Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

Aims:  To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters.
Methods and Results:  One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery.
Conclusions:  The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment.
Significance and Impact of the Study:  This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.     

Typical metabolic traits of two Oenococcus oeni strains isolated from Valpolicella wines

AIMS: Physiological comparison of two indigenous Oenococcus oeni strains, U1 and F3 isolated in the same area (Valpolicella, Italy) in order to select a performant starter for MLF in wine. METHODS AND RESULTS: Growth rate, sugar and malate metabolism in FT80 media at pH 5.3 and 3.5 were analysed. The amount of total protein synthesized and the level of expression of the small Hsp Lo18 were evaluated by radiolabelling and immunodetection experiments after heat (42 degrees C), acid (pH 3.5) and ethanol (12% v/v) stresses. Strain U1 showed significantly lower specific growth rate and growth yield in acid conditions than strain F3. However, strain U1 had a higher malate consumption capacity at pH 3.5 than strain F3, in relation with an higher malolactic activity determined on whole cells. Strain U1 exhibited about half the total protein synthesis level than strain F3, but both strains expressed Lo18 similarly. Evaluation of malolactic fermentation (MLF) performance by microvinification trials was carried out. Strain U1 was able to complete MLF, whereas strain F3 degraded malic acid partially when inoculated in Amarone wine. CONCLUSIONS: Considering its performances in microvinifications experiments, strain U1 could be a good candidate for malolactic starter as an alternative to deficient commercial starters.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking

Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar environments. 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 obligately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. Moreover, 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.     

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.     

Effect of ethanol and fatty acids on malolactic activity ofLeuconostoc oenos

Medium-chain fatty acids (C₆ to C₁₂), produced by yeast metabolism during alcoholic fermentation, are known to be inhibitory to lactic acid bacteria. The purpose of this work was to clarify the effect of both ethanol and decanoic and dodecanoic acids on the growth and malolactic activity of aLeuconostoc oenos strain isolated from Portuguese red wine. Ethanol in concentrations up to 12% had no significant effect on malolactic activity but strongly inhibited cell growth. The fatty acids decanoic acid, in concentrations up to 12.5 mg l^–1, and, dodecanoic acid up to 2.5 mg l^–1 seemed to act as growth factors stimulating also malolactic activity; at higher concentrations they exerted an inhibitory effect. We found clear pH dependence between pH 3.0 and pH 6.0, between decanoic acid concentration and its effect on malolactic activity, indicating that the undissociated molecule is the active form. At pH 3.0 the results can be explained by considering that fatty acids enter the cell as protonated molecules and dissociate in the cytoplasm due to the higher internal pH, leading to increased intracellular hydrogenous concentration. This may be the basis of two different effects that contribute to the observed inhibition: decrease in the intracellular pH and dissipation of the transmembrane proton gradient, thus inhibiting intracellular enzymes and ApH-dependent transport systems.     

Characterization of an Unusual Fluoride-Resistant Streptococcus mutans Isolate

A fluoride-resistant Streptococcus mutans isolate NCH105 was characterized and compared with wild-type strain UA130. The growth and lactic acid production of strain NCH105 were found to be unaffected by the presence of fluoride at the initial medium pH values of 6.5 and 6. In addition, NCH105 was found to be capable of lowering the pH of the medium to approximately 5.5, which is the critical level where enamel demineralization begins in vivo. Lactic acid production, glucose uptake at pH 6.5 and 6, as well as ATPase activity at pH 5 were found to be unaffected by fluoride. Finally, strain NCH105 is capable of binding as well as the wild-type bacterium to artificial tooth pellicles. These results are unusual when compared with previously isolated fluoride-resistant mutants and suggest that NCH105 may have the ability to colonize the tooth surface and initiate dental caries.     

Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach

Aims:  To study the effect of ethanol on Oenococcus oeni activity at the single cell level.
Methods and Results:  The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l -malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells.
Conclusion:  From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni .
Significance and Impact of the Study:  The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Oxidative phosphorylation in yeast VI. ATPase activity and protein synthesis in mitochondria isolated from nuclear mutants deficient in cytochromes

1. The oligomycin-sensitive Mg²⁺-dependent ATPase activity of mitochondria isolated from wild-type yeast Saccharomyces cerevisiae was only slightly inhibited by atractyloside at concentrations which entirely prevented oxidative phosphorylation. This indicated that most of the ATPase in these mitochondrial preparations was located outside the atractyloside-sensitive barrier and did not participate in the energy-transfer process.

2. ATPase activity of mitochondria isolated from nuclear gene mutants deficient in a single cytochrome, a, b, or c, respectively, was strongly inhibited by oligomycin. The mitochondria from these mutants, like those from the wild-type strain, were able to incorporate amino acids into protein.

