1 Unité Recherche Laitière et Génétique Appliquée, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
2 Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
Correspondence
Pascale Serror
serror{at}jouy.inra.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the ISLre1 sequence is AF449484.
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INTRODUCTION |
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We have previously shown that several plasmids were capable of replication in L. bulgaricus (Serror et al., 2002). Two of them, pG+host4 (Maguin et al., 1992
) and pGB305
(Le Chatelier et al., 1993
), were derived from two plasmids whose replication was previously reported to be thermosensitive in various bacteria. Plasmid pG+host1 (also published as pVE6002), a thermosensitive derivative of the lactococcal rolling circle plasmid pGK12 (Kok et al., 1984
), was shown to be thermosensitive in certain Gram-positive bacteria (Maguin et al., 1992
). Plasmid pGB305
was derived from pIP501, a theta replicating plasmid. pIP501 and several of its derivatives have been described as thermosensitive in several Gram-positive bacteria (Hershfield, 1979
; Evans & Macrina, 1983
; Pujol et al., 1998
) including lactobacilli (Luchansky et al., 1988
; Thompson & Collins, 1988
; Bhowmik et al., 1993
; Bates et al., 1989
). Therefore, we tested the thermosensitivity of these two plasmids in L. bulgaricus.
In order to develop a transposition mutagenesis system for L. bulgaricus, we also had to identify a transposon or an insertion sequence (IS) active in this host. Transposable elements are powerful tools for molecular genetic studies when they integrate at random into the genome (Berg & Berg, 1996; Hamer et al., 2001
). Efficient insertion mutagenesis systems based on transposons or ISs have been successfully used in Gram-positive (Youngman, 1993
) and Gram-negative (Berg & Berg, 1996
) bacteria. Transposons Tn916 and Tn917 were widely used for transposon mutagenesis of various Gram-positive bacteria, but showed a preference for hot-spot integration sites in certain species (Mullany et al., 1991
; Youngman et al., 1983
). A transposon derived from Tn10 was also used in Gram-positive bacteria (Petit et al., 1990
). ISs are small transposable elements flanked by inverted repeats and encode their own transposition functions (Chandler & Mahillon, 2002
). ISs are widely spread among lactic acid bacteria including lactobacilli (Chandler & Mahillon, 2002
). Several of them have been characterized, and could constitute a good basis for the development of random insertion mutagenesis systems (Maguin et al., 1996
; Mills, 2001
). Transposition can occur via two major mechanisms which are known as replicative and non-replicative. Replicative transposition leads to the fusion of the donor molecule and the target DNA, resulting in a structure often referred to as co-integrate (Craig, 1996
) corresponding to the donor backbone flanked by duplicated ISs in the target molecule (i.e. plasmid flanked by two copies of the IS). Non-replicative transposition, also called cut and paste, leads to the integration of the IS only into the target molecule. Although occurring at lower frequency, this mechanism can also lead to the integration of the delivery plasmid flanked by duplicated ISs. This may happen if plasmid dimers or tandem copies of the IS are the initial substrates of non-replicative transposition (Craig, 1996
; Chandler & Mahillon, 2002
; Rousseau et al., 2002
). The use of an IS delivery vector carrying an antibiotic marker allows easy detection of co-integrates through the detection of antibiotic-resistant cells. Selection for co-integrates should allow the determination of IS functionality in L. bulgaricus, irrespective of the transposition mechanism involved.
In this work, we describe the identification of two thermosensitive plasmids in L. bulgaricus. Using one of them, we tested the activity of one endogenous and five heterologous ISs in L. bulgaricus. Two of them, IS1223 (Walker & Klaenhammer, 1994) and IS1201 (Tailliez et al., 1994
), were shown to transpose in L. bulgaricus.
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METHODS |
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Plasmids used in this work were pG+host4 (Maguin et al., 1992), pG+host9 (Maguin et al., 1996
), pGB305
(Le Chatelier et al., 1993
), pVI1055 (M. van de Guchte, unpublished), pLEM3 (Fons et al., 1997
), pRL1 (Le Bourgeois et al., 1992
), pRTK371 (Walker & Klaenhammer, 1994
), pIL895 (Tailliez et al., 1994
), pNST73 (Guedon et al., 1995
) and pSKII (Stratagene). pVI1055 (M. van de Guchte, unpublished) is an E. coliL. bulgaricus shuttle which contains the ColE1 origin, the ampicillin-resistance gene and the multicloning site derived from pSKII and the replication functions and the erythromycin-resistance gene of pGB3631 (Brantl et al., 1994
).
