Physical and genetic map of the Weissella paramesenteroides DSMZ 20288T chromosome and characterization of different rrn operons by ITS analysis

Ivo M. Chelo, Líbia Zé-Zé, Lélia Chambel and Rogério Tenreiro

Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Edifício ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal

Correspondence
Rogério Tenreiro
rptenreiro{at}fc.ul.pt


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Weissella paramesenteroides DSMZ 20288T chromosome was analysed by pulsed-field gel electrophoresis, enabling the construction of a physical and genetic map. A total of 21 recognition sites of the restriction enzymes AscI, I-CeuI, NotI and SfiI were mapped on the chromosome, which was found to be circular with an estimated size of 2026 kb. This is believed to constitute the first study into the genomic organization of a strain of this genus, addressing the localization of important chromosomal regions such as oriC and terC. A total of 23 genetic markers were mapped, including eight rrn operons that were precisely assigned in 37 % of the W. paramesenteroides chromosome. The transcription direction of rrn loci was determined and three different rrn clusters were recognized regarding the presence/absence of tRNA genes in ITS regions.


Abbreviations: CHEF, counter-clamped homogeneous electric field; ITS, internal transcribed spacer; LAB, lactic acid bacteria

The GenBank accession numbers for the sequences reported in this paper are given in Tables 1 and 2 and in the legend of Fig. 3.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Weissella paramesenteroides (formerly Leuconostoc paramesenteroides), described by Garvie (1986), is one of the predominant lactic acid bacteria (LAB) species in fresh vegetables and can even be found in processed meat substrates such as fermented sausages and dry salami (Björkroth et al., 2002). Although it was initially placed in the genus Leuconostoc, DNA hybridization studies (Garvie, 1976) and phylogenetic analysis (Martinez-Murcia & Collins, 1990) soon revealed a closer relationship with some heterofermentative lactobacilli rather than with the other Leuconostoc species. The genus Weissella (Collins et al., 1993) was created to include L. paramesenteroides and five species of atypical heterofermentative lactobacilli, namely Lactobacillus viridescens (the type species), Lactobacillus confusus, Lactobacillus minor, Lactobacillus kandleri and Lactobacillus halotolerans.

Until recently, information regarding the structure and organization of LAB genomes was scarce, mainly due to the lack of appropriate genetic tools (Le Bourgeois et al., 1993, 2000). The introduction of ‘top-down’ approaches by the use of macrorestriction and pulsed-field gel electrophoresis techniques provided the means to estimate genome sizes and to construct physical and genetic maps. The structure of the chromosome of several LAB has already been obtained in this way. Physical and genetic maps of the facultative heterofermentative lactobacilli Lactobacillus sakei (Dudez et al., 2002), Lactobacillus plantarum (Chevallier et al., 1994) and Oenococcus oeni strains PSU-1 (Zé-Zé et al., 1998) and GM (Zé-Zé et al., 2000) were reported and may constitute the closest genome organization to that of Leuconostoc or Weissella. The knowledge of LAB genomes has been greatly enhanced by sequencing projects. In fact, by 2002 five genomes of LAB had already been sequenced and 29 more projects were under way all around the globe in private and public institutions (Klaenhammer et al., 2002). Although the number of completely sequenced LAB genomes has more than doubled (data from NCBI, JGI and other databases), no sequencing project for Weissella species has come to our attention.

In this study we analysed the chromosome structure and the localization of some relevant genes of Weissella paramesenteroides DSMZ 20288T. The first physical and genetic map of a Weissella strain was constructed using restriction enzymes AscI, I-CeuI, NotI and SfiI and electrophoretic separation by PFGE. The localization of several genetic markers was accomplished by Southern hybridization.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Type strains of Weissella paramesenteroides (DSMZ 20288T), Weissella confusa (DSMZ 20196T) and Leuconostoc pseudomesenteroides (CECT4027T) were grown in MRS medium at 30 °C. Escherichia coli JM109 and XL-1 Blue MRF' (Stratagene) were grown at 37 °C in LB broth, supplemented with 100 µg ampicillin ml–1 when required.

