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Distribution of Oenococcus oeni populations in natural habitats
The fate of O. oeni would have been very different if the benefits of performing MLF in wine had not been perceived in the middle of the 20th century (Davis, Wibowo et al. 1985). O. oeni would have been ranked as a minor LAB species barely detectable in the natural environment and more often in fruit juices when they start to ferment. It would also have been considered as a contaminant occurring in wine during aging or storage (Lonvaud-Funel 1999). However, since the 1950s it has been recognized that MLF improves the quality of wine (Davis, Wibowo et al. 1985). MLF has become an essential step for producing all red wines and numerous white wines. In the same time O. oeni, which is the best adapted species in wine, has gained much attention, not only as the key actor in MLF, but also as an industrial product marketed to better control MLF and as one of the most studied LAB species (Bartowsky 2005).
The main transformation that O. oeni achieves during MLF is the conversion of L-malate to L-lactate and carbon dioxide (Lonvaud-Funel 1999, Versari, Parpinello et al. 1999), where wine is deacidified and gains a softer taste (Figure 1.1). MLF lasts a few days, weeks or months, depending on wine making practices. During this period, bacteria metabolize other organic acids, sugars, amino acids, aroma precursors and diverse compounds (Figure 1.2). This improves the microbiological stability of wine by removing potential substrates that harmful microorganisms could use to grow, while increasing the aromatic complexity (Davis, Wibowo et al. 1985, Liu 2002, Bartowsky 2005, Sumby, Grbin et al. 2014).
O. oeni is one of three species of Oenococcus described to date, but the only one detected in wine. Although wine is its preferred environment, it is also a predominant species in other fermented beverages such as cider or kombucha. The first genome sequence showed that O. oeni has a rare genetic characteristic: It is hypermutable due to the absence of the DNA mismatch repair system, MutSL, which most likely contributed to its rapid adaptation to the fluctuating wine environment (Marcobal, Sela et al. 2008). There is a great diversity of strains more or less well adapted to wine. Their diversity has long been studied by various molecular methods, although their distribution in different regions and types of wine remained puzzling. Recently, comparative genomics based on genomes of many strains has shed new light on genetic characteristics, species diversity, and adaptation of O. oeni in wines or other habitats (Bartowsky and Borneman 2011, Bartowsky 2017).
O. oeni: The wine LAB
The first strains of O. oeni were isolated from wine in the late 19th and early 20th century when it was understood that malic acid was converted to lactic acid and carbon dioxide by wine bacteria during a « second fermentation », which is now called the MLF (Bartowsky 2005). The bacteria were tentatively attributed to species such as Leuconostoc gracile, Bacterium gracile, Leuconostoc citrovorum or Leuconostoc mesenteroides. In 1967 the species was described for the first time by comparing 19 LAB strains isolated during MLF of wines produced in California, France, and Australia (Garvie 1967). The strains had similar morphological and metabolic characteristics despite being isolated from distant regions, indicating not only that they belonged to the same species, but also that this species predominated during MLF in most wines. The species was named “Leuconostoc oenos” owing to phenotypic similarities with Leuconostoc species. It is a diplococcus that sometimes forms chains, Gram positive, microaerophilic, obligatory heterofermentative, producing D-lactate from glucose (along with CO2 and ethanol or acetate), acidophilic and more tolerant to low pH than all other Leuconostoc species. In 1995 it was reclassified in a newly created genus “Oenococcus” on the basis of molecular analyses that demonstrated its phylogenetical divergence from the genus Leuconostocs (Dicks, Dellaglio et al. 1995). The first genomic sequence was produced in 2005 from strain PSU-1 (Figure 1.3) (Mills, Rawsthorne et al. 2005). Although more than 200 genomes are now available, that of PSU-1 has remained the only complete genome published until very recently (Iglesias, Valdés La Hens et al. 2018). It is a rather small genome (1.8 Mb), which has undergone a reductive evolution, losing many biosynthetic pathways for amino acid, vitamins or cofactors. This denotes a strong specialization for nutrient-rich environments, in agreement with its prevalence in wine. The genome contains only two copies of the rRNA operon, compared to the 4 to 9 copies usually encountered in LAB (Makarova, Slesarev et al. 2006). It is suggested that the rRNA copy number is more important in fast growing bacteria that require higher translation activity to develop in a fluctuating environment (Klappenbach, Dunbar et al. 2000). In agreement, O. oeni is notoriously a slow growth species and it is rarely detected in the natural environment, where it is outcompeted by other species.
