Unité Flore Lactique et Environnement Carné, INRA, Domaine de Vilvert, F-78350 Jouy-en-Josas, France1
Author for correspondence: Monique Zagorec. Tel: +33 1 34 65 22 89. Fax: +33 1 34 65 21 05. e-mail: zagorec{at}diamant.jouy.inra.fr
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ABSTRACT |
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Keywords: lactic acid bacteria, meat, PFGE
Abbreviations: LM-PCR, ligation-mediated polymerase chain reaction
The GenBank accession numbers for the sequences reported in this paper are given in Table 2 and the legend to Fig. 3.
a Present address: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
b Present address: Unité Ecologie et Physiologie du Système Digestif, INRA, Domaine de Vilvert, F78350 Jouy-en-Josas, France.
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INTRODUCTION |
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The present study reports the determination of the chromosome size of a plasmid-cured L. sakei strain (Berthier et al., 1996 ). Several loci previously cloned from various L. sakei strains were used to construct a genetic map but additional probes were necessary to complete the map. For this purpose a collection of plasmids containing DNA fragments randomly cloned from the L. sakei chromosome (Leloup et al., 1997
) was used, as well as additional L. sakei probes obtained by various strategies.
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METHODS |
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PFGE analysis.
Bacteria were harvested from MRS cultures when the OD600 reached 0·5. Cells were collected from 1 ml culture samples by centrifugation at room temperature, suspended in 0·5 ml TEE buffer (Tris/HCl 10 mM, pH 9·0, EDTA 100 mM, EGTA 10 mM), incubated for 10 min at 37 °C and embedded in an equal volume of 2 % low-melting-point agarose. Bacteria were lysed by incubating plugs for 2 h at 37 °C in TEE buffer containing lysozyme (5 mg ml-1) and N-lauroylsarcosine (0·05%) and then overnight at 55 °C in TEE buffer containing proteinase K (1 mg ml-1) and SDS (1%). Plugs were dialysed for 1 h at room temperature in TE buffer (Tris/HCl 10 mM, pH 8·0, EDTA 10 mM) containing PMSF (0·1 mM), then twice in TE buffer, and kept at at 4 °C in EDTA (0·5 M) until use. For restriction enzyme digestion the plugs were first dialysed against sterile water, then equilibrated for 3 h in 200 µl of specific buffer and at the temperature recommended by the supplier for each enzyme. Restriction enzymes (20 U, or 5 U for I-CeuI) were added and the plugs were incubated at 4 °C for 4 h (overnight for I-CeuI) and then overnight (2 h for I-CeuI) at the temperature recommended for each enzyme. The restriction enzymes AscI and I-CeuI, NotI and SfiI were from New England Biolabs, MBI Fermentas and Boehringer, respectively. After digestion, the plugs were rinsed for 13 h in Tris/HCl 10 mM, pH 8·0, EDTA 100 mM and then submitted to PFGE on a CHEF-DR III apparatus (Bio-Rad). PFGE was performed in 1% GTG SeaKem agarose in 0·5x TBE buffer (Sambrook et al., 1989 ). To separate the large DNA fragments, migration was carried out at 14 °C for 22 h at 6 V cm-1 and an angle of 120°. The switch time was 50 s to 90 s. Then, a second migration was performed for 13 h, with the same parameters except a switch time of 1 s to 12 s, allowing the separation of small fragments. The molecular mass markers used were yeast chromosomal DNA (2·03194 kb) and
concatemers (48·5727 kb). After migration, gels were stained in ethidium bromide and photographed under UV light, or analysed on a FluorImager (Molecular Dynamics), with a filter at 488 nm, after staining with Vistra-Green (Amersham) according to the recommendation of the manufacturer.
DNA manipulation and hybridization.
Plasmid DNA was prepared from E. coli by classical methods (Sambrook et al., 1989 ). Chromosomal DNA used for PCR amplification was prepared from L. sakei 23K by the method of Anderson & McKay (1983)
. PCR amplification of DNA probes was performed on 1 µg chromosomal DNA, as previously described (Stentz et al., 1997
). Southern hybridization experiments were performed on chromosomal DNA transferred to Nytran-N nylon membranes (Schleicher & Schuell). DNA probes were labelled by the use of the ECL kit from Amersham or with [
-32P]dCTP by random priming (Sambrook et al., 1989
). When probes were PFGE restriction fragments, bands were excised from PFGE gels, extracted from agarose and ethanol-purified before labelling. For large fragments, DNA in agarose bands cut from gels was digested with EcoRI before extraction.
Ligation-mediated PCR of the rrn flanking regions.
