1 Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, PO Box 5003, N-1432 Ås, Norway
2 Matforsk, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway
Correspondence
Lars Axelsson
lars.axelsson{at}matforsk.no
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AJ628941 (p256), AY557622 (pLPV111) and AY557620 (pELS100).
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INTRODUCTION |
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Genetic work on LAB has led to the identification and characterization of several plasmids, which may be divided into two classes reflecting their mode of replication: (I) rolling-circle (RC) plasmids, whose replication involves single-stranded intermediates (Aleshin et al., 1999; Cocconcelli et al., 1996
; Leer et al., 1992
), and (II) theta plasmids, whose replication proceeds via a bi- or uni-directional replication fork (Alpert et al., 2003
; Biet et al., 2002
; Seegers et al., 2000
; Wyckoff et al., 1996
). Both these replication mechanisms generally require a replication initiation protein called Rep. Plasmids from both classes have been used to construct plasmid vectors (Aleshin et al., 1999
; Biet et al., 2002
; Leer et al., 1992
; Wyckoff et al., 1996
). In general, plasmids of the RC type from Gram-positive bacteria have a broad host range, with some replicating even in Gram-negative bacteria, while the theta plasmids have a narrower host range. Plasmids with a narrow host range are less likely to be horizontally transferred to other bacterial species than plasmids with broad-host-range replicons. This safety aspect is important with regard to the potential use of live recombinant DNA organisms in, for example, food products (Pouwels & Leer, 1993
). In addition, the single-stranded intermediates of RC plasmids are often associated with plasmid instability (Meijer et al., 1995
). Theta-replicating plasmids can contain larger insert sizes than RC-replicating plasmids, and still show structural and segregational stability (Kiewiet et al., 1993
); hence, species-specific theta plasmids with a narrow host range are of interest for the construction of food-grade vectors, i.e. vectors to be used for genetically modifying LAB strains used in food fermentations.
Lactobacillus plantarum is common in spontaneous food fermentations, and is also frequently found as one of the dominating Lactobacillus species in the small intestine of man (Ahrné et al., 1998; Johansson et al., 1993
). Different strains of L. plantarum are marketed commercially as starter cultures for vegetable and meat fermentations (Daeschel et al., 1987
; Hammes et al., 1990
), and as probiotics (Johansson et al., 1998
). Several small, cryptic plasmids from L. plantarum, such as pC30il (Skaugen, 1989
), p8014-2 (Leer et al., 1992
) and pA1 (Vujcic & Topisirovic, 1993
), have been analysed at the molecular level. These are of the RC type, and there is a high degree of similarity among them. Vectors have been developed from p8014-2 only; thus, there is a need for stable, narrow-host-range plasmids that can be used as a starting point for the development of new vectors for lactobacilli.
It has been shown that L. plantarum NC7 harbours a single 7·2 kb plasmid called p256 (Cosby et al., 1989). Using a transposon Tn917-tagged variant of p256 (Cosby et al., 1989
), we noted that this plasmid showed a high degree of stability when subjected to various plasmid-curing regimes (L. Axelsson, unpublished observations). The remarkable stability of p256 suggested that this plasmid could have special features, and indicated that it could be a good starting point for the development of new vectors for lactobacilli. In this report, we give a detailed analysis of p256, with respect to its DNA sequence, replication determinant, and host range. The results show that p256 replicates by a Rep-independent theta-replication mechanism that hitherto has not been observed in LAB. In addition, we present results indicating that the high segregational stability of p256 is due to a putative toxinantitoxin (TA) locus, which represents a new plasmid maintenance system in LAB. Finally, we show that the replication determinants of p256 may be used to construct new expression vectors for lactobacilli.
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METHODS |
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DNA techniques and transformation.
Routine DNA techniques were performed according to Sambrook et al. (1989). Total DNA from L. plantarum NC7 was isolated as described previously (Axelsson & Lindgren, 1987
). Plasmid DNA from L. plantarum NC7 and L. plantarum NC8 was isolated by a modified alkaline lysis method (Sambrook et al., 1989
), as described previously (Axelsson et al., 1993
). Plasmid DNA from E. coli was isolated using a commercial kit (Qiagen). Transformation of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus sakei, Lactococcus lactis, Streptococcus thermophilus and Carnobacterium sp. was done by electroporation according to various protocols (Ahrné et al., 1992
; Aukrust & Blom, 1992
; Holo & Nes, 1989
; Jacobs et al., 1995
; Luchansky et al., 1988
; Varmanen et al., 1998
). Bacillus subtilis was transformed according to Gryczan et al. (1978)
. E. coli strains (DH5
, Gibco-BRL; XL10 Gold, Stratagene) were transformed either by electoroporation (Hanahan et al., 1991
) or according to the manufacturer's instructions (Stratagene).
