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Applied and Environmental Microbiology, March 2009, p. 1291-1300, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02563-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multilocus Sequence Typing of Oenococcus oeni: Detection of Two Subpopulations Shaped by Intergenic Recombination{triangledown} ,{dagger}

Eric Bilhère, Patrick M. Lucas,* Olivier Claisse, and Aline Lonvaud-Funel

UMR 1219, Université de Bordeaux, INRA, ISVV, Talence 33405, France

Received 10 November 2008/ Accepted 21 December 2008

Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.

* Corresponding author. Mailing address: Université Bordeaux 2, Faculté D'Oenologie, ISVV, 210 Chemin de Leysotte, CS 50008, 33882 Villenave d'Ornon, France. Phone: 33 5 57575833. Fax: 33 5 57575813. E-mail: patrick.lucas{at}u-bordeaux2.fr

{triangledown} Published ahead of print on 29 December 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, March 2009, p. 1291-1300, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02563-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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