JOURNAL OF BACTERIOLOGY, Nov. 1986, p. 728-733 Vol. 168, No. 2 0021-9193/86/110728-06$02.00/0 Copyright C 1986, American Society for Microbiology Methylation-Dependent Controls Replication of the CloDF13 cop-i(Ts) Mutant ARNOLD J. VAN PUTTEN,1* RONALD DE LANG,' EDUARD VELTKAMP,' H. JOHN J. NIJKAMP,' PIET VAN SOLINGEN,' AND JOHAN A. VAN DEN BERG2 Department of Genetics, Biological Laboratory, Vrije Universiteit, 1081 HV Amsterdam,1 and Gist-brocades, 2600 MA Delft,2 The Netherlands Received 1 April 1986/Accepted 10 July 1986

The CloDF13 cop-l(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42°C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-l(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42°C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA H synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.

The bacteriocinogenic plasmid CloDF13 (9,957 base pairs) creased in comparison with that of the wild-type plasmid. originates from Enterobacter cloacae but is also stably However, at 42°C the plasmid copy number increases dras- maintained in Escherichia coli. In this latter host the plasmid tically. At this latter temperature the mutant plasmid shows is present to the extent of about 10 copies per cell (27). A a "runaway" behavior. Furthermore, cells harboring this small DNA region located between 1.8 and 11.5% of the mutant plasmid are killed at 42°C. CloDF13 genome is essential for autonomous replication (25, We have studied the implications of the cop-i(Ts) muta- 31). This region contains the (oriV) and tion at the molecular level. Our data show that the regulation the information for two transcripts involved in plasmid of DNA replication is altered owing to increased RNA II replication, namely, a preprimer RNA of about 580 nucleo- synthesis. This increase in transcription results in a decrease tides (RNA II) and a small 108-nucleotide repressor RNA in the amount of active RNA I repressor molecules synthe- (RNA I) (30). RNA I is complementary to RNA II and is sized from the opposite strand. involved in the control of the processing of RNA II (11). The Recently, we noticed that the runaway replication of control of replication of CloDF13 strongly resembles that of CloDF13 cop-J(Ts) derivatives was quite variable in dif- other small multicopy like ColEl (28) and pMB1 ferent hosts. We demonstrate that in the absence of Dam (8), particularly with respect to the RNA I-RNA II interac- methylation no increase in plasmid copy number and no tion. Moreover, convergent transcription has been described killing effect is observed at elevated temperatures, and that also for other plasmid replication systems (32). the regulation of DNA replication is restored in this back- Several nonconditional copy number mutants of CloDF13 ground. Analysis of the region surrounding the cop-i(Ts) and related plasmids have been described. The majority of mutation reveals the presence of three putative Dam these mutations map within the region essential for autono- methylation sites (GATC) (10), one of which is disrupted by mous replication, particularly in the part encoding RNA I the cop-J(Ts) mutation (24) (Fig. 1). As shown in this paper (12, 17, 18, 24, 33). However, in the case of ColEl mutations the absence of the Dam methylase leads at 42°C to a outside this region were found that influenced the plasmid decreased primer synthesis, attended with a restoration of copy number (29). It turned out that the presence of a the regulation by RNA I. The significance of Dam methyla- replication regulatory protein, designated Rop (9), on the tion for CloDF13 plasmid replication is discussed. pMB1 and ColEl genome was responsible for this effect. In the case of CloDF13 no evidence could be obtained for the presence of a Rop-like regulatory protein (A. J. van Putten MATERIALS AND METHODS and G. Cesareni, unpublished results). Bacterial strains. The E. coli K-12 strains used in this Beside nonconditional mutants, also some conditional study were N3012 (thr leu thi lacY rpsL minA minB supE copy control mutants