Molecular characterization of three plasmids from Bifidobacterium longum

Plasmid 51 (2004) 87–100 www.elsevier.com/locate/yplas

Molecular characterization of three plasmids from Bifidobacterium longum b

Nathalie Corneau,a,1 Eric and Gisele LaPointec,* Emond, a b c

Department of Biochemistry and Microbiology, Universit
e Laval, Canada Chr. Hansen, Inc., 9015 West Maple Street, Milwaukee, WI 53214, USA STELA Dairy Research Centre, Universit
e Laval, Que., Canada G1K 7P4 Received 17 April 2003, revised 18 December 2003

Abstract The complete nucleotide sequences for pNAC1 (3538 bp) from strain RW048 as well as for pNAC2 (3684 bp) and pNAC3 (10,224 bp) from strain RW041 of Bifidobacterium longum were determined. The largest ORF (repB) of pNAC1 encodes a putative protein similar to those involved in a rolling-circle (RC) replication mechanism, which was confirmed by demonstration of single-strand intermediates in the host cell. The putative RepB gene product of pNAC2 is most similar to the replication protein of pDOJH10L and pKJ36. A second gene (mob) is similar to mobilization proteins involved in conjugation. Plasmid pNAC3 is the largest bifidobacterial plasmid to be sequenced to date. Of the eight putative gene products coded by pNAC3, one is similar to replication proteins (RepB), and another (Orf2) to putative transfer proteins (Tra). Bifidobacterial plasmids were divided into five groups based on Rep amino acid sequence homology and the results suggest a new plasmid family for B. longum. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Bifidobacterium longum; Replication initiator protein; Iterons; Replication screening; Rolling circle

1. Introduction Bifidobacteria are natural members of the human intestinal microflora (Moore et al., 1969; Simon and Gorbach, 1986; van der Werf and Venema, 2001). These bacteria are thought to have a positive impact on the health of the host (Mit* Corresponding author. Fax: +1-418-656-3353. E-mail address: [email protected] (G. LaPointe). 1 Present address: Health Canada, Bureau of Microbial Hazards, Food Directorate, Sir F.G. Banting Research Centre, Postal Locator 2204A2, Ottawa, Ont., Canada K1A 0L2.

suoka, 1990; Salminen et al., 1999; Tannock, 1999). Bifidobacteria have been claimed to improve lactose digestion, reduce the duration and incidence of diarrhea and modulate the immune response (Sanders and Huis inÕt Veld, 1999; Tannock, 1999). In mice, Bifidobacterium longum was shown to inhibit colon, mammary, and liver carcinogenesis induced by a food mutagen (Reddy and Rivenson, 1993). Due to these possible healthpromoting properties, bifidobacteria have become a common adjunct culture in many fermented dairy products, and a common component of human and animal health products.

0147-619X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2003.12.003

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Understanding the probiotic mechanisms of bifidobacteria is essential to support their purported role in functional foods (Kullen and Klaenhammer, 2000). For this purpose, suitable gene transfer systems are required. Plasmids are rarely found in Bifidobacterium species except for B. longum and B. breve. In addition, they are usually small in size (Sgorbati et al., 1982). Six plasmids from B. longum have been completely sequenced to date: pMB1, pKJ50, pKJ36, pDOJH10L, pBLO1, and pB44. Four plasmids have been sequenced that come from other Bifidobacterium species: pAP1 (B. asteroides); pCIBb1, pNBb1 (B. breve) (OÕRiordan and Fitzgerald, 1999; Park et al., 1999; Rossi et al., 1996); and p4M (B. pseudocatenulatum). The significance of plasmids in this genus remains unclear since most open reading frames (ORFs) encode for proteins of unknown functions. Plasmid pMB1, the first to be fully sequenced, was used to construct recombinant plasmids (Rossi et al., 1996), and the first generation of cloning vectors (Missich et al., 1994; Rossi et al., 1996). However, little is known of its mechanism of replication. Increased knowledge of these autonomous replication elements will contribute to developing a new generation of cloning vectors, including food grade gene delivery systems. This study describes the genetic organization and molecular characterization of three cryptic plasmids (pNAC1, pNAC2, and pNAC3) from B. longum RW041 and B. longum RW048. Comparison of the putative replication initiation proteins of known Bifidobacterium plasmids enabled their classification into distinct families, as well as proposing mechanisms of replication.