The DNA regions flanking rrn loci were amplified by PCR as described by Prodhom et al. (1998) . The 16S-specific primer OLS1 (5'-CCGCCACTCACTCAAATGTTTATCAATGAG-3') was derived from the 16S rRNA gene of L. sakei available in the GenBank database (accession number M58829) and the 5S-specific primer OLS2 (5'-GAAGGATACACCTGTTCCCATGCCGAACAC-3') was derived from a conserved region in the consensus sequence of an alignment of known 5S rRNA genes from lactobacilli (Lactobacillus brevis, X02026; L. casei partial 5S rRNA gene sequence, AF098108; L. plantarum, X12886; Lactobacillus delbrueckii, X15246; and Lactobacillus viridescens, X01950). The EcoRI linker was composed of the two primers OLS36 (5'-TAGCTTATTCCTCAAGGCACGAGC-3') and OLS37 (5'-AATTGCTCGTGC-3'). Chromosomal DNA (100 µg) was digested with EcoRI, purified, and subsequently ligated to the EcoRI linker (generated by OLS36+OLS37) by incubating for 1 h at 16 °C. PCR amplifications were then conducted with OLS36/OLS1 or OLS36/OLS2 pairs of primers and OLS36 or OLS1/2 alone as controls. The Expand Long Template PCR system (Roche Diagnostics) was used. PCR products were purified by Geneclean kit (Bio 101) and cloned in pGEMt-easy vector (Promega).
Cloning or PCR amplification of the ackB, gal, argF, pur, glpK, pdc and gpm clusters.
Cloning of the ackB partial sequence and upstream region was previously described by Stentz (1998) . Briefly, a 100 bp PCR product obtained with degenerate primers based on an alignment of several bacterial ack genes was used as probe to clone a 950 bp NsiI fragment in pBluescriptSK+II (pBSKII+, Stratagene). Further inverse PCR experiments, performed with primers deduced from the sequence of the NsiI fragment, allowed the amplification of two overlapping fragments corresponding to the upstream (2·3 kb) and downstream (4·8 kb) regions. The two strategies yielded together a 6890 bp region of the ackB cluster. The galETM cluster was cloned in pBSKII+ as a 2·4 kb EcoRIHindIII DNA fragment which hybridized with a probe comprising the galT gene from L. casei (Bettenbrock & Alpert, 1998
), yielding plasmid pRV522. The argF gene cluster was cloned in pBSKII+ as a 4·3 kb EcoRV fragment (plasmid pRV411) by hybridization to a 100 bp PCR probe obtained with degenerate primers 5'-TTC(T)ATGCAC(T)TGC(T)TTA(G)CC-3' and 5'-(A,T,C,G)CGG(A)TTT(C)TC(A,T,C,G)GCT(C)TC-3'. These two primers were based on conserved motifs of ornithine transcarbamylases. The purB, purFMN and glpK clusters were cloned in pBSKII+ as a 1·55 kb HindIII fragment (plasmid pRV30), a 2·3 kb ClaI fragment (pRV29) and a 0·4 kb HindIII fragment (pRV31), respectively. These plasmids were isolated as false-positive clones hybridizing to rbsK and rbsR probes (Stentz & Zagorec, 1999
). The pdc cluster was isolated as a 940 bp PCR fragment. The gpm cluster was cloned by hybridization in pBSKII+, as a 3·8 kb EcoRV fragment, adjacent to a peptidase gene (plasmid pRV416, M.-C. Champomier-Vergès, unpublished results).
Construction of a mini-shotgun library.
Genomic DNA from cells harvested at the end of the exponential growth phase was diluted to a concentration of 200 ng µl-1 in ice-cold TE buffer and sonicated briefly. Several time-pulses were tested in order to produce several ranges of DNA fragment sizes (from 0·12 kb to 110 kb). DNA from these experiments was pooled, then treated with RNase A for 20 min at 37 °C, purified by phenol/chloroform/isoamyl alcohol extraction and precipitated with ethanol. End-repairing of sheared DNA was performed in several batches of 40 µl in T4 DNA polymerase buffer (Biolabs) containing 4 µg DNA. The end-repairing reaction was performed with dNTPs at final concentration of 0·075 mM and a mixture of T4 DNA polymerase (6 U)/Klenow enzyme (10 U) for 30 min at room temperature. The polymerization reaction was stopped by heating the samples at 75 °C for 15 min followed by chilling on ice for 15 min. T4 polynucleotide kinase (10 U) and ATP (final concentration 0·04 mM) were then added and the samples were incubated for 30 min at 37 °C. DNA was further purified by phenol/chloroform/isoamyl alcohol extraction and precipitated with ethanol before loading on agarose gel for size selection. The range from 1 to 2 kb was excised from the gel and purified with Geneclean purification kit. The blunt-ended DNA was then ligated to a 10-fold excess of BstXI adaptators (InVitrogen). The ligation mixture was loaded on to agarose gel to purify the 12 kb fraction from unligated adaptators or adaptor doublet. The 12 kb fragments were finally ligated to BstXI-digested pcDNA2.1 vector (InVitrogen) and transformed into E. coli DH5-. Ten white clones were chosen randomly and named pRV322, pRV323, pRV326, pRV327, pRV328, pRV329, pRV330, pRV331, pRV370 and pRV371.
