PLASMID 21,242-246 (1989)

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Replication Properties of Mini-Rtsl Derivatives Deleted for DnaA Boxes in the Replication Origin

YOSHIFUMI ITOH’ AND YOSHIRO TERAWAKI~

Depanment of Bacteriology, Shinshu University School of Medicine, Asahi 3-l-1, Matsumoto 390, Japan

Received November 10, 1988; revised March 17, 1989

Mini-Rtsl was found to be unable to replicate in a dna/l-null mutant. However, a mini-Rtsl derivative lacking entire tandem DnaA boxes in the replication origin retained the replication ability in a dnaA+ host although its copy number was about half that of the mini-Rtsl having complete DnaA boxes. Mini-Rtslcopl that contains a high copy number mutation in repA was found to replicate more efficiently than mini-Rtsl of wild repA when DnaA boxes were deleted. In addition, the copy number of mini-Rtslcopl without DnaA boxes increased 1.5fold upon removal of incl iterons, whereas that of mini-Rtsl without DnaA boxes did not increase after the iterons were deleted. These indicate that the RepAcopl protein can initiate the replication of mini-Rtsl efficiently even when DnaA boxes are absent from the origin of replication. 0 1989

Academic Pms, Inc.

The minimal regions for the autonomous replication, minireplicon, of Rts 1, F, and P 1 have analogous structures (Kamio et al., 1984; Murotsu et al., 1981; Abeles et al., 1984; No- zue et al., 1988). They contain a gene, rep, encoding the Rep protein essential for repli- cation. Upstream of the rep gene there exists the replication origin, ori, about 300 bp long. The ori region carries three to five copies of repeating sequences of ca. 20 bp (ori iterons) and a duplicated DnaA box, a 9-bp sequence for binding of DnaA protein (Fuller et al., 1984) at an end distal from rep. These mini- replicons also have five or nine repeating units (inc iterons) downstream of rep consisting of a sequence quite similar to that of the ori it- erons. The ori iterons are indispensable for the origin activity, whereas the inc iterons are nonessential, functioning only as a negative regulator and exerting incompatibility (Kamio and Terawaki, 1983; Chattoraj et al., 1984; Tsutsui et al., 1983; Austin et al., 1985; Dis- qu&Kochem et al., 1986; Itoh et al., 1987).

’ Present address: Genetic Engineering Laboratory, Di- vision of Applied Microbiology, National Food Research Institute, Ministry of Agriculture., Forestry and Fisheries, Yatabe, Tsukuba, Ibaragi 305, Japan.

2 To whom correspondence should be addressed.

Recently, Rep proteins of F, P 1, and Rts 1 were purified and shown to bind to both ori and inc iterons (Tokino et al., 1986; Abeles 1986; Kamio et al., 1988). Binding of Rep proteins to the inc iterons supports the idea that the iterons titrate the Rep protein to limit the amounts of the protein involved in repli- cation (Tsutsui et al., 1983; Kamio and Ter- awaki, 1983; Chattoraj et al., 1984) or that the Rep protein-bound DNA regions, the inc and ori, would pair and hinder the replication ini- tiation (Pal and Chattoraj, 1988). The Rep proteins are also shown to bind to the pro- moter region of rep, resulting in autoregulation of the Rep protein synthesis (Rokeach et al., 1985; Chattoraj et al., 1985; Terawaki et al., 1988). Mutants, cop, having a mutation in rep that causes an increase in the copy number of the plasmid have been isolated in mini-F (Kline and Trawick, 1983), mini-P 1 (Froehlich and Scott, 1988), and mini-Rtsl (Kamio et al., 1984; Terawaki and Itoh, 1985) as well as R6K (Stalker et al., 1983; Inuzuka and Wada, 1985). Thus, the replication frequency, i.e., the copy number, appears to be controlled by the amount and quality of Rep proteins in these plasmids (Chattoraj et al., 1985; Swack et al., 1987; Froehlich and Scott, 1988; Pal and Chattoraj, 1988).