Isolation and cleavage of chromosomal DNA, and DNA fragment nomenclature.
Intact genomic DNA was prepared in agarose plugs and single or double digested with the restriction enzymes AscI, I-CeuI, NotI and SfiI as previously described (Tenreiro et al., 1994; Zé-Zé et al., 1998, 2000). Restriction fragments produced by digestion with a single enzyme are indicated by the initial letter of the endonuclease. Those obtained by double digestion are designated using the names of the respective single-digestion overlapping fragments joined by a hyphen. All fragments are numbered in size order, from the largest to the smallest. Co-migrating fragments are numbered with sequential numbers; when presenting hybridization results, following the enzyme letter, the numbers of the fragments are separated by a slash (indicating the possibility of hybridization of both or just one of the fragments). DNA extraction and purification for general purposes was carried out as described by Pitcher et al. (1989). Some DNA fragments were recovered after gel electrophoresis using the Jet Quick–Gel extraction spin Kit (Genomed).

PFGE.
PFGE was carried out in the Gene Navigator system (Pharmacia) with contour-clamped homogeneous electric field (CHEF) as previously described (Zé-Zé et al., 1998). The mean size of each fragment was estimated from at least two (fragments larger than 1000 kb) or 15 (fragments smaller than 1000 kb) runs by linear interpolation with two or more flanking size standards using KODAK 1D 2.0 software (Kodak). Lambda ladder and Low-Range PFG Markers (New England Biolabs) were used as molecular mass standards as well as intact chromosomes of Saccharomyces cerevisiae (Bio-Rad).

PCR conditions.
PCR was performed in a Thermocycler (Stratagene). The reaction mixtures contained 100–500 ng template DNA, 50 pmol each primer, 200 µM each dNTP, 1·5 mM MgCl2 and 2·5 U Taq polymerase in the buffer supplied (all from Invitrogen). A list of conditions and primers used for DNA amplification can be found in Table 1.


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Table 1. PCR conditions used to amplify DNA sequences used directly as probes or in plasmid construction

 
Plasmid construction.
Plasmids containing fragments used as probes are listed in Table 2. Standard cloning procedures were used as described by Sambrook et al. (1989). Plasmids containing PCR-generated inserts (pICP) were based on pGEMT-easy (Promega) or pKSII (Stratagene) cloning vectors. T-vector construction used for pICP3, pICP4 and pICP5 was described previously (Marchuk et al., 1991). In the construction of pICR plasmids, all the cloned fragments were obtained through genomic restriction. A total of 5–10 µg genomic DNA was digested with EcoRI, EcoRI/NotI and XhoI/NotI and cloned into plasmid vectors digested with the same set of enzymes (see Table 2).


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Table 2. Plasmids

 
DNA probes.
All probes used in this work (see Table 5) were labelled with digoxigenin using PCR Dig labelling mix (Roche). Most PCR probes were amplified using specific primers for conserved regions in gene sequences. The specific probe for rrl was made using PCR-specific primers for the amplification of the downstream sequence from the I-CeuI restriction site in the 23S gene up to the 5S gene. harosynth and hrpoA were amplified with different primers designed for dnaA amplification in Lactococcus lactis and LAB, respectively. The GACA3 PCR product was obtained from fragment A3 with a single primer generally used for RAPDs (random amplified polymorphic DNA) and cloned into modified plasmid pKSII. The hypothetically corresponding genes or gene products can be found in Table 5. For the labelling of cloned probes, plasmid primers such as T3, T7 and SP6 were used in the PCR reaction.


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Table 5. DNA sequences used as probes in this study and their location on the W. paramesenteroides DSMZ 20288T chromosome

 
Southern blotting.
In Southern blotting, gel pre-treatment and capillary transfer was done using standard procedures (Sambrook et al., 1989). The supplier's procedures (Roche) were observed during all the stages of hybridization, stringency washes, immunochemical detection and chemiluminescent visualization. Results were observed by autoradiographic exposure, and treated with Kodak imaging software. Hybridizations were performed at 40 °C using formamide at 50 %, for high stringency (homologous probes) in pre-hybridization and hybridization solutions, and at 25 % for low stringency (heterologous probes). Probes from W. confusa and L. pseudomesenteroides were used as homologous probes.