2. Materials and methods 2.1. Bacterial strains, plasmids, and media Bacterial strains and plasmids used in this study are listed in Table 1. B. longum strains were generously provided by Dr. D. Roy (FRDC, Agriculture and Agri-Food Canada, St-Hyacinthe, Que). Bifidobacterium strains were grown in MRS broth (Difco Laboratories, Detroit, MI) supple-

mented with 0.05% cysteine (MRSc) to maintain a low redox potential in the medium. Escherichia coli strains were grown in Luria broth (Sambrook et al., 1989). Lactococcus lactis strains were grown in M17 (Terzaghi and Sandine, 1975) supplemented with 0.5% glucose (GM17) at 30 °C. Bifidobacteria and E. coli were incubated at 37 °C. Antibiotics for selection and plasmid maintenance were used at the following concentrations; Bifidobacterium (5 lg of chloramphenicol/ml), E. coli (100 lg of ampicillin, 10 lg chloramphenicol, 150 lg of erythromycin, 50 lg of kanamycin, and 50 lg of carbenicillin/ml). 2.2. DNA isolation and manipulation Plasmid DNA from E. coli was isolated as described by Sambrook et al. (1989). Plasmid DNA from Bifidobacterium was isolated using the method of OÕSullivan and Klaenhammer (1993) with a number of modifications. First, lysozyme was used at a concentration of 40 mg/ml, with incubation at 37 °C for 60 min. Second, instead of isopropanol precipitation, plasmid DNA was recovered using 200 ll of a silica solution (# S5631 Sigma, Oakville, Ont., Canada prepared in ddH2 O), incubating the mixture for 10 min at room temperature (Vogelstein and Gillespie, 1979). The silica pellet (centrifugation at 16,000g, 5 s) was washed twice with an ethanol solution (EtOH 50%, 0.1 M NaCl, 1 mM EDTA, and 10 mM Tris–HCl, pH 7.5). After drying for 5 min, the plasmid DNA was eluted by incubating at 55 °C for 5 min with 50 ll of 10 mM Tris–HCl, pH 8. All DNA manipulations were carried out as described by Sambrook et al. (1989). Competent E. coli cells were prepared and transformed with the Gene Pulser apparatus as described by the manufacturer (Bio-Rad Laboratories, Mississauga, ON). For the preparation of competent Bifidobacterium cells and electrotransformation, the method of Missich et al. (1994) was used. 2.3. DNA sequencing and analysis Plasmid pNC1, a vector that allows screening for functional replicons in Gram-positive bacteria, was constructed by cloning the chloramphenicol

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Table 1 Bacterial strains and plasmids used in this study Bacterial strain or plasmid Bacterial strains B. longum RW041 B. longum RW048 B. longum RW048.1 B. longum ATCC 15708 B. animalis ATCC 27536 L. lactis LM0230 L. lactis SMQ17 E. coli DH5a E. coli DPWC E. coli BW26 Plasmids pEBM3 pECM2 pGK12 pNAC1 pNAC2 pNAC3 pNC1 pNC2 pNC3 pNC4 pNC6 pNC8 pNC11 pNC12 pNC13 pNC14 pUC18

Relevant characteristicsa

Reference(s) or source

Host of pNAC2 and pNAC3 Host of pNAC1 Plasmid free variant of RW048 cured of pNAC1 Plasmid free, recipient strain for electroporation Plasmid free recipient strain for electroporation Plasmid and restriction/modification system (R/M) free LM0230 (pSRQ700) R/Mþ supE44 DlacU169 (U80 lacZ DM15) hsdR17 recA endA1 gyrA96 thi-1 relA1 supE42 DrecA [SstII–EcoRI] srl::TN10-[TetS ], Fþ F
et Kmr

D. Royb D. Royb This study ATCC, Manassas, VA ATCC, Manassas, VA McKay et al. (1972) S. Moineauc Invitrogen, Burlington, ON

9.6-kb; Kmr , Cmr ; Corynebacterium glutamicum 10.3-kb; Kmr , Cmr ; C. glutamicum 4.4-kb vector, source of chloramphenicol resistance gene, Cmr , Emr 3.5-kb wild-type plasmid 3.7-kb wild-type plasmid 10.2-kb wild-type plasmid Replicon screening vector, cat gene (0.9 kb) from pGK12 cloned into unique AatII site of pUC18, Apr , Cmr pNAC1 linearized with BamHI, cloned into pNC1; Apr , Cmr pNAC3 linearized with HindIII, cloned into pNC1; Apr , Cmr pNAC1 linearized with SacI, cloned into pNC1; Apr , Cmr 2.8-kb EcoRI–BamHI from pNAC3 cloned into pNC1; Apr , Cmr 4.1-kb EcoRI–HindIII from pNAC3 cloned into pNC1; Apr , Cmr 3.3-kb HindIII–BamHI from pNAC3 cloned into pNC1; Apr , Cmr 1.4-kb PvuII from pNAC2 cloned into pNC1; Apr , Cmr 2.3-kb PvuII from pNAC2 cloned into pNC1; Apr , Cmr pNAC2 linearized with BglII, cloned into pNC1; Apr , Cmr Cloning vector, Apr , 2.8 kb