Growth conditions and transformation procedures.
L. bulgaricus was cultured in MRS broth (Difco) (De Man et al., 1960) at 37 or 42 °C. Plates were incubated under anaerobic conditions in jars containing GasPak (Oxoid). E. coli was cultured on LuriaBertani medium (Difco) (Sambrook et al., 1989
) at 37 °C. Erythromycin was used at 7·5 µg ml-1 and 150 µg ml-1 for L. bulgaricus and E. coli, respectively. Ampicillin was used at 75 µg ml-1 for E. coli. Electrocompetent E. coli and L. bulgaricus cells were prepared and transformed as previously described (Dower et al., 1988
; Serror et al., 2002
).
DNA techniques.
Plasmid DNA (Sambrook et al., 1989) and L. bulgaricus chromosomal DNA were prepared as previously described (Serror et al., 2002
). Procedures for DNA manipulations were performed essentially as described in Sambrook et al. (1989)
. All enzymes for DNA modification were used according to the manufacturer's specifications. The oligonucleotides used in this work were synthesized on a DNA synthesizer oligo 1000M system (Beckman). DNA probes were labelled with [
-32P]dCTP (ICN Biomedicals) by a random priming kit (Boehringer). Fluorescent DNA sequencing was performed according to Perkin Elmer Biosystems recommendations. DNA chromosomal sequencing was performed as described by C. Heiner (http://www.genome.ou.edu/big_dyes_bact.html; Heiner et al., 1998
) using the coding strand primer pLB45 (5'-GGCTTTGGTCAGGTTCAGGACACC-3') upstream of the right end of IS1201.
Plasmid constructions.
ISS1 was cloned as a blunt-ended PCR product obtained after amplification with the coding strand primer 5'-GGAGAGAATGGGTTCTGTTGCAAAGTTTTCTGATAAGTCTA-3' and the complementary-strand primer 5'-GCTCTAGAGCATTCTCTGGTTCTGTTGCAAAGTTTAAAAATCAAA-3' using plasmid pRL1 as DNA template into the EcoRV site of pBluescript SKII. ISL3, IS1223 and ISLre1 were cloned after PCR amplification with primers containing additional ClaI and EcoRI sites in the coding-strand and complementary-strand primers, respectively. The coding-strand primers were OLB-137 (5'-AATCGATTTTTCGTGGCTCTA-3'), OLB-83 (5'-CTTATCGATAATGAAGTGCCTCATA-3') and OLB-85 (5'-CTTATCGATCTCTATTGAGTGACG-3') for ISL3, IS1223 and ISLre1, respectively. The complementary-strand primers were OLB-138 (5'-CAGGAATTCACGAAAAAGGCTCTTTG-3'), OLB-84 (5'-CAGGAATTCATTATGAAGTGCCATAGA-3') and OLB-86 (5'-CAGGAATTCACCATCGTCAGATTTGA-3') for ISL3, IS1223 and ISLre1, respectively. DNA templates used for PCR amplification of ISL3, IS1223 and ISLre1 were chromosomal DNA of CNRZ208, plasmid pRTK371 and plasmid pLEM3, respectively. After restriction, the PCR fragments were cloned in the ClaI and EcoRI sites of pSKII. The integrity of all PCR products was verified by sequencing. IS1191 was cloned as an AflIIScaI fragment of pNST73 encompassing IS1191 surrounded by 208 and 126 bp into the EcoRV site of pSKII. Next, the IS-containing fragment of each pSKII-IS derivative, including pIL895, was obtained by BssHII restriction and cloned in pVI1055 previously digested with BssHII. The resulting plasmids pVI45, pVI47, pVI49, pVI50, pVI52 and pVI55 carried IS1191, ISS1, IS1223, ISLre1, IS1201 and ISL3, respectively (Table 1).
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Structural analysis of the integrants.