DNA sequencing.
All probes used in this work, with the exception of the rrs probe, were sequenced in an automated DNA capillary sequencer CEQ 2000-XL (Beckman Coulter) by a dye-labelled dideoxy termination method (DTCS, Dye Terminator Cycle sequencer start kit, Beckman Coulter). DNA sequences were compared with National Center for Biotechnology Information GenBank entries using the BLAST algorithm (Altschul et al., 1990).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Macrorestriction fragments and genome size of W. paramesenteroides DSMZ 20288T
The restriction of W. paramesenteroides chromosome with AscI, NotI, SfiI and the homing endonuclease I-CeuI yielded three, six, four and eight fragments, respectively. The estimated mean sizes of the fragments produced by each enzyme are listed in Table 3 and restriction profiles are shown in Fig. 1. Restriction with SfiI sometimes also gave rise to two fragments of 820 kb and 650 kb (mean observed sizes). These fragments were considered to be the result of partial digestion because of their occasional appearance and lower intensity when compared to others. In I-CeuI restriction, the presence of a double fragment of about 640 kb was revealed by the following: the apparent ‘loss' of genome size relative to restriction with other enzymes; the production of two fragments in double digestion analysis with AscI, one of 640 kb (supposedly C1) and another of 563 kb (resulting from a AscI restriction site within C2 fragment; see Table 4); and the results from hybridizing probes which indicate that the target fragment C1/2 can be associated with different locations in the chromosome.


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Table 3. Sizes (kb) of restriction fragments obtained by cleavage of the W. paramesenteroides DSMZ 20288T chromosome

The observed (Obs.) sizes are mean values, calculated from 15–17 determinations, except for fragments A1 and S1 (2–5 determinations). The maximal deviation in size was estimated to be less than 2·6 %. The map sizes are theoretical sizes of fragments calculated by placing restriction sizes on the map.

 


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Fig. 1. Macrorestriction fragments of W. paramesenteroides DSMZ 20288T. Lane 1, Lambda Ladder PFG Marker (sizes in kb). Lanes 2–5, W. paramesenteroides DNA digested with AscI, I-CeuI, NotI and SfiI, respectively.

 

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Table 4. AscI, NotI, SfiI and I-CeuI double restriction fragments of the W. paramesenteroides chromosome

 
The genome size of W. paramesenteroides DSMZ 20288T was determined to be 2026 kb, the mean of the sizes obtained with the four enzymes.

Double digestions and co-localization of restriction sites
The analysis of double digestions provides the relative localization of almost all restriction sites in the W. paramesenteroides DSMZ 20288T chromosome. The order of AscI fragments is arbitrary, since there are only three fragments, and was considered to be A1-A2-A3 (clockwise orientation in circular map; Fig. 2). The presence of two partial digests of 820 kb and 650 kb in SfiI restriction resolves for itself the order of SfiI fragments. The 650 kb fragment was attributed to the sum of the fragments S2 (505 kb) and S4 (151 kb), and the fragment of 820 kb to the sum of S2 (505 kb) and S3 (315 kb). In this way the linkage S3-S2-S4 was deduced, leading to the linkage of S1 to S3 and S4. The relative position of AscI and SfiI fragments was deduced from double-restriction analysis (Table 4) and Southern hybridization with rpoC that placed S4 in fragment A1. The lack of AscI restriction of S3 indicated that A1 overlapped with S1 (A1-S1, 857 kb) and S2 (A1-S2, 222 kb). Once SfiI mapping of A1 was done, S3 was assigned to A2 (confirmed by hybridization with the hred/htransp probe). Since the order of SfiI fragments had already been obtained, the relative location of AscI and SfiI fragments and restriction sites was achieved. The clockwise linkage of SfiI fragments is thus S1-S3-S2-S4, the location of A3 in S2 being established.



