| 1. |
Sievers M,
Uermösi C,
Fehlmann M,
Krieger S,
( 2003 ) Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria. PMID : 14529177 : Abstract >>
The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.
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2. |
Bourdineaud JP,
Nehmé B,
Tesse S,
Lonvaud-Funel A,
( 2003 ) The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress. PMID : 12732516 : DOI : 10.1128/aem.69.5.2512-2520.2003 PMC : PMC154493 Abstract >>
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.
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3. |
Fortier LC,
Tourdot-Maréchal R,
Diviès C,
Lee BH,
Guzzo J,
( 2003 ) Induction of Oenococcus oeni H+-ATPase activity and mRNA transcription under acidic conditions. PMID : 12770702 : DOI : 10.1016/S0378-1097(03)00299-4 Abstract >>
The profiles of Oenococcus oeni IOB84.13 H(+)-ATPase activity under various conditions of growth were studied. Cells growing at low pH 3.5 had a 1.6-fold higher H(+)-ATPase activity compared to control cells grown at pH 5.3. While the pH of the growth medium was shown to be stable in the presence of malic acid, a drastic decrease in pH from 5.3 down to 3.9 during growth in the absence of malic acid induced an increase in H(+)-ATPase activity by 1.5-fold. This induction was even greater when the initial pH was 3.5. Partial cloning of the genes encoding the beta-subunit and the epsilon-subunit of the H(+)-ATPase suggested a typical F(1)F(0)-ATPase genetic organization in O. oeni. The atp mRNA was detected by slot blots. Cells shocked at acidic pH were shown to contain higher levels of atp mRNA compared to the control cells grown at pH 5.3. Taken together, these results indicate that the H(+)-ATPase of O. oeni is induced at low pH and that regulation seems to occur at the level of transcription. This agrees with the role of this enzyme in the regulation of the cytoplasmic pH and in the acid tolerance of O. oeni.
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4. |
Divol B,
Tonon T,
Morichon S,
Gindreau E,
Lonvaud-Funel A,
( 2003 ) Molecular characterization of Oenococcus oeni genes encoding proteins involved in arginine transport. PMID : 12631210 : Abstract >>
This work was carried out to complete the sequence of the arc cluster involved in arginine catabolism in Oenococcus oeni, and particularly to characterize the genes encoding proteins involved in arginine transport. Using molecular cloning, two loci encoding proteins involved in the arginine-ornithine antiport were isolated. Their expression patterns were monitored by RT-PCR to study the influence of arginine on their transcription. Polycistronic mRNAs were detected. PCR performed directly on colonies with primer pairs specific of arc genes was used to discriminate strains able/unable to degrade arginine. Oenococcus oeni contains two arcD loci encoding similar proteins. Their expression is not influenced by arginine and polycistronic messengers were detected. The inability to use arginine is due to a lack of genetic information encoding proteins of the arginine deiminase pathway. The constitutive expression of arcD genes points to the positive role of arginine on O. oeni cell growth. The occasional presence of all the arc ABCD genes together in O. oeni strains might provide insights into the growth rate variability within this species.
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5. |
Tonon T,
Bourdineaud JP,
Lonvaud-Funel A,
( 2001 ) The arcABC gene cluster encoding the arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene. PMID : 11605985 : Abstract >>
Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.
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6. |
Mesas JM,
Rodríguez MC,
Alegre MT,
( 2001 ) Nucleotide sequence analysis of pRS2 and pRS3, two small cryptic plasmids from Oenococcus oeni. PMID : 11591140 : DOI : 10.1006/plas.2001.1537 Abstract >>
Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2.
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7. |
Garmyn D,
Jobin MP,
( 1999 ) The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase. PMID : 10542163 : PMC : PMC94126 Abstract >>
Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42 degrees C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5' end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli.