The sister species O. kitaharae and O. alcoholitolerans
O. oeni has long been the only known representative of the genus Oenococcus, although two other species were more recently identified. In 2006, Oenococcus kitaharae was isolated from composting distillation residues of Shochu, a Japanese spirit produced by distillation of fermented rice, sweet potato, barley and other materials (Endo and Okada 2006). O. kitaharae is phylogenetically close from O. oeni, but it has different properties such as a higher pH optimum of growth, the inability to convert malic acid into lactic acid and CO2 and a different sugar consumption profile (Endo and Okada 2006, Cibrario, Peanne et al. 2016). Its genome has a similar size as O. oeni, but contains genetic elements suggesting adaptation to a different environment (Borneman, McCarthy et al. 2012). O. kitaharae carries genes for arginine and histidine biosynthesis, which are rarely present in O. oeni, probably because these amino acids are among the most abundant in wine. It has a different repertoire of sugar utilization genes, which correlates with different carbohydrate sources present in wine and in vegetables or cereals used for Shochu production. Surprisingly, orthologues of the 3 genes of the malolactic pathway, which is required for producing MLF, are present in O. kitaharae, but a stop codon prematurely interrupts the gene of the malolactic enzyme. This prevents the bacterium from consuming malate and suggests that it is not adapted to wine. O. kitaharae possesses genes for production of bacteriocins and other antimicrobials, a CRISPR system to fight against phages and other defense genes that are hallmarks of a species that develops in a competitive environment where it must fight against other microorganisms. These elements are absent or rarely present in O. oeni strains, presumably because it prefers to develop in wine, which hosts very few competitors (Borneman, McCarthy et al. 2012). To date, only 6 strains of O. kitaharae have been isolated, all from the same sample of composting residues of Shochu (Endo and Okada 2006). The species was presumably detected in Spanish wine (Gonzalez-Arenzana, Lopez et al. 2013) and Brazilian kefir (Zanirati, Abatemarco et al. 2015), but this was not confirmed by isolating strains. On the other hand, O. oeni has not been detected in Shochu distillation residues or during its production. Although they are evolutionarily close, it is clear that these two species have evolved to adapt to different environments.
O. alcoholitolerans is the third and most recently described species of the genus. The only 4 currently known strains were isolated in 2014 from sugarcane fermentation vats of Brazilian distilleries producing bioethanol and cachaça (Badotti, Moreira et al. 2014). Like in shochu, malolactic fermentation is not a feature in the sugarcane fermentations; in fact, the LAB are regarded as contaminants in the process (Badotti, Moreira et al. 2014). A small draft genome of 1.2 mb was assembled of one O. alcoholitolerans strain, which showed an almost 25% reduction in coding sequences compared to the other species in the genus. Unlike O.kitaharae, the malolactic operon appeared to be intact in O. alcoholitolerans, although the ability to degrade malate was not verified experimentally. The species is more sensitive to acidity than O. oeni and grow at higher ethanol levels than O. kitaharae (Badotti, Moreira et al. 2014). An adaptation to the sugarcane fermentation environment appears to have taken place, as it is able to metabolise sucrose, fructose and raffinose very well in contrast to O. oeni, but has reduced or no ability to degrade maltose, ribose and trehalose (Badotti, Moreira et al. 2014, Cibrario, Peanne et al. 2016).
It is not yet understood why the three Oenococcus species are associated with different alcohol-containing environments, but they have different genetic and metabolic properties that favor their predominance in one product over another.