DNA sequencing.
All plasmids and PCR products were sequenced with BigDye terminators according to the manufacturer (Perkin-Elmer). For chromosomal DNA sequencing, genomic DNA was first denatured for 5 min at 100 °C and chilled on ice. The DNA was then treated with RNase A for 20 min at 37 °C prior to purification with phenol/chloroforme/isoamyl alcohol extraction and precipitation with ethanol. The DNA was then washed with ethanol (70%), dried at room temperature and solubilized in sterile water. Sequencing reaction mixtures contained 6 µg high-quality treated DNA, 20 pmol 25-mer primers and 16 µl BigDye terminator mix in a total volume of 40 µl. The cycle conditions were an initial denaturation at 95 °C for 5 min followed by 99 cycles (95 °C for 30 s, 55 °C for 20 s and 60 °C for 4 min). Excess dye terminators were removed by passage through a Sephadex G50 column and reaction mixtures were dried in a SpeedVac system.
Bioinformatics.
Assembling projects of all clusters were carried out with the phred/phrap/consed software package (Gordon et al., 1998 ; http://bozeman.mbt.washington.edu). Annotation of the various clusters was done with the Artemis annotation tool from the Sanger Centre (Rutherford et al., 2000
; http://www.sanger.ac.uk/Software/artemis). These softwares were run on a LINUX workstation. DNA and protein properties were analysed with the STADEN-2000 software package and/or GCG programs via the genome server from the INRA. tRNA genes were detected with the program tRNAscan-SE (Lowe & Eddy, 1997
; http://genome.wustl.edu/eddy/tRNAscan-SE).
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RESULTS |
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Identification of the rrn flanking regions
Eleven fragments corresponding to the flanking regions of the rrn operons were isolated in order to orientate these clusters with respect to the origin of replication (Cole & Saint-Girons, 1994 ). As described above, the backbone of the L. sakei 23K chromosome was first defined by the position of the rrn loci given by the I-CeuI cutting sites. To isolate the seven rrn flanking regions we used the technique of ligation-mediated PCR (LM-PCR) based on the use of an EcoRI-digested DNA ligated to an EcoRI adapter (see Methods). This adapter-ligated DNA was then used in long-range PCR experiments with either primer OLS1, pointing out of the 16S rRNA gene and thus designed for upstream regions, or primer OLS2, pointing out of the 5S rRNA gene, for downstream regions. Results of the LM-PCR and cloning experiments are shown in Fig. 3
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LM-PCR with OLS1 also resulted in the amplification of seven products. However, only two products containing the rrn upstream regions could be cloned. The other products were then directly sequenced but only two of them were identified as an rrn upstream region, whereas the others were atypical amplification products (OLS36/OLS36). More LM-PCR experiments were conducted with OLS1 and DNA digested with other restriction enzymes but they did not result in the characterization of additional 16S rRNA gene upstream regions. Sequencing of the 2·5 kb OLS1-derived PCR product or chromosomal DNA sequencing of this region was tedious and only 500 bp could be identified upstream of the 16S rRNA gene. Thus, only five of the rrn upstream regions could be identified out of seven, including the small C7 region in between two close rrn loci. These regions were subsequently included in the hybridization experiments to confirm the orientation of the rrn operons determined with the use of the seven rrn downstream regions. Furthermore, the flanking regions of the rrn genes were sequenced, allowing the determination of new genetic clusters (see below).
Construction of the gene map
To assemble the AscI-, NotI- and SfiI-digested fragments on the I-CeuI backbone, we hybridized the digested fragments with unique gene probes. This strategy helped to localize genetic markers in addition to the construction of the whole genome map. For this study we used 11 gene clusters which had been previously cloned and characterized from L. sakei 23K or other strains. The probes specific for these loci were either plasmids, when the DNA originated from L. sakei 23K, or PCR fragments amplified from the L. sakei 23K chromosome with primers designed from the published sequences when these were derived from other strains. The lacLM locus (Obst et al., 1995 ) could not be mapped as several bands hybridizing to this probe were detected. In addition, we used 10 randomly cloned fragments from a mini-shotgun library of L. sakei 23K, 15 loci obtained from our laboratory plasmid collection or by collection of PCR products of L. sakei 23K, and the 11 probes corresponding to rrn flanking regions. Details of the gene probes used for this purpose, with data summarizing sequencing results of new loci characterized in this study, are listed in Table 2
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As described in Table 2, we could identify duplicated acetate kinase genes. The two paralogues, ackA and ackB, which display only 65% identity at the DNA level, did not show cross-hybridization under stringent hybridization conditions and were therefore localized at two distinct positions on the genome map.