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8. |
Garmyn D,
Diviès C,
Guzzo J,
( 1999 ) Expression of the Oenococcus oeni trxA gene is induced by hydrogen peroxide and heat shock. PMID : 10376841 : DOI : 10.1099/13500872-145-5-1245 Abstract >>
Sequencing of the DNA region located upstream of the alpha-acetolactate synthase and decarboxylase (alsS-alsD) cluster of Oenococcus oeni allowed identification of an ORF, named trxA. This encodes a protein of 104 amino acids very similar to known thioredoxins. The protein encoded by the cloned fragment was able to complement Escherichia coli strains lacking a functional thioredoxin. Considering the results of protein sequence comparisons and complementation experiments, it was concluded that the trxA gene encodes a functional thioredoxin. Studies of trxA expression showed that the abundance of trxA mRNA was similar during all growth stages. A significant increase in trxA mRNA levels was observed in the presence of hydrogen peroxide in the medium or after heat shock. A single transcriptional start site was determined with total RNA isolated from cells subjected or not subjected to oxidative stress or heat shock. In each case the same promoter region was identified and shown to have a high similarity to the consensus promoter sequence of Gram-positive bacteria, as well as to that of E. coli and the previously mapped promoters from O. oeni.
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9. |
Rodríguez MC,
Mesas JM,
( 1999 ) Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni. PMID : 10087217 : Abstract >>
A new cryptic plasmid, pRS1, from an Oenococcus oeni strain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid of O. oeni. Common features in other plasmids from O. oeni (i.e., pLo13 and pOg32) have been found in pRS1. They have three major ORFs in the same strand; the putative encoded proteins by two of these ORFs exhibit homology with the replication (Rep) and the recombination (Pre) proteins, respectively, of the pT181 plasmid family and related gram-positive bacteria plasmids; these plasmids contain the DNA sequences required for plasmid replication by the rolling circle mechanism and for recombination (i.e., double-strand origin, DSO; single-strand origin, SSO; recombination-specific sites, RSA and RSB); and finally, all these plasmids have a third ORF of unknown function. These features suggest that pRS1 could constitute together with pLo13 and pOg32 a family of small cryptic plasmids of O. oeni.
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10. |
Augagneur Y,
Ritt JF,
Linares DM,
Remize F,
Tourdot-Maréchal R,
Garmyn D,
Guzzo J,
( 2007 ) Dual effect of organic acids as a function of external pH in Oenococcus oeni. PMID : 17406856 : DOI : 10.1007/s00203-007-0230-0 Abstract >>
In this study we analyzed under various pH conditions including low pH, the effects of L-malic acid and citric acid, combined or not, on the growth, the proton motive force components and the transcription level of selected genes of the heterolactic bacterium Oenococcus oeni. It is shown here that L-malate enhanced the growth yield at pH equal or below 4.5 while the presence of citrate in media led to a complete and unexpected inhibition of the growth at pH 3.2. Nevertheless, whatever the growth conditions, both L-malate and citrate participated in the enhancement of the transmembrane pH gradient, whereas the membrane potential decreased with the pH. These results suggested that it was not citrate that was directly responsible for the inhibition observed in cultures done at low pH, but probably its end products. This was confirmed since, in media containing L-malate, the addition of acetate substantially impaired the growth rate of the bacterium and slightly the membrane potential and pH gradient. Finally, study of the expression of genes involved in the metabolism of organic acids showed that at pH 4.5 and 3.2 the presence of L-malate led to an increased amount of mRNA of mleP encoding a malate transporter.
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11. |
Marcobal A,
de las Rivas B,
Moreno-Arribas MV,
Muñoz R,
( 2006 ) Evidence for horizontal gene transfer as origin of putrescine production in Oenococcus oeni RM83. PMID : 17056681 : DOI : 10.1128/AEM.01213-06 PMC : PMC1694244 Abstract >>
The nucleotide sequence of a 17.2-kb chromosomal DNA fragment containing the odc gene encoding ornithine decarboxylase has been determined in the putrescine producer Oenococcus oeni RM83. This DNA fragment contains 13 open reading frames, including genes coding for five transposases and two phage proteins. This description might represent the first evidence of a horizontal gene transfer event as the origin of a biogenic amine biosynthetic locus.