DNA sequencing and sequence analysis.
The p256 sequence was determined by sequencing directly on p256 plasmid DNA and on standard PCR products (Expand High Fidelity PCR System Polymerase, Roche Diagnostics). Sequencing was carried out using an ABI Prism 3100 Genetic Analyser, with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, following the manufacturer's recommendations (Applied Biosystems). The BioEdit sequence alignment editor (Hall, 1999) was used for mapping and for identification of putative ORFs. The putative ORFs were assessed manually for start codon usage, potential ribosome-binding sites and promoters. Basic Local Alignment Search Tool (BLAST) analysis (Altschul et al., 1990
) was used to perform homology searches. Putative terminator structures and their free energies were analysed using Mfold, version 3.1 (Zuker, 2003
). The EMBOSS palindrome software was used to identify inverted repeats, and the GCG software (Wisconsin Package version 10.1, Genetics Computer Group) was used to localize direct repeats in the p256 sequence.
Plasmid constructions.
The plasmids used and constructed in this study are listed in Table 1. pLPV100 was constructed by replacing the 0·7 kb ClaI fragment of p256 (Fig. 1
) with a chloramphenicol-resistance gene (cat) from pIP501 (Trieu-Cuot et al., 1992
). Several fragments of p256 were cloned into pUC-C19N, and tested for replication in L. plantarum NC8. pUC-C19N is a variant of pUC19 (Yanisch-Perron et al., 1985
) containing an NcoI (instead of SalI) site in the polylinker, and with the ampicillin-resistance gene replaced by cat from pIP501. pUC-C19N, with the 1·8 kb BclIXmaI fragment (Fig. 1
), contained the replication region, and was called pLPV103 (Fig. 2
a). pLPV109 (Fig. 3
) was constructed by ligating the EcoRIHindIII replicon fragment of p256 (256rep) from the pLPV103 Erase-a-base-derivative pLPV-F72 (see below; Figs 2b and 3
) to the erythromycin-resistance gene (ermB) from L. reuteri (Axelsson et al., 1988
; GenBank accession no. AY556392). ermB was initially cloned as a blunt-ended fragment into the SmaI site of pGEM-7Zf(+) (Promega), and cleaved out again as an EcoRIHindIII fragment. The ligation mixture was electrotransformed directly into L. plantarum NC8. To construct pLPV111, pLPV109 was digested with XbaI and ClaI, which was essentially a linearization since these restriction sites were part of the polylinker (Fig. 3
), and the ends were blunted. This fragment was ligated to a 2·1 kb AvaIISspI (blunted) fragment from pGEM-7Zf(+) to create pLPV111 (Fig. 3
). Thus, the shuttle vector pLPV111 was essentially a pGEM-7Zf(+) derivative in which the ampicillin-resistance gene was replaced by the 256rep fragment and the ermB gene. pELS100 was a variant of pLPV111 in which the E. coli replicon part [derived from pGEM-7Zf(+)] was replaced by a fragment from the cloning vector pJB2 (Blatny et al., 1997
) containing the low-copy-number RK2 replicon.
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A fragment encompassing orf2 and orf3 of p256 (Fig. 1; see also Table 2
) was PCR amplified (Expand High Fidelity), and translationally fused to the regulated promoter in the inducible expression vector pSIP300 (Sørvig et al., 2003
) using an NcoI site. The introduction of an NcoI site in the start codon of orf2 did not change the nucleotide sequence of orf2. The resulting plasmid was called pSIP300TA.
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Frame-shift mutation of orf9.
A frame-shift mutation in orf9 (Fig. 1, Table 2
) was introduced by gene SOEing (Horton & Pease, 1991
) using primers delimiting the 688 bp minimal replicon, and a pair of fusion primers introducing a HindIII site in position 56355638, creating the sequence (HindIII site underlined) 5'-CTCAAGCTTTAATA-3' (original sequence at position 56325643: 5'-CTCAACTTAATA-3'). This frame-shift introduces a stop codon (in italics above), which results in truncation of the putative Orf9 protein after 17 aa. The fragment containing the mutation was then cloned in both orientations in pUC-C19N, and the resulting plasmids were tested in L. plantarum NC8 for replication.
