Plasmid 54 (2005) 184–189

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Short communication

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

Junghyun Park, Mihye Lee, Joohwa Jung 1, Jeongho Kim ¤

Department of Biological Sciences, Inha University, Inchon 402-751, Republic of Korea

Received 14 September 2004, revised 19 March 2005Available online 17 May 2005

Communicated by Grzegorz Wegrzyn

Abstract

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid con-tains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-typedouble strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highestsimilarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. Ashuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistancegene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillusplantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation ofLeuconostoc strains. 2005 Elsevier Inc. All rights reserved.

Keywords: Leuconostoc citreum; Lactic acid bacteria; Kimchi; Plasmid

Leuconostoc citreum has been isolated fromplant sources such as cabbage and garlic (Farrowet al., 1989; Lee et al., 2000). This gram-positivebacterium, therefore, plays important roles in thefermentation of kimchi, a cabbage product and isactively being used as a starter culture for kimchifermentation (Choi et al., 2003). However, mecha-

* Corresponding author. Fax: +82 32 874 6737.E-mail address: jhokim@inha.ac.kr (J. Kim).

1 Present address: Coreana Cosmetics Co. Cheonan, Republicof Korea.

0147-619X/$ - see front matter 2005 Elsevier Inc. All rights reservdoi:10.1016/j.plasmid.2005.03.003

nisms underlying the kimchi fermentation arepoorly understood. Plasmid vectors that enable totransform L. citreum may contribute to under-stand the mechanism of vegetable fermentationand related microbial physiology. So far two typesof rolling circle plasmids from Leuconostoc specieswere characterized. A broad host range crypticplasmid, pCI411 from Leuconostoc lactis has apE194-type replication origin and has been shownthat it is able to transform Leuconostoc mesentero-ides subsp mesenteroides and Weissella paramesen-teroides (CoVey et al., 1994). Second, a pT181-type

ed.

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cryptic plasmid, pFR18 from L. mesenteroidessubsp mesenteroides is able to transformLeuconostoc cremoris, L. mesenteroides subsp. dex-tranicum, and Lactobacillus sakei (Biet et al., 1999).pC194-type or pSN2-type rolling circle plasmids(Gruss and Ehrlich, 1989; Novick, 1989) remain tobe isolated from Leuconostoc strains.

Here, we characterized a small cryptic plasmid,pIH01, which is stably maintained in the L. citreumIH3 strain that was isolated from kimchi (Choiet al., 2003). Leuconostoc strains and other lacticacid bacteria were grown in MRS medium at30 °C. Exponentially growing cultures(OD600 D 0.7) were used for extraction of plasmids.Cells were harvested and resuspended in 200 �lTHMS (30 mM Tris–HCl, 3 mM MgCl2, and 25%sucrose, pH 8.0) containing 30 mg/ml lysozyme and10 �g/ml RNase for 1 h at 37 °C. The followingdenaturation and neutralization steps are same asthe alkaline lysis method (Birnboim and Doly,1979; O’sullivan and Klaenhammer, 1993).

The 1.8-kb sized plasmid from this strain thatharbors more than three plasmids was designated aspIH01. pIH01 carries a unique HindIII site, whichwas used for cloning into HindIII treated Esche-richia coli pUC19 vector, resulting in pHO301

(Table 1). Nucleotide sequence of pIH01 was deter-mined on both strands by using several walkingprimers and was deposited in the GenBank databaseunder Accession No. AY737645. The 39% G+ Ccontent of pIH01 is same as that of L. citreum (38–40%; Farrow et al., 1989; Takahashi et al., 1992).The nucleotide sequences of pIH01 showed highsimilarity with that of pFR18 from L. mesenteroides(69.1%; Biet et al., 1999), while some regions (posi-tion 200–480; Fig. 1) were much highly conserved(88% similarity) compared to other regions includ-ing Rep coding region, implying that this region isimportant for DNA replication. Prediction of openreading frames (ORF) and database searches wereperformed with the ORF Finder and BLAST algo-rithm (Altschul et al., 1990) provided by theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov), respectively. ORFprediction showed that a single open reading frame(position 735–1762) spans 56% of the wholesequence. Other small sized ORFs (less than 65amino acid residues) were neglected because theyshowed no homology to known proteins and addi-tionally they preceded no putative ribosome-bindingsites. This predicted ORF encodes a polypeptide of342 amino acid residues and shows homology with

