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Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector
FEMS Microbiology Letters 179 (1999) 375^383
Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector Franck Biet a
a;1;
*, Yves Cenatiempo a , Christophe Fremaux
b
Institut de Biologie Mole¨culaire et d'Inge¨nierie Ge¨ne¨tique, CNRS-ESA 6031, Universite¨ de Poitiers, 40 avenue du recteur Pineau, 86022 Poitiers, Cedex, France b Texel, groupe Rhoªne-Poulenc, Z.A. de Buxie©res B.P. 10, 86220 Dange¨ Saint-Romain, France Received 12 July 1999; received in revised form 25 August 1999; accepted 26 August 1999
Abstract A 1.8-kb cryptic plasmid pFR18 was isolated from Leuconostoc mesenteroides ssp. mesenteroides FR52 and characterized. The identification of single-stranded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replication of pFR18 is directed by a rolling-circle mechanism (RCR). Sequence analysis revealed a single open reading frame (rep18) encoding a putative 335-amino acid protein homologous to the pT181 replicase. Furthermore, a putative double strand origin similar to that of the pT181 plasmid family was identified. A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential region of the plasmid. The resulting construction was able to transform Lactobacillus sake and various species of Leuconostoc. It was stable in L. mesenteroides, however, the segregational stability of a pFR18 derivative containing large Escherichia coli DNA fragments was affected. Nevertheless, the new RCR plasmid pFR18 may be useful for the construction of food grade vectors. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Plasmid ; Rolling-circle replication ; Vector; Leuconostoc
1. Introduction Bacteria of the genus Leuconostoc represent a diversi¢ed group of heterofermentative organisms
* Corresponding author. Tel.: +33 (3) 2087 1155; Fax: +33 (3) 2087 1158; E-mail: [email protected] 1 Present address: INSERM U447, Institut Pasteur de Lille, 1 rue du Prof. Calmette, BP 245, 59019 Lille, Cedex, France.
commonly used in food industry. Among lactic acid bacteria (LAB), Leuconostoc contributes to £avor development, food preservation and is used for starter cultures. In dairy fermentations, this organism plays an important role in £avor development because of its ability to produce carbon dioxide and C4 aroma compounds through lactose heterofermentation and citrate utilization [1]. Several Leuconostoc species, such as Leuconostoc mesenteroides ssp. mesenteroides FR52 isolated from cow milk, produce bacteriocin [2]. The genome analysis of these organ-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 3 7 - 1
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isms has been of growing interest, providing the base of biotechnological exploitation. As many other LAB, Leuconostoc species harbor one or several plasmids of various sizes [3] without any known function for most of them. Gene cloning in LAB relies classically on the development of vectors from cryptic or drug resistance plasmids. Cloning vectors derived from plasmids of Gram-positive bacteria replicate either by rolling-circle replication (RCR) or by a replication mechanisms [4,5]. RCR-type vectors such as pWV01 derivatives have been extensively studied and widely used, because of their broad replication host-range [6]. However, several studies have indicated that the RCR mechanism leads to plasmid segregational instability [4^7]. a-type plasmids are also used to design vectors, the best known being pAML1 [8], a broad host-range plasmid. In this report, we describe the cloning and sequence analysis of pFR18, a small cryptic plasmid from L. mesenteroides ssp. mesenteroides FR52. Its genetic organization and mode of replication are described, as well as its host-range and stability. The potential of pFR18 to provide a `food grade' cloning vector for general use in LAB is examined.
2. Materials and methods 2.1. Bacterial strains and plasmids Bacterial strains and plasmids used in this study are listed in Table 1. Leuconostoc ssp. strains and Pediococcus acidilactici P120 were grown in MRS (Difco) broth or agar (1.2%, w/v) at 30³C. MRS medium supplemented with 1% glucose was used for Lactobacillus sake 23K propagation at 30³C. When appropriate, erythromycin 5 Wg ml31 and rifampicin 100 Wg ml31 were added. Escherichia coli strains were propagated at 37³C in LB [9] broth or on agar (1.5%, w/v). Ampicillin and erythromycin were used at ¢nal concentrations of 100 and 150 Wg ml31 , respectively. Erythromycin resistant transformants of E. coli were selected on brain heart infusion agar plates (Difco). 2.2. DNA isolation, manipulation and sequence determination General genetic techniques used were as previously described [9,10]. Plasmid DNA was digested with