Plasmid-copy-number determination.
Estimation of plasmid copy number was performed in a slot-blot experiment by hybridizing a p256-specific probe, encompassing the minimal replicon of p256, to known amounts of total DNA isolated from L. plantarum NC7, and known amounts of purified p256 DNA. By comparing the amounts of total DNA and plasmid DNA giving equivalent hybridization signals, the amount of plasmid DNA in a sample of total DNA could be estimated. By knowing the respective sizes of chromosomal and plasmid DNA, the number of p256 plasmids per chromosome could be calculated (Gardner et al., 2001).
Segregational stability.
To determine the segregational stability of L. plantarum NC8 containing p256-2 and pLPV100, the two cultures were inoculated into MRS medium containing appropriate antibiotics, and incubated overnight at 30 °C. The selective medium was removed by washing the cells three times with MRS. Subsequently, the cultures were subcultured (0·1 % inoculum) daily into MRS without antibiotics, and incubated at 30 °C. Under these conditions, one round of subculturing was estimated to correspond to 10 generations. Samples were removed at regular intervals, and plated on selective and non-selective MRS agar medium to determine numbers of antibiotic-resistant cells and total viable cells.
L. plantarum NC8/pSIP300TA was tested for segregational stability as described above, with some modifications. By varying the amount of added inducing peptide (SapIP), which is necessary for inducing gene expression in pSIP300-derived plasmids (Sørvig et al., 2003), the level of expression of the cloned orf2orf3 operon was controlled. The following three conditions were employed in the subculture medium throughout the experiment: (I) no SapIP; (II) 1 ng SapIP ml1 and (III) 25 ng SapIP ml1. For reference, the strain L. plantarum NC8/pSIP300 was tested for segregational stability with no SapIP, and with 25 ng SapIP ml1.
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RESULTS |
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orf1 encodes a putative integrase/recombinase showing more than 90 % sequence identity with several integrase/recombinase proteins from different LAB (Alpert et al., 2003; Cuozzo et al., 2000
; Daming et al., 2003
; Motlagh et al., 1994
).
BLAST analysis of the orf2 and orf3 gene products indicated that they belong to the PemI and PemK family of plasmid maintenance proteins. The two proteins (Orf2 and Orf3) show 22 and 24 % identity, and 49 and 46 % similarity (conservative amino acid changes allowed), to PemI and PemK from E. coli, respectively (Tsuchimoto et al., 1988; GenBank accession nos P13975 and P13976). They are also highly similar (97 and 99 % identity, respectively) to PemI- and PemK-like proteins encoded by genes found on plasmid pSMB74 from Pediococcus acidilactici (Motlagh et al., 1994
; GenBank accession no. PA02482). The orf2 and orf3 genes are translationally coupled, and are followed by an inverted repeat structure (
G=18·8 kcal mol1; 78·7 kJ mol1) that is likely to function as a transcriptional terminator. Thus, the two genes seem to be organized as an operon.
orf4 encodes a putative transposase that is >97 % identical to transposases identified in L. plantarum (Danielsen, 2002; Nicoloff & Bringel, 2003
) and Pediococcus pentosaceus (K. K. J. Leenhouts, A. A. Bolhuis, J. J. Kok & G. G. Venema, GenBank accession no. Z32771). This ORF forms part of an insertion sequence (IS) element spanning bp 22683316 (Fig. 1
), which is 98 % identical to IS element ISLpl1 from L. plantarum FB335 (Nicoloff & Bringel, 2003
). Compared to the functional transposase TraISLpl1 from L. plantarum FB335, however, the putative transposase encoded by orf4 in p256 is truncated after 257 aa due to a point mutation leading to a TGA stop codon. Similar to orf3, orf4 has an inverted repeat structure (
G=16·7 kcal mol1; 69·9 kJ mol1) following the stop codon, which is a potential transcriptional terminator.
orf5 encodes a protein of 125 aa that is 85 % identical to prophage Lp3 protein 20 from L. plantarum WCSF1 (Kleerebezem et al., 2003). orf6, downstream of orf5, encodes a peptide of 66 aa showing similarity to putative cold-shock proteins (CSPs) previously detected in L. plantarum, Lactobacillus delbrueckii and Enterococcus faecalis (Derzelle et al., 2000
; Kleerebezem et al., 2003
; Paulsen et al., 2003
; P. Serror, R. Dervyn, S. D. Ehrlich & E. Maguin, GenBank accession no. AY094622). orf6 is succeeded by a potential transcriptional terminator, although the hairpin structure appears to be less strong (
G=6·2 kcal mol1; 25·9 kJ mol1) than the other putative terminator structures identified on p256.