0147-619X/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

242

SHORT COMMUNICATIONS 243

In mini-F and mini-P1 it has been dem- onstrated both in vivo and in vitro that the replication of these plasmids depends on the dnaA liutction (or DnaA protein) (Hansen and Yarmolinsky, 1986; Kline et al., 1986; Mu- raiso et al., 1987; Murakami et al., 1987; Wickner and Chattoraj, 1987). It was further shown that deletions in the DnaA boxes of the mini-F origin abolish the replication ability (Murakami et al., 1987). By contrast, the ori(Rts1) lacking DnaA boxes could function although its replication ability was lower than that of the origin with the complete DnaA boxes (Itoh et al., 1987). We describe here the replication function of mini-Rts 1 derivatives that contain a combination of the elements affecting the copy number of the plasmid, i.e., cop1 mutation with or without the DnaA box and inc iterons. We also examined the repli-

cation of mini-Rts 1 derivatives in a dnatl-null mutant.

The deletion derivatives, pTW506, pTW507, and pTW508, of pTW602 (mini- Rtsl plus pBR322) lacking a part or the entire DnaA boxes were obtained by using exonu- clease BAL3 1 as previously described (Itoh et ul., 1987). pTW506, having the mini-Rtsl co- ordinates 1 to 1395, has lost 8 bp of the outer DnaA box, pTW507 (coordinates 1 to 1389) has a deletion that proceeded into 6 bp of the inner box, and pTW508 (coordinates 1 to 138 1) has no consensus DnaA box in the or- igin region. These deletion derivatives could be established in the polA host JG112 (Miller et al., 1978) as a plasmid. To verity dispens- ability of the DnaA boxes for replication of Rts 1, we ligated the mini-Rts 1 fragments from pTW506, pTW507, and pTW508 with the 2.5-

TABLE 1

CONSTITUENTS AND COPY NUMBERS OF MINI-Rtsl DERIVATIVES

Plasmid Mini-Rtsl

coordinates”

Composition of mini-Rtsl sequence Sp resistance

level” incl repA DnaA box k/ml) Copy number’

pTW519 l-1441 + Wild + 100 7.3 + 0.9 pTW5 19AE/HII 217-1441 - Wild + 200 20.2 * 2.4 pTW522 l-1381 + Wild 50 3.1 + 0.2 pTW522AE/HII 217-1381 - Wild - 50 3.4 + 0.3 pTW563 l-1441 + cop1 + 400 22.0 f 2.0 pTW563AE/HII 217-1441 - cop1 + 800 50.6 + 5.1 pTW565 l-1381 + cop1 - 200 13.6 + 1.4 pTW565AEfHII 217-1381 - cop1 - 400 19.4 + 0.8 pKPl013d 25 l-2

Note. pTW5 19 and pTW522 were constructed by ligation of the mini-Rtsl fragments from pTW505 (Itoh et al., 1987) and pTW508 (see the text), respectively, with the 2.5-kb spectinomycin (Sp) resistance fragment of pTW601 (Itoh et al., 1982). The incl deletion derivatives (pTW519AE/HII and pTW522AE/HII) were obtained by replacing the XbaI (at the coordinate 5 12~IIindIIl fragment of pTW604 (Kamio and Terawaki, 1983) with the relevant fragments of pTW505 and pTW508, respectively. The 2.1&b XmnI fragment containing a part of the Sp fragment ( 1.3 kb) plus the 0.7 kb mini-RtsI sequence of pTW601-1 (Kamio et al., 1984) and the 1.85-kb XmnI fragment containing the 1.3- kb Sp fragment plus the 0.5-kb mini-Rtsl sequence of pTW601-1 AE/HII (Terawaki and Itoh, 1985) were combined with the 2.0-kb XmnI fragment of pTW519AE/HII and pTW522AE/HII, giving rise to pTW563, pTW563AE/HII, pTW565, and pTW565AE/HII. The host strain used was JC1569 (rec4).

’ Mini-Rts I coordinates were taken from Ramio et al., 1984. b Single colony r&stance level to Sp was determined as previously (Terawaki and Itoh, 1985). ‘Copy number was determined by measuring the amount of plasmid DNA as previously (Terawaki and Itoh, 1985)

and expressed as per chromosome equivalent. The values are averages of at least two independent determinations. dA mini-F plasmid carrying the same Sp fragment as mini-Rtsl derivatives (Maki et al., 1983).