Furthermore, the annotation of plasmid pRV3002 indicated the presence of an IS3-like insertion element. An internal fragment of the IS was amplified by PCR and used subsequently as probe in order to estimate the copy number of recurrent elements. The IS3-like element hybridized with three AscI and three I-CeuI fragments (A1, A2 and A4; C1, C2 and C3), four NotI fragments (N2, N4, N7 and N11) and five SfiI fragments (S1, S2, S4, S5 and S6). According to the hybridization pattern of this experiment, the IS3-like element is located in at least five different positions on the genome.
Positioning of AscI, NotI, SfiI and I-CeuI skeletons
Some of the AscI, NotI and SfiI restriction sites were placed on the I-CeuI backbone by using double digestion of C2, C3 or C5 fragments. The results are shown in Table 3. Some of the double-restricted fragments could be combined unambiguously to produce AscI, NotI or SfiI fragments overlapping I-CeuI bands. Bands corresponding to N7, N8 and N11 could be identified amongst the fragments produced by NotI digestion of C2. Moreover, the 150 kb fragment could be combined with the 20 kb NotI digestion of C5 to give a fragment of 170 kb which could be N5. Indeed, the fragment order established by gene probe hybridization indicated that C2 and C5 are contiguous and that N5 might overlap the two I-CeuI bands. Similarly, the 85 kb fragment obtained with NotI digestion of C5 could be combined with the 50 kb fragment of NotI-cut C3 band to produce a fragment of 135 kb which could be N6. Band N8bis could be identified in the NotI digestion products of C4. Another 5 kb product could be combined with the 20 kb fragment obtained with the NotI digestion of C2 to give a 25 kb fragment which could be N11 (located within the C2C4 region according to hybridization results). The remaining 70 kb fragment resulting from NotI digestion of C4 is presumably a fraction of the broad N1 fragment. Also, the remaining 130 kb fragment of NotI digestion products of C3 is presumably a fraction of the broad N2 fragment. Bands A10, A11 and A12 could be identified in the AscI digest of C5, together with a 10 kb fragment which is a part of either A7 or A1 (see Table 1
). Finally, bands S5 and S11 could be identified in the SfiI digest of C2. In the same digest, a 75 kb fragment appeared broader and with a higher staining intensity than the 130 kb S5 band, indicating that it might be a doublet or a triplet. This 75 kb band could correspond to S6 and to other partial bands of higher size flanking the C2 region (such as S4 and S2; see Table 1
). From these data, the position of AscI, NotI, SfiI and I-CeuI skeletons could be precisely determined. Consequently, the physical and genetic map of L. sakei 23K strain was constructed (Fig. 4
). The origin of replication was arbitrarily placed at the beginning of band S6, which is located in middle of band C2.
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DISCUSSION |
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Ten coding sequences had no homologies with any proteins from databases, 10 were similar to proteins of unknown function, and 17 had homologies with hypothetical proteins such as ABC transporters, hypothetical membrane proteins or transcriptional regulators.
One IS3-like element was observed in our plasmid library. It is present in at least five copies in the chromosome. Some other IS3-like transposons have already been described in L. sakei as mobile elements responsible for mutations in bacteriocin-production operons (Skaugen & Nes, 1994 , 2000
). Further studies would be necessary to establish the exact copy number of these elements in the L. sakei chromosome. From all the DNA sequences used in this work, a mean G+C content of the chromosome of 42 mol% was found, with a minimal value of 35·4 mol% and a maximal value of 50·1 mol%, which is in accordance with the G+C content previously estimated (Hammes & Vogel, 1995
).
Finally, the observation of seven copies of rrn clusters in a 1845 kb genome might indicate that L. sakei could be designated as a fast-growing organism according to Krawiec & Riley (1990) . This high copy number of rrn clusters might be linked to the bacteriums ability to colonize the meat environment, hence reflecting an ecological and physiological role for rrn copies as was demonstrated for E. coli (Condon et al., 1995
) and soil bacteria (Klappenbach et al., 2000
). The upstream and downstream regions flanking the rrn gene clusters were sequenced. Four of the seven regions located downstream from the 5S rRNA genes were shown to encode tRNAs. In total 45 tRNAs were identified, corresponding to 19 of the 20 amino acids.
The genetic map, as well as the sequence of the 47 loci used in this study, will be of great help for the final assembly of the sequences for the genome sequencing project that has been undertaken in our laboratory.
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ACKNOWLEDGEMENTS |
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Received 30 July 2001;
revised 8 October 2001;
accepted 12 October 2001.