The Oenococcus oeni genome: physical and genetic mapping of strain GM and comparison with the genome of a ‘divergent’ strain, PSU-1

Líbia Zé-Zé1,2, Rogério Tenreiro1,2 and Helena Paveia1,2

Departamento de Biologia Vegetal, FCUL, Campo Grande, 1749-016 Lisboa, Portugal1
Centro de Genética e Biologia Molecular, UL, 1749-016 Lisboa, Portugal2

Author for correspondence: Rogério Tenreiro. Tel: +351 21 7500000. Fax: +351 21 7500048. e-mail: rpat{at}fc.ul.pt


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The physical and genetic maps of the Oenococcus oeni strains GM and PSU-1, which represent two genomic divergent groups on the basis of macrorestriction and ribotyping analysis, were compared. To achieve this comparison, the GM maps were constructed and the PSU-1 maps, already established, were improved. All the recognition sites of the restriction enzymes AscI, I-CeuI, FseI, NotI and SfiI were located in both chromosomes and the position of 26 genetic markers, including two rrn operons and 14 new putative oenococcal genes, were allocated to the restriction fragments generated by the five enzymes. The comparative analysis of O. oeni GM and PSU-1 genomes revealed extensive conservation of loci order. As for the differences encountered in the locations of restriction sites, they seem to be a reflection of the differences in restriction fragment sizes, explainable by insertion/deletion events and point mutations. No evidence for major genomic rearrangements was found. The genomic conservation between the two strains is in agreement and suggests homogeneity within the species, which was not unexpected in view of the restricted ecological niche of O. oeni. Further comparisons of physical maps, both of O. oeni strains and related species, will certainly help to assess whether O. oeni is really an homogeneous species and physical mapping is suitable for taxonomic purposes, both at the supra- and intraspecific levels.

Keywords: Oenococcus oeni, genomic maps, comparative genome analysis


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Oenococcus, first described by Dicks et al. (1995) , contains a sole species, Oenococcus oeni, formerly known as Leuconostoc oenos. O. oeni has a restricted ecological niche (wine and related habitats), a peculiar acidophilic nature and viability at high ethanol levels (Garvie, 1986 ) and, although its dissimilarity to other leuconostocs is fully accepted, there is some controversy regarding both the evolution rate of the species (Martínez-Murcia et al., 1993 ; Morse et al., 1996 ) and its diversity (Dicks et al., 1990 ; Zavaleta et al., 1996 ). Based on metabolic/physiological criteria, splitting of O. oeni into two species was once proposed (Peynaud & Domercq, 1968 ), but the levels of DNA–DNA homology (73–110%; Dicks et al., 1990 ) are in agreement with the genomic concept of bacterial species. More recently, the characterization of 30 strains of O. oeni of different origins on the basis of macrorestriction profiles and ribotyping, clustered them in two groups and led to the suggestion that the species might be divided into two subspecies (Tenreiro et al., 1994 and unpublished results).

To determine the validity of this assumption and gain insight into the genomic variability within O. oeni, we report here the comparison of the genomes of strains GM and PSU-1. These strains were chosen since, besides being representative of the above-mentioned clusters, they are used as starters for induction of the malolactic fermentation of wines and are therefore industrially important. To achieve this comparison, we constructed a physical and genetic map of the O. oeni GM chromosome and improved the existing physical and genetic maps of O. oeni PSU-1 (Zé-Zé et al., 1998 ).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Growth conditions for O. oeni strains GM (Microlife Techniques) and PSU-1 (R. Kunkee, University of California at Davis, USA) were described by Tenreiro et al. (1993) . Escherichia coli JM109 and XL-1 Blue (Stratagene) were grown at 37 °C in LB broth, supplemented with 100 µg ampicillin ml-1 when required.

Isolation and restriction 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, NotI, FseI and SfiI as described previously (Tenreiro et al., 1994 ; Zé-Zé et al., 1998 ). I-CeuI digestion proceeded for 10–12 h with 0·1–0·2 U enzyme per agarose plug. All the enzymes were purchased from New England Biolabs.

Restriction fragments produced by digestion with a single enzyme are indicated by the initial letter of the endonuclease. Those obtained by double digestion were designated using the letters of the first and second digestions joined by a hyphen. All fragments were numbered in size order, from the largest to the smallest. Co-migrating fragments were numbered with sequential numbers.