244 SHORT COMMUNICATIONS

kbp spectinomycin resistance fragment of plasmid NR 1 containing no replication func- tion (Itoh et al., 1982) giving rise to pTW520, pTW52 1, and pTW522, respectively. These plasmids without the complete DnaA boxes could be introduced into the recA host JC 1569 (Clark et al., 1966). However, the copy num- ber of pTW522 was about half that of pTW5 19 which has complete DnaA boxes (Table 1). pTW520 and pTW521 also had lower copy numbers than did pTW522 (data not shown).

To examine whether the mutations that make the copy number increase, such as the cop1 mutation which is due to a single amino acid residue alteration in the RepA protein We142 --* Lys) (Kamio et al., 1984) and dele- tion of ind iterons, could overcome the rep- lication ability deficiency in the DnaA box negative mini-Rts 1, we constructed a series of derivatives from pTW519 and pTW522. As shown in Table 1, pTW563 carrying the cop1 mutation had a copy number 3-fold higher than that of the wild-type pTW5 19. The effect of cop1 mutation along with the deletion of ind iterons on the copy number was additive as shown with pTW563AE/HII (Table 1) and as reported previously (Terawaki and Itoh, 1985). It should be noted that the removal of

incl iterons from pTW565 (repAcop1 and no DnaA box) caused a 1.5-fold increase in the copy number, whereas the deletion of the it- erons in pTW522 (wild repA and no DnaA box) caused no effect on the copy number of the plasmid (Table 1). Although the altered nature of the RepAcopl protein is not yet well analyzed, we presume that the RepAcopl pro- tein might bind strongly to some ori sequence or interact efficiently with host replication fac- tors, such as the DnaA protein at the origin, to lead to an elevated frequency of the repli- cation initiation.

Our present results indicate that mini-Rts 1 could replicate without DnaA boxes in ori (Rtsl). We hence examined whether mini- Rtsl derivatives, especially pTW565AE/HII which has no DnaA box but does replicate well, could replicate in a dnacl-null mutant. The results are presented in Table 2. The mini- Rts 1 derivatives were introduced successfully into CM3400 (rnh-244 dnaA+) as well as CM3438 (m/z+ dnaA+) but were not estab- lished in the &rail-null strain CM3452 (rnh- 244 dnaA854: :TnlO) under the same condi- tions where pBR322 transformed the CM3452 strain. When active DnaA protein was sup- plied in tram by the dnaA+ resident plasmid

TABLE 2

REQUIREMENT OF THE dnaA FUNCTION FOR THE REPLICATION OF THE MINI-Rtsl DERIVATIVES

No. of transformants/pg DNA

Plasmid CM3438 CM3440 CM3452 CM3452(pHB9)

(mh+ dnaA+) (mh-244 dnaA+) (mh-244 dnaA854::TnIO) (rnh-244 dnaA854::TnlO/dnaA+)

pTW519 1.1 x lo5 1.4 x lo5 pTW5 19AE/HII 1.1 x lo5 2.1 x lo5 pTW563 1.5 x IO5 6.5 X lo4 pTW563AE/HII 1.6 X lo5 7.4 x lo4 pTW565 3.9 x IO4 1.3 x lo5 pTW565AE/HII 5.4 x lo4 1.2 x lo5 pKP1013 2.4 x lo4 2.8 x IO4 pBR322 1.2 x lo5 1.6 x 10’

<50 <50 t50 <50 <50 <50 <50

3.6 X lo3

1.4 x lo4 2.2 x lo4 3.2 x lo4 3.3 x lo4 2.4 X lo4 2.3 x lo4 2.3 X lo4

nt

Note. The Escherichia co/i strains, gifts from T. Kogoma, were described by von Meyenburg et al.. 1987. pHB9 carrying the dnaA gene (Ohmori et al., 1984) was supplied by H. Ohmori. The mini-F plasmid pKPlOl3 (Maki et al., 1983) and pBR322 (Bolivar et al., 1977) were used as controls for the dnuA-dependent and dnukindependent plasmid. Transformation was performed as described by Maniatis et al., 1982, and transformants were selected on an L-agar containing either 25 pg spectinomycin/ml or 30 pg ampicillin/ml. nt, not tested.