Table 1 Bacterial strains and plasmids Strains or plasmids E. coli DH5K L. mesenteroides ssp. mesenteroides FR52 L. mesenteroides ssp. dextranicum DSM20484 Leuconostoc cremoris LC L. lactis IL1403 RD230 L. sake 23K P. acidilactici P120 Plasmids pBSSKII pFR18 pGhost9:ISS1 pFBYC018 pFBYC018E pFBYC18E
Relevant characteristics
Source or reference
recA endA1 gyrA96 thi-1 hsdR17 supE44 vlac U169(x80 dlacZvM15) deoR F3 V3
Gibco BRL
Wild-type strain
[2]
Wild-type strain
DSM
Wild-type industrial strain
Texel
Lab strain Wild-type industrial strain
[23] Texel
Plasmid cured
[13]
Wild-type industrial strain
Texel
pBluescript SK II 2.96-kb cloning vector, ApR 1.8-kb cryptic plasmid from L. mesenteroides FR52 pWV01 derivative replicon, EmR pBSSKII : :1.8-kb HindIII pFR18, 4.8 kb, ApR pFBYC018: :1.1-kb BamHI pGhost9:ISS1, 5.9 kb, ApR , EmR pFBYC018E v 2.5-kb PvuII pBSSKII , 3.5 kb, EmR
Stratagene This study [22] This study This study This study
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restriction enzymes (Gibco BRL) and recombinant DNA was obtained using T4 DNA ligase (Gibco BRL) according to the supplier's recommendations. In-gel plasmid DNA digestions were performed using SeaPlaque GTG agarose (FMC BioProducts) according to the manufacturer's instructions. DNA restriction fragments were puri¢ed from agarose gels using the Prep-a-Gene kit (Bio-Rad). E. coli competent cells were prepared and transformed according to Hanahan [11]. Preparation of electro-competent LAB cells and electroporation were as described previously [12,13]. DNA sequences were determined using the Auto-read1 sequencing kit (Pharmacia) and appropriate primers with an automated DNA sequencer (ALF, Pharmacia). Sequence analyses were performed with the GCG sequence analysis software package (University of Wisconsin). 2.3. Detection of single-stranded DNA (ssDNA) in whole-cell lysates of Leuconostoc Cells were grown in MRS medium. When required, rifampicin was added at 100 Wg ml31 , in order to inhibit RNA polymerase [6]. Whole-cell lysate preparation was as described previously [10,14]. S1 nuclease (Gibco BRL) treatment was performed according to Noirot-Gros and Ehrlich [14]. ssDNA was detected by non-alkaline Southern blot hybridization. DNA transfer from an agarose gel to a Hybond-N nylon membrane (Amersham) was adapted from [9], the DNA denaturation step was omitted. DNA probes consisted in puri¢ed DNA restriction fragments labelled by using a random priming kit (Gibco BRL) and [K-32 P]dCTP (Amersham). Hybridization of the transferred DNA with the labelled probes was performed according to te Riele et al. [15]. 2.4. Plasmid stability analysis Single colonies of Leuconostoc transformants carrying various plasmids were used to inoculate MRS broth without erythromycin and successive cultures were performed in order to obtain approximately 100 generations. The plasmid stability was then estimated by comparison of culture numeration on selective (5 Wg ml31 erythromycin) and non-selective (without antibiotic) agar plates. Plasmid DNA pre-
377
pared from erythromycin resistant colonies cultured over 100 generations without selection pressure was analyzed by a Southern blot to insure the plasmid structural stability.
3. Results and discussion 3.1. Isolation of pFR18 and derivatives construction L. mesenteroides ssp. mesenteroides FR52 was isolated from cow milk [2]. It harbors four plasmids of approximately 38, 16, 14 and 1.8 kb (not shown). The smallest of these plasmids, pFR18 (1.8 kb), was cloned into the unique HindIII site of pBSSKII to yield pFBYC018. This construct was used for sequence determination and replicon analysis. In order to develop a plasmid amenable to Gram-positive bacteria, the BamHI erythromycin cassette originating from pGhost9:ISS1 was inserted into the unique BamHI site of pFBYC018 to give pFBYC018E. Finally, pFBYC18E was obtained by deleting the 2.4kb PvuII fragment of pFBYC018E containing the ColE1 origin of replication. 3.2. Nucleotide sequence and general features of pFR18 The complete nucleotide sequence of pFR18 (1828 bp) was determined and is shown in Fig. 1. The G+C content in pFR18 is 37%, in accordance with the G+C % of Leuconostoc ssp. chromosomes and plasmids previously described [16^18]. The plot of GC/ ATGC in the pFR18 sequence revealed the presence of a large 281-bp G+C rich region (above 50% G+C), located between nucleotides 178 and 459 (Fig. 1). Within this region, three palindromic structures noted IRII, IRIII and IRIV were identi¢ed (see Fig. 1). IRII and IRIV could form hairpin loops, with calculated free energies of formation, vG³, of 35.8 and 38 kcal mol31 , respectively. A fourth inverted repeat noted IRI in Fig. 1 was detected at positions 47^66 (vG³ = 314 kcal mol31 ). In addition, two 11-bp direct repeats, DRI and DRII, were identi¢ed upstream from the G+C rich region and between IRII and IRIV, respectively (Fig. 1). Sequence analysis revealed a single open reading frame (rep18), spanning 55% of the plasmid sequence. The rep18
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Fig. 1.