Wine: the favorite habitat
Wine is undoubtedly the favorite habitat of O. oeni. Since the first description of strains isolated from Californian, Australian and French wines, it has been reported as the predominant species during MLF in wines produced in all regions, at times the only species detected. However, each wine is different and more or less favorable to bacterial growth, which includes the growth of O. oeni. It grows better than other LAB because of a superior tolerance to the low pH that is encountered in most wines (typically pH 2.9-3.6) (Davis, Wibowo et al. 1988). However, when pH exceeds 3.4-3.6, O. oeni is challenged by various species of Lactobacilli and Pediococci, which grow faster and may become predominant and perform MLF (Lonvaud-Funel 1999).
The LAB population in grape must is about 102 to 104 cells/ml depending on climate conditions and grape quality at harvest time (Lonvaud-Funel 1999). O. oeni is only a small part of it. During AF when alcohol content starts to exceed 5 or 6% and becomes a significant stress that adds to that already caused by the low pH, most LAB die and their total population decreases (Figure 1.4). O. oeni resists better and starts to develop towards the end or after AF, when yeast autolysis releases the essential nutrients that it needs (Lonvaud-Funel, Joyeux et al. 1991). The degradation of L-malate becomes perceptible when the O. oeni population reaches 106 cells/ml. It can increase up to 107-10E8 cells/ml until the end of MLF when all malate has been exhausted. O. oeni cells are then removed by adding sulfur dioxide and using oenological practices such as decantation, filtration, etc. When sulfur dioxide is not used, the O. oeni population decreases progressively, but it can negatively affect the wine quality by removing desirable aromas or by producing undesirable compounds such as harmful biogenic amines,mousy off-flavor, or bitterness (Bartowsky 2009). Many studies have been carried out to unravel the diversity of O. oeni strains during wine production. There are always many strains in the fermenting grape must, but a selection occurs during the course of AF. On average, 2 to 6 strains are present during MLF, but not necessarily during all of the MLF because there may be a succession of strains from the beginning to the end (Reguant and Bordons 2003, Cappello, Stefani et al. 2008, Mesas, Rodriguez et al. 2011, Gonzalez-Arenzana, Santamaria et al. 2012, El Khoury, Campbell-Sills et al. 2017). The type of wine and winemaking practices modulate not only the LAB species and population, but also the strains of O. oeni (Gonzalez-Arenzana, Lopez et al. 2013). A remarkable example is the presence of strains belonging to two different genetic lineages which preferentially develop in the French white wines of Burgundy and Champagne or in the red wines of Burgundy (Campbell-Sills, El Khoury et al. 2017). The main difference of the two lineages is that they tolerate better the low pH of white wines or in contrast phenolic compounds in red wines (Breniaux, Dutilh et al. 2018). However, it would be simplistic to consider that there is a strain type for each wine type. Even in the previous example, the strains of the two genetic lineages were isolated from wines in which other strains belonging to different genetic lineages were present (El Khoury, Campbell-Sills et al. 2017).
Vineyard and cellar: the origin of wine strains
Wine is a seasonal environment that permits the development of microorganisms only for a few months a year. The O. oeni strains that develop in wine originate from the surface of grapes in the vineyard, or from the cellar where they can persist by producing exopolysaccharides and biofilms at the surface of tanks, barrels and other cellar’s equipment (Dimopoulou, Vuillemin et al. 2014, Bastard, Coelho et al. 2016). Nevertheless, O. oeni is a minor species in the oenological environment as soon as it is not in wine. It was not isolated from the vineyard (Bae, Fleet et al. 2006, Yanagida, Srionnual et al. 2008), except in a recent study in which several strains were isolated from grapes of the Priorat region (Catalonia, Spain) (Franquès, Araque et al. 2017). For the first time, this study describes the same strains on grape and in wine, thus confirming the role of the vineyard as a source of strains that colonize wine. The role of the cellar’s equipment has not been directly established, but it is possible to detect commercial strains in cellars where they have been used in the past, suggesting that they were present in the cellar or its immediate environment (Gonzalez-Arenzana, Lopez et al. 2014, El Khoury, Campbell-Sills et al. 2017, Franquès, Araque et al. 2017). The same “wild” strains are sometimes detected in wines of the same cellar during several consecutive vintages, but this does not indicate whether they are residents of the vineyard or the cellar (Reguant and Bordons 2003, Franquès, Araque et al. 2017).