SHORT COMMUNICATIONS 245

pHB9 (Ohmori et al., 1984), the mini-Rtsl derivatives were established in the &u&null host. Thus, the dnaA function appears to be a prerequisite for the replication of Rtsl but the replication could be initiated even when the recognition sequence for the DnaA protein is absent from ori(Rts1). As a possibility, a 9- bp sequence (TTTTCCACA, 1175- 1167) (Kamio et al., 1984) which contains one base substitution with the DnaA consensus (Fuller et al., 1984) and overlaps with the - 10 se- quence of the repA promoter that is located outside of ori(Rts1) (Itoh et al., 1987) could compensate the absence of the DnaA box in the origin for initiating the replication. Al- though we presently have no experimental data to exclude the possibility, the present study should provide a clue to understanding the mode of interaction between RepA and DnaA proteins at the replication origin of Rtsl.

ACKNOWLEDGMENTS

We thank H. Ohmori for supplying pHB9 and T. Ko- goma for providing the bacterial strains. This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.

REFERENCES

ABELES, A. L. (1986). Pl plasmid replication. Purification and DNA-binding activity of the replication protein RepA. J. Biol. Chem. 261, 3548-3555.

ABELES, A. L., SNYDER, K. M., AND CHAI-~ORAJ, D. K. (1984). Pl plasmid replication: Replicon structure. J. Mol. Biol. 173, 301-324.

AUSTIN, S. J., MURAL, R. J., CHAI-~ORAJ, D. K., AND ABELES, A. L. (1985). Truns- and c&acting elements for the replication of PI miniplasmids. J. Iwo/. Biof. 183, 195-202.

BOLIVAR, F., RODRIGUEZ, R. L., GREEN, P. J., BETLACH, M. C., HEYNECKER, H. L., BOYER, H. W., AND FAL- KOW, S. (1977). Construction and characterization of new cloning vehicles. II. A multiple cloning system. Gene 2,95-113.

CHATTORAJ, D. K., CORDES, K., AND ABELES, A. (1984). Plasmid Pl replication: Negative control by repeated DNA sequences. Proc. Null. Acad. Sci. USA 81,6456- 6460.

CHATTORAJ, D. K., SNYDER, K. M., AND ABELES, A. L. (1985). PI plasmid replication: Multiple functions of RepA protein at the origin. Proc. Natl. Acad. Sci. USA 82,2588-2592.

CLARK, A. J., CHAMBERLIN, M., AND BOYCE, R. P. ( 1966). Abnormal metabolic response to UV light of a recom- binant deficient mutant of E. coli K-12. J. Mol. Biol. 130, 161-173.

DISC&KOCHEM, C., SEIDAL, U., HELSBERG, M., AND EICHENLAUB, R. (1986). The repeated sequences (incB) proceeding the protein E gene of plasmid mini-F are essential for replication. Mol. Gen. Genet. 202, 132- 135.

FROEHLICH, B. J., AND SCOTT, J. R. (1988). A single amino acid difference between Rep proteins of PI and P7 a&& plasmid copy number. Plasmid 19, 121-133.

FULLER, R. S., FUNNELL, B. E., AND KORNBERG, A. (1984). The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38, 889-900.

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 83,4423-4421.

INUZUKA, M., AND WADA, Y. (1985). A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K. EMBO J. 4,2301-2307.

ITOH, Y., KAMIO, Y., FURUTA, Y., AND TERAWAKI, Y. (1982). Cloning of the replication and incompatibility regions of a plasmid derived from R&l. Plasmid 8,232- 243.

ITOH, Y., KAMIO, Y., AND TERAWAKI, Y. (1987). Essential DNA sequence for the replication of Rts 1. J. Bacterial. 169, 1153-1160.

KAMIO, Y., ITOH, Y., AND TERAWAKI, Y. (1988). Pm% fication of Rtsl RepA protein and binding of the protein to mini-Rtsl. J. Bacterial. 170,441 l-4414.

KAMIO, Y., TABUCHI, A., ITOH, Y., KATAGIRI, H., AND TERAWAKI, Y. (1984). Complete nucleotide sequence of mini-Rtsl and its copy mutant. J. Bacterial. 158, 307-312.

KAMIO, Y., AND TERAWAKI, Y. (1983). Nucleotide se- quence of an incompatibility region of miniRts1 that contains five direct repeats. J. Bacterial. 155,1185- 119 1.