Chromosomal DNA of pVI45, pVI49 and pVI52 integrants was digested with the restriction enzyme BglI. DNA restriction fragments were separated by electrophoresis and the IS was revealed by Southern hybridization (Southern, 1975). Similar experiments were carried out for the analysis of pVI55 integrants using the restriction enzyme SalI.
Excision of pVI52 after transposition.
pVI52 (erythromycin-resistant) integrants were streaked on MRS-erythromycin and incubated at 44 °C (non-permissive temperature) for 42 h. Isolated colonies were grown in MRS without erythromycin at 44 °C until the OD600 reached between 0·5 and 0·9. The culture was then diluted 103104-fold and incubated at 37 °C until saturation. This step was repeated one to three times. The saturated culture was diluted 105106-fold and incubated at 44 °C (to allow the loss of the excised plasmid) until saturation. Serial dilutions were then plated on selective or non-selective medium and incubated at 44 °C for 36 h. Excised clones were identified as erythromycin-sensitive colonies.
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RESULTS |
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Identification of functional IS in L. bulgaricus
Five different IS elements, previously characterized in lactic acid bacteria, and ISLre1 (Table 1) were cloned in pVI1055, a pIP501 derivative (Table 1
and Methods). The resulting plasmids (Table 1
) were introduced by electrotransformation in L. bulgaricus and their presence as free plasmid forms was confirmed by hybridization with an IS-specific probe (data not shown).
The use of an IS delivery vector carrying an antibiotic marker facilitates the detection of transposition events leading to the formation of co-integrates (i.e. plasmid flanked by duplicated ISs; Fig. 2), which are easily selected as antibiotic-resistant cells. In order to test whether our plasmids were able to integrate into the chromosome, pVI1055 (used as a control) and its derivatives carrying the IS elements were established in VI104 at the permissive temperature (37 °C). Cells were then cultivated at the non-permissive temperature (44 °C) under non-selective conditions (MRS) to allow the loss of the plasmids. At different times cells were plated at 44 °C with and without antibiotic. The ratio of these colony counts is an estimation of the integration efficiency since antibiotic-resistant cells are expected to contain an integrated plasmid.
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IS1191 integrants.
Chromosomal DNA of 44 integrants of pVI45 obtained from two independent experiments was analysed. IS1191 and vector specific probes revealed a single band of about 10 kb. This analysis revealed the absence of duplicated ISs surrounding the integrated vector, therefore we concluded that the erythromycin-resistant cells did not result from transposition but from recombination at a single site (Fig. 3a). Since hybridization with an IS1191 probe did not reveal any endogenous IS1191 in the strain, it is likely that pVI45 integration resulted from an illegitimate or homologous recombination event involving the 208 and 126 bp regions surrounding IS1191 in the plasmid insert.
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IS1223 integrants.
Analysis of chromosomal DNA of 15 integrants of pVI49 with an IS1223 probe revealed four patterns exhibiting two bands of variable sizes (data not shown). As VI104 does not carry any IS1223, these two bands correspond to the junctions between the chromosome and the integrated plasmid. They indicated that the IS has been duplicated as expected during replicative transposition. Since four different patterns were observed, we concluded that transposition of IS1223 occurred at different chromosomal sites. However, the limited number of patterns suggested that early transposition events had been enriched or that IS1223 had transposition hot spots. We analysed 73 additional pVI49 integrants isolated from five independent experiments. They displayed eight different hybridization patterns with one to three patterns in each independent experiment (data not shown). The fact that hybridization patterns differed more from one experiment to another than among the clones resulting from one experiment is consistent with the enrichment of transposants and against the hypothesis of transposition hot spots. Enrichment of Tn917 insertions has been observed in L. lactis using a Tn917-delivery plasmid which exhibits segregational instability under normal growth conditions (Israelsen & Hansen, 1993). One approach to circumvent the enrichment of Tn917 integrants has been to eliminate the subculturing steps aiming at plasmid loss. In order to confirm that an enrichment occurred during our previous experiments, we eliminated the 48 h incubation at 44 °C prior to plating (i.e. selection of the integrants). The cells, grown at 37 °C, were directly plated at 44 °C to select for integrants. Seventeen integrants were analysed: they displayed 10 different hybridization patterns (Fig. 4a
). Nine of these 10 patterns undoubtedly resulted from replicative transposition: (i) six exhibited two (lanes 3, 4, 7, 15 and 16) or four (lane 17 corresponding to a mixture of two clones) bands with variable sizes as expected after mono-copy transposition and (ii) the other three, which displayed an additional third band (lanes 1) or a doublet (lanes 5 and 9) at
8 kb (size of pVI49), resulted from tandem transposition. The last pattern corresponded to a single band of
8 kb which was observed in 8 of the 17 integrants (Fig. 4a
, lanes 2, 6, 8 and 1014). It may correspond to (i) linear pVI49 that was not completely eliminated from the cells, or (ii) a transposition event that led to two junctions of identical size after BglI restriction. The increased diversity of hybridization patterns confirmed that in previous experiments few transposants were enriched.