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12. |
Renouf V,
Claisse O,
Miot-Sertier C,
Lonvaud-Funel A,
( 2006 ) Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis. PMID : 16942997 : DOI : 10.1016/j.fm.2005.01.019 Abstract >>
Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.
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13. |
Werning ML,
Ibarburu I,
Dueñas MT,
Irastorza A,
Navas J,
López P,
( 2006 ) Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages. PMID : 16416914 : DOI : 10.4315/0362-028x-69.1.161 Abstract >>
Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.
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14. |
Beltramo C,
Desroche N,
Tourdot-Maréchal R,
Grandvalet C,
Guzzo J,
( 2006 ) Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. PMID : 16171980 : DOI : 10.1016/j.resmic.2005.07.006 Abstract >>
The tolerance of the lactic acid bacterium Oenococcus oeni to hostile wine conditions is essential for the success of malolactic fermentation (MLF). In this study, reverse transcription quantitative PCR (RT-qPCR) was used to quantify the transcript level of 13 genes that could play a role in adaptation of O. oeni in wine. To optimize survival and growth in wine, cells were adapted during growth at low pH (3.5) prior to inoculation into wine. The level of gene expression was analyzed after growth at pH 3.5 in a rich medium and during MLF in a wine-like medium. RT-qPCR analyses exhibited different expression ratios of stress genes. The data obtained showed that determination of mRNA levels could constitute a new approach to studying the stress response of O. oeni after adaptation at low pH and during growth in a wine-like medium.
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15. |
de Las Rivas B,
Marcobal Ae,
Gómez A,
Muñoz R,
( 2005 ) Characterization of ISLpl4, a functional insertion sequence in Lactobacillus plantarum. PMID : 16278055 : DOI : 10.1016/j.gene.2005.09.005 Abstract >>
A Lactobacillus plantarum strain, CECT 4645, was found to have insertions of a sequence (985 bp in length) at least in eight loci in its genome. The prototype copy (Lp1) of this insertion sequence (named ISLpl4) has one open reading frame encoding a putative protein that is 292 amino acids in length with significant levels of similarity with IS982 family transposases. Perfect 16-bp inverted repeats were found at its termini. Upon transposition, generates 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. The ISLpl4 pattern changed over many generations on the CECT 4645 strain. This finding strongly supports our hypothesis that ISLpl4 is a functional element in L. plantarum. Some of these elements may be cryptic, since point mutation or 1-nucleotide deletions were found in their transposase encoding genes. ISLpl4 copies have been detected in Leuconostoc mesenteroides, Oenococcus oeni, and Lactobacillus sakei. An ISLpl4 copy of O. oeni contained a +1 nucleotide insertion on its transposase encoding gene and, by using an experimental system, we were able to demonstrate that this specific sequence originates a +1 programmed translational frameshifting. Although the frameshifting process reported here operates at a low rate, this description might represent the first case of a functional +1 frameshifting among IS.
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16. |
Spano G,
Rinaldi A,
Ugliano M,
Moio L,
Beneduce L,
Massa S,
( 2005 ) A beta-glucosidase gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses. PMID : 15752331 : DOI : 10.1111/j.1365-2672.2004.02521.x Abstract >>
Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative beta-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. The coding region of the putative beta-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oenibeta-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61.19 kDa. Furthermore, the expression of the beta-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. The beta-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. The inhibitory effect of temperature and ethanol on the L. plantarumbeta-glucosidase gene may be useful to explain the differences found in beta-glucosidase activity reported in wines by several authors.
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17. |
Nehmé B,
Ganga MA,
Lonvaud-Funel A,
( 2006 ) The arginine deiminase locus of Oenococcus oeni includes a putative arginyl-tRNA synthetase ArgS2 at its 3'-end. PMID : 16088344 : DOI : 10.1007/s00253-005-0095-6 Abstract >>
Oenococcus oeni is the most important lactic acid bacteria of the winemaking process involved in malolactic fermentation. Most O. oeni strains are able to catabolyze arginine via the arginine deiminase (ADI) pathway. The arcR, A, B, C, D1, and D2 cluster of O. oeni bacteria has been characterized. Here, we completed the ADI locus sequence. Downstream of arcD2 gene, we found an additional gene which encodes a putative arginyl-tRNA synthetase (argS2). It is not the same arginyl-tRNA synthetase which was sequenced in O. oeni MCW strain. Transcriptional analyses have shown that argS2 was induced by arginine. In addition, systematic polymerase chain reaction amplification of each arc gene and argS2 has provided a characteristic feature of the ADI locus within the O. oeni species: all genes of ADI locus are present or absent according to the strains.