PFGE.
PFGE and two-dimensional PFGE were carried out in the Gene Navigator system (Pharmacia) with contour-clamped homogeneous field (CHEF) or Geneline (Beckman) with transverse alternating field (TAFE), as previously described (Zé-Zé et al., 1998 ). The mean size of each fragment was estimated from several gels by linear interpolation with two flanking size standards (Heath et al., 1992 ) using KODAK 1D 2.0 software (Kodak). Saccharomyces cerevisiae chromosomes (Bio-Rad), lambda DNA, mid-range and low-range PFG ladders (New England Biolabs) were used as size markers.

DNA probes.
Preparation of [{alpha}-32P]dCTP-labelled DNA probes and Southern hybridization conditions were as previously described (Zé-Zé et al., 1998 ).

The DNA sequences used as probes in this study are reported below and listed in Table 3. The restriction fragments generated from O. oeni PSU-1 and GM chromosomes by the endonucleases AscI, FseI, NotI and SfiI were described previously (Zé-Zé et al., 1998 ).


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Table 3. DNA sequences used as probes in this study and their location on the O. oeni GM chromosome

 
Gene sequences obtained by PCR, and primers and amplification conditions are listed in Table 1. PCR was performed in a Robocycler (Stratagene) and the reaction mixtures contained 50 pmol each primer (purchased from Gibco-BRL), 250 µM each dNTP, 1·5 mM MgCl2 and 2·0 U Taq DNA polymerase, in the supplied buffer (Gibco-BRL). Reactions were performed with approximately 200 ng O. oeni PSU-1 genomic DNA, except for hexB from Lactococcus lactis (cloned in plasmid pDU190-1; Duwat et al., 1997 ), where 100 ng plasmid was used as template. RAPD 1.2 is a randomly amplified polymorphic DNA fragment of ~1·2 kb from strain PSU-1.


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Table 1. PCR conditions used to amplify gene sequences used as probes

 
Most of the plasmids harbouring sequences from PSU-1 have already been described (Zé-Zé et al., 1998 ). The plasmid pZAR4.7 was constructed by subcloning, in pBluescript KS II (pKS II; Stratagene), a 4·7 kb EcoRI fragment from a clone of a genomic library of O. oeni PSU-1 in lambda DASH II (Stratagene) which hybridized with araA from Bacillus subtilis (pSNL4, Sá-Nogueira et al., 1997 ). pMIR0.5 was constructed by subcloning in pKS II a 0·5 kb PstI fragment from pLB3 (pMK4 with an insert of a 5·1 kb Sau3AI fragment from strain PSU-1 that confers resistance to mitomycin C; Brito, 1996 ). Plasmids pZN11 and pZN12 were obtained by shotgun cloning of PSU-1 DNA in pKS II, both digested with NotI. Plasmids p-4 and lc-36 contain NotI linking clones of O. oeni PSU-1 in pGEM5Z (Promega) and pKS II, respectively (Zé-Zé et al., 1998 ). Plasmid p-20 was isolated when screening for PSU-1 NotI linking clones in pGEM5Z vector.

DNA sequencing and analysis.
Non-automated DNA sequencing was performed by the dideoxynucleotide triphosphate chain termination method with [{alpha}-35S]dATP and Sequenase version 2.0 kit (USB, Amersham) or with [{gamma}-32P]dATP by the dsDNA Cycle Sequencing system (Gibco-BRL). Automated sequencing was purchased from the ‘4 base lab’ (Reutlingen) which uses the ALF Sequencer (Pharmacia).

DNA sequences were analysed by DNASIS (Hitachi) and, for database searches, the BLAST programs (Altschul et al., 1997 ) were used on the NCBI Sequence Database (http://www.ncbi.nlm.nih.gov).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Physical mapping of the O. oeni GM chromosome
In a previous study, a physical map of the O. oeni PSU-1 chromosome was constructed using the enzymes AscI, FseI, NotI and SfiI (Zé-Zé et al., 1998 ). The same rare cutting endonucleases were employed to construct the GM physical map (Fig. 1) and the intron-encoded endonuclease I-CeuI sites were added to both maps.