3. Mitochondrial ATPase activity of single nuclear gene mutants deficient in both cytochromes a and b was only slightly inhibited by oligomycin. These mitochondria were incapable of incorporating amino acids into protein. The mitochondria from these nuclear mutants thus resembled mitochondria of cytoplasmic respiration-deficient mutants.

4. The results suggest that mitochondrial cytochromes may be coded by nuclear genes and that product(s) of mitochondrial protein synthesis may be required for integrating the cytochromes a and b and the components of the oligomycin-sensitive ATPase complex into the mitochondrial membranes.     

Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB1832 in Saccharomyces cerevisiae

Wine production is characterized by a primary alcoholic fermentation, conducted by Saccharomyces cerevisiae, followed by a secondary malolactic fermentation (MLF). Although most lactic acid bacteria (LAB) have the ability to metabolize L-malate, only a few species survive the high ethanol and SO2 levels in wine. Wines produced in colder viticultural regions have a lower pH than wines produced in warmer regions. The decarboxylation of L-malate in these wines leads to an increase in pH, more organoleptic complexity and microbiological stability. MLF is, however, difficult to control and problems often occur during filtering of such wines. Pediococcus spp. are known to occur in high pH wines and have strong malolactic activity. However, some pediococci synthesize exocellular polysaccharides, which may lead to abnormal viscosity in wine. In this study, the malolactic gene from Pediococcus damnosus NCFB1832 (mleD) was cloned into S. cerevisiae and co-expressed with the malate permease gene (mae1) of Schizosaccharomyces pombe. Expression of the mleD gene was compared to the expression of two other malolactic genes, mleS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni Lal1. The genetically modified strain of S. cerevisiae decreased the level of L-malate in grape must to less than 0.3 gl(-1) within 3 days. This is the first expression of a malolactic gene from Pediococcus in S. cerevisiae.     

Evidence of distinct populations and specific subpopulations within the species Oenococcus oeni

Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.     

Subunit D (Vma8p) of the yeast vacuolar H+-ATPase plays a role in coupling of proton transport and ATP hydrolysis

To investigate the function of subunit D in the vacuolar H(+)-ATPase (V-ATPase) complex, random and site-directed mutagenesis was performed on the VMA8 gene encoding subunit D in yeast. Mutants were selected for the inability to grow at pH 7.5 but the ability to grow at pH 5.5. Mutations leading to reduced levels of subunit D in whole cell lysates were excluded from the analysis. Seven mutants were isolated that resulted in pH-dependent growth but that contained nearly wild-type levels of subunit D and nearly normal assembly of the V-ATPase as assayed by subunit A levels associated with isolated vacuoles. Each of these mutants contained 2-3 amino acid substitutions and resulted in loss of 60-100% of proton transport and 58-93% of concanamycin-sensitive ATPase activity. To identify the mutations responsible for the observed effects on activity, 14 single amino acid substitutions and 3 double amino acid substitutions were constructed by site-directed mutagenesis and analyzed as described above. Six of the single mutations and all three of the double mutations led to significant (>30%) loss of activity, with the mutations having the greatest effects on activity clustering in the regions Val(71)-Gly(80) and Lys(209)-Met(221). In addition, both M221V and the double mutant V71D/E220V led to significant uncoupling of proton transport and ATPase activity, whereas the double mutant G80D/K209E actually showed increased coupling efficiency. Both a mutant showing reduced coupling and a mutant with only 6% of wild-type proton transport activity showed normal dissociation of the V-ATPase complex in vivo in response to glucose deprivation. These results suggest that subunit D plays an important role in coupling of proton transport and ATP hydrolysis and that only low rates of turnover of the enzyme are required to support in vivo dissociation.     

Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance

Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.     

Lactic acid bacteria associated with wine grapes from several Australian vineyards

AIMS: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. METHODS AND RESULTS: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. CONCLUSIONS: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.     

Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation

Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link dynein with cargo and activate dynein motor function. The mechanisms by which dynactin regulates dynein activity are not clear. In this study, we examine the role of dynactin in regulating dynein ATPase activity. We show that dynein-microtubule binding and ATP-dependent release of dynein from microtubules are reduced in dynactin null mutants, Deltaro-3 (p150(Glued)) and Deltaro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, but not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produced in a Deltaro-3 mutant has approximately 8-fold reduced ATPase activity relative to dynein isolated from wild-type. However, dynein ATPase activity from wild-type is not reduced when dynactin is separated from dynein, suggesting that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Deltaro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon lambda protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation of dynein light chains.     

Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity

The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue. The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein. Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2. To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue. The new [Asn160]recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein. This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain. Although it is an active single-stranded DNA-dependent ATPase, the [Asn160]recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro. Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the [Asn160]recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E. coli protein. To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the [Asn160]recA protein.