FEMS Microbiology Reviews 29 (2005) 465–475 www.fems-microbiology.org Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking David A. Mills a,*, Helen Rawsthorne a,1, Courtney Parker a,2, Dafna Tamir a,3, Kira Makarova b a Department of Viticulture and Enology, University of California, Davis, CA 95616, United States b National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, United States Received 24 February 2005; accepted 23 April 2005 First published online 28 August 2005 Abstract Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar envi- ronments. O. oeni is commonly responsible for the malolactic fermentation in wine and due to its positive contribution is frequently used as a starter culture to promote malolactic fermentation. In collaboration with the Lactic Acid Bacteria Genome Consortium the genome sequence of O. oeni PSU-1 has been determined. The complete genome is 1,780,517 nt with a GC content of 38%. 1701 ORFs could be predicted from the sequence of which 75% were functionally classified. Consistent with its classification as an obli- gately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. More- over, genes related to flavor modification in wine, such as malolactic fermentation capacity and citrate utilization were readily identified. The completion of the O. oeni genome marks a significant new phase for wine-related research on lactic acid bacteria in which the physiology, genetic diversity and performance of O. oeni starter cultures can be more rigorously examined. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Oenococcus oeni; Genome; Physiology; Genetic diversity Contents 1. Introduction . 466 2. Oenococcus oeni.......................................................................... 466 2.1. Genomic analysis of Oenococcus oeni strain PSU-1 ............................................. 466 2.2. General genome description ............................................................. 467 3. Metabolism related to growth in wine – selected topics . 468 3.1. Carbohydrate metabolism ............................................................... 468 3.2. Organic acids – malate ................................................................. 468 * Corresponding author. Tel.: +1 530 754 7821; fax: +1 530 752 0382. E-mail address: [email protected] (D.A. Mills). 1 Present address: Department of Bioscience and Biotechnology, Drexel University, 32nd and Chestnut Street, Philadelphia, PA 19104-2875, United States. 2 Present address: Osel, Inc., 1800 Wyatt Dr. Suite 14, Santa Clara, CA 95054, United States. 3 Present address: Department of Plant Pathology and Microbiology, Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. 0168-6445/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsre.2005.04.011 466 D.A. Mills et al. / FEMS Microbiology Reviews 29 (2005) 465–475 3.3. Organic acids – citrate ................................................................. 469 3.4. Nitrogen metabolism .................................................................. 470 3.5. Amino acid catabolism ................................................................. 470 3.6. Bacteriophage........................................................................ 471 3.7. Stress-related genes . .................................................................. 471 3.8. Phylogeny and systematics .............................................................. 472 4. Future work . 472 Acknowledgments . 472 References. 473 1. Introduction relatively nutrient-limited, and contains inhibitory com- pounds such as polyphenolics. To make microbial The production of wine involves an amalgam of growth more challenging, winemakers typically add sul- microbial transformations comprising a complex succes- fur dioxide to grape must to control indigenous yeast sion of various yeast and bacterial species. The initial and bacterial populations. Thus while many LAB are conversion of grape must is an alcoholic fermentation able to perform the MLF, few can thrive sufficiently to carried out by one or more strains of Saccharomyces complete the fermentation in a desired fashion. Oeno- (typically Saccharomyces cerevisiae), either purposely coccus oeni is the agent most commonly associated with inoculated or naturally present in the grape/winery envi- the MLF in the production of wine and for that reason ronment. This primary fermentation produces ethanol has attracted considerable attention. O. oeni is consid- and creates the anaerobic conditions that limit growth ered a desirable bacterium because it can grow in the of other yeast