfer genes traS, traT, and traZ si tuated distally to the p romote r are identical to those of R538. Thus, by recombinat ion a- cross the middle of its transfer genes, ColV2-K94 probab ly acquired the terminal por t ion of its tra operon and the adjacent RepA replicon from R538. By sequence evolution within the copA gene, this RepA region evolved into the compat ible system Repl.

Acknowledgements. We thank Dr. Soren Molin for his recombinant plasmid containing the RepA and CopB functions of RI. Thanks are also due to Dr. Barry P. Rosen for help with the DNA sequence data analysis. This research was supported by Public Health Service grant AI21273.

References

Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant DNA. Nucleic Acids Res 7:1513-1523

Cooper P (1971) Interaction of a colicinogenic factor with a resis- tance factor and with the fertility factor F in Escherichia coli K 12. Genet Res Camb 17 : 151-159

Molin S, Stongaard P, Light J, Nordstrom M, Nordstrom K (1981) Isolation and characterization of new copy mutants of plasmid RI, and of a polypeptide involved in copy number control. Mol Gen Genet 183 : 323-330

Nordstrom K, Molin S, Aagaard-Hansen H (1980) Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region. Plasmid 4:215-227

Nordstrom D, Molin S, Light J (3984) Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. Plasmid 32:71 90

Nordstrom M, Nordstrom K (3985) Control of replication of FII plasmids: comparison of the basis replicons and of the copB systems of plasmids R100 and R1. Plasmid 13:83-87

Ryder TB, Davidson DB, Rosen JI, Ohtsubo E, Ohtsubo H (3982) Analysis of plasmid genome evolution based on nucleotide- sequence comparison of two related plasmids in Escherichia coli. Gene 17:299-310

324

Scott JR (1984) Regulation of plasmid replication. Microbiol Rev 48 : 153

Stougaard P, Molin S, Nordstom K, Hansen FG (1981) The nucle- otide sequence of the replication control region of the resistance plasmid Rldrd-19. Mol Gen Genet 181:116-122

Weber PC (1986) Ph.D. Thesis, Wayne State University Weber PC, Palchaudhuri S (1986) An inverted repeat sequence

of the IncFI plasmid ColV2-K94 increases multimerization-me- diated plasmid instability. J Gen Microbiol 132: 989-995

Weber PC, Mitra G, Palchaudhuri S (1984) Second replicon in

ColV2-K94 mediates the stable coexistence of two incompatible plasmids. J Bacteriol 160:245-250

Willets N, Skurray R (1980) The conjugation system of F-like plasmids. Annu Rev Genet 14:41 76

Communicated by H. Hennecke

Received April 22, 1989 / September 25, 1989