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18. |
Desroche N,
Beltramo C,
Guzzo J,
( 2005 ) Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni. PMID : 15649534 : DOI : 10.1016/j.mimet.2004.10.010 Abstract >>
The expression gene pattern reflects, in part, mechanisms involved in adaptation to environmental conditions. Thus, we established and validated a method that enables relative transcript quantification in different conditions in the lactic acid bacteria Oenococcus oeni, notably in a technological medium. First, we determined an internal control in our conditions by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the SYBR Green I technology. Among the seven presumed housekeeping tested genes, the ldhD gene was retained for further experiments. Then, the PCR reproducibility was verified in our conditions and the comparative critical threshold (2deltadeltaC(T)) method was applied to quantify the transcript level of genes. The quantification of transcript levels of several stress genes already studied in our laboratory by Northern blot after a heat shock and at the entry of stationary phase allowed us to validate this method. RT-qPCR appeared as a powerful tool to study O. oeni response in stress conditions and wine mimetic conditions.
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19. |
de Las Rivas B,
Marcobal A,
Muñoz R,
( 2004 ) Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes. PMID : 15574919 : DOI : 10.1128/AEM.70.12.7210-7219.2004 PMC : PMC535203 Abstract >>
Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.
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20. |
Marcobal A,
de las Rivas B,
Moreno-Arribas MV,
Muñoz R,
( 2004 ) Identification of the ornithine decarboxylase gene in the putrescine-producer Oenococcus oeni BIFI-83. PMID : 15476968 : DOI : 10.1016/j.femsle.2004.08.036 Abstract >>
We report here the identification of an ornithine decarboxylase (ODC) gene in the putrescine-producer Oenococcus oeni BIFI-83 strain. The gene contains a 2,235-nucleotide open reading frame encoding a 745-amino acid residues protein with a deduced molecular mass of 81 kDa. The primary structure of the ODC deduced from the nucleotide sequence has a consensus sequence containing the pyridoxal-5-phosphate (PLP) binding domain, and the critical amino acids residues involved in enzymatic activity are also conserved. As determined by BLAST analysis, the deduced amino acid sequence of the odc gene shares a 67% identity with the ODC protein from Lactobacillus 30a. The odc gene appears to be rarely present in the genome of O. oeni, since in a screening for the presence of this gene in 42 oenococcal strains none of the strains possessed an odc gene copy.
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21. |
São-José C,
Santos S,
Nascimento J,
Brito-Madurro AG,
Parreira R,
Santos MA,
( 2004 ) Diversity in the lysis-integration region of oenophage genomes and evidence for multiple tRNA loci, as targets for prophage integration in Oenococcus oeni. PMID : 15231388 : DOI : 10.1016/j.virol.2004.04.029 Abstract >>
The central genomic regions of Oenococcus oeni phages fOg30 and fOgPSU1 have been compared with the equivalent regions of oenophages fOg44 and phi 10MC. In all cases, an almost identical endolysin gene was followed by one of two orfs, encoding putative holins (orf117 and orf163). The fOg44 endolysin was established as a secretory protein when expressed in Lactococcus lactis. Orf117 (from fOg44) promoted lysis of Escherichia coli cultures upon induction of a defective lambda Sam7 prophage, but Orf163 (from fOg30) failed to elicit a lysis response in this system. fOg44 and fOgPSU1 were shown to integrate at the 3' end of a tRNA(Glu) and a tRNA(Lys), respectively. Searching the available sequence of the O. oeni MCW genome for attP-like elements, two other tRNA targets could be proposed for prophage establishment. Between the lysis and integration elements, a diverse cluster of genes (absent in phi 10MC) was observed. One common gene in this "lysogenic conversion cluster" was experimentally confirmed as a transcriptional repressor, affecting the expression of a putative permease gene.