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Fig. 1. Physical map of the chromosome of O. oeni GM using the enzymes AscI, I-CeuI, FseI, NotI and SfiI. Radiating out from the centre, the five annuli show the restriction sites for the five enzymes, respectively. The locations of the genetic markers are also shown, although the order of markers in a single fragment is arbitrary. Direction of transcription of rrn operons is indicated by arrows. Scale is in kb.

 
I-CeuI cuts the genome of both strains twice, identifying two rrn operons in O. oeni located at the limits of the generated fragments (GM C1, 1200 kb; C2, 732 kb; PSU-1 C1, 1166 kb; C2, 691 kb). Under different digestion conditions, i.e. more than 0·2 U enzyme per agarose plug or longer incubation times, restriction with I-CeuI gave rise either to four reproducible fragments or to degradation of the DNA. As the presence of extra rrl gene sequences could be discarded, taking in account the two precisely located rrn operons on the O. oeni PSU-1 physical map (Zé-Zé et al., 1998 ) and the hybridization analysis with rrl and rrs probes (data not shown), the four-fragment profiles must be artefacts. Although I-CeuI is able to cleave the rrl sequence in most bacteria and lower eukaryotes, these results suggest that the presence of an rrn operon based solely on the cleavage by I-CeuI (Liu et al., 1993 , 1999 ; Marshall & Lemieux, 1992 ) cannot be taken for granted and must be confirmed by hybridization of I-CeuI profiles with rrl and rrs probes. To our knowledge, reproducible extra cutting sites for I-CeuI were never reported. Nevertheless, the absence of I-CeuI cleavage of the rrl gene due to a single base deletion in the recognition sequence has been described in Mycobacterium tuberculosis (Philipp et al., 1996 ).

Restriction of the O. oeni GM circular chromosome with AscI, FseI, NotI and SfiI produced 3, 7, 12 and 18 fragments, respectively; their estimated mean sizes are presented in Table 2. All fragments were resolvable under the PFGE conditions used, except for three SfiI bands (S7/S8, S11/S12 and S13/S14) which were assumed to be doublets. The size of the O. oeni GM chromosome was estimated to be 1932 kb.


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Table 2. Sizes (kb) of restriction fragments obtained by cleavage of the O. oeni GM chromosome

 
The establishment of relationships between restriction fragments and the mapping of all 42 restriction sites was accomplished by a reasoning similar to the one described for the O. oeni PSU-1 physical map (Zé-Zé et al., 1998 ).

The experimental strategy involved a combined approach using the analysis of double digestions, two-dimensional PFGE and Southern hybridization. Besides the assessment of fragment linkage, cross-hybridization with PSU-1 linking clones and restriction fragments both from GM and PSU-1, as well as the use of probes for genetic markers (plasmid cloned genes and PCR products), were essential to the confirmation of the relative order of restriction sites (see Table 3).

The integration of these data and comparison to the O. oeni PSU-1 physical map (Fig. 2) led to the physical map of strain GM presented in Fig. 1.



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Fig. 2. Comparison of the physical and genetic maps of O. oeni strains GM and PSU-1. Restriction sites for AscI, I-CeuI, FseI, NotI and SfiI are indicated. The circular genomes are shown linearized from a common NotI site, identified by the linking clone p-4. {triangleright} represents an insertion and {triangleleft} a deletion event in the GM chromosome. |— and —| indicate genetic markers assigned to endonuclease recognition sites.

 
Assignment of genetic markers to the O. oeni GM physical map
The chromosomal locations of 26 genetic markers, namely 23 gene markers, two linking clones and a PSU-1 RAPD fragment, were determined (Fig. 1). The probes used, listed in Table 3, assigned the corresponding sequences to the restriction fragments they hybridized with, although their exact positions remain unknown and, when in the same fragment, the order is arbitrary. Only the markers containing restriction site(s) were precisely located (Fig. 1).

The exact location of the two rrn operons in the O. oeni GM chromosome was determined by hybridization of rrs and rrl probes to the I-CeuI and FseI restriction profiles (Table 3). As in strain PSU-1 (Zé-Zé et al., 1998 ), these operons have opposite orientations in the genome, rrnA being transcribed clockwise and rrnB anticlockwise (Figs 1 and 2).