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Fig. 2. Physical and genetic map of W. paramesenteroides DSMZ 20288T using the enzymes AscI, I-CeuI, NotI and SfiI. The chromosome map was oriented based on the location of dnaA. Direction of transcription of rrn operons is indicated by an arrow. The scale is in kb.

 
Regarding the physical mapping of NotI fragments, only N5 and N6 did not have any AscI or SfiI restriction site. Allocation of N5 to A1 and S2 was possible by the hybridization of the h5'nucl probe, and the assignment of N6 to A1 and S1 was due to hpolC hybridization. Fragment N1 was not cut by AscI and due to its size (870 kb) it had to be located within A1. In SfiI digestion N1 generated a fragment of 615 kb that represented an overlap of N1 and S1. The hybridization of rpoC in N1 and S4 (which had no NotI site) allowed the precise location of N1 in SfiI and AscI maps. The remainder of S1 (apart from N6) was found to be a fragment of 396 kb shared also by N3, which was confirmed by hybridization with dnaK and harosynth. These hybridizations revealed also that N3 and A2 partially overlapped (fragment A2-N3 of 214 kb). With this, the clockwise linkage of N3-N2, that provided the NotI site in S3, was also established, taking into consideration the assignment of hred/htransp to both A2 and N2. N2 extended over to S2 (195 kb) and had in common with A3 a fragment of 88 kb. N4, which was not restricted by SfiI, was partially allocated to A3 by double digestion analysis and GACA3 hybridization. With this last identification of a NotI restriction site in A3, the order of NotI fragments was completely assessed as being N1-N6-N3-N2-N4-N5, starting from N1 and in a clockwise direction. Similar analysis was applied to I-CeuI mapping, providing the location of all but one (C8) I-CeuI fragments. C1, C3, C5, C6 and C7 have no AscI restriction site and, with the exception of C7, were allocated to A1. By double restriction with I-CeuI, A2 was found to be constituted by a fragment of 563 kb common to C2 (confirmed by hybridizations of rrl, dnaK and hred/htransp probes), an A2-C4 fragment of 28 kb and C7. This could also be confirmed by the fact that C7 had to be placed in N2 since it was not cleaved by NotI, pZN12 showed hybridization to both fragments and A3 had no I-CeuI restriction site. Assignment of A3 to C4 and therefore the clockwise linkage C7-C4 was confirmed by the hybridization of GACA3 (a probe generated from A3). I-CeuI restriction of fragment N4 into two fragments common to C4 (98 kb) and C1 (59 kb) provided the evidence of C4-C1 linkage. A clockwise linkage order of C1-C6-C3 and C3-C5 was deduced both from double digests of I-CeuI/NotI (in which N5 and N6 are not restricted) and from the allocation of the hctpsynth probe in C3 and N1. The clockwise order of I-CeuI fragments was thus determined to be C1-C6-C3-C5-C2-C7-C4.

At this point the only unknown location was that of the C8 fragment, since due to its very small size (8 kb) it could be adjacent to any I-CeuI restriction site. This was resolved later using a specific PCR amplification, as described below under ‘rrn organization’.

Mapping of genetic markers
The probes used have been assigned to the restriction fragments to which they hybridized in Southern experiments (Table 5, Fig. 2). In most cases their exact positioning and transcription direction in the fragment remained unknown. Their mapping position corresponds to the median position in the smaller fragment where they were located, be it from single or double digestion. Since h5'nucl and hred/htransp were obtained through genomic restriction with NotI, they could be precisely located in the chromosome. Although the hpolC probe was obtained the same way as h5'nucl, its precise location remains unknown as it can be at either extremity of N6 (N1-N6 or N6-N3, clockwise), since N6 is not restricted by any of the other enzymes. The need to obtain a probe that hybridized to A3 fragment led to the use of a random amplification approach, which resulted in the amplification of a 580 bp product (GACA3 probe).

