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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Oenococcus oeni, genomic maps, comparative genome analysis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1012 h with 0·10·2 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 [-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
).
|
|
DNA sequencing and analysis.
Non-automated DNA sequencing was performed by the dideoxynucleotide triphosphate chain termination method with [-35S]dATP and Sequenase version 2.0 kit (USB, Amersham) or with [
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
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
.
|
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
.
|
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 rrnBN5N6 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brito, L. (1996). Molecular analysis in Leuconsotoc oenos. PhD thesis, Technical University of Lisbon.
Cole, S. T. & Saint Girons, I. (1994). Bacterial genomics. FEMS Microbiol Rev 14, 139-160.[Medline]
Dicks, L. M. T., Vuuren, H. J. J. & Dellaglio, F. (1990). Taxonomy of Leuconostoc species, particularly Leuconostoc oenos, as revealed by numerical analysis of total soluble cell protein patterns, DNA base compositions and DNA-DNA hybridizations. Int J Syst Bacteriol 40, 83-91.
Dicks, L. M. T., Dellaglio, F. & Collins, M. D. (1995). Proposal to reclassify Leuconostoc oenos as Oenococcus oeni [corrig.] gen. nov., comb. nov. Int J Syst Bacteriol 45, 395-397.[Abstract]
Duwat, P., Ehrlich, S. D. & Gruss, A. (1992). A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction. J Bacteriol 174, 5171-5175.[Abstract]
Duwat, P., Cochu, A., Erlich, S. D. & Gruss, A. (1997). Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition. J Bacteriol 17, 4473-4479.
Garmyn, D., Monnet, C., Martineau, B., Guzzo, J., Cavin, J.-F. & Diviès, C. (1996). Cloning and sequencing of the gene encoding -acetolactate decarboxylase from Leuconostoc oenos. FEMS Microbiol Lett 145, 445-450.[Medline]
Garvie, E. (1986). Genus Leuconostoc. In Bergeys Manual of Systematic Bacteriology , pp. 1071-1075. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore, MD:Williams & Wilkins.
Heath, J. D., Perkins, J. D., Sharma, B. & Weinstock, G. M. (1992). NotI cleavage map of Escherichia coli K12 strain MG1655. J Bacteriol 174, 5171-5175.[Abstract]
Johansen, E. & Kibenich, A. (1992). Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria. Plasmid 27, 200-206.[Medline]
Karlyshev, A. V., Henderson, J., Ketley, J. M. & Wren, B. W. (1998). An improved physical and genetic map of Campylobacter jejuni NCTC 11168 (UA580). Microbiology 144, 503-508.[Abstract]
Labarre, C., Diviès, C. & Guzzo, J. (1996). Genetic organization of the mle locus and identification of mleR-like gene from Leuconostoc oenos. Appl Environ Microbiol 62, 4493-4498.[Abstract]
Ladefoged, S. A. & Christiansen, G. (1992). Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis. J Bacteriol 174, 2199-2207.[Abstract]
Liu, S.-L., Hessel, A. & Sanderson, K. E. (1993). Genomic mapping with I-CeuI, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli and other bacteria. Proc Natl Acad Sci USA 90, 6874-6878.[Abstract]
Liu, S.-L., Schryvers, A. B., Sanderson, K. E. & Johnston, R. N. (1999). Bacterial phylogenetic clusters revealed by genome structure. J Bacteriol 181, 6747-6755.
Marshall, P. & Lemieux, C. (1992). The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene. Nucleic Acids Res 20, 6401-6407.[Abstract]
Martínez-Murcia, A. J., Harland, N. M. & Collins, M. D. (1993). Phylogenetic analysis of some leuconostocs and related organisms as determined from large-subunit rRNA gene sequences: assessment of congruence of small- and large-subunit rRNA derived trees. J Appl Bacteriol 74, 532-541.[Medline]
Morse, R., Collins, M. D., OHanlon, K., Wallbanks, S. & Richarson, P. T. (1996). Analysis of the ß' subunit of DNA-dependent RNA polymerase does not support the hypothesis inferred from 16S rRNA analysis that Oenococcus oeni (formerly Leuconostoc oenos) is a tachytelic (fast-evolving) bacterium. Int J Syst Bacteriol 46, 1004-1009.[Abstract]
Peynaud, E. & Domercq, S. (1968). Etude de quatre cents souches de conques heterolactiques isolés de vins. Ann Inst Pasteur 19, 159-169.
Philipp, W. J., Nair, S., Guglielmi, G., Lagranderie, M., Gicquel, B. & Cole, S. T. (1996). Physical mapping of Mycobacterium bovis BCG Pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis. Microbiology 142, 3135-3145.[Abstract]
St. Jean, A. & Charlebois, R. L. (1996). Comparative genomic analysis of the Haloferax volcani DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement. J Bacteriol 178, 3860-3868.[Abstract]
Sá-Nogueira, I., Nogueira, T. V., Soares, S. & de Lencastre, H. (1997). The Bacillus subtilis l-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology 143, 957-969.[Abstract]
Sesma, F., Gardiol, D., Holgado, A. P. R. & de Mendoza, D. (1990). Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli. Appl Environ Microbiol 56, 2099-2103.[Medline]
Tenreiro, R., Santos, R., Brito, L., Paveia, H., Vieira, G. & Santos, M. A. (1993). Bacteriophages induced by mitomycin C treatment of Leuconostoc oenos strains from Portuguese wines. Lett Appl Microbiol 16, 207-209.
Tenreiro, R., Santos, M. A., Paveia, H. & Vieira, G. (1994). Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA. J Appl Bacteriol 77, 271-280.[Medline]
Yamamoto, S. & Harayama, S. (1995). PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61, 1104-1109.[Abstract]
Zavaleta, A. I., Martínez-Murcia, A. J. & Rodríguez-Valera, F. (1996). 16S23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous. Microbiology 142, 2105-2114.[Abstract]
Zavaleta, A. I., Martínez-Murcia, A. J. & Rodríguez-Valera, F. (1997). Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analysis. Appl Environ Microbiol 63, 1261-1267.[Abstract]
Zé-Zé, L., Tenreiro, R., Brito, L., Santos, M. A. & Paveia, H. (1998). Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers. Microbiology 144, 1145-1156.[Abstract]
Received 30 May 2000;
revised 18 August 2000;
accepted 22 August 2000.