of nonconjugative plasmids were de- [2]), C600 (14), 71/18 [A(lac pro) F lacJi ZAM15 pro' (16)]; scribed previously. Those mutants are thermosensitive for GM48 (dam-3 dcm-6 leu thi thr lacY galK galT ara fnuA their control of plasmid replication (3, 17, 33). A conditional tsx-78 supE44 [13]). E. coli HB101 was used for plasmid CloDF13 copy mutation, designated cop-J(Ts), is located constructions. within the region involved in the initiation of preprimer Media, buffers, and chemicals. Cells were grown in synthesis, a region that overlaps with the transcription Lennox broth (LB) medium. LA plates contain LB medium terminator of the cloacin operon (3, 24) (Fig. 1). At 30°C the with 1 to 2% agar. Antibiotics were added at the following plasmid copy number of this mutant is only slightly in- final concentrations: ampicillin (50 to 100 jig/ml) and tetra- cycline (15 ,ug/ml). * Corresponding author. Plasmids and phages. The phages used in this study were 728 VOL. 168, 1986 METHYLATION CONTROLS CloDF13 PLASMID REPLICATION 729

, T -35 homology fT CX G T A T A --'-G-C cop mutation-I(TSA)* A - T T-A * C-G T - A

T - A 3' 669 terminator RNA 694 T-A5A744 * *o - H S* CTAGTTTGGTGGAGGGGTCCACCAAAAAAGCAAATGTCCCGTTTTCTAATGCGCGTCTTTT CTAGAAAAGATGACTTGG initiation RNA II -10 homology terminator cloacin operon FIG. 1. Nucleotide sequence of the region surrounding the RNA II promoter. The terminator structures of the cloacin operon and the RNA I transcript are also shown. Symbols: (*) Dam methylation site, (0) Dcm methylation site.

X132 (9) and XBG37 and 4BG38 (this paper). The plasmids (kb) BglII-BamHI-PvuII DNA fragment cloned into used are listed in Table 1. pBR322. Plasmid pPA153-209 containing the cop-J(Ts) ori- In vitro construction and characterization of recombinant gin of plasmid CloDF13 was constructed by P. M. Andreoli phages and plasmids. For the construction of the recombi- by ligating the 1.74-kb PstI-AvaI fragment of plasmid nant phages 4BG37 and 4)BG38 as well as the plasmids pVU209 (3) to the 2.2-kb PstI-AvaI fragment of plasmid pVU401, pVU402, and pVU403 we made use of standard pAT153 (29). Plasmid pGB210 was constructed by inserting recombinant DNA techniques. DNA fragments containing a 0.4-kb chromosomal E. coli DNA fragment into the PstI the promoter-terminator to be studied were cloned either in site of plasmid pPA153-209. Ampicillin-susceptible trans- HindIll-linearized X132 DNA or SmaI-linearized pKG1800 formants were selected by the method of Boyko et al. (6). DNA. Plaques obtained from the transfection of X132 DNA Plasmid pDAMA was constructed by deleting the 0.5-kb were screened by the method of Benton and Davis (4). BamHI-BamHI fragment from plasmid pDAM118. This frag- Occasionally, clones that were known to contain the pro- ment is a central part of the dam gene. Relevant restriction moter fragments in the right orientation were identified by maps and characteristics of the plasmids used in this study directly plating on McConkey plates and screening for red are given in Fig. 2. plaques. Recombinant plasmids were analyzed after the Determination of I8-galactosidase and galactokinase activi- isolation of plasmid DNA and subsequent digestion with ties. The P-galactosidase activity was determined by the restriction enzymes. The orientation of the cloned fragments procedure of Miller (16). The galactokinase activity in cells was verified by DNA sequence analysis by using the dideoxy harboring pKG1800 derived plasmids was assayed by the chain-termination method (22). We observed no influence of method of Adhya and Miller (1). the cloning of promoter-terminator fragments on plasmid Quantitation of plasmid amplification. The amount of plas- copy number or growth rate of plasmid-containing cells. mid amplification was analyzed by comparing the standard Plasmid pDAM118 containing the DNA adenine methyl- plasmid preparation with and without induction at the ase gene (dam) was described by Brooks et al. (7). Plasmid nonpermissive temperature. The following procedure was pDCM containing the