Table 1Bacterial strains and plasmids

a KCTC, Korean Collection for Type Cultures.b DSMZ, German collection of Microorganisms and Cell Cultures.

Strains or plasmids Characteristics Sources

DH5� recA endA1 gyrA96 thi-1 hsdR17 supE44 �lac U169(�80 dlacZ�M15)deoR F-�

Gibco-BRL

Leuconostoc citreum IH3 This studyLeuconostoc citreum IH22 Choi et al. (2003)Leuconostoc mesenteroidessubsp. mesenteroides KCTC 3505T Type strain KCTCa

Lactobacillus plantarum IH357 This studyLactococcus lactis subsp. lactis DSM 20481T Type strain DSMZb

PlasmidspUC19 2.7 kb, ApR NEBpGEM-T 3.0 kb, ApR PromegapGK12 4.4 kb, EmR, CmR Kok et al. (1984)pIH01 1.8 kb cryptic plasmid from L. citreum IH3 This studypHO301 pUC19::1.8 kb HindIII pIH01, 4.5 kb, ApR This studypHO302 pGEM-T::1.8 kb pIH01, 4.8 kb, ApR This studypHO303 pHO302::1.2 kb ermC gene BamHI/NcoI pGK12,

6.0 kb, ApR, EmRThis study

pHO304 pHO303 �1.1 kb ApaI/ScaI pGEM-T, 4.9 kb, EmR This study

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Fig. 1. (A) Nucleotide sequence of plasmid pIH01 and predicted amino acid sequence of repC gene. Inverted repeats (IR) sequences aredisplayed as horizontal dashed arrows. Ribose binding site (RBS), a putative double-stranded origin (DSO), the ¡10 region of putativepromoter of repC gene, and HindIII restriction site are underlined. Tyrosine residue (Y) involved in the initiation of replication is indi-cated as a bold letter and some surrounding conserved resides are underlined. Starting and stop codons were italicized. (B) Nucleotidesequence comparison of the putative pIH01 DSO. The hairpin structure that includes the possible nick site (vertical arrow) is under-lined and indicated by a dashed horizontal arrow. (C) Unrooted neighbor-joining tree based on the amino acid sequences of Rep pro-teins showing relationships between pHI01 and other rolling circle replicons. Unrooted evolutionary trees were inferred by using twotreeing algorithms, namely, the Fitch–Margoliash (Fitch and Margoliash, 1967) and neighbor-joining (Saitou and Nei, 1987) methods.The PAM matrices (DayhoV et al., 1978) were used to generate an evolutionary distance matrix. The distance model numbers at thenodes indicate levels of bootstrap (Felsenstein, 1993) support based on neighbor-joining analyses of 500 resampled datasets.

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the replication initiator protein (Rep; 335 aa) ofpFR18 (64.0% identity and 78.7% similarity), apT181 family member (Khan and Novick, 1983).Even though this Rep protein showed very lowsimilarity to RepC protein of pT181 (24%), wefound that active site sequence of Rep (IRLYDK)is strictly conserved (Fig. 1A; Biet et al., 1999), sug-gesting that this Rep is a RepC family member andpIH01 employs a rolling circle mechanism as a rep-lication mode, because all Rep proteins of pT181family contain a conserved tyrosine residue (Y) inthe active site (Novick, 1989). In addition, theneighbor-joining tree based on Rep proteinsimplies that pIH01 has evolutionary relationshipsto pT181 family (Fig. 1C). Detailed phylogeneticanalysis methods were described previously (Kimet al., 2000). Since Rep binds double strand origin(DSO) of rolling circle plasmids, a DSO sequencewould require each corresponding Rep proteinwith speciWc binding aYnity (Novick, 1989). Apossible ¡10 region (TATAAT) and a ribosome-binding sequence (RBS) were predicted for thisrepC gene. Several putative sigma factor bindingsequences were found at ¡35 promoter region.Finally, the hairpin structure (�Go D ¡6.7 kcal/mol) and accompanying UUU (IR4; Fig. 1A) atthe downstream of the TAG stop codon of repCgene imply an involvement of �-independent tran-scription termination.