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Characterization of IS1201 transposition targets
The integrants obtained with pVI52 contain duplicated IS1201 sequences flanking the plasmid backbone. To sequence the IS1201 transposition sites, we first excised the transposed vector (pVI52) to obtain stable mutants carrying a single copy of IS1201 into their chromosomes (excised clones). Excision was achieved by homologous recombination between the two copies of IS1201 during growth at permissive temperature (37 °C) without selection. Excised clones were then screened as erythromycin-sensitive bacteria at 44 °C (see Methods). The efficiency of plasmid loss estimated by the frequency of erythromycin-sensitive cells at 44 °C varied from 50 to 95 % between integrants. The chromosomal sequence flanking the right end of IS1201 in the excised clones was determined by direct chromosomal sequencing using an oligonucleotide corresponding to part of IS1201. The transposition target of IS1201 was previously defined as an 8 bp sequence (Tailliez et al., 1994). The sequences of the first 8 bp following the IS1201 in the excised clones were random although thymine and/or adenine residues seemed favoured at positions 3, 4, 6 and 7 of the targets (Table 3
). Analysis of longer sequence stretches and homology searches showed that four of the seven insertions occurred in different putative open reading frames (Table 3
), thus confirming that transposition of IS1201 in L. bulgaricus can generate mutants.
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DISCUSSION |
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Several observations underline that plasmid thermosensitivity cannot be predicted for a given host. For example, pG+host derivatives are not thermosensitive in Lactobacillus sakei (Gory et al., 2001) and Lactobacillus plantarum (strain CCM 1904; P. Horvath & B. Kammerer, personal communication). In contrast, they are thermosensitive in (i) Lactobacillus casei but at a temperature which is hardly compatible with growth (V. Monedero & G. Perez-Martinez, personal communication) and (ii) Lactobacillus acidophilus and Lactobacillus gasseri, two species in which pGK12, the parental non-thermosensitive plasmid of pG+host, is unstable at high temperature (Russell & Klaenhammer, 2001
). In L. bulgaricus, we found that pG+host9 was unstable at 44 °C. However, more than 20 generations under restrictive conditions were necessary to observe less than 0·1 % of plasmid-containing cells whereas in L. lactis, a similar result required only about 11 generations. Since we previously established that pG+host plasmids have a low copy number in L. bulgaricus (Serror et al., 2002
), we propose that residual replication occurs at high temperature and results in the observed slower kinetics of loss than in L. lactis. It may be due to a variety of host factors which can alter the thermosensitivity of the replication protein.
The instability of pIP501 and derivatives at high temperature has been established in various thermotolerant Gram-positive hosts (Hershfield, 1979; Evans & Macrina, 1983
; Pujol et al., 1998
; Luchansky et al., 1988
; Thompson & Collins, 1988
; Bhowmik et al., 1993
). We showed that this phenomenon also occurs in L. bulgaricus. With more than 99 % of plasmid-free cells after about 24 generations at 44 °C in L. bulgaricus, pIP501 derivatives appear to be lost at a similar rate as in several Lactobacillus helveticus strains (Thompson et al., 1999
). Since the upper temperature limit for L. bulgaricus growth is 45 °C, it was impossible to improve the rate of plasmid loss by increasing the non-permissive temperature. To improve plasmid loss, it may ultimately be necessary to isolate new mutants of pG+host or pIP501 derivatives with an increased thermosensitivity as compared to the original plasmids. Such plasmids would be useful to decrease the incubation time at the restrictive temperature and avoid the enrichment problems observed in some of our experiments.