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22. |
Bourdineaud JP,
Nehmé B,
Tesse S,
Lonvaud-Funel A,
( 2004 ) A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine. PMID : 15033264 : DOI : 10.1016/S0168-1605(03)00162-4 Abstract >>
The wine lactic acid bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, non-optimal growth temperatures, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins. We here describe characterisation and cloning of the O. oeni omrA gene encoding a protein belonging to the ATP-binding cassette superfamily of transporters. The OmrA protein displays the highest sequence similarity with the subfamily of ATP-dependent multidrug resistance (MDR) proteins, most notably the bacterial Lactococcus lactis LmrA homologue of the human MDR1 P-glycoprotein. The omrA gene proved to be a stress-responsive gene since its expression was increased at high temperature or under osmotic shock. The OmrA protein function was tested in Escherichia coli, and consistent with the omrA gene expression pattern, OmrA conferred protection to bacteria grown on a high salt medium. OmrA also triggered bacterial resistance to sodium laurate, wine and ethanol toxicity. The homologous LmrA protein featured the same stress-protective pattern than OmrA when expressed in E. coli, and the contribution to resistance of both OmrA and LmrA transporters was decreased by verapamil, a well-known inhibitor of the human MDR1 protein. Genes homologous to omrA were detected in other wine lactic acid bacteria, suggesting that this type of genes might constitute a well-conserved stress-protective molecular device.
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23. |
Beltramo C,
Grandvalet C,
Pierre F,
Guzzo J,
( 2004 ) Evidence for multiple levels of regulation of Oenococcus oeni clpP-clpL locus expression in response to stress. PMID : 15028706 : DOI : 10.1128/jb.186.7.2200-2205.2003 PMC : PMC374425 Abstract >>
A locus containing the clpP and clpL genes in the lactic acid bacterium Oenococcus oeni was studied. Real-time reverse transcription-PCR analysis revealed different induction factors involved in expression of these genes during stress. According to the conditions, clpP and clpL genes could be transcripted as two distinct transcripts or cotranscripted. The clpP promoter depended on the CtsR regulator, but surprisingly the clpL promoter did not. The amount of the clpL transcript depended on mRNA stability. This clp ATPase gene is at least controlled at the posttranscriptional level.
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24. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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25. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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26. |
Bonnin-Jusserand M,
Grandvalet C,
David V,
Alexandre H,
( 2011 ) Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni. PMID : 21819657 : DOI : 10.4315/0362-028X.JFP-10-466 Abstract >>
Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35�XC. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U�Pmg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.
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27. |
Michlmayr H,
Schümann C,
Kulbe KD,
del Hierro AM,
( 2011 ) Heterologously expressed family 51 alpha-L-arabinofuranosidases from Oenococcus oeni and Lactobacillus brevis. PMID : 21169445 : DOI : 10.1128/AEM.01385-10 PMC : PMC3067215 Abstract >>
Putative �\-L-arabinofuranosidases of Oenococcus oeni and Lactobacillus brevis were heterologously expressed and characterized. We report the basic functional properties of the recombinant enzymes in comparison to those of a commercial family 51 arabinosidase of Aspergillus niger.
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28. |
Naouri P,
Chagnaud P,
Arnaud A,
Galzy P,
( 1990 ) Purification and properties of a malolactic enzyme from Leuconostoc oenos ATCC 23278. PMID : 2097345 : Abstract >>
The malolactic enzyme of Leuconostoc oenos ATCC 23278 was purified 136fold. The molecular weight was estimated at 132,000 when determined by gel filtration. The enzyme contained two identical subunits (Mw = 66,000 using sodium dodecyl sulfate gel electrophoresis). The malolactic enzyme catalyzes the NAD(+)- and Mn(+)-dependent reaction L-malate----L-lactate + CO2. The apparent Km values for malic acid, NAD+, and Mn2+ were 17 mM, 0.044 mM, and 0.017 mM, respectively. The optimal pH and the optimal temperature for activity were 5.0, and 37 degrees C, respectively and the isoelectric point was pH 4.30. L-lactate and ethanol were non-competitive inhibitors, whereas succinate, citrate, and D-tartrate showed competitive type inhibitions.
