isolation and characterization of plasmid psw200 from erwinia stewartii

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Isolation and Characterization of Plasmid pSW200 from Erwinia stewartii Jen-Fen Fu, Jer-Ming Hu, Yu-Sun Chang, and Shih-Tung Liu 1 Molecular Genetics Laboratory, Department of Microbiology and Immunology, Chang-Gung University, Kwei-Shan, Taoyuan, 333, Taiwan Received December 16, 1997; revised April 1, 1998 The nucleotide sequence of pSW200 of Erwinia stewartii SW2 was determined. This plasmid is 4367 bp long, consisting of four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. The plasmid also contains a region of forty-one 15-bp repeats. Deleting this region does not affect the stability or the copy number when maintained as sole plasmid in the cell. However, the plasmid is rapidly lost when a homoplasmid with the intact repeat region is introduced into the cell. The function of this region may provide pSW200 an advantage in competing with an incompatible plasmid in the cell. © 1998 Academic Press Key Words: Erwinia stewartii; ColE1-type plasmid; mobilization; plasmid competition. Erwinia stewartii, also called Pantoea stew- artii subsp. stewartii, is a pathogenic bacterium of corn that causes necrosis and systemic wilt- ing, known as Stewart’s wilt. This organism usually contains a large number of plasmids, ranging in size from 4 to 318 kb (Coplin et al., 1981). E. stewartii is a member of Enterobac- teriaceae, accounting for why many of these plasmids can replicate in Escherichia coli (Frederick and Coplin 1986 Fu et al., 1995, 1996, 1997). Although the total length of these plasmids in a cell may constitute 20 to 25% of the bacterial genome, no function, except for plasmid replication, mobilization, and transfer, has been identified (Coplin et al., 1981; Fu et al., 1995, 1996). Studies have shown that these plasmids are not related to virulence, carbohy- drate utilization, heavy-metal resistance, antibi- otic resistance, and bacteriocin production (Co- plin et al., 1981; Frederick and Coplin 1986). Four of the thirteen plasmids in strain SW2, i.e., pSW100, pSW500, pSW1200, and pDC250 (pSW800), have been partially characterized. Plasmid pSW100 is the smallest plasmid in E. stewartii SW2, containing a replicon similar to those of ColE1, p15A, and ColA (Fu et al., 1995). The replicon of pSW500 has been se- quenced and the region required for plasmid replication is located in a 1.6-kb region, which contains a repA gene and seven iterons (Fu et al., 1996). Plasmid pSW1200 is incompatible with P1 and belongs to the IncY group (Fu et al., 1997). In addition, plasmid pDC250 (pSW800) is conjugative and can mobilize pCR1 and pSW100 from E. stewartii to E. coli (Coplin et al., 1985; Fu et al., 1995). In this study, we isolate and characterize a 4-kb plas- mid of E. stewartii SW2, pSW200. This plas- mid contains a replicon and mob genes homol- ogous to those of ColE1-type plasmids. MATERIALS AND METHODS Bacterial strains, plasmids, and media. E. stewartii strain SW2 was obtained from D. Co- plin (Coplin et al., 1981). E. coli strains used in this study were HB101 (F - , hsdS20, supE44, recA13, ara14, proA2, rpsL20, xyl-5, mtl-1) (Boyer and Roulland-Dessoix, 1969) and ATCC 23739 [K-12, Hfr (Hayes) prototrophic (l) - ]. Plasmids used in this study are listed in Table 1. LB 2 broth and agar (Miller, 1972) were used as general-purpose media. Cells containing pMAK705 were cultured at 30°C (Hamilton et al., 1989). Chloramphenicol (Cm) (30 mg/ml), 1 To whom correspondence should be addressed. Fax: 886-3-328-0292. E-mail: [email protected]. 2 Abbreviations used: LB, Luria–Bertani; Km, Kanamy- cin; Cm, chloramphenicol; Ap, ampicillin; Tc, tetracycline; Sm, streptomycin; nt, nucleotide(s); ORF, open reading frame. 100 0147-619X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. PLASMID 40, 100 –112 (1998) ARTICLE NO. PL981350

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Isolation and Characterization of Plasmid pSW200 from Erwinia stewartii

Jen-Fen Fu, Jer-Ming Hu, Yu-Sun Chang, and Shih-Tung Liu1

Molecular Genetics Laboratory, Department of Microbiology and Immunology,Chang-Gung University, Kwei-Shan, Taoyuan, 333, Taiwan