entire EcoRII modification system was used: transformants were grown overnight at 28°C in 10 ml of kindly provided by T. Gingeras. It consists of a 5.4-kilobase LB medium and suspended in 100 ml of fresh LB medium at an optical density at 600 nm of 0.2. The cells were grown at TABLE 1. Plasmids used in this study Eco RI Bam HI/Bgill Source or EcoRI Plasmid Description reference PstI PM pVU206 Clo DF13 cop-l(Ts) 24 derivative pGB 210 Pst (4.3 kb) p DCM pPA153-209 Hybrid plasmid containing P. M. Andreoli kb) Cop_tS1 ~~~(8.1 the CloDF13 cop-l(Ts) Amps, S.Tet tR,cop-i Is Aval AMpR, Tets, dcmr origin pGB210 This study pDAM118 pBR322 derivative 7 copit containing the DNA adenine Eco RI Eco RI methylase gene Bam Hi pDAMA& This study Barn Hi pDCM pBR322 derivative T. Gingeras containing the EcoRII modification system pDAM18 Bar HI PsIl pDAM A pKO-1 Promoter study plasmid 14 (3.8 kb) | (3.3 kb) Arnp,Tet.darn pKG1800 Terminator study plasmid 14 AMPR0, TetlS. darn Pu pKG1820 1* pKG1800 containing the A 21 X t01* readthrough terminator Pvuo pVU401, pVU402, This study FIG. 2. Schematic representation of the plasmids used in this pVU403 study. _2\\;XSP~~~~~~~~~~~~~~~vuD 730 VAN PUTTEN ET AL. J. BACTERIOL.

28°C to an optical density at 600 nm of 1.0 (ca. 109 cells per Plasmid Construction Units galactokinase ml). Half ofthe culture was then shifted to 38°C, and the rest Strain C600 Strain GM48 was incubated at 28°C. After incubation for 150 min an dae dcm dam' dcfn equivalent amount of cells with an optical density at 600 nm 30%C 420C 42%C of 35 was harvested by centrifugation. Plasmid DNA was galK isolated by the method of Birnboim and Doly (5). The pKO-1 f- 23 S0 23 plasmid preparation was dissolved in a volume of 250 ,l of 10 mM Tris hydrochloride [pH 8.01-1 mM EDTA. Before pK6 1800 Pga A 621 613 505 agarose gel electrophoresis the plasmids were linearized by pKG 1120 1x b Pgal T1x - 373 324 digestion with restriction endonuclease EcoRI. pVU 401 Pgal T-P.,a, - 506 568 219

RESULTS pVU 402 Pgai TrnazI -| 72 196 30

Implications of the cop-i(Ts) mutation at the molecular 403 Pgal Trnai(wt)., 75 95 28 level. In a first approach we compared the efficiency of pVU initiation of both the wild-type and the mutant primer (RNA FIG. 4. Galactokinase activities of different plasmids derived II) promoters. For that purpose we made use of the phage from the terminator study plasmid pKG1800. The values listed are lambda vector described by Cesareni et al. (9). This vector mean values ofthree independent experiments. The variation is less includes an unique HindIII site preceding a functional ,B- than 5%. The assay of plasmid pKG1820 1* was performed at 37c galactosidase structural gene that lacks its natural promoter. as recommended by J. Sutiphong. When this vector is inserted into the bacterial chromosome, ,B-galactosidase expression is dependent on the activity of DNA. The resulting plasmids, designated pVU401 and any upstream promoter sequence inserted. To determine the pVU402, harbor the mutant cloacin terminator region and activity of the primer promoter, we have cloned a 160-base- the RNA I terminator, respectively, between the gal pro- pair TaqI-HpaII fragment (nucleotides 651 through 811 [24]) moter and the structural galactokinase gene. As a control, derived from the wild type and the cop-l(Ts) mutant plasmid plasmid pKG1800 was used. C600 (dam' dcm+) cells har- into this vector. After the sticky ends were filled in with boring the different plasmids were assayed for their Klenow polymerase and HindIlI linkers were added, this galactokinase activity (Fig. 4). frgment was cloned into the unique HindIII site of phage We observed that cloning of the cop-1(Ts) promoter- X132 DNA. Subsequently, the 3-galactosidase activity of the terminator region in plasmid pKG1800, resulting in plasmid resulting fusions 4BG37 and 4)BG38 was determined at both pVU401, only leads to a weak reduction ofthe galactokinase 30 and 42°C (Fig. 3). These data demonstrate that de novo expression (Fig. 4). We infer that the galactokinase