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29. |
Bridier J,
Claisse O,
Coton M,
Coton E,
Lonvaud-Funel A,
( 2010 ) Evidence of distinct populations and specific subpopulations within the species Oenococcus oeni. PMID : 20935119 : DOI : 10.1128/AEM.01544-10 PMC : PMC2988583 Abstract >>
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.
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30. |
De Bruyne K,
Camu N,
De Vuyst L,
Vandamme P,
( 2010 ) Weissella fabaria sp. nov., from a Ghanaian cocoa fermentation. PMID : 19801391 : DOI : 10.1099/ijs.0.019323-0 Abstract >>
Two lactic acid bacteria, strains 257(T) and 252, were isolated from traditional heap fermentations of Ghanaian cocoa beans. 16S rRNA gene sequence analysis of these strains allocated them to the genus Weissella, showing 99.5 % 16S rRNA gene sequence similarity towards Weissella ghanensis LMG 24286(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and biochemical tests confirmed their unique taxonomic position. DNA-DNA hybridization experiments towards their nearest phylogenetic neighbour demonstrated that the two strains represent a novel species, for which we propose the name Weissella fabaria sp. nov., with strain 257(T) (=LMG 24289(T) =DSM 21416(T)) as the type strain. Additional sequence analysis using pheS gene sequences proved useful for identification of all Weissella-Leuconostoc-Oenococcus species and for the recognition of the novel species.
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31. |
Bon E,
Delaherche A,
Bilhère E,
De Daruvar A,
Lonvaud-Funel A,
Le Marrec C,
( 2009 ) Oenococcus oeni genome plasticity is associated with fitness. PMID : 19218413 : DOI : 10.1128/AEM.02194-08 PMC : PMC2663225 Abstract >>
Oenococcus oeni strains are well-known for their considerable phenotypic variations in terms of tolerance to harsh wine conditions and malolactic activity. Genomic subtractive hybridization (SH) between two isolates with differing enological potentials was used to elucidate the genetic bases of this intraspecies diversity and identify novel genes involved in adaptation to wine. SH revealed 182 tester-specific fragments corresponding to 126 open reading frames (ORFs). A large proportion of the chromosome-related ORFs resembled genes involved in carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication, recombination, and repair. Six regions of genomic plasticity were identified, and their analysis suggested that both limited recombination and insertion/deletion events contributed to the vast genomic diversity observed in O. oeni. The association of selected sequences with adaptation to wine was further assessed by screening a large collection of strains using PCR. No sequences were found to be specific to highly performing (HP) strains alone. However, there was a statistically significant positive association between HP strains and the presence of eight gene sequences located on regions 2, 4, and 5. Gene expression patterns were significantly modified in HP strains, following exposure to one or more of the common stresses in wines. Regions 2 and 5 showed no traces of mobile elements and had normal GC content. In contrast, region 4 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhances the fitness of O. oeni strains.
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32. |
Bilhère E,
Lucas PM,
Claisse O,
Lonvaud-Funel A,
( 2009 ) Multilocus sequence typing of Oenococcus oeni: detection of two subpopulations shaped by intergenic recombination. PMID : 19114515 : DOI : 10.1128/AEM.02563-08 PMC : PMC2648160 Abstract >>
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.
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33. |
Renouf V,
Vayssieres LC,
Claisse O,
Lonvaud-Funel A,
( 2009 ) Genetic and phenotypic evidence for two groups of Oenococcus oeni strains and their prevalence during winemaking. PMID : 19151972 : DOI : 10.1007/s00253-008-1843-1 Abstract >>
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.