Received December 16, 1997; revised April 1, 1998

The nucleotide sequence of pSW200 ofErwinia stewartiiSW2 was determined. This plasmid is4367 bp long, consisting of four mobilization genes,mobCABD,and an origin of replicationhomologous to those of ColE1-type plasmids. The plasmid also contains a region of forty-one 15-bprepeats. Deleting this region does not affect the stability or the copy number when maintained as soleplasmid in the cell. However, the plasmid is rapidly lost when a homoplasmid with the intact repeatregion is introduced into the cell. The function of this region may provide pSW200 an advantage incompeting with an incompatible plasmid in the cell.© 1998 Academic Press

Key Words: Erwinia stewartii; ColE1-type plasmid; mobilization; plasmid competition.

Erwinia stewartii,also calledPantoea stew-artii subsp.stewartii, is a pathogenic bacteriumof corn that causes necrosis and systemic wilt-ing, known as Stewart’s wilt. This organismusually contains a large number of plasmids,ranging in size from 4 to 318 kb (Coplinet al.,1981).E. stewartii is a member of Enterobac-teriaceae, accounting for why many of theseplasmids can replicate inEscherichia coli(Frederick and Coplin 1986 Fuet al., 1995,1996, 1997). Although the total length of theseplasmids in a cell may constitute 20 to 25% ofthe bacterial genome, no function, except forplasmid replication, mobilization, and transfer,has been identified (Coplinet al., 1981; Fuetal., 1995, 1996). Studies have shown that theseplasmids are not related to virulence, carbohy-drate utilization, heavy-metal resistance, antibi-otic resistance, and bacteriocin production (Co-plin et al., 1981; Frederick and Coplin 1986).Four of the thirteen plasmids in strain SW2, i.e.,pSW100, pSW500, pSW1200, and pDC250(pSW800), have been partially characterized.Plasmid pSW100 is the smallest plasmid inE.stewartii SW2, containing a replicon similar tothose of ColE1, p15A, and ColA (Fuet al.,1995). The replicon of pSW500 has been se-quenced and the region required for plasmid

replication is located in a 1.6-kb region, whichcontains arepA gene and seven iterons (Fuetal., 1996). Plasmid pSW1200 is incompatiblewith P1 and belongs to the IncY group (Fuetal., 1997). In addition, plasmid pDC250(pSW800) is conjugative and can mobilizepCR1 and pSW100 fromE. stewartiito E. coli(Coplin et al., 1985; Fuet al., 1995). In thisstudy, we isolate and characterize a 4-kb plas-mid of E. stewartii SW2, pSW200. This plas-mid contains a replicon andmobgenes homol-ogous to those of ColE1-type plasmids.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. E.stewartiistrain SW2 was obtained from D. Co-plin (Coplin et al.,1981).E. coli strains used inthis study were HB101 (F-, hsdS20, supE44,recA13, ara14, proA2, rpsL20, xyl-5, mtl-1)(Boyer and Roulland-Dessoix, 1969) andATCC 23739 [K-12, Hfr (Hayes) prototrophic(l)-]. Plasmids used in this study are listed inTable 1. LB2 broth and agar (Miller, 1972) wereused as general-purpose media. Cells containingpMAK705 were cultured at 30°C (Hamiltonetal., 1989). Chloramphenicol (Cm) (30mg/ml),

1 To whom correspondence should be addressed. Fax:886-3-328-0292. E-mail: [email protected].

2 Abbreviations used: LB, Luria–Bertani; Km, Kanamy-cin; Cm, chloramphenicol; Ap, ampicillin; Tc, tetracycline;Sm, streptomycin; nt, nucleotide(s); ORF, open readingframe.

1000147-619X/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

PLASMID 40, 100–112 (1998)ARTICLE NO. PL981350

kanamycin (Km) (50mg/ml), ampicillin (Ap)(50 mg/ml), tetracycline (Tc) (12.5mg/ml), andstreptomycin (Sm) (25mg/ml) were added tothe medium for selection.

Plasmid manipulation.Plasmids were pre-pared according to the alkaline-lysis method ofBirnboim and Doly (1979) and followed byCsCl–ethidium bromide centrifugation (Sam-brooket al.,1989). Plasmid screening was car-ried out using the method of Kado and Liu(1981).E. coli was transformed by the methodof Cohenet al., (1972).E. stewartiiSW2 wastransformed by electroporation according to amethod described elsewhere (Fuet al., 1995).