expres- RNA synthesis from the mutant promoter is three- to four- sion by plasmid pVU401 represents de novo transcription fold increased as compared with its wild-type equivalent. from the RNA II promoter present on this fragment as well Furthermore, we observed that this increased activity oc- as putative readthrough transcription from the gal promoter. curs at both temperatures. We conclude, based on the fact At elevated temperatures the galactokinase expression of that identical fragments were cloned as well as the low pVU401 was only slightly increased (Fig. 4). The cloning of background of transcription from any upstream promoter the RNA I terminator in the appropriate position between sequence (see X132), that the elevated P-galactosidase ex- the gal promoter and the structural galactokinase gene pression is almost entirely due to an increased activity ofthe (pVU402) gives rise to a drastic reduction of the galK mutant RNA II promoter. Although the cop-l(Ts) mutation expression (Fig. 4). In the construction of pVU4O2 interfer- thus affects the initiation ofprimer RNA, the nonconditional ence might occur between the opposite directed transcripts character of this increased activity provides no adequate initiated from the gal and the mutant RNA II promoter, explanation for the conditional, temperature-sensitive phe- which is comparable to the convergent transcription ofRNA notype of the mutant. I and RNA II within the CloDF13 replication region. Since Since the primer promoter overlaps with the region in- besides the gal promoter no transcription-initiating signals volved in the termination of transcription of the cloacin that might direct the expression of the galK gene are present operon, we have studied whether read-through transcription on plasmid pVU402, we conclude that the remaining galK also contributes to the mutant phenotype. Therefore, after expression is entirely due to readthrough transcription. the protruding ends were filled in with Klenow polymerase, Analysis of the RNA I terminator at 42°C revealed that the the 160-base-pair TaqI-HpaII fragment originating from the efficiency of termination is diminished at this temperature. mutant plasmid was cloned into SmaI-linearized pKG1800 Apparently, the temperature-sensitive phenotype of the cop- I(Ts) mutant is caused by a decrease in the RNA I termina- tion efficiency, which may result in the formation of inactive Phage / construction UnitsP galactosidase repressor molecules (see Discussion). To determine whether there is a correlation between the 30 C 42° increased convergent transcription from the mutant RNA II promoter and the occurrence of readthrough transcription at H3 Ioc Z the RNA I terminator in pVU402 we performed the following A132 26 26 experiment. The cognate TaqI-HpaII fragment comprising T Pwt the RNA I terminator and the wild-type primer promoter- p BG 37 7Z.zz7 239 171 cloacin terminator region was isolated from the wild-type T PCOP -1 ( TS) plasmid pEV22. The fragment was cloned in pKG1800, and yBG 38 903 651 the resulting plasmid was designated pVU403. The orienta- FIG. 3. ,B-Galactosidase activities of different phage constructs. tion of the fragment in pVU403 is such that the RNA I The values listed are mean values of three independent experiments. terminator is located in the appropriate position between the The variation is less thanM10%. gal promoter and the galK gene. Convergent transcription in VOL. 168, 1986 METHYLATION CONTROLS CloDF13 PLASMID REPLICATION 731