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34. |
Dols-Lafargue M,
Lee HY,
Le Marrec C,
Heyraud A,
Chambat G,
Lonvaud-Funel A,
( 2008 ) Characterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus and Oenococcus oeni, two bacterial species commonly found in wine. PMID : 18469121 : DOI : 10.1128/AEM.00673-08 PMC : PMC2446535 Abstract >>
Ropiness is a bacterial alteration in wines, beers, and ciders, caused by beta-glucan-synthesizing pediococci. A single glucosyltransferase, Gtf, controls ropy polysaccharide synthesis. In this study, we show that the corresponding gtf gene is also present on the chromosomes of several strains of Oenococcus oeni isolated from nonropy wines. gtf is surrounded by mobile elements that may be implicated in its integration into the chromosome of O. oeni. gtf is expressed in all the gtf(+) strains, and beta-glucan is detected in the majority of these strains. Part of this beta-glucan accumulates around the cells forming a capsule, while the other part is liberated into the medium together with heteropolysaccharides. Most of the time, this polymer excretion does not lead to ropiness in a model medium. In addition, we show that wild or recombinant bacterial strains harboring a functional gtf gene (gtf(+)) are more resistant to several stresses occurring in wine (alcohol, pH, and SO(2)) and exhibit increased adhesion capacities compared to their gtf mutant variants.
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35. |
González-Arenzana L,
Pérez-Martín F,
Palop ML,
Seseña S,
Santamaría P,
López R,
López-Alfaro I,
( 2015 ) Genomic diversity of Oenococcus oeni populations from Castilla La Mancha and La Rioja Tempranillo red wines. PMID : 25846918 : DOI : 10.1016/j.fm.2015.02.001 Abstract >>
The Oenococcus oeni populations of Tempranillo wines from Castilla La Mancha and La Rioja winemaking regions were analysed from one to three years and up to ten wineries. The objective was to evaluate the genetic variability and the O. oeni population structure. For this purpose a MLST scheme based on four loci (gyrB, purK, pgm and recP genes) and PFGE with SfiI restriction enzyme were developed for later combination. The results showed an O. oeni population completely adapted to winemaking regions. A purifying selection influenced the genes evolution, especially recP that along with purK were the most interesting loci to analyse the genetic variability of the isolates. In this way linkage disequilibrium and intergenic and intragenic recombination were determined between isolates. PFGE typing with UPGMA data were not coincident with the phylograms assessed for MLST by Maximum likelihood and combination of both techniques differentiated all the isolates as strains. Those results led the research to conclude that O. oeni population from CM and LR was a panmictic population with a slight clonal evolution, so subpopulations could not be described. A broader study including more winemaking regions with different grape varieties and distinct ways of elaborating would be interesting to complete the knowledge about O. oeni populations.
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36. |
Wang T,
Li H,
Wang H,
Su J,
( 2015 ) Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Oenococcus oeni from different wine-producing regions of China. PMID : 25625911 : DOI : 10.1016/j.ijfoodmicro.2015.01.006 Abstract >>
The present study established a typing method with NotI-based pulsed-field gel electrophoresis (PFGE) and stress response gene schemed multilocus sequence typing (MLST) for 55 Oenococcus oeni strains isolated from six individual regions in China and two model strains PSU-1 (CP000411) and ATCC BAA-1163 (AAUV00000000). Seven stress response genes, cfa, clpL, clpP, ctsR, mleA, mleP and omrA, were selected for MLST testing, and positive selective pressure was detected for these genes. Furthermore, both methods separated the strains into two clusters. The PFGE clusters are correlated with the region, whereas the sequence types (STs) formed by the MLST confirm the two clusters identified by PFGE. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics.