Isolation of pSW200. E. stewartiiSW2 plas-mids were introduced intoE. coli HB101 byindirect selection (Kretschmeret al., 1975) ac-cording to the method previously described (Fuet al., 1995). Briefly,E. coli HB101 was trans-formed with a plasmid mixture containing totalplasmids of E. stewartii SW2 (50 mg) and

pMAK705 (0.1 mg), a chloramphenicol-resistant temperature-sensitive derivative ofpSC101 (Hamiltonet al., 1989). The transfor-mants resistant to chloramphenicol were cul-tured at 30°C and were screened for the pres-ence of anE. stewartiiplasmid. Cells containingboth pSW200 and pMAK705 were subse-quently cultured at 42°C to cure pMAK705.

DNA sequencing.DNA sequencing was per-formed according to the chain-terminationmethod of Sangeret al., (1977), using double-stranded plasmid DNA as a template.

Mobilization assay.Plasmid mobilizationwas performed by separately culturing the do-nor and recipient cells in LB broth to 0.3A600.The cells were then mixed at a 1:1 ratio andincubated at 30°C overnight without shaking.After conjugation, the cell mixture was platedon LB agar containing appropriate antibiotics.

Plasmid stability and incompatibility. E. coliHB101 (pSW203) was cultured overnight at 37°C

TABLE 1

Plasmids Used in This Study

Plasmid Characteristics Source

pMAK705 A pSC101 derivative, temperature sensitive, Cm resistant Hamiltonet al., 1989pRK415 A pRK290 derivative, Tc resistant Dittaet al., 1985pUCK4-KISS A ColE1 derivative; Km and Ap resistant Okaet al., 1981pSW100 The smallest plasmid ofE. stewartiiSW2; 4272 bp Fuet al., 1995pSW106 A plasmid consisting of the 0.9-kbSspI fragment of pSW100 and an

ampicillin-resistance geneFu et al., 1995

pSW102 A derivative of pSW100 containing a kanamycin-resistance gene at theSacI site

Fu et al., 1995

pBR322 A ColE1 derivative; ampicillin and tetracycline resistant Bolivaret al., 1977pACYC184 A p15A derivative; chloramphenicol and tetracycline resistant Chang and Cohen, 1978pSW200 A plasmid ofE. stewartiiSW2; 4367 bp This studypSW201 A derivative of pSW200 containting a kanamycin-resistance gene

inserted at thePstI site; 5.6 kbThis study

pSW203 A plasmid consisting of the 1.3-kbTaqI fragment (nt 172 to nt 1473)of pSW200 and a kanamycin-resistance gene; 2.6 kb

This study

pSW207 A plasmid consisting of the 3.4-kbDraI–SspI fragment of pSW200 anda tetracycline-resistance gene; 5.6 kb

This study

pSW206 A derivative of pSW200 containing a tetracycline-resistance geneinserted at theDraI site; 6.4 kb

This study

pSW210 A derivative of pSW201 that lacks theBglII fragment (nt 3981 to nt380); 4.8 kb

This study

pSW219 A derivative of pSW201 that lacks theHincII fragment (nt 3388 to nt3943); 5.1 kb

This study

pSW235 A derivative of pSW210 that lacks theSspI–SpeI fragment (nt 3212 tont 3356); 5.0 kb

This study

101PLASMID PSW200 FROMErwinia stewartii

in LB–Km broth and, then, was inoculated in LBbroth at a density of 23 106 CFU/ml. The cellswere subcultured every 12 h for a total of 140generations. Cells were plated on LB agar and theproportion of plasmid-free cells in the populationwas determined by replica plating on LB–Kmagar. To study plasmid incompatibility,E. coliHB101 harboring pSW203 and pBR322, pSW203and pACYC184, or pSW203 and pSW106 wascultured in LB broth containing the appropriateantibiotics to maintain the plasmids. Cells wereinoculated and cultured for 140 generations in anantibiotic medium that preferentially selected forthe cells containing pBR322, pACYC184, orpSW106. Finally, loss of pSW203 was examinedby replica plating.