Palindromic sequence analysis (Hofacker, 2003)predicted Wve signiWcant palindromic structures(IR1, IR2, DSO, IR3, and IR4). Like pFR18, fourpalindromic structures (IR1, IR2, DSO, and IR3)of pIH01 reside at the high G + C content region(54%; position 231–462; Fig. 1A.). Even though theputative DSO sequence of pIH01 is slightly diVer-ent from those of pT181 or pFR18 (Fig. 1B; Grussand Ehrlich, 1989; Novick, 1989), the size of thestem and loop structure and nicking site is strictlyconserved, indicating that most Rep recognizesalmost the same sized hairpin structure that con-tains the nick site in the hairpin loop (Fig. 1B).However, SSO is not found in pIH01 plasmid, onthe basis of sequence analysis.

To determine whether pIH01 replicates throughthe rolling circle mechanism, we examined the exis-tence of single-stranded DNA (ssDNA) in themini-prep of plasmid DNA using Southern blotanalysis. If necessary, DNA samples were preparedfrom exponentially growing cells treated with rif-ampicin (100 �g/ml) for 1 h. Electrophoresis ofDNA in 1% agarose gel was followed by capillarytransfer of these DNA to a charged nylon mem-brane (Amersham) by using 10£ SSC buVer. ADNA probe containing EcoRI-digested DNA frag-ments from pHO302 was labeled by using an ECLdirect nucleic acid labeling and detection system(Amersham). We found that this probe hybridized

Fig. 1. (continued)

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to a single fast migrating band (Fig. 2, lane 1). Withthe addition of S1 nuclease (200 U/ml; Riele et al.,1986), this band completely disappeared, suggest-ing that this band might represent a single-strandedDNA intermediate produced during the rolling cir-cle replication of pIH01 (Fig. 2, lane 2). WeconWrmed that this band migrated faster than dou-ble-stranded DNA using alkaline Southern blotanalysis (data not shown). We also showed that likepIH01, two shuttle vectors, namely pHO303 andpHO304 (described below) also generated ssDNAin E. coli (Fig. 2, lanes 3 and 5), indicating that inaddition to theta replication by ColE1 origin, roll-ing circle replication simultaneously exists in E.coli. However, we could not conWrm that pHO303or pHO304 generated ssDNA in L. citreum or Lac-tobacillus plantarum. We suspect that the amountof ssDNA may be lower than that of detection limitby Southern blot, because we could not even detectthe double-stranded vectors isolated from thesehosts by alkaline Southern blot. However, thegrowth rate of both L. citreum IH3 and L. planta-

Fig. 2. Detection of single-stranded pIH01, pHO303, andpHO304 DNA. Agarose gel electrophoresis of DNA isolatedfrom L. citreum IH3 (lanes 1 and 2) or E. coli containingpHO303 (lanes 3 and 4) or pHO304 (lanes 5 and 6) was per-formed and DNA samples that were treated (lanes 2, 4, and 6) ornot treated (lanes 1, 3, or 5) with nuclease S1 were transferred tonitrocellulose membrane by using 10£ SSC. Single-strandedDNA was detected by the hybridization of peroxidase-labeledprobes. M, 1 kb ladder (Promega). The positions of each double-stranded DNA that were deWned by ethidium bromide stainingand alkaline Southern blot analysis were marked by arrows.

rum IH357 containing pHO303 in the presence oferythromycin (5�g/ml) is almost same as those ofthe corresponding original host strains, implyingalmost same segregational stability of pHO303 asthat of pIH01.