Sch€afer et al. (1994) Tauch et al. (1994) Kok et al. (1984) This study This study This study This study

Strathmann et al. (1991) Stratagene, La Jolla, CA

This study This study This study This study This study This study This study This study This study Yanisch-Perron et al. (1985)

a

Apr , ampicillin resistance; Cmr , chloramphenicol resistance; Kmr , kanamycin resistance. FRDC, Agriculture and Agri-food Canada, St-Hyacinthe, Canada. c Department of Biochemistry and Microbiology, Universit
e Laval, Que., Canada. b

resistance (Cmr ) cassette from pGK12 (Kok et al., 1984) into the single AatII restriction site of pUC18. The Cmr cassette was amplified by PCR from pGK12 using two primers; NC1: 50 GAC GTC GAC GTC GTG GTC TTT ATT CTT CAA CTA AAG C and NC2: 50 GAC GTC GAC GTC CCT TCT TCA ACT AAC GGG GCA GG. DNA fragments from the Bifidobacterium plasmids pNAC1, pNAC2, and pNAC3 were cloned into pNC1. The Tn1000 strategy was used according to the manufacturerÕs instructions (Stratagene, La Jolla, CA) for sequencing some of

the subclones. All other DNA sequencing was performed using the M13 mp forward and reverse primers. Both strands were sequenced using an ABI Prism DNA sequencer (Applied Biosystems, Foster City, CA) at the Institut Armand Frappier (Laval, Que.). Fragment assembly at junction points between restriction fragments was confirmed by sequencing through the junction regions using specific primers and the wild-type plasmids as template. The Genetic Computer Group sequence analysis package (version 10.0, Madison, Wisconsin) was used to assemble and analyse the

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DNA sequences. Searches for open reading frames were performed using GenBank release 127.0, EMBL release 69.0, PIR-Protein release 71.0, and SWISS-PROT release 40.7. Conserved motifs were identified using Prosite (release 16.0), and protein hydrophobicity was calculated using TMHMM (release 1.0, Sonnhammer et al., 1998). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.0 (Kumar et al., 2001). Comparative DNA sequence alignments of pNAC1 and pNAC2 with related plasmids (pKJ36 and pKJ50) were created with the SIM Alignment tool and Lalnview (Huang and Miller, 1991; http:// ca.expasy.org/). 2.4. Detection of intermediates of plasmid replication Single-stranded plasmid DNA was isolated, treated with nuclease S1 (Roche Diagnostics) and separated by agarose gel electrophoresis as described by Noirot-Gros and Ehrlich (1994).

The majority of the ORFs were preceded by putative Shine–Dalgarno sequences (AGG motif) homologous to the 30 end of the 16S rRNA of Bifidobacterium (Tanaka et al., 2000). No upstream sequences similar to known promoters preceded these ORFs. Furthermore, many of the predicted protein sequences did not show significant similarity to other proteins in the databases (Table 2). The putative protein product of Orf3 from pNAC1 did share some similarity with previously reported proteins (Table 2). Orf3 encodes a protein with similarity to the carboxyterminus of the EcoRII endonuclease (Reuter et al., 1999). Based on the predictive model of the TMHMM software (Sonnhammer et al., 1998), Orf3, Orf7, and Orf8 from pNAC3 could be a series of membrane proteins. Inverted repeats similar to rho independent terminators (Lewin, 2000) were present on pNAC1, pNAC2, and pNAC3 (Fig. 1). A direct repeat of 24 bp was present downstream of orf2 in pNAC1, and was repeated twice. The functional significance of this feature is unknown.

2.5. Nucleotide sequence accession numbers The GenBank accession numbers for the plasmid DNA sequences are AY112724 for pNAC1, AY112723 for pNAC2, and AY112722 for pNAC3.

3. Results and discussion Bifidobacterium longum strain RW041 was chosen for two reasons. It carries the largest plasmid described to date, and it harbours two plasmids, thus providing two compatible replicons for vector construction. Strain RW048 was chosen because of the presence of a small plasmid of similar size, in order to investigate the diversity of small plasmids. 3.1. General features of pNAC1, pNAC2, and pNAC3 The GC content of pNAC1 (3538 bp), pNAC2 (3684 bp), and pNAC3 (10,224 bp) were 58.5, 65, and 62%, respectively. The open reading frames identified on the plasmids are listed in Table 2.