Apple cider: the second home
Apple cider is also a suitable environment for O. oeni. This is not very surprising given that cider and wine are close in terms of production process (AF and MLF), microbial diversity (yeasts and LAB), and composition (low pH, presence of ethanol, phenolic compounds, malic acid, etc.) (Cousin, Le Guellec et al. 2017). O. oeni is one of the main LAB contributing to MLF in cider. It has always been detected along with other LAB species (Salih, Drilleau et al. 1988, Sánchez, Rodríguez et al. 2010, Sanchez, Coton et al. 2012, Dierings, Braga et al. 2013). This contrasts with its predominance in wine, probably because cider has lower alcohol content (1.2-8%) and sometimes a higher pH than wine, which makes it more suitable for the growth of non-O. oeni species. The microbial biodiversity of cider is still incompletely described and, given the wide variety of ciders produced around the world, it is possible that O. oeni is absent in some of them, or on the opposite predominant. Interestingly, cider and wine are two different environments that not only influence the biodiversity of LAB species, but also O. oeni strains. As discussed below, strains that preferentially develop in wine or cider are different and belong to different genetic lineages (El Khoury, Campbell-Sills et al. 2017).
Other natural habitats
While the presence of O. oeni in wine and cider is well documented, it has recently been identified as the main LAB species of a third fermented beverage (Coton, Pawtowski et al. 2017). Kombucha is a traditional Asian drink that has become popular and industrially produced in North America and Europe. It is obtained by spontaneous fermentation of sweetened black or green tea by an indigenous microbiota composed of yeasts, acetic acid bacteria and LAB. During fermentation the pH drops down to 3.5-3.3 with the production of organic acids, and traces of alcohol may be produced (up to 1%). In a recent analysis of industrial production of French kombucha, O. oeni was not only detected in all fermentation tanks, but it was also the main LAB species (~105 CFU/ml) (Coton, Pawtowski et al. 2017). It is clear that this environment is as favorable as wine and cider for O. oeni, although it remains to be determined which parameters, in addition to the low pH, can benefit O. oeni. In addition, as mentioned above for cider and wine strains, those isolated from kombucha form a distinct phylogenetic lineage, which suggests a specific adaptation of the species to this product (Lorentzen et al, in review).
O. oeni may be a minor species in other fermented beverages such as Brazilian kefir, where it has been detected (Zanirati, Abatemarco et al. 2015). It may be part of the natural microbiota that develops on rotting fruits or in fruit juices, such as mango juice (Ethiraj and Suresh 1985) or stone fruits (Bridier, Claisse et al. 2010), from which it has been isolated, but its presence is probably sporadic and minor. Nevertheless, all fermented products that might be appropriate for O. oeni have not yet been investigated. The recent examples of kombucha, but also Shochu for O. kitaharae and Cachaça for O. alcoholitolerans, suggest that it is still possible to identify new products that O. oeni has colonized.