ITS assignment
Amplification of the ITS was done using primers PS1490 and PL132 (Massol-Deya et al., 1995), revealing three different products of about 600 bp, 500 bp and 400 bp. This difference in ITS sizes was determined, by sequencing, to be due to the presence/absence of different tRNAs. The larger product has both tRNAIle and tRNAAla genes, the 500 bp ITS has only tRNAAla and the smallest one has no tRNA gene. PCR amplification with specific I-CeuI fragments allowed us to assign to each ribosomal operon its specific ITS. This was possible because, apart from C1, which had two 16S rRNA genes, and C2, which contained no 16S rRNA gene (explained in the following section), all the other I-CeuI fragments contained only one 16S rRNA gene and only one ITS region. Additionally, the ITS amplification of C1 gave rise to only the smallest ITS, and so the two ITS present in C1 were considered to be of this type. As can be seen in Fig. 2, tRNAIle and tRNAAla genes are present in rrn operons B and D, and rrn operons E and G contain a tRNAAla gene, whereas rrn operons A, C, F and H contain no tRNA gene.

rrn organization
Since the W. paramesenteroides chromosome has eight I-CeuI restriction sites, this was considered to be the number of rrn operons present in the genome. This result was confirmed by hybridizations with rrs and rrl probes. Hybridization with rrn probes provided not only the confirmation of I-CeuI restriction sites in the other restriction fragments but also the organization and direction of transcription of ribosomal operons. Since the rrl probe is in fact specific for the I-CeuI downstream sequence of the 23S gene (see Methods), any I-CeuI fragment that hybridizes to both rrs and rrl probes indicates the presence of two transcriptionally co-orientated rrn operons (assuming the classical 16S-23S-5S arrangement). This was the case for all I-CeuI fragments obtained, with the exception of C1 and C2. In fact, the observable fragment C1/2 hybridized to both probes but, from the hybridization of I-CeuI double digests with AscI and SfiI, we can deduce that C1 hybridizes only with rrs and C2 only with rrl (see Table 5). Taking into account the position of I-CeuI restriction sites in other fragments, the overall rrn positioning and orientation presented in Fig. 2 was determined.

The localization of C8 was obtained from the result of the hybridization of a C8-specific probe and the analysis of the ITS of the rrn operon located upstream. Since the observed size for C8 fragment was 8 kb, and C8 hybridized with both rrl and rrs probes, the presence of two transcriptionally co-orientated operons no more than 8 kb apart was indicated. Two primers were designed (5Sf and 16Sr; see Table 1 and Fig. 3) which allowed the amplification of the entire region between the 5S rRNA gene and the downstream 16S rRNA gene. This amplification gave rise to a 2·8 kb fragment (intC8) that was used as a probe and also confirmed the estimated size of C8 as 8 kb (considering the 16S, 23S and 5S sizes, as well as the ITS mean size of 500 bp). Hybridization of intC8 in NotI fragments revealed the localization of C8 in N3. Fragment N3 also contains C5, and so C8 could be either upstream or downstream from C5 (orientation regarding the direction of rrn transcription). Using primers 16Sr and PS1490 (see Fig. 3), an amplification product of about 5·7 kb was obtained, corresponding to the region between the beginning of the 16S gene present in C8 and the 3' end of the 16S gene from the I-CeuI fragment located upstream (intC8us in Fig. 3). A nested PCR using this last fragment of DNA as template and primers PS1490 and PL132 resulted in the amplification of the 400 bp specific ITS allocated to C3 instead of C5 (which was determined to contain an ITS of 500 bp). This result allowed the exact location of fragment C8. The determination of the ITS in C8 as being of 600 bp was obtained following an identical PCR strategy but using primers 5Sf and PL132 (intC8ds in Fig. 3) in the first PCR reaction.