3.2. The replication regions of pNAC1, pNAC2, and pNAC3 The regions likely involved in plasmid replication of pNAC1, pNAC2, and pNAC3 have been identified using DNA sequence comparison. The rep genes of these plasmids show a uniform distribution of conserved nucleotides. Moreover, the open reading frames coding for these peptides are found downstream of the iterons, which is a typical structure for an origin of replication (Del Solar et al., 1998). For pNAC1, the putative RepB protein demonstrated 66% identity with the RepA protein from pDOJH10L and the Rep protein of pKJ50 (Park et al., 1999); both plasmids are from B. longum. For pNAC2, the predicted RepB has 99% identity with RepB, the replication initiator of pDOJH10L. The putative replication protein of pNAC3 has 56% identity with the B. asteroides RepA protein of pAP1. Moreover, this protein contains a consensus sequence for an aminoacyl-tRNA synthetase which is not present in the pAP1 Rep. The conserved sequence belongs to a Pfam conserved domain (Ac-

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Table 2 General features of the putative open reading frames of pNAC1, pNAC2, and pNAC3 as well as comparison with databases Plasmid and ORF

pI

Free energy of binding SD sequence 16S rRNA (kcal/mol)

Function or similarity

Percentage of identity (aa)

Accession No.

pNAC1 repB

10.20

)9.0

orf2 orf3 orf4

8.55 9.91 12.92

)3.7 Not found Not found

B. longum, B. longum, B. longum, B. longum, B. longum, B. longum, B. longum, Unknown

RepA pDOJH10L Rep pKJ50 Rep pNAC2 Rep pBLO1 RepB pKJ36 hypothetical protein type II restriction enzyme

66 (170/257) 66 (167/253) 52 (154/295) 47 (149/315) 46 (113/245) 53 (55/102) 100 (142/142)

NC_004252 U76614 AY112723 AF540971 AF139129 NC_004307 NC_004307

B. B. B. B. B. B. B. B.

RepB pDOJH10L Rep pB44 RepB pKJ36 Rep pKJ50 RepB pNAC1 MobA unknown unknown

99 92 91 58 52 85 92 82

(295/297) (278/299) (208/227) (136/232) (154/295) (483/565) (158/170) (101/123)

NC_004252 NC_004443 AF139129 U76614 AY112724 AF540971 NC_004252 AF538868

56 (162/285) 54 (157/290) 31 (71/228)

Y11549 NC_004252 AF201825

32 (133/409)

AF164956

pNAC2 repB

8.95

)5.4

mob orf3

9.32 5.14

)7.0 )5.4

pNAC3 repB

6.75

)12.3

orf2

5.36

)8.7

orf3 orf4

4.29 9.69

)10.7 )7.2

orf5

10.75

Not found

orf6 orf7 orf8

5.95 10.27 9.34

)6.5 )4.1 )9.2

longum, longum, longum, longum, longum, longum, longum, longum,

B. asteroides, RepA pAP1 B. longum, Replicase pDOJH10L X. campestris, RepA pXV2 Aminoacyl-transfer RNA synthetases class-I signature C. glutamicum TraA ATP-/GTP-binding site motif A (P-loop) Unknown Prokaryotic membrane lipoprotein lipid attachment site Neutral zinc metallopeptidases, zinc-binding region signature Unknown Unknown Prokaryotic membrane lipoprotein lipid attachment site

SD, Shine–Dalgarno; B. asteroides, Bifidobacterium asteoides; B. longum, Bifidobacterium longum; X. campestris, Xanthomonas campestris; C. glutamicum, Corynebacterium glutamicum.

cession No. PF01336) which represents the nucleic acid-binding domain found in aminoacyl-tRNA synthetase, replication factor A as well as some DNA repair and polymerase enzyme subunits. The second most similar protein at 54% is the replicase coded by pDOJH10L, also from B. longum, but no other orf showed high similarity with those of pDOJH10L. The RepB of pNAC3 contains two

tyrosine residues, of which the second (position 238) is conserved among all plasmid replication proteins of the same cluster, as well as within the Pfam RepA_C domain (Accession No. PF04796). However, the function in replication of proteins belonging to this family is poorly understood at this time, although DNA-binding activity has been demonstrated (Basu et al., 2002).

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Fig. 1. Circular map of plasmids pNAC1, pNAC2, and pNAC3 depicting unique restriction sites. ORFs are represented by arrows and cis regions by boxes (ori, origin of replication; t, terminator; DR, direct repeat; IR, inverted repeat; and oriT, origin of transfer).