The next ORF, orf7, is homologous to orfX-type proteins of unknown function from L. lactis and L. sakei (Alpert et al., 2003; Emond et al., 1998
; Gravesen et al., 1995
). orf8 is homologous to the N-terminal part of a protein of unknown function encoded by a gene on the L. plantarum plasmid pLP9000 (Daming et al., 2003
). For the two remaining ORFs (orf9 and orf10), we were not able to detect significant similarity with known or predicted protein sequences.
Identification of the minimal replicon
The cloning of p256-derived fragments in pUC-C19N showed that a 1·8 kb BclIXmaI fragment contained the replicon. pUC-C19N containing this 1·8 kb fragment was called pLPV103 (Fig. 2a). The Erase-a-base deletion analysis of pLPV103 is summarized in Fig. 2(b)
. The minimal replicon was defined as the minimal fragment giving normal transformation frequencies in L. plantarum NC8 [approx. 106 c.f.u. (µg DNA)1]. The minimal replicon was delimited by Erase-a-base derivatives pLPV-F72 and pLPV-R17 (Figs 2b and 4
), showing that it is confined to a 688 bp fragment representing bp 55606247 in the p256 sequence. This 688 bp fragment was amplified, and then confirmed to contain replication functions by cloning and transformation into L. plantarum NC8, and it has subsequently been used in various vector constructions (Axelsson et al., 2003
; Sørvig et al., 2003
). The identified 688 bp minimal replicon region contained one ORF, putatively encoding a protein of 59 aa (orf9, Fig. 1
, Table 2
). The deduced product of this ORF did not share homology with any known proteins. A frame-shift mutation in orf9 (see Methods) did not affect the replication function of the fragment (results not shown).
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Host range and vector construction
pLPV100 was used in transformations of several LAB (number of strains in parentheses): L. acidophilus (1), L. casei (1), L. curvatus (1), L. reuteri (2), L. fermentum (1), L. plantarum (4), L. sakei (3), L. lactis (2), S. thermophilus (1), Carnobacterium sp. (1). Transformation of pLPV100 into E. coli (2) and B. subtilis (1) was also attempted. The vector pVS2 (Von Wright et al., 1987), containing the same cat marker gene as pLPV100, was used as a control, and it gave 102107 c.f.u. (µg DNA)1 depending on the strain and species. Transformants of pLPV100 were obtained only from L. curvatus, L. plantarum and L. sakei.
The p256 replicon was used to construct E. coliL. plantarumL. sakei shuttle vectors. The first vector constructed was pLPV111 (Fig. 3, Table 1
), which is based on the Erase-a-base plasmid pLPV-F72. Thus, it contains a slightly larger p256-derived fragment than the minimal replicon (see Fig. 2b
). pLPV111 contains lacZ with the pGEM-7Zf(+) polylinker, permitting regular bluewhite screening in E. coli. The copy number of pLPV111 in E. coli is high (similar to the pGEM vectors). A low-copy-number variant was created by replacing the high-copy-number E. coli replicon part in pLPV111 with the RK2 replicon, creating pELS100 (Table 1
).
Copy number
By comparing the hybridization signal of a p256-specific probe in known amounts of total DNA and purified plasmid, and by using the sizes of the chromosome and the plasmid, the copy number of p256 in L. plantarum NC7 could be deduced. The chromosome size of L. plantarum NC7 was set to 3·3 Mb (Kleerebezem et al., 2003). The copy number of p256 was estimated to be between five and ten copies per chromosome for L. plantarum NC7.
Plasmid maintenance system
The similarity of the putative products of orf2 and orf3 to PemI- and PemK-like proteins suggested that these genes could encode a TA system with plasmid maintenance functions. A variant of p256, p256-2, was created by replacing the 1·6 kb EcoRIPvuII fragment of p256 (orf2, orf3 and approximately 1000 bp downstream of orf3) with the erythromycin-resistance gene ermB (Axelsson et al., 1988; GenBank accession no. AY556392). The segregational stability under non-selective conditions was tested, and approximately 2000 colonies were counted. After 80 generations, 8·8 % of the L. plantarum NC8/p256-2 cells harboured the p256-2 plasmid, as assessed by erythromycin resistance. The construction of a second variant of p256, in which the 0·9 kb ClaIPvuII fragment was to be replaced by ermB (thus similar to p256-2, but with intact orf2 and orf3), was attempted numerous times. However, for unknown reasons, this was not successful. For comparative purposes, pLPV100 (intact orf2 and orf3, and 0·7 kb ClaI fragment replaced by cat) was tested for segregational stability instead. pLPV100 appeared completely stable since 100 % of the L. plantarum NC8/pLPV100 cells were chloramphenicol resistant after 80 generations under non-selective conditions.