Determination of plasmid copy number. E.coli HB101 containing both pSW200 and pRK415was obtained by transforming pRK415 intoE.coli HB101 (pSW200).E. coli HB101 con-taining pRK415 and pSW201 or pRK415 andpSW203 was obtained by cotransformation.After they were cultured overnight in LB me-dium containing appropriate antibiotics, cellswere subcultured in the same medium to mid-log phase. Plasmids in the cells were isolatedfrom approximately 13 109 cells accordingto the method of Kado and Liu (1981) andwere separated by agarose gel electrophore-sis. The gel was stained by ethidium bromideand photographed over an UV transillumina-tor (Foto/Analyst Archiver Electronic Docu-mentation system, Fotodyne Incorporated).The image of the gel was saved and the in-tensity of the DNA bands was determinedwith Macintosh 7200 computer with softwareGel-Pro Analyzer (Media Cybernetics). Theratio between the readings of pRK415 and ofpSW200 or its derivatives was calculated andthe value was further divided by 5, the copynumber of pRK415 (Dittaet al., 1985), toobtain the copy number of pSW200 and itsderivatives.

RESULTS

Isolation of pSW200

Plasmid pSW200 was isolated by indirectselection according to the method previously

described (Fuet al., 1995). The transformantswere selected on LB–Cm agar and the plasmidsin the cells were examined by plasmid screen-ing. One of the colonies examined containedpMAK705 and a 4-kb plasmid, pSW200. Plas-mid pMAK705 was then cured by culturing thecells at 42°C. Plasmid pSW200 was subse-quently purified and characterized. The restric-tion map of pSW200 is shown in Fig. 1.

Sequence of pSW200

Sequencing results indicate that pSW200 is4367 bp long (Fig. 2) (GenBank Accession No.:L42525), 95 bp longer than the smallest plasmidof E. stewartiiSW2, pSW100 (Fuet al., 1995).Herein, we designate the uniqueDraI site as nu-cleotide (nt) 1 of the plasmid (Fig. 1). The regionbetween nt 380 and nt 960 is 88.4, 72.2, and71.8% homologous to the regions controlling thereplication of p15A, ColE1, and pSW100, respec-tively. The length of RNAII of pSW200 is 518 nt(Fig. 2) and the length of RNAI is 107 nt (Fig. 2).As depicted in Fig. 3, the sequence of the loopregions of RNAI was different from those regionsin pSW100, p15A and ColE1. Between nt 1344and nt 3156, plasmid pSW200 contains a se-quence homologous to the mobilization genes(mobCABD)of ColE1 (Chanet al.,1985; Boydetal., 1989) and pSW100 (Fuet al.,1995) (Fig. 2),with homologies of 63, and 92.7%, respectively.Unlike the mobAof pSW100, which contains anonsense mutation at the 59 portion of the gene(Fu et al., 1995), the MobA protein encoded bypSW200 remains intact and has a length compa-rable to that of ColE1 (Fig. 4). The amino acidsequences of MobC, MobB, and MobD proteinsof pSW100 and pSW200 closely resemble eachother and have sequence identities of 91, 85, and84%, respectively (Fig. 4). Plasmid pSW200 alsocontains a putativeoriT and abomregion (nt 1017to nt 1300) (Fig. 2) which is 68 and 83% homol-ogous to those of ColE1 and pSW100, respec-tively. The regions between nt 3312 to nt 3917and nt 3275 to nt 35 contain two open readingframes (ORFs), ORF-F (606 bp) and ORF-R(1128 bp) (Fig. 2). These two ORFs are partiallycomplementary and include forty-one 15-bp re-peats (Fig. 2) with a consensus sequence of CAG-

102 FU ET AL.

GTTAGC(A/T)T(C/T)GCT. This region is nothomologous with any sequences in GenBank.

Mobilization of pSW200

This study also usesE. stewartii SW2(pSW201) and SW2 (pSW206) to examine thefunction of themob genes of pSW200. InE.stewartii SW2 (pSW201), pSW200 was curedby incompatibility; in E. stewartii SW2(pSW206), both pSW200 and pSW206 werepresent if the cells were cultured on mediumcontaining tetracycline. In pSW201,mobAwasdisrupted by inserting a kanamycin-resistancegene at thePstI site (Table 1), whereas, inpSW206, themobgenes remained intact (Table1). According to our results, pSW206 could bemobilized into E. coli HB101, probably by

pDC250 (Coplinet al.,1985), at a frequency of6.8 3 1026, i.e., a value comparable to thefrequency for the mobilization of pCR1 bypDC250 (Coplin et al., 1981). Contrary topSW206, the mobilization frequency forpSW201 was less than 53 1028. These resultsdemonstrated that disruption ofmobA ofpSW200 significantly decreased the mobiliza-tion frequency, suggesting that themobgenes ofpSW200 are indeed functional. Our previousstudy revealed that pSW100 could be mobilizedby pDC250 fromE. stewartii SW2 to E. coliHB101, despite the fact that pSW100 does nothave an intactmobA (Fu et al., 1995). There-fore, in that study, we suggested that the MobAencoded by pSW200 may be responsible for themobilization of pSW100. In this study, we used

FIG. 1. Map of pSW200. Plasmid pSW200 consists of an RNAI–RNAII region andmobgenes homologousto those of ColE1 and pSW100. Arrows denote the direction of transcription.