A phylogenetic tree was generated by aligning 18 Rep protein sequences in order to study the relationship among bifidobacterial and other related plasmids. Replication proteins of plasmids isolated from bifidobacterial strains were classified into five separate groups with a minimum similarity of 50% (Fig. 2). The Rep proteins of pNAC1 and pNAC2 (group 1) are closely related to other known plasmids from B. longum (pDOJH10L, pB44, pBLO1, pKJ50, and pKJ36; Fig. 2), all sharing the conserved Pfam domain Rep_3 (Accession No. PF01051). The Rep_3 conserved

domain identified is also found in initiator replication proteins of many plasmids from lactic acid bacteria, for which a number have been shown to replicate by a rolling circle mechanism (reviewed by Del Solar et al., 1998). The replication protein from pNAC3 is more closely related to that of pAP1 from B. asteroides and to a replicase coded by pDOJH10L, forming a second group (group 2) containing the conserved Pfam domain RepA_C (Accession No. PF04796) also found in a small number of sequences. The replication protein of p4M from B. pseudocate-

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Fig. 2. Phylogenetic tree comparing 18 Rep proteins from various Gram-positive bacteria generated by the MEGA2 program using pdistance values by the neighbor-joining method (Kumar et al., 2001). The alignments were generated using ClustalW (Thompson et al., 1994) with a gap insertion penalty of 8 and a gap extension penalty of 2. The numbers associated with the branches represent the bootstrap values as a proportion of 500 replicates in which the branch point occurred. The bar represents the number of substitutions per residue and branch lengths are significant only for branch points over 95%. The plasmid, host species and protein accession numbers of the Rep proteins used are pNAC1 (AAM66786), pNAC2 (AAM66783), pNAC3 (AAM66775), pKJ50 (AAD00258.1), pKJ36 (AAG43280.1), pBLO1 (AAN31777.1), pB44 (NP_758541.1), pDOJH10L (RepA—NP_694594.1, RepB—NP_694602.1, Rep— NP_694598.1), and pMB1 (X84655) from B. longum; pAP1 (CAA72313.1) from Bifidobacterium asteroides; p4M (AAM00235.1) from Bifidobacterium pseudocatenulatum; pCIBb1 (NP_052879.1), pNBb1 (GenBank Accession No. E17316) from Bifidobacterium breve; pXZ10142 (NP_052176.1) from Corynebacterium glutamicum; pBL-A8 (CAA72653.1) from Brevibacterium linens; ColE2-GEI602 (BAA06295) from E. coli. The complete alignment and details used to construct this tree are available upon request from the corresponding author.

nulatum is the only member of the third group (group 3), containing the Pfam domain Viral_Rep (Accession No. PF02407) that is found in numerous viral replication proteins.The Rep protein sequences from B. breve represent a fourth group of bifidobacterial plasmids (group 4), all of which contain the conserved Pfam domain Rep (Accession No. PF01446). However, these show lower similarity to other bifidobacterial plasmids. The Rep of plasmid pMB1 from B. longum forms a fifth group (group 5), showing some similarity (less than 50%) to Rep proteins of plasmids from Brevibacterium linens and Corynebacterium glu-

tamicum, but sharing the conserved Pfam domain Replicase (Accession No. PF03090). The Rep protein of pMB1 has thus been grouped with a family of small replicons showing similarity to ColE2, a theta-replicating plasmid from E. coli (De Mot et al., 1997). Although the replication mechanism has not been demonstrated experimentally for pMB1, motifs revealed by sequence comparison suggest a theta mechanism. Rep sequence comparison is a simple method to establish plasmid similarity groups (Del Solar et al., 1998). The 13 plasmids for which sequences are available in GenBank can be classified into five

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similarity groups. Plasmid pDOJH10L codes for three types of potential replication proteins, all found within B. longum. Only one type has also been found in another species, B. asteroides (pAP1). The Rep protein of pNAC3, along with that of pAP1 and the replicase of pDOJH10L, form a new family that is distinct from all other groups of bifidobacterial replication proteins. The remaining two groups of bifidobacterial plasmids appear constrained to a single species, but further work is necessary to determine host specificity. In general, bifidobacterial Rep proteins are related to each other within their respective phylogenetic group, but to a very low level with those of plasmids from other bacteria. Within group 1, two subgroups of higher similarity can be distinguished, justifying a nucleotide

sequence comparison extending along the whole length of these plasmids. The comparative similarity plot shows that variation in structural organization and similarity levels can be observed between subgroups (Fig. 3). Plasmids pNAC2 and pKJ36 (pB44 is 98% identical to pKJ36) have highly similar organization of all elements while only the Rep and ori regions are similar in the subgroup containing pNAC1 and pKJ50 (Fig. 3). Between the subgroups, the Rep proteins share similarity while the ori regions do not. Plasmid pDOJH10L appears to have ORFs similar to three different groups of Rep proteins; RepA, RepB, and a replicase. RepA belongs to the pNAC1 similarity subgroup while RepB belongs to the pNAC2 subgroup, and the replicase to the pNAC3 group. The complexity of the organization of