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Fig. 3. Fragment C8 and PCR products that allowed its positioning relative to C3 and C5 and identification of its specific ITS. ITS accession numbers are AY454527, AY454528, AY454529 for the ITS of 592 bp (with tRNAIle and tRNAAla genes), 495 bp (with tRNAAla gene) and 399 bp (with no tRNA), respectively.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Through a ‘top-down’ approach, using macrorestriction and PFGE analysis as well as hybridization of genetic probes, a physical and genetic map of W. paramesenteroides DSMZ 20288T was obtained (Fig. 2). From the overall characteristics of this chromosome some important features regarding genome structure and organization can be observed. The W. paramesenteroides chromosome is circular and has an estimated size of 2026 kb. Restriction with endonucleases AscI, NotI and SfiI, which recognize eight GC-pair sequences, resulted in a somewhat reduced number of fragments (13). When comparing with results from strains of phylogenetically close genera such as Oenococcus oeni PSU-1 and O. oeni GM with 32 and 36 fragments each (Zé-Zé et al., 1998, 2000) and Lactobacillus sakei 23K with 38 (Dudez et al., 2002), the difference becomes evident. However, this cannot be related to genome characteristics such as total GC content, which is about 40 % both in W. paramesenteroides and O. oeni (Schillinger et al., 1989), or overall gene number, since the number of restriction sites differs considerably, depending on the enzyme. Compared to O. oeni strain GM, with 18 SfiI and 3 AscI fragments, more than a fourfold decrease in SfiI restriction sites but the same number of AscI fragments is observed. The physical map of W. paramesenteroides presented here has a mean fragment size of 96·5 kb, with few fragments of more than 200 kb. This fact imposes limitations on the precise localization of some genetic markers, namely those situated in the larger fragments. Nevertheless, in chromosomal regions with a concentration of restriction sites, such as the S2 fragment, the map resolution is greatly enhanced (mean fragment size of 56 kb). This variation in map resolution obtained with restriction enzymes that recognize eight GC-pair sequences suggests a non-random distribution of GC regions and may indicate the presence of chromosomal regions with different gene content.

Taking into consideration a general association of gyrB, dnaA and the chromosomal localization of the origin of replication oriC (Gasc et al., 1998; Krause et al., 1997; Zawilak et al., 2001), positioning of both genes in C1 indicates fragment C1-S1 (279 kb) as the most probable site of the chromosomal origin of replication. The terminus of replication terC, which is usually about 180° from oriC, is most likely in fragment C2. This functional arrangement description is reinforced by rRNA operon organization and transcription direction (Cole & Saint Girons, 1994). Ribosomal operons are usually more or less symmetrically disposed around oriC, with their transcription being towards terC (Le Bourgeois et al., 2000). The presence of eight rrn operons, given by the number of I-CeuI fragments, may indicate an adaptation to an unstable environment with periodic resource fluctuations, where high growth rates are advantageous (Klappenbach et al., 2000).

Apart from its practical use in determining I-CeuI fragment order, the analysis and discriminative amplification of ITS regions could be useful in determining homology with rRNA operons present in species of closely related genera such as Lactobacillus, Leuconostoc and Oenococcus. This is based in the fact that in Leuconostoc and Oenococcus strains only ITS with tRNAAla have been found (Nour, 1998a) whereas tRNAIle+Ala and no tRNA ITS seem to be prevalent in Lactobacillus species (Nour, 1998b; Tannock et al., 1999). Nevertheless, the apparently random disposition of the different ITS regions could be the result of intra-chromosomal recombination events between different rRNA operons (Hashimoto et al., 2003), which would undermine any homology assessment. Although the identification of conserved structures in the W. paramesenteroides chromosome cannot be done at this point, beyond the disposition of oriC, terC and rrn, the relative order of some genes such as gyrB, rpoC, recA, tgt/clpB and the probable oriC region seems to be similar to that seen in the PSU-1 and GM strains of O. oeni (Zé-Zé et al., 2000).

The physical map of W. paramesenteroides DSMZ 20288T reported here represents the first study into the genomic mapping of a Weissella strain. Further studies in evolutionarily close species may help in clarifing the common and distinctive features in genomic organization of lactic acid bacteria, in strain improvement, and in the establishment of evolutionary relationships.


   ACKNOWLEDGEMENTS
 
I. Chelo and L. Zé-Zé are the recipients of research grants from FCT SFRH/BD/10675/2002 and SFRH/BPD/3653/2000, respectively.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 8 July 2004; revised 16 September 2004; accepted 17 September 2004.



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