Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

A β-glucosidase from <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ATCC BAA-1163 with potential for aroma release in wine: cloning and expression in <Emphasis Type="Italic">E. coli</Emphasis>

Lactic acid bacteria (LAB) are responsible for olfactory changes in wine during malolactic fermentation (MLF). A side characteristic of MLF is the release of grape derived aroma compounds from their glycosylated precursors by β-glycosidase activities of these bacteria. Apart from Oenococcus oeni, which is regarded as the most promising species for MLF, glycosidic activities have also been observed in wine related members of the genera Lactobacillus and Pediococcus. Nevertheless, information on the involved enzymes including their potential use in winemaking is limited. In this study we report that β-glucosidases with similar protein sequences can be identified in the genomes of Lactobacillus brevis, O. oeni and Leuconostoc mesenteroides. TTG serves as start codon for the glucosidase gene of O. oeni. The β-glucosidase of O. oeni ATCC BAA-1163 was expressed in E. coli and partially characterized. The enzyme displayed characteristics similar to β-glucosidases isolated from L. brevis and L. mesenteroides. A pH optimum between 5.0 and 5.5, and a K _m of 0.17 mmol L⁻¹ pNP-β-d-glucopyranoside were determined. A glycosyltransferase activity was observed in the presence of ethanol. The enzyme from O. oeni was capable to hydrolyze glycosides extracted from Muskat wine. This study also contains a report on glycosidase activities of several LAB species including Oenococcus kitaharae.     

Intraspecific diversity of Oenococcus oeni strains determined by sequence analysis of target genes

Using molecular techniques and sequencing, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium involved in red winemaking. A relationship between the phenotypic and genotypic characterization of 16 O. oeni strains isolated from wine with different levels of enological potential was shown. The study was based on the comparative genomic analysis by subtractive hybridization between two strains of O. oeni with opposite enological potential. The genomic sequences obtained from subtractive hybridization were amplified by polymerase chain reaction and sequenced for the 16 strains. A considerable diversity among strains of O. oeni was observed.     

Genomic variations of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strains and the potential to impact on malolactic fermentation and aroma compounds in wine

Malolactic fermentation (MLF) is the bacterially driven decarboxylation of l-malic acid to l-lactic acid and carbon dioxide, and brings about deacidification, flavour modification and microbial stability of wine. The main objective of MLF is to decrease wine sourness by a small increase in wine pH via the metabolism of l-malic acid. Oenococcus oeni is the main lactic acid bacterium to conduct MLF in virtually all red wine and an increasing number of white and sparkling wine bases. Over the last decade, it is becoming increasingly recognized that O. oeni exhibits a diverse array of secondary metabolic activities during MLF which can modify the sensory properties of wine. These secondary activities include the metabolism of organic acids, carbohydrates, polysaccharides and amino acids, and numerous enzymes such as glycosidases, esterases and proteases, which generate volatile compounds well above their odour detection threshold. Phenotypic variation between O. oeni strains is central for producing different wine styles. Recent studies using array-based comparative genome hybridization and genome sequencing of three O. oeni strains have revealed the large genomic diversity within this species. This review will explore the links between O. oeni metabolism, genomic diversity and wine sensory attributes.     

Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.     

The ftsH Gene of the Wine Bacterium Oenococcus oeni Is Involved in Protection against Environmental Stress

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.     

Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N₂-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps_{T. erythraeurm} protein (Dps_tery) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps_tery, and we found that Dps_tery, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps_tery binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps_tery indicated that it has an iron core that resembles that of horse spleen ferritin.     

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines

Aims: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain‐specific primers. Strains were further grouped using a multiplex RAPD‐PCR analysis. Then, six strains were inoculated in two wine‐like media with two different ethanol concentrations (11 and 13% vol/vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT‐PCR) approach was adapted to monitor the physiological state of the strains selected. Conclusion: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. Significance and Impact of the Study: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.     

Correlation between indigenous <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strain resistance and the presence of genetic markers

This study reports on monitoring Oenococcus oeni intraspecific diversity evolution during winemaking. Three different wines were monitored. The proportion of O. oeni species was determined by species-specific PCR and O. oeni strains were distinguished by multiplex PCR-RAPD. Each strain was tested by PCR for 16 significant markers revealed by a previous genetic comparison between a strong oenological potential strain and one with poor oenological potential. Population levels and diversity changed according to winemaking stages, oenological practices and the chemical properties of the wine. In all situations, O. oeni was the best-adapted species. Within the O. oeni group, intraspecific strain diversity decreased and the malolactic fermentation was the result of the most resistant strains with the highest number of markers.     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

Isolation and characterization of Oenococcus oeni from Aglianico wines

A technological characterization of Oenococcus oeni strains isolated from Aglianico wines was performed to select starter cultures for malolactic fermentation (MLF). One hundred and fifty six O. oeni isolates were extracted from Aglianico wines, and identified by using species-specific PCR. Malolactic activity (MLA), sulphur dioxide (SO₂) resistance, acetaldehyde metabolism and other technological characteristics were tested. Differences in the technologically relevant characteristics were observed. All O. oeni strains were able to grow at low temperature and none in presence of 14% of ethanol. About 80% of O. oeni degraded more than 80% of acetaldehyde, producing ethanol and acetic acid as final products. Among nine O. oeni chosen, four isolates were sensitive to 60 mg of SO₂ l⁻¹, while the other five had high resistance. Considering their technological characteristics, five O. oeni strains could be selected starter cultures for MLF in Aglianico.     

High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 10⁷ CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 10³ CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 10³ per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.     

Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation

Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5–10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l⁻¹ was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.     

Genetic and phenotypic evidence for two groups of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strains and their prevalence during winemaking

Polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis was the most relevant method to follow the diversity of lactic acid bacteria during winemaking. By targeting the rpoB gene, two types of Oenococcus oeni strains were distinguished resulting from a single mutation in the rpoB region targeted in PCR and generating two different electrophoresis profiles. The first one prevailed during fermentation and the second during ageing. Some strains of each type were isolated during winemaking and were studied using several genetic methods (real-time PCR, PCR-random amplified polymorphic DNA, multiple locus sequence typing and the presence of gene markers). Physiological characters related to environmental conditions were examined. The results confirmed the relevance of the rpoB mutation for characterising the two O. oeni subgroups. The relationship between the physiological response to stress and the rpoB genetic groups raised the question of O. oeni intraspecies grouping. A possible division within this species, of great technological interest to the wine industry, was also raised.     

Molecular Cloning,Expression and Characterization of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Priming Glycosyltransferases

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni,a Wine-Associated Lactic Acid Bacterium

Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.