Fig. 3. Comparative DNA sequence analysis of B. longum plasmids pNAC1, pNAC2, pKJ50, and pKJ36. The predicted ORFs are identified by boxes. The length of the box is proportional to the length of the predicted ORFs. The colored bars give the percentage of nucleotide identity between two sequences as defined in the color code at the top of the figure. The vertical lines indicate the transition zones between two plasmids. The figure was created with the SIM Alignment tool and Lalnview (Huang and Miller, 1991; http:// ca.expasy.org/).

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pDOJH10L could not be represented in the same figure as the smaller plasmids, but homologous orfs similar to those of pNAC1/pKJ50 as well as to those of pNAC2/pKJ36 can be found within the sequence of pDOJH10L. This suggests that one or more cointegration events occurred to form pDOJH10L with plasmids of the subgroups of pNAC1 and pNAC2. However, most orfs of pDOJH10L differ from those found in pNAC3. Several conserved regions are thus shared by the B. longum plasmid sequences in the largest similarity group, including pNAC1 and pNAC2. The positions of the similar ORFs differ between pNAC2 and pKJ36. As the differences in the nucleic acid sequence are equally distributed over the whole DNA molecule, divergence has led to a notable variation in ORF structure. Overall, the most conserved elements in this group are involved in the replication and mobilization processes. This is not surprising, since these mechanisms ensure the distribution and the maintenance of these selfreplicating molecules. Each plasmid contained a putative cis-acting DNA structure typical of an origin of replication located upstream of the proposed repB genes (Fig. 4). The ori of pNAC1 contains three direct repeats of 22 bp, similar to the iterons involved in theta-type replication mechanisms and those of some rolling circle (RC) plasmids (Del Solar et al., 1998; Khan, 1997). Even though the iterons were not perfect, a consensus sequence (CCCTgagTT)

95

was determined. In addition, an A + T rich region located at the proper distance upstream of the iterons (Del Solar, 1998) and possibly involved in the initial opening of the dsDNA in a replication cycle was identified. In pNAC2, the ori region contained four perfect direct repeats of 22 bp (Fig. 4). In this case, an inverted repeat was found with the same consensus sequence as the iteron, near the start codon of the putative Rep protein. Similarly, an A + T rich region comparable to that found in the sequence of pNAC1 is also located upstream of the iteron. The inverted repeat (IR) located in this region is well positioned to constitute a RC nick site (dso) (Del Solar et al., 1998). However, no similarity was found between the IR and the dso consensus sequence of other RC plasmids. In pNAC3, the iteron consists of three direct repeats of 21 bp (Fig. 4), which can also be found upstream of the replicase of pDOJH10L. Once again, an A + T rich region is located upstream of the iteron (Fig. 4). The alignment of this ori region with a comparable region from pAP1 of B. asteroides did not show any significant similarity (data not shown). Potential origins of replication containing iterons are thus located upstream of each Rep protein. Plasmid replication (strand denaturation) could be initiated upstream of the iterons, where A + T rich sequences preceded by G + C rich sequences were identified. The thermal stability induced by the G + C rich region increases the flexibility of

Fig. 4. Organization of the ori region of the three B. longum plasmids sequenced in this study. Boxed arrows represent the beginning of the Rep protein while the direction of each repeat region is designated by line arrows. Regions rich in A + T are indicated by open rectangles.