GM48(pGB210, pDAM118) we prepared plasmid DNA from the cotransformant by the alkaline denaturation method. Plasmid DNA was incubated with restriction endonuclease Sau3A, which cuts DNA irrespective of methylation, and with restriction endonuclease MboI, which splits only nonmethylated DNA. Plasmid pGB210 was only resistant to MboI digestion when plasmid pDAM118 was also present in E. coli GM48, showing that pDAM118 indeed was expressed in E. coli GM48(pGB210, pDAM118) and that pGB210 had been methylated (Table 2). Methylation-regulated RNA transcription is involved in the expression of the mutant phenotype. Since methylation sites are found in the cloacin operon terminator-RNA II promoter region and the RNA I terminator (Fig. 1), we investigated the role of methylation in the functioning of these regulatory signals. C600 (dam' dcm+) and GM48 (dam dcm) cells were FIG. 5. Effect of DNA methylation on runaway replication of a transformed with the plasmids mentioned in Fig. 4. The CloDF13 cop-i(Ts)-derived plasmid, pGB210. E. coli GM48 (dam-3 difference in termination efficiency in both hosts was deter- dcm-6) isolates containing the plasmids pGB210 and pDAM118 mined by assaying the galactokinase activity at 42°C, the (lanes 2 and 3), plasmids pGB210 and pDAMA (lanes 4 and 5), or nonpermissive temperature of the runaway mutant. Figure 4 plasmids pGB210 and pDCM (lanes 6 and 7) were grown at 30°C shows a comparison of the enzyme activities measured in (lanes 2, 4, and 6) or shifted to 38°C for 150 min (lanes 3, 5, and 7), These and standard plasmid preparations were prepared (see Materials and C600 and GM48 cells harboring the different plasmids. Methods). Samples were linearized with restriction endonuclease 'data show that the absence of methylation only slightly EcoRI and analyzed by agarose gel electrophoresis. Other lanes: 1, affects the galactokinase expression in cells harboring the 1-kb DNA molecular weight markers (Bethesda Research Labora- plasmid pKG1800. However, the amount of galactokinase tories, Inc.); 8, control plasmids pDCM, pGB210, pDAM118, and found in pVU401-containing cells is significantly reduced in pDAMA linearized by EcoRI digestion. the methylation-deficient background. It is not clear whether this is the result of a decreased promoter activity or an increased termination efficiency. We determined whether pVU403 occurs between the gal promoter and the wild-type the absence of methylation also leads to a restoration of the RNA II promoter. There was no difference in galactokinase RNA I transcription termination. The efficiency of termina- expression between the plasmids pVU402 and pVU403 at tion of the RNA I terminator was drastically increased in the 30°C (Fig. 4), which indicates that both terminators function absence of methylation at 42°C (Fig. 4). with comparable efficiencies. However, at elevated temper- To gain insight in the sequence specificity of this phenom- atures the efficiency of termination of the wild-type RNA I enon, we have determined the influence of methylation on terminator is, in contrast to its mutant equivalent, only the termination properties of another read-through termina- slightly affected (Fig. 4). We conclude that the increased tor, lambda tol* (21). From the nucleotide sequence of this primer synthesis by the mutant RNA II promoter leads at terminator, it is known that it contains neither Dam nor Dcm elevated temperatures to a decrease in the efficiency of the methylation sites. We have transformed C600 and GM48 terminator structure involved in the termination of the cells with the plasmid pKG1820 1*, which contains this convergent RNA I transcript. terminator. Subsequently we have analyzed these cells for Influence of methylation on the expression of the mutant their galactokinase activity. Assays were performed at 37°C, phenotype. We observed that the expression of the cop-l (Ts) as recommended by J. Sutiphong (personal communication). phenotype was abolished in the host GM48 lacking the DNA adenine and DNA cytosine methylation enzymes (13). To investigate which of the DNA methylation enzymes was TABLE 2. Effect of DNA methylation on plasmid amplification in E. GM48 (dam-3 runaway replication of plasmid and cop-J(Ts) effect of plasmid pGB210 coli responsible for the loss of dcm-6) pGB210, this plasmid was cotransformed into E. coli GM48 (dam-3 dcm-6) with plasmids containing the DNA adenine Growtha at: Digestability of methylation gene (pDAM118) and the DNA cytosine meth- Plasmid DNA withb: Amplification of ylation (pDCM) gene, respectively. 