103PLASMID PSW200 FROMErwinia stewartii

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104 FU ET AL.

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105PLASMID PSW200 FROMErwinia stewartii

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106 FU ET AL.

an E. coli Hfr strain, ATCC 23739, to investi-gate the role of pSW200 in mobilizing apSW100 derivative, pSW102. We found thatpSW102 was mobilized intoE. coli HB101 at afrequency of 2.33 1025 when a pSW200 de-rivative pSW207 that containsmobAwas alsopresent in the cell. According to our results, themobilization frequency decreases approxi-mately 50-fold at 5.03 1027 if pSW207 isabsent from the cell. These results suggest thatthe defectivemobAgene of pSW100 could becomplemented by themobAof pSW200.

Stability, Incompatibility, and Copy Number

E. coli HB101 (pSW203) was cultured in LBbroth and subcultured every 12 h for a total of140 generations. Results indicated pSW203could be stably maintained in LB broth for atleast 140 generations. Nearly 100% of the cellsretained pSW203. Our previous study alsofound that a pSW100 derivative, pSW103 couldbe stably maintained for at least 120 generations

in an antibiotic-free medium (Fuet al., 1995).Neither pSW100 or pSW200 contains a se-quence homologous to thecer of ColE1 (Sum-mers and Sherratt, 1984); therefore, these twoplasmids may use mechanisms other than themechanism used by ColE1 to stabilize the plas-mids in E. stewartiiand inE. coli. Our resultsalso demonstrated that the stability of pSW200is unrelated to the region of 15-bp repeats sincepSW203 does not contain this region and isstably maintained inE. coli. Furthermore, weexamined the incompatibility between pSW203and the other ColE1-type plasmids, includingpBR322, pACYC184, and pSW106. As gener-ally known, pBR322 is unstable when culturedin a medium without antibiotic (Summers andSherratt, 1984), accounting for why we addedampicillin to maintain pBR322. Similarly, tet-racycline and ampicillin were also added tomaintain pACYC184 and pSW106, respec-tively, to examine if these plasmids were com-patible with pSW203. We have cultured the

FIG. 3. Comparison of the RNAI sequences of pSW200, pSW100, p15A, and ColE1.

107PLASMID PSW200 FROMErwinia stewartii

cells for a total of 140 generations. Resultsindicated that pSW203 could be stably main-tained in the cells and was compatible with

these ColE1-type plasmids. Sequence analysisrevealed that the loop regions of RNAI ofpSW200 are different from those of pSW100,

FIG. 4. Amino acid sequence comparison among the mobility proteins of ColE1, pSW100, and pSW200. TheMobA protein of pSW100 is truncated at the C terminus due to the presence of a nonsense mutation in the gene.

108 FU ET AL.

FIG. 4—Continued

109PLASMID PSW200 FROMErwinia stewartii

ColE1, and p15A (Fig. 3), which may explainwhy these plasmids are compatible. The copynumber of pSW200, pSW201, and pSW203 inE. coli was estimated by agarose gel electro-phoresis and was approximately equal to that ofpSW100 (Fuet al., 1995), i.e., about 10 copiesper cell (Fig. 5).

Plasmid Exclusion

Our results indicated that the repeat region ofpSW200 confers the function of plasmid exclu-sion by transformation. Plasmid pSW207 was aderivative of pSW200, in which forty of theforty-one 15-bp repeats were removed. Thisplasmid could be transformed intoE. coliHB101 at a frequency of 2.33 105 transfor-mants/mg DNA and, also, could be stably main-tained in the cells without antibiotic selection.However, the transformation frequency forpSW207 decreased approximately 100-foldwhen the host already contained pSW201 (Ta-ble 2). On the other hand, we were able totransform pSW201 intoE. coli HB101