In our system, ISS1, IS1191, ISLre1 and ISL3 appeared inactive in L. bulgaricus VI104. ISS1 has been successfully used for random insertional mutagenesis of various Gram-positive bacteria (Ward et al., 2001; Boyd et al., 2000
; Rallu et al., 2000
) and some observations had indicated that ISL3 might be active in L. bulgaricus (Germond et al., 1995
). For the other ISs, there was no evidence for their functionality, although the detection of IS1191- and ISLre1-related elements in various lactic acid bacteria including L. bulgaricus (Guedon et al., 1995
; Roussel et al., 1997
) and Lactobacillus fermentum strains (M. Fons, personal communication), respectively, suggested that these ISs are active. The simplest interpretation of our results is that ISS1 and ISLre1 are not functional or exhibit very low transposition activity in our system. The interpretation is slightly different in the case of ISL3 and IS1191. We observed a high integration rate of the ISL3-containing plasmid (pVI55, 10-1) and all 12 EryR integrants analysed resulted from a recombination event with the endogenous ISL3 (a 1·5 kb region). Therefore, ISL3 may be inactive or transpose at a frequency lower than 8x10-3. Similarly for IS1191, the delivery plasmid pVI45 integrated with a frequency of 10-4. This integration may have happened (i) via a recombination event involving the short regions (208 and 126 bp) which flanked this IS in our construct or (ii) through a hot spot of illegitimate recombination. The fact that the frequency of homologous recombination decreases with the size of the homologous regions (Biswas et al., 1993
) is in agreement with the integration frequencies obtained for pVI45 (10-4) and pVI55 (10-1).
In our tests, IS1223 and IS1201 were found to be active in L. bulgaricus VI104. IS1223 was isolated as a functional element from Lactobacillus johnsonii (Walker & Klaenhammer, 1994). Since IS1223 belongs to the IS3 family, it is likely that its transposition mechanism will share common features with members of this family. Several members of the IS3 family including IS911 generate circles that are integrated into the targets during the transposition process (Ton-Hoang et al., 1998
; Sekine et al., 1999
; Lewis & Grindley, 1997
). The resulting structures can result from direct cut and paste insertions of the IS but also be co-integrates' (integration of the plasmid between duplicated ISs) when the IS3 family members are delivered on plasmids (Turlan et al., 2000
). Further experiments will be needed to determine whether the co-integrates selected in this work have been formed by a cut and paste or a replicative event of transposition. Our results, including the characterization of seven transposition targets, provide the first evidence for IS1201 random transposition. In that context, it is interesting to mention that IS1201, which was first characterized in L. helveticus (Tailliez et al., 1994
), has since been found in many L. helveticus strains (de los Reyes-Gavilan et al., 1992
; Reinheimer et al., 1996
) as well as in Lactobacillus amylovorus, Lactobacillus gallinarum and Lactobacillus crispatus strains (P. Tailliez, unpublished results). As previously suggested (Tailliez et al., 1994
), our results confirm that IS1201 favours A+T-rich targets. Thymine and/or adenine residues appeared to be favoured at positions 3, 4, 6 and 7 of the 8 bp targets. IS1201 belongs to the IS256 family. Although little is known about the transposition mechanism of the IS256 family members (for review see Chandler & Mahillon, 2002
), recent data suggest that IS256 may transpose by the so-called cut and paste mechanism using a circular intermediate (Ziebuhr et al., 1999
; Prudhomme et al., 2002
). According to our observations, we propose that the events (co-integrates) selected in this work using plasmids with IS1223 or IS1201 result from cut and paste transposition. Further experiments will be designed in order to compare the efficiency of co-integrate formation versus direct IS integration during transposition of IS1223 and IS1201 in L. bulgaricus. Genome sequences of three L. bulgaricus strains and of other lactobacilli are currently being determined (Klaenhammer et al., 2002
). These projects stress the need for genetic tools adequate to develop functional analysis studies of L. bulgaricus and other lactobacilli. The identification of thermosensitive plasmids and functional ISs constitutes an essential step in developing efficient molecular tools leading to random insertional mutagenesis, targeted gene disruption and gene replacement.
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ACKNOWLEDGEMENTS |
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Received 24 June 2002;
revised 19 December 2002;
accepted 25 February 2003.
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