30°C 42°C Sau3A MboI at 38°CC Cotransformation of plasmid pDAM118 carrying the + + + + + cloned DNA adenine methylation gene of E. coli clearly pGB210 pGB210 + - + - + + + + + restored the runaway replication behavior of plasmid pDAM pGB210 (Fig. 5, lane 3). Concurrently with the restoration of pGB210 + + + + + + the runaway replication, growth at 43°C was also inhibited. pDAMA Cotransformation with plasmid pDAMA containing a deleted pGB210 + + + + + + + DNA adenine methylation gene (Fig. 5, lane 5) and with pDCM plasmid pDCM carrying the EcoRII modification gene (lane a Colonies were streaked onto LA plates and incubated at 30 and 42°C. 7) did not restore the runaway replication of plasmid b Plasmid DNA was extracted (see Materials and Methods) and digested pGB210. This shows that DNA cytosine methylation has no with restriction endonuclease Sau3A, which cuts GATC sequences irrespec- effect on the runaway replication of plasmid pGB210. More- tive of adenine methylation, and with restriction endonuclease Mbol, which over, the effect of pBR322 sequences acting in trans can be only splits nonmethylated GATC sequences. c Plasmid DNA was extracted according to the standard plasmid procedure ruled out. (see Materials and Methods). The extent of plasmid amplification was To demonstrate that the cloned DNA adenine methylation determined by scanning photographs of agarose gels of standard plasmid gene was indeed expressed in the cotransformant E. coli preparations (Fig. 5). 732 VAN PUTTEN ET AL. J. BACTERIOL.

The readthrough transcription, as a result of the decreased dam dEm termination efficiency ofthis terminator, was hardly affected tlo DF13 AC C T T C G AT C AA AC C AC C T C I C C AG G T G G T T T T T T C G T T T by the absence of methylation (Fig. 4). _ T G G T Col El T C C G G G A A A C A A A C C A C C G T T A G C G G T G G T T T T T T T G T T T DISCUSSION GG T Our experiments provide more insight into the implica- pM141 T C C G G C A A A C A A AC C A C C G C T A G C G G T G 0 T T T T T T 0 T T T tions at the molecular level of the CloDF13 cop-l(Ts) muta- tion and the role of host functions in the expression of the G C T A mutant phenotype. The CloDF13 cop-l(Ts) mutation is C C T A T C C* T G C G located in an area involved in both the initiation of primer T A G C G C C G C 6 C G transcription and the termination of the cloacin operon C G C 6 C G A T A T A T transcript. This region comprises several putative Dam C C G C G C 6 C G C G methylation sites. The mutation, a G-to-A transition, may A T A T A T A T A T A T destabilize the stem-loop structure that could be formed in * A T A T A A T this region, but also disturbs one of the two opposite Dam G A T C T T T A A A C T T T A A A c T T T methylation sites within this palindrome. The host-encoded Clo DF13 Col El pm18 Dam methylase is essential for the expression of the mutant FIG. 6. Comparison of the DNA regions surrounding the RNA I phenotype. In a methylation-deficient background (dam- terminator of CloDF13, ColEl, and pMB1. dcm-) no increased plasmid copy number and no kill effect on host cells at elevated temperatures was observed. The mutation results in a dam' dcm+ host in a nonconditional stem-loop structure (Fig. 1) are methylated. The absence of increased primer synthesis due to an elevated primer pro- methylation here has no or little effect on the functioning of moter activity, which interferes at elevated temperatures this region. However, in the cop-1(Ts) mutant plasmid only with the termination of the convergent overlapping RNA I one GATC sequence is present, which results in an asym- transcript. Panayotatos (19) performed experiments in which metric methylation. Apparently, the presence of only one priming of replication was effected by constitutive or induc- methylation site leads to a disturbance of the structure and ible promoters. Although the increased primer synthesis has function of this region. The absence of methylation in a dam some influence on the copy number of these ColEl-derived host restores the regulation of plasmid replication to the