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Fig. 1. Complete nucleotide sequence of pFR18 linearized at its unique HindIII site, showing the deduced amino acid sequence for replicase Rep18. The arrows indicate inverted (IRI, IRII, IRIII and IRIV) and direct repeats (DRI and DRII). Putative DSO, a potential ribosome binding site (RBS) and a HindIII restriction site are underlined. The large G+C rich region is indicated in bold letters. The EMBL accession number for the sequence reported in this paper is Z99436.
gene extends from position 740 to 1745 and is capable to encode a polypeptide of 335 residues. A potential ribosome binding site GGAGA is located 8 bp upstream of the putative ATG start codon (Fig. 1). Scanning data banks for sequences homologous to that deduced from rep18 (Rep18) revealed stringent homology with the replication initiation protein RepC of pT181 (20% identity and 38% similarity) [19]. This plasmid isolated from Staphylococcus aureus de¢nes one of the plasmid families described by Novick [20]. Plasmids of the pT181 family all encode replication initiator proteins (Rep) with high degrees of identity ranging from 63 to 80% [21]. Projan and Novick de¢ned two domains within the Rep proteins [21]. The ¢rst domain contains an active tyrosine and is involved in the nicking of the leading strand origin termed double strand origin (DSO). A similar domain including a tyrosine residue was found within Rep18 (see Fig. 2A). The second domain is less conserved within the pT181 Rep family and plays a role
in the DSO recognition. DSOs are very similar within the pT181 family (see Fig. 2B). However, this region is slightly divergent in pFR18 and located outside of the rep18 gene (Fig. 2B), in contrast to the pT181 family DSOs which are located in the replicase genes. This may explain the low degree of homology observed between the Rep18 domain involved in the recognition of the DSO and the homologous region of other Reps. On the basis of sequence comparison, no pT181 single strand origin (SSO) was identi¢ed in pFR18. This element is probably speci¢c to the bacterial genus. 3.3. Evidence for ssDNA intermediate Plasmids of the pT181 family replicate via an RCR mechanism. Thus, pFR18 was likely to use a similar replication mechanism. The RCR implicates the formation of a single stranded (ssDNA) intermediate of the plasmid [15]. Therefore, the ability of pFR18 to generate ssDNA was examined using
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whole-cell DNA of L. mesenteroides ssp. dextranicum DSM20484 containing pFBYC18E. First, a DSM20484 transformant containing pGhost9:ISS1, a well described RCR plasmid [22], was used in a control experiment (Fig. 3A). Whole-cell DNA prepared from DSM20484 (pGhost9:ISS1) grown in MRS containing rifampicin was hybridized to a pGhost9:ISS1 speci¢c probe. In addition to the open circular form (OC) and covalently close circular form (CCC) of the plasmid, the probe hybridized to a fast migrating band, suspected to be a ssDNA intermediate (SS, Fig. 3A, lane 1). Its sensitivity to nuclease S1 demonstrated its single-stranded nature (Fig. 3A, lane 2). This form was barely detectable in extracts from cells grown in the absence of rifampicin (data not shown). Similarly, whole-cell DNA from DSM20484 containing pFBYC18E was tested
using pFR18 as a probe (Fig. 3B). A single-stranded form of plasmid was clearly detected (SS, Fig. 3B, lanes 1 and 3) as proved by the nuclease S1 sensitivity of the fasted migrating band (Fig. 3B, lanes 2 and 4). In contrast to pGhost9:ISS1, pFBYC18E generated an accumulation of ssDNA intermediate in DSM20484 grown with or without the addition of rifampicin to the medium. This could be due to the cloning of the DNA fragment containing the erythromycin resistance cassette, in an SSO or in a locus implicated in the ssDNA intermediate conversion. This observation is not surprising since the insertion of foreign DNA in RCR plasmids has been shown to disturb their replication and favor the accumulation of ssDNA intermediate, thus enhancing segregational instability [7]. The detection of ssDNA intermedi-
Fig. 2. (A) Amino acid comparison between the active site region of replicases encoded by the pT181 plasmid family (upper part of the ¢gure) and the putative Rep18 replicase encoded by pFR18. Asterisks indicate amino acid identity and dots indicate conserved residues between RepC, encoded by pT181, and Rep18. The conserved tyrosine residue involved in the DNA nicking activity is indicated by a vertical arrow. (B) Nucleotide sequence comparison of the DSO of pT181-type plasmids and putative pFR18 DSO. The stem-loop structure that includes the replication nick site (vertical arrow) is indicated by a horizontal arrow. Some gaps were allowed for maximal alignment. The putative pFR18 DSO was aligned according to the potential nick site located within the potential stem-loop structure.
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Fig. 3. Detection of single-stranded intermediates of pGhost9:ISS1 (A) and pFBYC18E (B) in L. mesenteroides ssp. dextranicum DSM20484. Non-alkaline Southern blot analysis was performed with radio-labelled pGhost9:ISS1 (A) and pFBYC18E (B) probes. Whole-cell DNA was prepared from cells grown in MRS containing rifampicin (lanes 1 and 2) or in MRS without rifampicin (lanes 3 and 4). DNA preparations were treated with endonuclease S1 in lanes 2 and 4. OC, OC DNA; CCC, CCC DNA; SS, ssDNA.