Since very few genes of O. oeni have been described, gene-like probes obtained by PCR using degenerate primers (Table 1) and some shotgun clones (Karlyshev et al., 1998 ) of O. oeni PSU-1 were further analysed by DNA sequencing, aiming to find homologies by database searches, a strategy that is suitable for locating essential genes in organisms with low G+C contents when using cloned fragments with G+C-rich restriction sites (Ladefoged & Christiansen, 1992 ). Therefore, the NotI linking clone p-4, plasmid pZAR4.7, as well as small cloned fragments produced by NotI digestion of O. oeni PSU-1 genomic DNA (pZN11, 10 kb; pZN12, 6 kb), were sequenced ~700 nucleotides in each direction using universal vector primers. The insert cloned in pMIR0.5 was sequenced on both strands and its precise size is 557 bp. The putative genes found are presented in Table 4.


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Table 4. Orthologous genes found after sequencing selected plasmid inserts and PCR fragments

 
Improvement of O. oeni PSU-1 physical and genetic maps
The construction of the GM physical map, and the comparative analysis of cross-hybridization results, enabled the mapping of two previously unlocalized SfiI sites in the PSU-1 map (Zé-Zé et al., 1998 and Fig. 2). The use of fragments S10PSU-1 and S17GM as probes allowed the assignment of the linkage S7–S10 (clockwise) in the PSU-1 chromosome. As fragment S16PSU-1 hybridized to the contiguous GM fragments S11/S12, and S16PSU-1 is located in the region S8–S11 (Zé-Zé et al., 1998 ), its position must lie between S11 and S14 (see Fig. 2).

All the genetic markers were also located on the O. oeni PSU-1 physical map, as shown in Fig. 2, except for the RAPD 1.2 fragment (hybridization experiment not performed).

Comparison of O. oeni GM and PSU-1 genomic maps
Comparison of the O. oeni GM and PSU-1 physical maps showed that, although there are some polymorphisms in restriction profiles with all enzymes used, in fragment length and number (Table 2, Fig. 2 and Zé-Zé et al., 1998 ), there is a high degree of conservation in cleavage sites (~40%) and in the order of loci. The fact that NotI-cloned linking sequences of PSU-1 are maintained in the GM physical map and the unambiguous results of cross-hybridization analysis (Table 3) support the assumption of global genetic linkage preservation.

The smaller region between the rrn operons, defined by fragments C2 or F1 in both GM and PSU-1, has an overall conservation of restriction sites, whereas the other region, defined by fragment C1, shows more variation. In terms of size, the C2/F1 region is 41 kb longer in strain GM. As the size difference between the O. oeni GM (1932 kb) and PSU-1 (1857 kb) chromosomes is 75 kb, a similar level of insertion/deletion events can be expected in both genomic regions.

Putting together the cross-hybridization results, relative dimension of homologous fragments and alignment of GM and PSU-1 physical maps, eight insertion events (in N1, N4, N7, N8, N10, S2, S3 and S16) and a deletion (in S4) were deduced in strain GM. For instance, the insertion of a 19 kb sequence in N4 was determined by cross-hybridization of N4PSU-1 and N4GM fragments. As N4GM (154 kb) fragment only hybridized with N4PSU-1 (135 kb), that region is expected either to contain a duplication or a sequence absent in PSU-1. Although the deletion event in S4 and the insertion in N1 have the same size (29 kb), there is no indication of an unequal crossing-over event involving the flanked region taking into account the maintenance of loci order relative to PSU-1. The conservation of several restriction fragments between strains GM and PSU-1 through the whole genome (e.g. N5 and N5, F7 and F8, S6 and S7, S9 and S10, N6 and N6) is also indicative of the absence of major recombination events, leaving point mutations and localized duplications or transpositions (involving regions smaller than the resolution of the presented map) as the most probable explanation for the observed restriction polymorphisms.

In terms of physical maps, and except for the presence of an extra SfiI site in the PSU-1 chromosome, the region defined by rrnB–N5–N6 seems to be largely conserved. Nevertheless, minor differences were detected in the genetic maps, namely the presence of an extra IS1165 sequence in strain PSU-1, and the different location of secA-like gene in the two chromosomes.