(pSW207) with a normal transformation fre-quency (1.73 105 transformants/mg DNA) (Ta-ble 2). Nevertheless, the copy numbers ofpSW207 was maintained at a low level if thecells were cultured on LB–Tc, Km medium(Fig. 6). On the other hand, plasmid pSW207was excluded at a frequency of 1.43 1021 pergeneration when the cells were cultured on anantibiotic-free medium. Similar phenomenawere also observed inE. stewartiiSW2; i.e., thecell could be transformed by pSW201 but notby pSW207 (Table 2). We also cotransformedtwo pSW200 derivatives lacking the repeat re-gion. One of the plasmids used in the cotrans-formation experiment was pSW219, which wasobtained by deleting the 555-bpHincII (nt 3388to nt 3943) (Fig. 1) of pSW201. Experiments

FIG. 4—Continued

FIG. 5. Comparison of copy number of pSW200 deriv-atives.E. coli HB101 containing (A) pRK415 and pSW200,(B) pRK415 and pSW201, and (C) pRK415 and pSW203.Plasmid DNA was prepared according to the method ofKado and Liu (1981).

TABLE 2

Plasmid Exclusion Phenomenon Exhibited by theDerivatives of pSW200

Host

Plasmid usedfor

transformationa

Transformationfrequency(cfu/mg)

E. coli HB101 pSW207 2.33 105

E. coli HB101 pSW201 3.03 105

E. coli HB101(pSW201) pSW207 2.03 103

E. coli HB101(pSW207) pSW201 1.73 105

E. stewartiiSW2 pSW207 ,101

E. stewartiiSW2 pSW201 4.03 104

a Transformation was carried out with 100 ng of plasmidDNA.

110 FU ET AL.

results showed that the copy numbers ofpSW207 and pSW219, when cotransformedinto E. coli HB101, were approximately equal(Fig. 6) if the cells were cultured in the mediumcontaining both kanamycin and tetracycline,showing that presence of the repeat region inone plasmid was required for the plasmid com-petition. We have also generated deletions inpSW201. One of the deletions removed the766-bpBg/II fragment (nt 3981 to nt 380) (Fig.1) which contains the initiation codon ofORF-R; the other deletion removed the 144-bpSpeI–SspI fragment (nt 3212 to nt 3356) (Fig. 1)which contains the initiation codon of orf-F.After the deletions, both ORF-F and ORF-R donot form new ORFs with the sequences up-stream. We found that a plasmid with either ofthe deletions, when cotransformed withpSW207, still conferred the exclusion pheno-type (Fig. 6), suggesting that the sequence of therepeats rather than the ORFs was required forplasmid competition. Until now, the mechanisminvolved in this competition phenomenon ofpSW200 still remains unknown.

Plasmid exclusion by competition has alsobeen observed in pT181 (Gennaro and Novick,1986, 1988) and pSC101 (Tuckeret al., 1984).Deletion incmplocus of pT181 and deletion ofone PR segment topar locus of pSC101 do notinfluence plasmid replication and stability whenthe plasmid is maintained singly in the cells.However, the mutant plasmid has a profounddisadvantage when coexisting with the parentplasmid containing an intactcmp or par(Gennaro and Novick, 1986; Miller and Cohen,

1993; Tuckeret al., 1984). According to thoseinvestigations, replication of plasmid pSC101and pT181 required initiator protein, Rep, andcmp or par loci can increase the interactionbetween replication origin and initiator protein(Gennaro and Novick, 1986; Zhanget al.,1997). In this case, both of the loci were thoughtto have functioned as the replication enhancers(Ohkubo and Yamaguchi, 1995; Miller and Co-hen, 1993; Henriquezet al.,1993). However, inthe case of pSW200, this plasmid may use adifferent mechanism for plasmid competitionsince the replication of pSW200 does not re-quire a replication initiator.

In summary, this study has isolated and char-acterized a ColE1-like plasmid ofE. stewartiiSW2. This plasmid contains a ColE1-type ori-gin, four mobilization genes, abomregion, anda region that confers the competition phenotypeof the plasmid. This plasmid has a copy numbersimilar to that of pSW100 and can be stablymaintained inE. stewartii SW2 and E. coliHB101. Plasmid pSW200 apparently does notcontain any genes that are essential for the sur-vival of the host. This plasmid, which is ex-tremely stable in cells, can be useful in con-structing expression vectors for producingrecombinant proteins by high-density fermenta-tion. Furthermore, the sequences in the repeatregion of pSW200 can be useful for diagnosingStewart’s wilt pathogens.

ACKNOWLEDGMENTS

This research was supported in part by a medical re-search grant, CMRP 720, from the Chang-Gung MemorialHospital and by a biological research grant, NSC 87-2314-B-182-006, from the National Science Council of the Re-public of China.

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111PLASMID PSW200 FROMErwinia stewartii

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Communicated by D. Chattoraj

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