O. oeni strains diversity: methods and applications
Since the first description of the species in 1967, numerous studies have investigated the biodiversity of O. oeni strains in wine regions, vineyards, cellars, wines, ciders and more recently kombucha. The first methods were used to differentiate strains by producing molecular fingerprints. This includes pulsed-field gel electrophoresis of large DNA fragments produced by restriction enzyme digestion of the bacterial chromosome (REA-PFGE). It was first used in 1993, and often afterwards, although it is difficult and time-consuming (Kelly, Huang et al. 1993, Tenreiro, Santos et al. 1994, Sato, Yanagida et al. 2001, Guerrini, Bastianini et al. 2003, López, Tenorio et al. 2007, Larisika, Claus et al. 2008, Gonzalez-Arenzana, Lopez et al. 2012, Gonzalez-Arenzana, Santamaria et al. 2012, Zapparoli, Fracchetti et al. 2012, Wang, Li et al. 2015, Vigentini, Praz et al. 2016). More simple and rapid methods based on the use of PCR were later developed and applied, such as RAPD or Rapid Amplification of Polymorphic DNA (Zavaleta, Martinez-Murcia et al. 1997, Zapparoli, Reguant et al. 2000, Reguant and Bordons 2003, Lechiancole, Blaiotta et al. 2006, Canas, Perez et al. 2009, Capozzi, Russo et al. 2010, Solieri, Genova et al. 2010, Marques, Duarte et al. 2011), Amplified Fragment Length Polymorphism (AFLP) (Viti, Giovannetti et al. 1996, Sato, Yanagida et al. 2000, Cappello, Stefani et al. 2008, Cappello, Zapparoli et al. 2010), or more recently Multiple Loci VNTR Analysis (MLVA), which targets genomic regions conserved among all strains but with different sizes as they contain a variable number of tandem repeats (VNTR) (Claisse and Lonvaud-Funel 2012, Claisse and Lonvaud-Funel 2014, Garofalo, El Khoury et al. 2015, Cruz-Pio, Poveda et al. 2017, El Khoury, Campbell-Sills et al. 2017, Franquès, Araque et al. 2017). The methods have revealed that there is a great diversity of strains in each region, several strains in each wine tank and generally 2 to 6 major strains during MLF; that strains present in the vineyard at the surface of grapes contribute to MLF in wines produced from these grapes; and that strains can persist in cellars for several years and thus contribute to MLF in wines produced during several consecutive vintages. They were also employed for assessing the biodiversity of cider strains (Sanchez, Coton et al. 2012), and they are still used today because they are simple, cost efficient and useful for analyzing large collections of strains or isolates. Nevertheless, these methods fail at providing data on the species population structure and phylogenetic proximity of the strains. Multilocus equence Typing (MLST), which is based on the sequence analysis of housekeeping genes, was developed and used for this purpose (de Las Rivas, Marcobal et al. 2004, Bilhere, Lucas et al. 2009, Bridier, Claisse et al. 2010, Bordas, Araque et al. 2013, Gonzalez-Arenzana, Santamaria et al. 2014, Wang, Li et al. 2015, Romero, Ilabaca et al. 2018). It has provided the first hints on the species population structure, showing that strains form at least two main genetic lineages, named groups A and B, and their incidence in regions and products. But nowadays the method of choice is genome sequencing and comparative genomics. Since the first genome of strain PSU-1 produced in 2005 by Sanger technology (Mills, Rawsthorne et al. 2005), next generation sequencing technologies have made it possible to compare genomic sequences of 14 strains in 2012 (Borneman, McCarthy et al. 2012), 57 in 2015 (Campbell-Sills, El Khoury et al. 2015), 196 in 2016 (Sternes and Borneman 2016) and more than 220 genomes are now available in databanks. Phylogenomics analyses have confirmed the population structure and phylogenetic lineages previously suggested by MLST (Figure 1.45. They have also revealed new strains lineages and allowed the discovery of some correlations with the regions or products of origin. Comparative genomics investigations have started to unravel the genetic characteristics of the strains, shedding new light on their adaptation to different environments.
Table of contents :
Introduction & Objectives 1 Background
I. Chapter 1 Distribution of Oenococcus oeni populations in natural habitats.
2. O. oeni: The wine LAB
3. The sister species O. kitaharae and O. alcoholitolerans
4. Wine: the favorite habitat
5. Vineyard and cellar: the origin of wine strains
6. Apple cider: the second home
7. Other natural habitats
8. O. oeni strains diversity: methods and applications
9. Diversity of strains in wine and other products
10. Diversity of strains in regions and the concept of microbial terroir
11. Diversity in the O. oeni Pan-Genome
II. Chapter 2 Expanding the biodiversity of Oenococcus oeni through comparative genomics of apple cider and kombucha strains
III. Chapter 3 Biodiversity of bacteria and Oenococcus oeni during alcoholic and malolactic fermentation in conventional and organic red wines
IV. Chapter 4 Comparative analysis of wine adaptation in Oenococcus oeni and Oenococcus alcoholitolerans.