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the adjacent area (Sobell et al., 1978) and could be used as a denaturation site to initiate plasmid replication. The balance between monomeric and dimeric forms of replication proteins contributes to the regulation of plasmid replication (Garcia de Viedma et al., 1996; Giraldo et al., 1989), and thus in controlling copy number. This balance is determined by inverted repeat sequences similar to the iterons, but located in the promoter region of the replication initiator. An inverted repeat, with partial similarity to the iteron consensus sequence, is located downstream of the iteron in pNAC2 but is missing in pKJ36. This structural difference between pNAC2 and pKJ36 could play an important role in the control of copy number of these plasmids. 3.3. The mobilization regions of pNAC2 and pNAC3 The second ORF of pNAC2 (Mob) is similar to other mobilization proteins (Table 2). A cis region was identified in the sequence of pNAC2 that resembles a putative origin of transfer, oriT, located upstream of the predicted mobilization protein and containing several inverted repeats (Fig. 1). Finally, a putative TraA protein (Orf2) was identified on pNAC3. The C-terminus of the predicted product of orf2 is similar to the nickase located at the N-terminus of TraA. Horizontal plasmid transfer allows different families of plasmids to spread from genus to genus. One of the most effective mechanisms for genetic information transfer is conjugation. This mechanism is initiated by cis (oriT) and trans (transfer proteins) elements found in mobilizable plasmids. Plasmids may not be able to promote their own conjugal transfer, but may remain able to be transferred if critical mobilization components are supplied by the host cell. For plasmid pNAC2, the position of the Mob protein and the characteristic cis elements suggests a mobilization potential. However, the consensus sequence for the nick site (TTCCTCTTG/C; Waters et al., 1991) in the cis element was not found. The Mob protein is similar to other mobilization proteins, where the activity has already been demon-

strated in L. lactis and T. ferroxidans (Dougherty et al., 1998; Drolet et al., 1990). The presence of other plasmids such as pNAC2, highly similar to pKJ36 and pB44, in other bifidobacteria does suggest active plasmid transfer, but evidence for this has yet to be presented. The orf2 in pNAC3 codes for a potential mobilization protein (155.79 kDa) similar to TraA found in Agrobacterium tumefaciens and Rhizobium spp. (Alt-M€ orbe et al., 1996; Farrand et al., 1996; Freiberg et al., 1997; Suzuki et al., 1998). Alt-M€ orbe et al. (Alt-M€ orbe et al., 1996) were the first to point out an internal start codon for traA preceded by a Shine–Dalgarno sequence. The analysis of TraA by separating the N-terminus and C-terminus revealed two distinct domains. The Nterminus moiety of TraA is a nickase essential for mobilization, and the second domain has helicase activity. Alt-M€ orbe et al. (Alt-M€ orbe et al., 1996) proposed that TraA results from the fusion of two separate genes (nickase and helicase). If so, the fusion has been inverted for Orf2, as the nickase domain is in the C-terminus and the helicase in the N-terminus. 3.4. Identification of ssDNA as an intermediate in the replication of pNAC1 Plasmid preparations isolated from B. longum strain RW048 were analysed for the presence of single-stranded DNA (ssDNA), which was confirmed by treatment with S1 nuclease, and Southern hybridization analysis of plasmid preparations with and without denaturation. Without denaturation, only ssDNA on the membrane hybridized with the probe (Fig. 5B). With denaturation, both the OC and CCC forms of plasmid pNAC1 were observed (data not shown). These results suggest that plasmid pNAC1 replicates by a rolling circle mechanism. The putative RepB proteins of pNAC1 and pNAC2 are similar to those encoded by pKJ50 and pKJ36, both of which likely replicate by the rolling circle mechanism (Park et al., 1999). The presence of ssDNA in B. longum RW048 confirms that pNAC1 replicates via a rolling circle mode. Although the replication mechanism of pNAC2 was not demonstrated, the similarity of RepB from

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Fig. 5. (A) Ethidium-bromide stained agarose gel of plasmid DNA from B. longum strains RW041 (lane 3) and RW048 (lane 4). Lane 1, 1-kb DNA ladder (Invitrogen, Burlington, ON); lane 2, supercoiled DNA ladder (Invitrogen). (B) Hybridization of the pNAC1 probe with single-stranded DNA intermediates of plasmid pNAC1 replication. DNA from strain RW048 was transferred under nondenaturing conditions. Lane 1 contains sample without S1 nuclease treatment while Lane 2 contains S1-nuclease-treated extract.

pNAC1 with the Rep of pKJ50 suggests a RC mode. 3.5. Isolation of the pNAC1 replicon Previous reports indicated the use of plasmids based on the ColE1 origin of replication to isolate functional replicons of Bifidobacterium in E. coli (Matsumura et al., 1997; Matteuzzi et al., 1990; Park et al., 1997; Park et al., 1999; Rossi et al., 1996). Since the vector pNC1 carries the cat gene as a functional marker in Bifidobacterium, the recombinant plasmid named pNC2 containing linearized pNAC1 cloned into pNC1 was also introduced by electroporation into B. longum ATCC 15708 and B. animalis ATCC 27536 to test for replication. The insertion site in pNC1 was selected to be far from the putative origin as well as from the orf coding for the RepB, in order to avoid interference with replication. Plasmid pNC1 was used as negative control and two Corynebacterium plasmids (pEBM3 and pECM2) replicating in Bifidobacterium (Sch€ afer et al., 1994; Tauch