As the p256-2 plasmid was unstable, it was of interest to investigate if the putative TA system would stabilize plasmids derived from the minimal replicon of p256. To test this, the orf2orf3 operon was inserted into the pSIP300 expression vector, yielding pSIP300TA (Table 1). This allowed peptide pheromone-dependent expression of the orf2orf3 operon. The segregational stability of pSIP300TA in L. plantarum NC8 was tested in cultures containing different concentrations of the inducing peptide SapIP, thus creating different levels of expression of orf2 and orf3. After 100 generations, 92 % of the L. plantarum NC8/pSIP300TA cells induced with 25 ng SapIP ml1, and 75 % of cells induced with 1 ng SapIP ml1, were erythromycin resistant. In comparison, less than 30 % of non-induced cells were erythromycin resistant after the same number of generations. L. plantarum NC8/pSIP300 (vector only) was also tested under both induced and non-induced conditions, and after 100 generations approximately 30 % of the cells harboured the plasmid. In this case, addition of peptide pheromone did not affect the outcome of the experiment.
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DISCUSSION |
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The region required for p256 replication was confined to a 688 bp fragment, containing one potential ORF (orf9) (Fig. 1; Fig. 4
). The deduced product of this ORF did not share homology with any known proteins, and a frame-shift mutation did not affect the replication function of the fragment. Therefore, orf9 does not appear to encode a protein that is relevant for plasmid replication. This conclusion is strengthened by the fact that the minimal replicon fragment starts only 19 bp upstream of the putative RBS of orf9. Thus, there appears to be limited space for a promoter connected to orf9. Since orf9 does not appear to be essential, it is presumably possible to narrow the minimal replicon fragment further, but this was not done in this study. Remarkably, sequence analysis of the other nine ORFs of p256 did not reveal any similarities with genes encoding proteins known to be involved in replication. One exception could be orf7, which is homologous to orfX-type proteins found in other LAB. These proteins are not essential for replication, but a possible role in the control of plasmid copy number has been found for the distantly related gene product RepB of pUCL287 (Benachour et al., 1997
). This role has not been confirmed for related proteins in other plasmids (Alpert et al., 2003
; Emond et al., 2001
). Our results clearly show that orf7 does not encode a protein that is necessary for the replication of p256, as it is not part of the minimal replicon fragment. Hence, it would seem that replication of p256 is not dependent on plasmid-encoded replication proteins, such as the Rep protein usually found encoded on other LAB plasmids.
RC and theta replication are the two main mechanisms of plasmid DNA replication. RC replicons encode a Rep protein that initiates DNA replication by nicking the supercoiled DNA at a target site termed the double-stranded origin (dso). A characteristic feature of RC-replicating plasmids is the formation of single-stranded intermediates (del Solar et al., 1998). The absence of a dso sequence and an ORF encoding a Rep protein, as well as the lack of single-stranded replication intermediates (L. Axelsson, unpublished observations), indicate that p256 does not belong to the RC plasmid family. Several features which are typical for theta-replicating plasmids are, however, found in the minimal replicon of p256, including an AT-rich region with multiple direct and inverted repeats, and four putative DnaA boxes. Fourteen putative DnaA boxes were found in other parts of the plasmid (results not shown). However, these were scattered over approximately 6500 bp, whereas the four boxes in the minimal replicon fragment were concentrated within 500 bp. The AT-rich region is the area where opening of the strands and assembly of host initiation factors occurs, and the DnaA boxes function as binding sites for host DnaA initiator proteins (del Solar et al., 1998
). These p256 features have been reported for other plasmids from Gram-positive bacteria that are assumed to use the theta mechanism of replication (Biet et al., 2002
; Meijer et al., 1995
). The lack of a Rep protein has not been reported for LAB plasmids, but Meijer et al. (1995)
have shown that plasmid pLS20 from B. subtilis replicates by a Rep-independent theta-replication mechanism. Due to similarities with this plasmid, not in DNA sequence, but in the features found in the minimal replicon region, and due to the lack of a Rep protein and single-stranded replication intermediates, it is very likely that p256 replicates by a theta mechanism.