ate demonstrated that pFR18 replicates using a RCR mechanism. 3.4. Construction of a cloning vector based on pFR18 pFR18 may be a good alternative for the development of Leuconostoc and other LAB cloning vectors, considering its small size, food grade origin and compatibility with the pWV01 replicon that served as a basis for most genetic tools devoted to LAB. The minimum replicon seems to be pFR18 itself. Indeed, by deleting di¡erent regions of pFBYC018E, we found that rep18 is involved in pFR18 replication, as well as the features contained within the G+C rich region (data not shown). RCR plasmids are known to be a¡ected in segregational stability when they contain foreign DNA [7] and we observed that pGhost9:ISS1 is not fully stable in DSM20484 (see Table 2). Similarly, the segregational stability of the E. coli/Leuconostoc shuttle vector pFBYC018E, which contains more than 4 kb of foreign DNA, is signi¢cantly a¡ected compared to that of
pFBYC18E, which contains a 1.6-kb insert (Table 2). Southern blot analysis of plasmid DNA prepared from erythromycin resistant colonies cultivated for 100 generations without selection pressure did not reveal any structural change in pFBYC018E nor pFBYC18E. The severe segregational loss observed with DSM20484 (pFBYC018E) growing with no selection pressure seems to be related to the size of the foreign DNA inserted into the pFR18 BamHI site as no plasmid loss was observed with DSM20484 (pFBYC18E). This region could play a role in the ssDNA duplication as a SSO or as a structure recTable 2 Stability of pFR18 derivatives during extended growth under non-selective conditions Plasmids
% of DSM20484 clones containing the plasmid after 100 generationsa
pGhost9:ISS1 pFBYC018E pFBYC18E
76 3 100
a
Average for duplicate experiments.
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ognized by host factors. To improve segregational stability of recombinant plasmids, other regions of pFBYC18E could be investigated as cloning sites. However, plasmid instability could be pro¢table in some cases, as shown for pIL252 [23,7]. pFBYC18E is able to replicate in various Leuconostoc species (cremoris, mesenteroides ssp. mesenteroides and mesenteroides ssp. dextranicum) as well as in L. sake, but not in Lactococcus lactis, P. acidilactici or E. coli (data not shown). Consequently, pFBYC18E constitutes a potential narrow host-range cloning vector. The pFR18 replicon is small and compatible with pWW01 derivatives and with at least three other plasmids harbored by L. mesenteroides ssp. mesenteroides FR52. In addition, legal issues on modi¢ed micro-organisms may require to use genetic material originating from the Leuconostoc genus for the genetic manipulation of Leuconostoc species. Thus pFR18 is an attractive new replicon for food grade vector construction.
4. Uncited references [24]
Acknowledgements This work has been achieved as part of the program `BIOAVENIR' (contract # 780227), supported by Rhoªne-Poulenc with the participation of the Ministe©re de la Recherche et de l'Espace and the Ministe©re de l'Industrie et du Commerce Exte¨rieur. We are grateful to Monique Zagorec for her contribution to the transfer of plasmids in L. sake. We thank Laurent Jannie©re for helpful discussion on ssDNA detection. We are grateful to the Laboratoire de Microbiologie Industrielle et Alimentaire, ENSAIA-INPL, Vandoeuvre, France, for supplying L. mesenteroides ssp. mesenteroides FR52.
References [1] Buckenhu«skes, H.J. (1993) Selection criteria to be used as starter cultures for various food commodities. FEMS Microbiol. Rev. 12, 253^271.
[2] Mathieu, F., Sudirman Suwhandi, I., Rekhif, N., Millie©re, J.B. and Lefebvre, G. (1993) Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52. J. Appl. Bacteriol. 74, 372^379. [3] Orberg, P.K. and Sandine, W. (1984) Common occurence of plasmid DNA and vancomycin resistance in Leuconostoc ssp.. Appl. Environ. Microbiol. 48, 1129^1133. [4] Gruss, A. and Ehrlich, D. (1989) The family of interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53, 231^241. [5] Jannie©re, L., Gruss, A. and Ehrlich, D.S. (1993) Plasmids. In: Bacillus subtilis and other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (Sonenshein, A.L., Hoch, J.A. and Losick, R., Eds.), pp. 625^643. American Society for Microbiology, Washington, DC. [6] Leenhouts, K.J., Tolner, B., Bron, S., Kok, J., Venema, G. and Seegers, J.F.M.L. (1991) Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWV01. Plasmid 26, 55^66. [7] Kiewiet, R., Bron, S., De Jonge, K., Venema, G. and Seegers, J.F.M.L. (1993) Theta replication of the lactococcal plasmid pWV02. Mol. Microbiol. 10, 319^327. [8] Jannie©re, L., Bruand, C. and Ehrlich, D.S. (1990) Structurally stable Bacillus subtilis cloning vectors. Gene 87, 53^61. [9] Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning : a Laboratory Manual. Cold Spring Harbor Laboratory Press, NY. [10] Muriana, P.M. and Klaenhammer, T.R. (1987) Conjugal transfert of plasmid-encoded determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Appl. Environ. Microbiol. 53, 553^560. [11] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmid DNA. J. Mol. Biol. 166, 557^580. [12] Raya, R.R., Fremaux, C., De Antoni, G.L. and Klaenhammer, T.R. (1992) Site-speci¢c integration of the temperate bacteriophage Padh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment site. J. Bacteriol. 174, 5584^ 5592. [13] Berthier, F., Zagorec, M., Champomier-Verge©s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) E¤cient transformation of Lactobacillus sake by electroporation. Microbiology 142, 1273^1279. [14] Noirot-Gros, M.-F. and Ehrlich, S.D. (1994) Detection of single-stranded plasmid DNA. Methods Mol. Genet. 3, 370^ 378. [15] te Riele, H., Michel, B. and Ehrlich, D.S. (1986) Singlestranded plasmid DNA in bacillus subtilis and Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 83, 2541^2545. [16] Garvie, E.I., Zezula, V. and Hill, V.A. (1974) Hybridization between deoxyribonucleic acids of some strains of heterofermentative lactic acid bacteria. Int. J. Syst. Bacteriol. 24. [17] Brito, L., Vieria, G., Santos, M.A. and Paveia, H. (1996) Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos. Plasmid 36, 49^54. [18] Co¡ey, A., Harrington, A., Kearney, K., Daly, C. and Fitzgerald, G. (1994) Nucleotide sequence and structural organ-