et al., 1994) were used as positive controls. A transformation efficiency of 1  104 /lg of DNA was obtained with the positive controls pEBM3 and pECM2 and the recombinant plasmid pNC2. Surprisingly, colonies were also obtained with the negative control pNC1. The identity of the transformants was confirmed by observation of cell morphology under the microscope. Following plasmid DNA extraction, however, no plasmid DNA for either pNC1 or pNC2 was detectable on an ethidium bromide stained agarose gel. To confirm the presence of plasmids, the extracts were transformed back into E. coli DH5a by electroporation. Transformants were obtained at high frequency and the plasmid DNA was isolated from these cells for further characterization. The plasmid DNA was of variable size, indicating that DNA rearrangements had occurred in Bifidobacterium (Fig. 6). In addition, Southern hybridization with probes consisting of pNC1 and pNAC1, respectively, showed that the DNA rearrangements completely deleted pNAC1 in pNC2 transformants while maintaining pNC1 (Fig. 6). These

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Fig. 6. Analysis of B. animalis plasmid content after retransformation into E. coli DH5a. Plasmid DNA electroporated in E. coli DH5a was previously isolated from B. animalis ATCC 27536. (A) DNA digested with AatII or BamHI and migrated in 0.8% agarose gel. (B) Autoradiogram obtained after Southern hybridization with pNC1 as probe. (C) Autoradiogram obtained after Southern hybridization with pNAC1 as probe (lanes 7–20 are negative). Top row: 1, 1-kb DNA ladder (Invitrogen, Burlington, ON); 2, k EcoRI/ HindIII DIG labeled (Invitrogen); 3, pNC1/AatII; 4, pNC2/BamHI; 5–15, pNC1 retransformation in E. coli/AatII; 16–20, pNC2 retransformation in E. coli/BamHI. Bottom row: 1, 1-kb DNA ladder (Invitrogen); 2, k EcoRI/HindIII DIG labeled (Invitrogen); 3, pNC2/BamHI; 4–20, pNC2 retransformation in E. coli/BamHI.

results clearly indicate that ColE1, from high copy number plasmid pUC18, can be rescued from B. longum and B. animalis. Loss of the chloramphenicol resistance phenotype was observed for 50% of the transformant colonies after incubation in MRSc liquid medium containing chloramphenicol at 5 lg/mL, indicating that the pUC18-ColE1 cannot be stably maintained in Bifidobacterium. The ColE1 origin of replication has been widely used to develop replicon probe vectors for bifidobacteria species (Matsumura et al., 1997; Matteuzzi et al., 1990; Park et al., 1997, 1999; Rossi et al., 1996) along with the cat resistance gene from pC194 (Matteuzzi et al., 1990; Park et al., 1999; Rossi et al., 1996, 1997, 1998). Furthermore, most of the vectors used to isolate replicons from Bifidobacterium plasmids are in fact based on the ColE1 origin (Matsumura et al., 1997; Matteuzzi et al., 1990; Missich et al., 1994; Park et al., 1997, 1999; Rossi et al., 1996). Future work should be directed at developing replicon screening vectors based on different types of E. coli origins of rep-

lication in order to surmount such potential incompatibility problems as were encountered with the use of pUC18.

4. Conclusions Plasmids from both strains RW048 and RW041 encode replication proteins that are closely related to Rep proteins of other plasmids in B. longum. The availability of a variety of plasmid replicons diversifies the choice for constructing vectors that may be compatible with resident plasmids of Bifidobacterium. The consumption of bifidobacteria has been claimed to provide beneficial effects on human health (Mitsuoka, 1990). As a consequence of this, dairy products supplemented with Bifidobacterium are now widely available. Fundamental studies are essential in order to develop the molecular tools to validate the proposed beneficial properties linked with these products. Knowledge of the structural organization of plasmids in Bifidobacterium allows

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their application in constructing vectors to carry out these fundamental studies. The identification of new plasmid replicons provides more options for compatibility with resident plasmids. Future studies will aim at developing this new generation of vectors.

Acknowledgments We thank the members of the plasmid group of the Canadian Network on Lactic Acid Bacteria for helpful discussion and Martin Kalmokoff for critical reading of the manuscript. We are very grateful to Dr. Denis Roy for kindly providing the bifidobacterial strains RW041 and RW048. We would like to thank Dr. Andreas Tauch for providing plasmids pECM2 and pEBM3. The authors acknowledge, for their financial support, the Natural Sciences and Engineering Research Council of Canada (Research Partnership Program-Research Network on Lactic Acid Bacteria), Agriculture and Agri-Food Canada, and Institut RosellLallemand Inc.

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