KLINE, B. C., KOGOMA, T., TAM, J. E., AND SHIELDS, M. S. (1986). Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance. J. Bacterial. 168,440-443.

KLINE, B. C., AND TRAWICK, J. (1983). Identification and characterization of a second copy number control gene in mini-F plasmids. Mol. Gen. Genet. 192,408-4 15.

MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. ( 1982). “Molecular Cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

MAKI, S., KURIBAYASHI, M., MIKI, T., AND HORIUCHI, T. (1983). An amber replication mutant of F plasmid mapped in the minimal replication region. Mol. Gen. Genet. 191,23 l-237.

MILLER, J., MANIS, J., KLINE, B., AND BISHOP, A. (1978). Nonintegrated plasmid-folded chromosome complexes. Plasmid 1, 273-283.

246 SHORT COMMUNICATIONS

MURAISO, K., TOKINO, T., MUROTSU, T., AND MATSU- BARA, K. (1987). Replication of mini-F plasmid in vitro promoted by purified E protein. Mol. Gen. Genet. 206, 519-521.

MURAKAMI, Y., OHMORI, H., YURA, T., AND NAGATA, T. (1987). Requirement of the Escherichiu coli dnaA gene function for ori-2dependent mini-F plasmid rep- lication. J. Bacterial. 169, 1724- 1730.

MUROTW, T., MATSUBARA, K., SUGISAKI, H., AND TAK- ANAMI, M. (1981). Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene 15, 257-27 1.

NOZUE, H., TSUCHIYA, K., AND KAMIO, Y. (1988). Nu- cleotide sequence and copy control function of the ex- tension of the incl region (incl-b) of Rtsl. Plasmid 19, 46-56.

OHMORI, H., K~MURA, M., NAGATA, T., AND SAKAKI- BARA, Y. (1984). Structural analysis of the dnaA and dnaN genes of Escherichia coli. Gene 8, 159- 170.

PAL, S. K., AND CHA~ORAJ, D. K. (1988). PI plasmid replication initiator sequestration is inadequate to ex- plain control by initiator-binding site. J. Bacterial. 170, 3554-3560.

ROKEACH, L. A., S&AARDANDERSEN, L., AND MOLIN, S. (1985). Two functions of the E protein are key ele- ments in the plasmid F replication control system. J. Bacterial. 164, 1262- 1270.

STALKER, D. M., FILUTOWICZ, M., AND HELINSKI, D. R. (1983). Release of initiation control by a mutational alteration in the R6K K protein required for plasmid DNA replication. Proc. Nat/. Acad. Sci. USA 80,5500- 5504.

SWACK, J. A., PAL, S. K., MASON, R. J., ABELES, A. L., AND CHATTORAJ, D. K. ( 1987). PI plasmid replication: Measurement of initiation protein concentration in vivo. J. Bacterial. 167, 3737-3742.

TERAWAKI, Y., HONG, Z., ITOH, Y., AND KAMIO, Y. (1988). Importance of the C terminus of plasmid Rtsl RepA protein for replication and incompatibility of the plasmid. J. Bucteriol. 170, 126 I-1267.

TERAWAKI, Y., AND ITOH, Y. (1985). Copy number of mini-Rtsl: Lowered binding affinity of mutated RepA protein to direct repeats. J. Bacterial. 162, 72-77.

TOKINO, T., MUROTSU, T., AND MATSUBARA, K. (1986). Purification and properties of the mini-F plasmid-en- coded E protein needed for autonomous replication control of the plasmid. Proc. Natl. Acad. Sci. USA 83, 4109-4113.

TSUTSUI, H., FUJIYAMA, A., MUROTSU, T., AND MAT- SUBARA, K. (1983). Role of nine repeating sequences of the mini-F genome for expression of F-specific in- compatibility phenotype and copy number control. J. Bacterial. 155, 337-344.

VON MEYENBURG, K., ROYES, E., SKARSTAD, K., KOPPES, L., AND KOGOMA, T. (1987). Mode of consitutive stable DNA replication in RNaseHdefective mutants of Escherichia coli K-12. J. Bacterial. 169,2650-2658.

WICKNER, S. H., AND CHATTORAJ, D. K. (1987). Repli- cation of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage PI and the dnaA gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 84,3668-3672.

Communicated by Donald R. Helinski