Replication properties of mini-Rts1 derivatives deleted for DnaA boxes in the replication origin

PLASMID

21,242-246 (1989)

SHORT COMMUNICATIONS 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, whereasthat 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; Nozue et al., 1988). They contain a gene, rep, encoding the Rep protein essential for replication. 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 sequencesof 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 minireplicons also have five or nine repeating units (inc iterons) downstream of rep consisting of a sequence quite similar to that of the ori iterons. 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; Disqu&Kochem et al., 1986; Itoh et al., 1987). ’ Present address:Genetic Engineering Laboratory, Division of Applied Microbiology, National Food Research Institute, Ministry of Agriculture., Forestry and Fisheries, Yatabe, Tsukuba, Ibaragi 305, Japan. 2To whom correspondence should be addressed. 0147-619X/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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 replication (Tsutsui et al., 1983; Kamio and Terawaki, 1983; Chattoraj et al., 1984) or that the Rep protein-bound DNA regions, the inc and ori, would pair and hinder the replication initiation (Pal and Chattoraj, 1988). The Rep proteins are also shown to bind to the promoter 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 causesan 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, appearsto 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).

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In mini-F and mini-P1 it has been demonstrated 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; Muraiso 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 (miniRtsl plus pBR322) lacking a part or the entire DnaA boxes were obtained by using exonucleaseBAL3 1 as previously described (Itoh et ul., 1987). pTW506, having the mini-Rtsl coordinates 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 1381) has no consensus DnaA box in the origin region. These deletion derivatives could be established in the polA host JG112 (Miller et al., 1978) as a plasmid. To verity dispensability 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 COPYNUMBERSOFMINI-Rtsl DERIVATIVES Composition of mini-Rtsl sequence Plasmid pTW519 pTW5 19AE/HII pTW522 pTW522AE/HII pTW563 pTW563AE/HII pTW565 pTW565AEfHII pKPl013d

Mini-Rtsl coordinates”

incl

repA

DnaA box

l-1441 217-1441 l-1381 217-1381 l-1441 217-1441 l-1381 217-1381

+ + + + -

Wild Wild Wild Wild cop1 cop1 cop1 cop1

+ + + + -

Sp resistance level” k/ml) 100 200 50 50 400 800 200 400 25

Copy number’ 7.3 + 20.2 * 3.1 + 3.4 + 22.0 f 50.6 + 13.6 + 19.4 + l-2

0.9 2.4 0.2 0.3 2.0 5.1 1.4 0.8

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 sequenceof pTW601-1 (Kamio et al., 1984) and the 1.85-kb XmnI fragment containing the 1.3kb Sp fragment plus the 0.5-kb mini-Rtsl sequenceof 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 expressedas per chromosome equivalent. The values are averagesof at least two independent determinations. dA mini-F plasmid carrying the same Sp fragment as mini-Rtsl derivatives (Maki et al., 1983).

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kbp spectinomycin resistance fragment of plasmid NR 1 containing no replication function (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 number 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 deletion of ind iterons, could overcome the replication ability deficiency in the DnaA box negative mini-Rts 1, we constructed a seriesof 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 iterons 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 protein might bind strongly to some ori sequence or interact efficiently with host replication factors, such as the DnaA protein at the origin, to lead to an elevated frequency of the replication initiation. Our present results indicate that mini-Rts 1 could replicate without DnaA boxes in ori (Rtsl). We hence examined whether miniRtsl derivatives, especially pTW565AE/HII which has no DnaA box but does replicate well, could replicate in a dnacl-null mutant. The results are presentedin Table 2. The miniRts 1 derivatives were introduced successfully into CM3400 (rnh-244 dnaA+) as well as CM3438 (m/z+ dnaA+) but were not established in the &rail-null strain CM3452 (rnh244 dnaA854: :TnlO) under the same conditions where pBR322 transformed the CM3452 strain. When active DnaA protein was supplied in tram by the dnaA+ resident plasmid

TABLE 2 REQUIREMENT OFTHE dnaA FUNCTIONFORTHE REPLICATIONOFTHE MINI-Rtsl DERIVATIVES

No. of transformants/pg DNA

Plasmid pTW519 pTW5 19AE/HII pTW563 pTW563AE/HII pTW565 pTW565AE/HII pKP1013 pBR322

CM3440 CM3452 CM3438 (mh+ dnaA+) (mh-244 dnaA+) (mh-244 dnaA854::TnIO) 1.1 x 1.1 x 1.5 x 1.6 X 3.9 x 5.4 x 2.4 x 1.2 x

lo5 lo5 IO5 lo5 IO4 lo4 lo4 lo5

1.4 x 2.1 x 6.5 X 7.4 x 1.3 x 1.2 x 2.8 x 1.6 x

lo5 lo5 lo4 lo4 lo5 lo5 IO4 10’

<50 <50 t50 <50 <50 <50 <50 3.6 X lo3

CM3452(pHB9) (rnh-244 dnaA854::TnlO/dnaA+) 1.4 x 2.2 x 3.2 x 3.3 x 2.4 X 2.3 x 2.3 X nt

lo4 lo4 lo4 lo4 lo4 lo4 lo4

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 usedas 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.

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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 sequencefor the DnaA protein is absent from ori(Rts1). As a possibility, a 9bp 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 sequence 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. Although 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. Kogoma for providing the bacterial strains. This work was supported by a grant-in-aid for scientific researchfrom 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.

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