plasmids, no runaway phenotype was observed. These data wild-type level. Based on these observations, we conclude indicate that at least in the case of ColEl an increased primer that not the G-to-A transition but the methylation ofonly one synthesis does not lead to a runaway phenotype. It was of the opposite GATC sequences within the stem-loop demonstrated by Tamm and Polisky (26) for ColEl that an structure is important for the expression of the mutant extended RNA I species as a result of readthrough transcrip- phenotype. tion is unable to perform its repressor function in vivo. We infer that readthrough transcription at the RNA I terminator ACKNOWLEDGMENTS at 42°C, resulting in an extended "inactive" RNA I tran- script, contributes to the expression of the runaway pheno- We thank G. Cesareni for his help and interest during the initiation type. of this work, M. Rosenberg and T. R. Gingeras for the generous gift of plasmids, and Karin Uyldert for typing the manuscript. We have investigated the functioning of the different This investigation was supported by the Netherlands Foundation transcription regulatory structures in the presence and ab- for Chemical Research with the financial aid from the Netherlands sence of methylation. From these experiments we obtained Organization for the Advancement of Pure Research. evidence that the absence of methylation leads to a reduced primer synthesis, either by an increased transcription termi- LITERATURE CITED nation of the cloacin terminator or a decreased transcription 1. Adbya, S., and W. Miller. 1979. Modulation of the two promot- initiation from the RNA II promoter or both. We observed ers ofthe galactose operon ofEscherichia coli. Nature (London) that this reduction in RNA II transcription is accompanied 279:492-494. by a restoration of the termination at the RNA I terminator. 2. Adler, H. I., W. D. Fisher, A. Cohen, and A. A. Hardigree. 1967. Although the RNA I terminator regions of CloDF13, ColEl, Miniature Escherichia coli cells deficient in DNA. Proc. Natl. and pMB1 show a high degree of conservation, differences Acad. Sci. USA 57:321-326. are found with respect to the presence of a Dam and a Dcm 3. Andreoli, P. M., R. A. Brandsma, E. Veltkamp, and H. J. J. might explain the Nijkamp. 1978. Isolation and characterization of a methylation site (Fig. 6). Those features CloDF13::Tn9O0 mutant with thermosensitive control of DNA difference in the regulation of plasmid replication between replication. J. Bacteriol. 135:612-621. CloDF13 and related plasmids. 4. Benton, W. D., and R. W. Davis. 1977. Screening Xgt recombi- Dam methylation sites have been identified in the replica- nant clones by hybridization to single plaques in situ. Science tion regions of several organisms (34). Recently, Messer and 196:180-182. co-workers (15) and Smith et al. (23) showed the importance 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction of methylation of GATC sequences in the efficient initiation procedure for screening recombinant plasmid DNA. Nucleic of DNA replication at the oriC of E. coli. Whether this Acids Res. 7:1513-1523. methylation is directly involved in the regulation of tran- 6. Boyko, W. L., and R. E. Ganschow. 1982. Rapid identification of as described for the mom gene of phage Mu (20) is Escherichia coli transformed by pBR322 carrying inserts at the scription PstI site. Anal. Biochem. 122:85-88. unknown. 7. Brooks, J. E., R. M. Blumenthal, and T. R. Gingeras. 1983. The In this manuscript we describe a clear example of the isolation and characterization of the DNA adenine methylase influence of methylation on the replication control of the (dam) gene. Nucleic Acids Res. 11:837-851. CloDF13 cop-1(Ts) mutant. We infer that in the wild-type 8. Cesareni, G. 1981. The target of the negative regulator of pMB1 plasmid both opposite GATC sequences in the stem of the replication overlaps with part of the repressor coding sequence. VOL. 168, 1986 METHYLATION CONTROLS CloDF13 PLASMID REPLICATION 733

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