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[19]
[20] [21] [22]
isation of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533. Microbiology 140, 2263^2269. Khan, S. and Novick, R.P. (1983) Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Plasmid 10, 251^259. Novick, R.P. (1989) Staphylococcal plasmids and their replication. Annu. Rev. Microbiol. 43, 537^565. Projan, S.J. and Novick, R.P. (1988) Comparative analysis of ¢ve related staphylococcal plasmids. Plasmid 19, 203^221. Maguin, E., Pre¨vost, H., Ehrlich, D.S. and Gruss, A. (1996)
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E¤cient insertional mutagenesis in Lactococci and other Gram-positive bacteria. J. Bacteriol. 178, 931^935. [23] Simon, D. and Chopin, A. (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70, 559^566. [24] Noguchi, N., Aoki, T., Sasatsu, M., Kono, M., Shishido, K. and Ando, T. (1986) Determination of the complete nucleotide sequence of pNS1, a staphylococcal tetracycline-resistance plasmid propagated in Bacillus subtilis. Appl. Environ. Microbiol. 48, 1129^1133.
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Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector Franck Biet a
a;1;
*, Yves Cenatiempo a , Christophe Fremaux
b
Institut de Biologie Mole¨culaire et d'Inge¨nierie Ge¨ne¨tique, CNRS-ESA 6031, Universite¨ de Poitiers, 40 avenue du recteur Pineau, 86022 Poitiers, Cedex, France b Texel, groupe Rhoªne-Poulenc, Z.A. de Buxie©res B.P. 10, 86220 Dange¨ Saint-Romain, France Received 12 July 1999; received in revised form 25 August 1999; accepted 26 August 1999
Abstract A 1.8-kb cryptic plasmid pFR18 was isolated from Leuconostoc mesenteroides ssp. mesenteroides FR52 and characterized. The identification of single-stranded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replication of pFR18 is directed by a rolling-circle mechanism (RCR). Sequence analysis revealed a single open reading frame (rep18) encoding a putative 335-amino acid protein homologous to the pT181 replicase. Furthermore, a putative double strand origin similar to that of the pT181 plasmid family was identified. A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential region of the plasmid. The resulting construction was able to transform Lactobacillus sake and various species of Leuconostoc. It was stable in L. mesenteroides, however, the segregational stability of a pFR18 derivative containing large Escherichia coli DNA fragments was affected. Nevertheless, the new RCR plasmid pFR18 may be useful for the construction of food grade vectors. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Plasmid ; Rolling-circle replication ; Vector; Leuconostoc
1. Introduction Bacteria of the genus Leuconostoc represent a diversi¢ed group of heterofermentative organisms
* Corresponding author. Tel.: +33 (3) 2087 1155; Fax: +33 (3) 2087 1158; E-mail: [email protected] 1 Present address: INSERM U447, Institut Pasteur de Lille, 1 rue du Prof. Calmette, BP 245, 59019 Lille, Cedex, France.
commonly used in food industry. Among lactic acid bacteria (LAB), Leuconostoc contributes to £avor development, food preservation and is used for starter cultures. In dairy fermentations, this organism plays an important role in £avor development because of its ability to produce carbon dioxide and C4 aroma compounds through lactose heterofermentation and citrate utilization [1]. Several Leuconostoc species, such as Leuconostoc mesenteroides ssp. mesenteroides FR52 isolated from cow milk, produce bacteriocin [2]. The genome analysis of these organ-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 3 7 - 1
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isms has been of growing interest, providing the base of biotechnological exploitation. As many other LAB, Leuconostoc species harbor one or several plasmids of various sizes [3] without any known function for most of them. Gene cloning in LAB relies classically on the development of vectors from cryptic or drug resistance plasmids. Cloning vectors derived from plasmids of Gram-positive bacteria replicate either by rolling-circle replication (RCR) or by a replication mechanisms [4,5]. RCR-type vectors such as pWV01 derivatives have been extensively studied and widely used, because of their broad replication host-range [6]. However, several studies have indicated that the RCR mechanism leads to plasmid segregational instability [4^7]. a-type plasmids are also used to design vectors, the best known being pAML1 [8], a broad host-range plasmid. In this report, we describe the cloning and sequence analysis of pFR18, a small cryptic plasmid from L. mesenteroides ssp. mesenteroides FR52. Its genetic organization and mode of replication are described, as well as its host-range and stability. The potential of pFR18 to provide a `food grade' cloning vector for general use in LAB is examined.