and bacterial species and thus protects hostile environment of wine and, among the wine- the wine from spoilage [1]. The malolactic fermentation related LAB, it is least associated with off flavors or (MLF) is a secondary fermentation that typically occurs other undesirable metabolites. at the end of alcoholic fermentation and is carried out by one or more populations of lactic acid bacteria (LAB). This secondary fermentation can be beneficial 2. Oenococcus oeni or detrimental, depending on the wine style. MLF de- acidifies wine by conversion of malate (L-malic acid) to In 1967, Garvie [8] first proposed a separate species, lactate (L-lactic acid) and is favored in high-acid wines Leuconostoc oenos, for wine-related leuconostoc strains. produced in cool-climate regions. Conversely, this pro- L. oenos was differentiated from other Leuconostoc spe- cess is less desired in warm-climate regions where al- cies primarily on the basis of tolerance to acidic condi- ready low-acid wines are further de-acidified by MLF. tions and a peculiar growth enhancement in media MLF stabilizes wines in a straightforward fashion, by containing tomato juice. Since that time numerous tax- consuming available nutrients and lowering the poten- onomic surveys of L. oenos strains have been under- tial for additional growth of other indigenous microbes. taken, first examining growth characteristics and later Finally MLF contributes to the organoleptic quality of using various molecular methods [9,10]. More recently, wines through direct production of flavor compounds Dicks et al. [11] proposed a reclassification of Leuconos- [2] or through indirect impact on other microorganisms toc oenos into a new genus Oenococcus, of which O. oeni present in the wine [3–5]. From a winemakerÕs perspec- is the only species. tive there is a need to control the MLF, allowing for pre- cise application, or prevention, to enhance the positive 2.1. Genomic analysis of Oenococcus oeni strain PSU-1 attributes or reduce potential negative impacts on the particular wine. Perhaps the greatest measure of control In order to advance the study of O. oeni, we gener- has been achieved through inoculation of starter cul- ated the complete genome sequence of the strain PSU- tures in order to perform the MLF. 1. It was isolated in 1972 by Beelman et al. [12] from a A number of lactic acid bacterial species have been spontaneous malolactic fermentation in an experimental isolated from the wine environment [6,7]. Most belong wine made in Pennsylvania (USA). Initial characteristics to the genera Pediococcus, Lactobacillus, Leuconostoc of the strain demonstrated strong similarity to another and Oenococcus and most possess the capacity to carry well-known oenococcal strain, ML-34, previously iso- out the malolactic fermentation. Wine is a particularly lated in California [13]. Ze-Ze and coworkers [14,15] harsh environment for growth as it possesses a low pH carried out an extensive PFGE analysis of PSU-1. (ranging from 2.5 to 4.0), and following the primary Indeed, we chose PSU-1 for genome sequencing on the fermentation, a high alcohol content. Wine is also basis of this available genetic map. D.A. Mills et al. / FEMS Microbiology Reviews 29 (2005) 465–475 467 Genomic sequencing was undertaken as part of a Jazz. Gap filling was carried out in collaboration with larger effort to generate publicly accessible genome a private company, Fidelity Systems, Inc. (Gaithers- sequence for a number of fermentation-associated bacte- burg, MD), using a direct whole genome sequencing ap- ria. This effort was coordinated by a working unit proach as previous described [18]. Annotation was termed the ‘‘Lactic Acid Bacteria Genome Consortium’’ performed at NCBI using the GeneMark program fol- (LABGC), the mission of which is to advance functional lowed by manual curation. genomic studies on the food grade LAB [16,17]. In 2002, the LABGC partnered with the Joint Genome Institute 2.2. General genome description (JGI), a high throughput sequencing facility run by the United States Department of Energy to generate draft At the time of this writing, manual curation of the sequence of the eleven bacterial genomes, five of which O. oeni PSU-1 genome, and comparison to other LAB (Lactobacillus casei, Lactobacillus brevis, Leuconostoc genomes, are ongoing. As such, what is presented is a mesenteroides, Pediococcus pentosaceus and Oenococcus preliminary report on the PSU-1 genome sequence with oeni) can be readily isolated from wines or musts. A high a focus on specific wine-related fermentation properties.