To develop a shuttle vector useful for both Leu-conostoc strains and E. coli, we ampliWed almost allthe region of pIH01 plasmid using two PCRprimers: 5�-ggatccctgaataaatcatgaatata-3� (position9–32 ; contains a BamHI site) and 5�-agcttcaaataattcgatcaa-3� (1804–1822). We next inserted this PCRproduct into a pGEM-T vector (Promega). Thisresultant plasmid, designated as pHO302 (Table 1)was also stably maintained in E. coli DH5�, eventhough it has two diVerent replication origins. Weconstructed pHO302 because aforementionedpHO301 appears to contain a non-functional repgene due to a HindIII digestion (position at1670 bp). To introduce a selection marker for Leuco-nostoc strains into the shuttle vector, we inserted anermC gene (EmR) that is ampliWed from pGK12plasmid (Kok et al., 1984) using two primers: 5�-aaaagccatgggaaacgtctcagaaacga-3� (NcoI site) and5�-cgcggatccacacaaaaacaagt-3� (BamHI site) into alinearized pHO302 by both BamHI and NcoI. ThisampliWed ermC product contains full length openreading frame as well as a 400 bp promoter regionand a 40bp downstream region from a TAA stopcodon. The resultant shuttle vector (pHO303;6.0 kb) has two replication origins and two antibioticmarkers, bla (ApR) and ermC genes. To reduce thesize of this shuttle vector, we deleted the 1.1 kbpGEM-T fragment containing part of a bla gene byApaI and ScaI double digestion followed by end Wll-ing and self-ligation, resulting in pHO304 (4.9kb).

For transformation of Leuconostoc strains, elec-trocompetent cells were made according to modiWedAukrust and Blom’s method (1992). Cells in MRSmedia containing 3% glycine and 40 mM L-threo-nine were cultured at 30 °C until OD600 D 0.6. Cellswere washed two times with distilled water and cellpellets were suspended in 30% polyethylene glycol1500 (1/10 volume of original cell culture). Compe-tent cells (100�l) were incubated with plasmids(10�l) on ice for 10min and were subjected to elec-tric pulse (2.5 kV, 200�, 25�F). Cells were incubatedfor 10 min on ice and were subsequently grown withthe addition of 1 ml MRS media at 30°C for 2 h.

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Transformants were selected on MRS plates con-taining 5�g/ml erythromycin. We found that theshuttle vectors pHO303 or pHO304 were able toreplicate in various lactic acid bacteria, namely, L.mesenteroides subsp mesenteroides KCTC 3505T,Lactococcus lactis KCTC 3528T, and L. plantarumIH357 as well as L. citreum. TransformationeYciency of pHO33 and pHO34 was low in L. citr-eum IH3 and IH22 (10 CFU/�g DNA), while it washigh in L. plantarum IH357 (103 CFU/�g DNA).Introduction of pHO303 or pHO304 into L. citreumIH3 did not change the innate plasmid distributions.

Then we tested the ability of plasmid mainte-nance with no selection forces. We found that thesmall-sized vector (pHO304) showed better segrega-tional stability than pHO303 in the absence oferythromycin. About 70% of total cells still carriedpHO304 after 50 generations in the absence oferythromycin, while 5% of total cells carriedpHO303. However, in the presence of erythromycin,these host strains carrying pHO303 grew as fast asthe host strain, indicating that this vector is stablymaintained with no segregational instability.

Taken together, in this study we showed that asmall cryptic plasmid, pIH01 replicates via rollingcircle replication mode and a basic shuttle vectorbased on pIH01 may be useful for further con-struction of diverse plasmid vectors for Leuconos-toc strains and other related species.

Acknowledgments

This work was supported by the Inha Univer-sity research grant. J. Park and M. Lee contributedequally to this work. We thank Seok-Ho Jung andHye-Ja Lee for assistance.

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