2. Materials and methods 2.1. Bacterial strains and plasmids Bacterial strains and plasmids used in this study are listed in Table 1. Leuconostoc ssp. strains and Pediococcus acidilactici P120 were grown in MRS (Difco) broth or agar (1.2%, w/v) at 30³C. MRS medium supplemented with 1% glucose was used for Lactobacillus sake 23K propagation at 30³C. When appropriate, erythromycin 5 Wg ml31 and rifampicin 100 Wg ml31 were added. Escherichia coli strains were propagated at 37³C in LB [9] broth or on agar (1.5%, w/v). Ampicillin and erythromycin were used at ¢nal concentrations of 100 and 150 Wg ml31 , respectively. Erythromycin resistant transformants of E. coli were selected on brain heart infusion agar plates (Difco). 2.2. DNA isolation, manipulation and sequence determination General genetic techniques used were as previously described [9,10]. Plasmid DNA was digested with
Table 1 Bacterial strains and plasmids Strains or plasmids E. coli DH5K L. mesenteroides ssp. mesenteroides FR52 L. mesenteroides ssp. dextranicum DSM20484 Leuconostoc cremoris LC L. lactis IL1403 RD230 L. sake 23K P. acidilactici P120 Plasmids pBSSKII pFR18 pGhost9:ISS1 pFBYC018 pFBYC018E pFBYC18E
Relevant characteristics
Source or reference
recA endA1 gyrA96 thi-1 hsdR17 supE44 vlac U169(x80 dlacZvM15) deoR F3 V3
Gibco BRL
Wild-type strain
[2]
Wild-type strain
DSM
Wild-type industrial strain
Texel
Lab strain Wild-type industrial strain
[23] Texel
Plasmid cured
[13]
Wild-type industrial strain
Texel
pBluescript SK II 2.96-kb cloning vector, ApR 1.8-kb cryptic plasmid from L. mesenteroides FR52 pWV01 derivative replicon, EmR pBSSKII : :1.8-kb HindIII pFR18, 4.8 kb, ApR pFBYC018: :1.1-kb BamHI pGhost9:ISS1, 5.9 kb, ApR , EmR pFBYC018E v 2.5-kb PvuII pBSSKII , 3.5 kb, EmR
Stratagene This study [22] This study This study This study
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restriction enzymes (Gibco BRL) and recombinant DNA was obtained using T4 DNA ligase (Gibco BRL) according to the supplier's recommendations. In-gel plasmid DNA digestions were performed using SeaPlaque GTG agarose (FMC BioProducts) according to the manufacturer's instructions. DNA restriction fragments were puri¢ed from agarose gels using the Prep-a-Gene kit (Bio-Rad). E. coli competent cells were prepared and transformed according to Hanahan [11]. Preparation of electro-competent LAB cells and electroporation were as described previously [12,13]. DNA sequences were determined using the Auto-read1 sequencing kit (Pharmacia) and appropriate primers with an automated DNA sequencer (ALF, Pharmacia). Sequence analyses were performed with the GCG sequence analysis software package (University of Wisconsin). 2.3. Detection of single-stranded DNA (ssDNA) in whole-cell lysates of Leuconostoc Cells were grown in MRS medium. When required, rifampicin was added at 100 Wg ml31 , in order to inhibit RNA polymerase [6]. Whole-cell lysate preparation was as described previously [10,14]. S1 nuclease (Gibco BRL) treatment was performed according to Noirot-Gros and Ehrlich [14]. ssDNA was detected by non-alkaline Southern blot hybridization. DNA transfer from an agarose gel to a Hybond-N nylon membrane (Amersham) was adapted from [9], the DNA denaturation step was omitted. DNA probes consisted in puri¢ed DNA restriction fragments labelled by using a random priming kit (Gibco BRL) and [K-32 P]dCTP (Amersham). Hybridization of the transferred DNA with the labelled probes was performed according to te Riele et al. [15]. 2.4. Plasmid stability analysis Single colonies of Leuconostoc transformants carrying various plasmids were used to inoculate MRS broth without erythromycin and successive cultures were performed in order to obtain approximately 100 generations. The plasmid stability was then estimated by comparison of culture numeration on selective (5 Wg ml31 erythromycin) and non-selective (without antibiotic) agar plates. Plasmid DNA pre-
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pared from erythromycin resistant colonies cultured over 100 generations without selection pressure was analyzed by a Southern blot to insure the plasmid structural stability.