Concerning the restriction sites, an uneven distribution can be observed both for specific enzymes and the overall G+C-rich sequences. In spite of the identical G+C content of their recognition sequences, FseI and SfiI cutting sites predominate in the large fragment defined by the two rrn operons (C1), whereas NotI sites are more frequent in the smaller region (C2), both for the GM and PSU-1 genomes. Taking into account the G+C content of the restriction sites, and normalizing their distribution according to the size difference of fragments C1 and C2, there is no doubt that GC-rich sequences seem to prevail in fragment C1 for strain GM and in fragment C2 for strain PSU-1. Nevertheless, the general agreement in the order of the genetic markers does not support the hypothesis of differential gene spreading in both genomes.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The physical map of O. oeni GM was constructed by a top-down approach (Cole & Saint-Girons, 1994 ) using the rare-cutting enzymes AscI, I-CeuI, FseI, NotI and SfiI, and the chromosome size was estimated as 1932 kb. The map has an average resolution of 46 kb with the majority of map intervals (83·3%) smaller than 100 kb and, therefore, is in the acceptable range for genetic mapping (Cole & Saint Girons, 1994 ). The cross-hybridization experiments were invaluable for the GM map construction. The success of this approach and the previous knowledge of the PSU-1 physical map (Zé-Zé et al., 1998 ), not only made GM mapping relatively easy but also enabled the completion of SfiI mapping in PSU-1.

Hybridization experiments allowed the localization of 26 genetic markers in both maps, including 14 new O. oeni putative genes: (i) four PCR products – ccpA-like, hexB, rpoC, secA-like; (ii) 10 ORFs identified by database searches of DNA sequences araA, celR-like, clpB, dedA-like, efp, gcg, gyrB, lytS, lytR and tgt. The known O. oeni genes related to the oenological properties of this species, mleA (Labarre et al., 1996 ) and alsS/alsD (Garmyn et al., 1996 ), as well as genes essential for replication and transcription (gyrB, rpoC), or involved in mismatch repair (hexB) and NotI linking clones were also mapped. The heterologous probes for citP and recA also enabled the localization of the putative orthologous sequences in O. oeni.

The precise location and opposite transcription direction of the two rrn operons (Zavaleta et al., 1996 ) was determined by Southern hybridization with rrs and rrl homologous probes, taking advantage of the fact that the sequence of these genes is cleaved by FseI and I-CeuI, respectively. The origin and terminus of replication of the O. oeni chromosome remains unknown. However, considering that in most known eubacteria oriC is located near gyrB and rpoC, and that the transcription of rrn operons usually occurs in the opposite direction to oriC (Cole & Saint Girons, 1994 ), the suggestion of its location in fragment C1, as proposed by Zé-Zé et al. (1998) , is reinforced.

In a study involving 30 O. oeni strains, Tenreiro et al. (1994) , chose strains GM and PSU-1 as representatives of two divergent genomic groups. The comparative analysis of the genetic maps of these strains presented here (Fig. 2), showed global similarity, in spite of the restriction fragment polymorphisms (62·5% of the fragments produced by the five enzymes have different sizes). Although we can speculate that some of the differences in the locations of NotI and SfiI restriction sites in the vicinity of rrnB operon (Figs 1 and 2) could be due to the IS1165 element, the cause for the majority of the variation remains unknown. As strain GM has no lysogenic derivatives and considering that the chromosomal regions corresponding to both attachment sites in PSU-1 genome (attB1, S8 and attB2, S11; Zé-Zé et al., 1998 ) presents no considerable difference in the two strains, the in and out movement of bacteriophage DNA cannot explain the overall diversity. As, except for secA-like and IS1165, the order of genetic markers is identical in the two O. oeni chromosomes and the small differences observed are apparently due to insertion/deletion events that do not affect the global genomic similarity, other differences on the restriction maps probably result from point mutations.

The extensive degree of conservation of the order of loci presented here supports the previously suggested homogeneous nature of O. oeni (Morse et al., 1996 ; Zavaleta et al., 1996 , 1997 ). As suggested by St. Jean & Charlebois (1996) , there is no objective measure to determine the degree of similarity between genomes and the maps can be preserved despite the potential for rearrangement. Nevertheless, the value of genomic maps per se, both at specific and subspecific levels, can only be ascertained by comparisons involving strains from different taxonomic ranks, in order to assess taxonomic divergence and homogeneity.


   ACKNOWLEDGEMENTS
 
We express our gratitude to M. A. Santos (Centro de Genética e Biologia Molecular) for providing primers to amplify some DNA probes. L.Z. was the recipient of a research grant from PRAXIS XXI (BD/4541/94).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 30 May 2000; revised 18 August 2000; accepted 22 August 2000.