The assumption that the AT-rich region containing repeats and DnaA boxes is important for replication is supported by the results of the Erase-a-base analysis (Figs 2b and 4). In the pLPV-R17 derivative, all DnaA boxes and inverted repeat structures are intact (Fig. 4
), and this plasmid replicates in a normal way in L. plantarum NC8, as judged by the number of transformants obtained [approx. 106 c.f.u. (µg DNA)1]. In contrast, the pLPV-R22 derivative, lacking a DnaA box and several of the repeats (Fig. 4
), did not replicate. pLPV-F72 gave similar transformation frequencies in the NC8 host to pLPV-R17, leading to the conclusion that the minimal replicon spans over 688 bp, running from the deletion point R17 to F72 (Figs 2b and 4
). Truncation of the minimal replicon from the F72 side yielded two plasmids (pLPV-F83 and pLPV-F92) that still gave low transformation frequencies [approx. 102 c.f.u. (µg DNA)1] in the NC8 host. The regions removed in the latter two plasmids do not have the highest concentration of repeat structures and DnaA boxes (Fig. 4
), and it is therefore possible that the most essential features are still intact. This could allow the plasmids to replicate, albeit in a suboptimal manner.
The p256 replicon failed to transform the majority of the bacterial strains tested, and this shows that p256 has a limited host range. It can be speculated that the limited host range is caused by the complete dependence of p256 on host factors for replication initiation, due to the lack of a Rep protein.
Many low-copy-number bacterial plasmids depend on different strategies to ensure their distribution (Nordstrom & Austin, 1989), and amongst them is the TA system. p256 contains a putative plasmid maintenance (TA) system consisting of orf2 and orf3. A TA locus typically consists of an antitoxin gene directly followed by a toxin gene. The inherent instability of the antitoxin leads to activation of the toxin in plasmid-free cells. The presence of a TA system, therefore, results in increased plasmid maintenance, since plasmid-free progeny have a much lower chance of survival than the plasmid-bearing cells (Gerdes, 2000
). The segregational stability of plasmid p256-2 was compared with that of pLPV100, and after 80 generations only 8·8 % of the L. plantarum NC8 cells harboured the p256-2 plasmid, whereas pLPV100 was 100 % stable. This result clearly indicates that orf2 and orf3 promote plasmid stability since, except for the antibiotic-resistance marker change, the difference between these plasmids is essentially the presence or absence of the orf2orf3 operon. We have currently no explanation as to why we were unable to obtain a p256 derivative that would be optimal for comparison with p256-2 (see Results). To test whether orf2 and orf3 contribute to maintaining p256-derived plasmids in general, the orf2orf3 operon was translationally fused to the regulated promoter in the inducible expression vector pSIP300 (Sørvig et al., 2003
), creating pSIP300TA. The studies with this construct clearly show that the orf2orf3 operon contributes to plasmid stability to an extent that is dependent on the expression level of these two genes. Putative TA systems similar to orf2orf3 have previously been identified in other LAB, such as species of Pediococcus, Enterococcus and Streptococcus (Ajdic et al., 2002
; Motlagh et al., 1994
; Paulsen et al., 2003
), but not in Lactobacillus. The systems found in other LAB are all identified based on sequence similarity, and no experimental evidence on function has been presented. Thus, to our knowledge, this is the first time a putative TA system with demonstrated plasmid maintenance function has been identified in LAB.
In conclusion, we have identified the minimal replicon of plasmid p256 from L. plantarum NC7, and shown that it is a replicon with a fairly low copy number and a narrow host range. It seems to represent a new type of replicon for lactobacilli, and it has successfully been used to construct a variety of E. coli shuttle vectors for use in L. sakei and L. plantarum, including effective expression vectors that have been shown to accept rather large inserts (78 kb) (Axelsson et al., 2003; Diep et al., 2003
; Johnsborg et al., 2003
; Mathiesen et al., 2004
; Sørvig et al., 2003
). Further development of vectors based on this replicon, e.g. the construction of food-grade derivatives, seems feasible. Preliminary results obtained in our laboratories indicate that the p256-derived pSIP vectors (Sørvig et al., 2003
) have copy numbers in the same range as p256. We have also identified a putative TA operon and, for the first time in LAB, demonstrated a plasmid maintenance function for such a system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 June 2004;
revised 30 September 2004;
accepted 15 October 2004.
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