3. Results and discussion 3.1. Isolation of pFR18 and derivatives construction L. mesenteroides ssp. mesenteroides FR52 was isolated from cow milk [2]. It harbors four plasmids of approximately 38, 16, 14 and 1.8 kb (not shown). The smallest of these plasmids, pFR18 (1.8 kb), was cloned into the unique HindIII site of pBSSKII to yield pFBYC018. This construct was used for sequence determination and replicon analysis. In order to develop a plasmid amenable to Gram-positive bacteria, the BamHI erythromycin cassette originating from pGhost9:ISS1 was inserted into the unique BamHI site of pFBYC018 to give pFBYC018E. Finally, pFBYC18E was obtained by deleting the 2.4kb PvuII fragment of pFBYC018E containing the ColE1 origin of replication. 3.2. Nucleotide sequence and general features of pFR18 The complete nucleotide sequence of pFR18 (1828 bp) was determined and is shown in Fig. 1. The G+C content in pFR18 is 37%, in accordance with the G+C % of Leuconostoc ssp. chromosomes and plasmids previously described [16^18]. The plot of GC/ ATGC in the pFR18 sequence revealed the presence of a large 281-bp G+C rich region (above 50% G+C), located between nucleotides 178 and 459 (Fig. 1). Within this region, three palindromic structures noted IRII, IRIII and IRIV were identi¢ed (see Fig. 1). IRII and IRIV could form hairpin loops, with calculated free energies of formation, vG³, of 35.8 and 38 kcal mol31 , respectively. A fourth inverted repeat noted IRI in Fig. 1 was detected at positions 47^66 (vG³ = 314 kcal mol31 ). In addition, two 11-bp direct repeats, DRI and DRII, were identi¢ed upstream from the G+C rich region and between IRII and IRIV, respectively (Fig. 1). Sequence analysis revealed a single open reading frame (rep18), spanning 55% of the plasmid sequence. The rep18
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Fig. 1.
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Fig. 1. Complete nucleotide sequence of pFR18 linearized at its unique HindIII site, showing the deduced amino acid sequence for replicase Rep18. The arrows indicate inverted (IRI, IRII, IRIII and IRIV) and direct repeats (DRI and DRII). Putative DSO, a potential ribosome binding site (RBS) and a HindIII restriction site are underlined. The large G+C rich region is indicated in bold letters. The EMBL accession number for the sequence reported in this paper is Z99436.
gene extends from position 740 to 1745 and is capable to encode a polypeptide of 335 residues. A potential ribosome binding site GGAGA is located 8 bp upstream of the putative ATG start codon (Fig. 1). Scanning data banks for sequences homologous to that deduced from rep18 (Rep18) revealed stringent homology with the replication initiation protein RepC of pT181 (20% identity and 38% similarity) [19]. This plasmid isolated from Staphylococcus aureus de¢nes one of the plasmid families described by Novick [20]. Plasmids of the pT181 family all encode replication initiator proteins (Rep) with high degrees of identity ranging from 63 to 80% [21]. Projan and Novick de¢ned two domains within the Rep proteins [21]. The ¢rst domain contains an active tyrosine and is involved in the nicking of the leading strand origin termed double strand origin (DSO). A similar domain including a tyrosine residue was found within Rep18 (see Fig. 2A). The second domain is less conserved within the pT181 Rep family and plays a role
in the DSO recognition. DSOs are very similar within the pT181 family (see Fig. 2B). However, this region is slightly divergent in pFR18 and located outside of the rep18 gene (Fig. 2B), in contrast to the pT181 family DSOs which are located in the replicase genes. This may explain the low degree of homology observed between the Rep18 domain involved in the recognition of the DSO and the homologous region of other Reps. On the basis of sequence comparison, no pT181 single strand origin (SSO) was identi¢ed in pFR18. This element is probably speci¢c to the bacterial genus. 3.3. Evidence for ssDNA intermediate Plasmids of the pT181 family replicate via an RCR mechanism. Thus, pFR18 was likely to use a similar replication mechanism. The RCR implicates the formation of a single stranded (ssDNA) intermediate of the plasmid [15]. Therefore, the ability of pFR18 to generate ssDNA was examined using
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whole-cell DNA of L. mesenteroides ssp. dextranicum DSM20484 containing pFBYC18E. First, a DSM20484 transformant containing pGhost9:ISS1, a well described RCR plasmid [22], was used in a control experiment (Fig. 3A). Whole-cell DNA prepared from DSM20484 (pGhost9:ISS1) grown in MRS containing rifampicin was hybridized to a pGhost9:ISS1 speci¢c probe. In addition to the open circular form (OC) and covalently close circular form (CCC) of the plasmid, the probe hybridized to a fast migrating band, suspected to be a ssDNA intermediate (SS, Fig. 3A, lane 1). Its sensitivity to nuclease S1 demonstrated its single-stranded nature (Fig. 3A, lane 2). This form was barely detectable in extracts from cells grown in the absence of rifampicin (data not shown). Similarly, whole-cell DNA from DSM20484 containing pFBYC18E was tested
using pFR18 as a probe (Fig. 3B). A single-stranded form of plasmid was clearly detected (SS, Fig. 3B, lanes 1 and 3) as proved by the nuclease S1 sensitivity of the fasted migrating band (Fig. 3B, lanes 2 and 4). In contrast to pGhost9:ISS1, pFBYC18E generated an accumulation of ssDNA intermediate in DSM20484 grown with or without the addition of rifampicin to the medium. This could be due to the cloning of the DNA fragment containing the erythromycin resistance cassette, in an SSO or in a locus implicated in the ssDNA intermediate conversion. This observation is not surprising since the insertion of foreign DNA in RCR plasmids has been shown to disturb their replication and favor the accumulation of ssDNA intermediate, thus enhancing segregational instability [7]. The detection of ssDNA intermedi-
Fig. 2. (A) Amino acid comparison between the active site region of replicases encoded by the pT181 plasmid family (upper part of the ¢gure) and the putative Rep18 replicase encoded by pFR18. Asterisks indicate amino acid identity and dots indicate conserved residues between RepC, encoded by pT181, and Rep18. The conserved tyrosine residue involved in the DNA nicking activity is indicated by a vertical arrow. (B) Nucleotide sequence comparison of the DSO of pT181-type plasmids and putative pFR18 DSO. The stem-loop structure that includes the replication nick site (vertical arrow) is indicated by a horizontal arrow. Some gaps were allowed for maximal alignment. The putative pFR18 DSO was aligned according to the potential nick site located within the potential stem-loop structure.
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Fig. 3. Detection of single-stranded intermediates of pGhost9:ISS1 (A) and pFBYC18E (B) in L. mesenteroides ssp. dextranicum DSM20484. Non-alkaline Southern blot analysis was performed with radio-labelled pGhost9:ISS1 (A) and pFBYC18E (B) probes. Whole-cell DNA was prepared from cells grown in MRS containing rifampicin (lanes 1 and 2) or in MRS without rifampicin (lanes 3 and 4). DNA preparations were treated with endonuclease S1 in lanes 2 and 4. OC, OC DNA; CCC, CCC DNA; SS, ssDNA.
ate demonstrated that pFR18 replicates using a RCR mechanism. 3.4. Construction of a cloning vector based on pFR18 pFR18 may be a good alternative for the development of Leuconostoc and other LAB cloning vectors, considering its small size, food grade origin and compatibility with the pWV01 replicon that served as a basis for most genetic tools devoted to LAB. The minimum replicon seems to be pFR18 itself. Indeed, by deleting di¡erent regions of pFBYC018E, we found that rep18 is involved in pFR18 replication, as well as the features contained within the G+C rich region (data not shown). RCR plasmids are known to be a¡ected in segregational stability when they contain foreign DNA [7] and we observed that pGhost9:ISS1 is not fully stable in DSM20484 (see Table 2). Similarly, the segregational stability of the E. coli/Leuconostoc shuttle vector pFBYC018E, which contains more than 4 kb of foreign DNA, is signi¢cantly a¡ected compared to that of
pFBYC18E, which contains a 1.6-kb insert (Table 2). Southern blot analysis of plasmid DNA prepared from erythromycin resistant colonies cultivated for 100 generations without selection pressure did not reveal any structural change in pFBYC018E nor pFBYC18E. The severe segregational loss observed with DSM20484 (pFBYC018E) growing with no selection pressure seems to be related to the size of the foreign DNA inserted into the pFR18 BamHI site as no plasmid loss was observed with DSM20484 (pFBYC18E). This region could play a role in the ssDNA duplication as a SSO or as a structure recTable 2 Stability of pFR18 derivatives during extended growth under non-selective conditions Plasmids
% of DSM20484 clones containing the plasmid after 100 generationsa
pGhost9:ISS1 pFBYC018E pFBYC18E
76 3 100
a
Average for duplicate experiments.
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ognized by host factors. To improve segregational stability of recombinant plasmids, other regions of pFBYC18E could be investigated as cloning sites. However, plasmid instability could be pro¢table in some cases, as shown for pIL252 [23,7]. pFBYC18E is able to replicate in various Leuconostoc species (cremoris, mesenteroides ssp. mesenteroides and mesenteroides ssp. dextranicum) as well as in L. sake, but not in Lactococcus lactis, P. acidilactici or E. coli (data not shown). Consequently, pFBYC18E constitutes a potential narrow host-range cloning vector. The pFR18 replicon is small and compatible with pWW01 derivatives and with at least three other plasmids harbored by L. mesenteroides ssp. mesenteroides FR52. In addition, legal issues on modi¢ed micro-organisms may require to use genetic material originating from the Leuconostoc genus for the genetic manipulation of Leuconostoc species. Thus pFR18 is an attractive new replicon for food grade vector construction.
4. Uncited references [24]
Acknowledgements This work has been achieved as part of the program `BIOAVENIR' (contract # 780227), supported by Rhoªne-Poulenc with the participation of the Ministe©re de la Recherche et de l'Espace and the Ministe©re de l'Industrie et du Commerce Exte¨rieur. We are grateful to Monique Zagorec for her contribution to the transfer of plasmids in L. sake. We thank Laurent Jannie©re for helpful discussion on ssDNA detection. We are grateful to the Laboratoire de Microbiologie Industrielle et Alimentaire, ENSAIA-INPL, Vandoeuvre, France, for supplying L. mesenteroides ssp. mesenteroides FR52.
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