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37. |
Cappello MS,
De Domenico S,
Logrieco A,
Zapparoli G,
( 2014 ) Bio-molecular characterisation of indigenous Oenococcus oeni strains from Negroamaro wine. PMID : 24929730 : DOI : 10.1016/j.fm.2014.02.004 Abstract >>
The variation in the coding capacity within Oenococcus oeni can have a significant impact on wine quality. The detection of several genes involved in important metabolic pathways (i.e. citrate, sulphur and arginine metabolisms) was performed on 10 indigenous O. oeni strains from Negroamaro wine, a red table wine (Apulia, Italy). These strains were selected from 95 isolates, collected during spontaneous malolactic fermentation, according to the results of an Amplified Fragment Length Polymorphism (AFLP) analysis. A total of 16 genes were screened, most (11) of which had never previously been assayed on O. oeni. All strains possessed 10 genes encoding enzymes such as malolactic enzyme (mleA), esterase (estA), citrate lyase (citD, citE and citF), citrate transporter (maeP), �\-acetolactate decarboxylase (alsD), �\- acetolactate synthase (alsS), S-adenosylmethionine synthase (metK) and cystathionine �]-lyase (metC) and resulted negative in the detection of genes encoding cystathionine �^-lyase (metB), ornithine transcarbamylase (arcB) and carbamate kinase (arcC). The sequence of PCR fragments of 11 genes of a representative strain (ITEM 15929) was compared to those of three reference O. oeni strains. The indigenous strain was phylogenetically more similar to PSU-1 and ATCC BAA1163 than AWRI B429. This study describes new genetic markers useful for detecting the genetic potential of O. oeni strains to contribute to aroma production and for investigating the population structure of the species.
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38. |
González-Arenzana L,
Santamaría P,
López R,
López-Alfaro I,
( 2014 ) Oenococcus oeni strain typification by combination of Multilocus Sequence Typing and Pulsed Field Gel Electrophoresis analysis. PMID : 24290654 : DOI : 10.1016/j.fm.2013.07.014 Abstract >>
Oenococcus oeni is usually the main lactic acid bacteria (LAB) responsible for conducting malolactic fermentation (MLF) in wines. Pulsed Field Gel Electrophoresis (PFGE) is one of the most common methods used to identify different genotypes among the wine LAB populations. Although PFGE is a powerful typing tool, it is time-consuming and its results are not easily exchangeable between laboratories so typing methods such as Multilocus Sequence Typing (MLST) have been developed. In this study, thirty O. oeni isolates from Rioja Tempranillo wines were characterized performing SfiI and ApaI PFGE and MLST with eight housekeeping genes. Using the latter technique, six new alleles have been described for five genes. PFGE was slightly more efficient than MLST because of the number of genotypes and of the index of diversity (ID) that each technique discriminated. This has been the first time that PFGE and MLST results have been combined to shape a unique dendrogram. Thus, the combination of results from both typing methods allowed the discrimination of twenty-two PFGE-ST genotypes showing the highest ID of these research (0.947). According to these results, the future application of the combination of PFGE and MLST results could be successful for reliable O. oeni strain typification.
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39. |
Miot-Sertier C,
Lonvaud A,
Le Marrec C,
Dols-Lafargue M,
( 2012 ) IS30 elements are mediators of genetic diversity in Oenococcus oeni. PMID : 22809637 : DOI : 10.1016/j.ijfoodmicro.2012.06.009 Abstract >>
Oenococcus oeni is responsible for the malolactic fermentation of wines. Genomic diversity has been recently established in the species and extensive attention is now being given to the genomic bases of strain-specific differences. We explored the role of insertion sequences (IS), which are considered as driving forces for novel genotypic and phenotypic variants in prokaryotes. The present study focuses on members of the IS30 family, which are widespread among lactic acid bacteria. An in silico analysis of the three available genomes of O. oeni in combination with the use of an inverse PCR strategy targeting conserved IS30-related sequences indicated the presence of seven IS30 copies in the pangenome of O. oeni. A primer designed to anneal to the conserved 3' end of the IS30 element was paired with each of the seven primers selected to bind to unique sequences upstream of each of the seven mobile elements identified. The study presents an overview of the abundance, and the genomic environment of IS30 elements in the O. oeni pangenome and shows that the two existing genetic sub-populations previously described in the species through multilocus sequence typing analysis (MLST) differ in their IS30 content. Possible IS30 impacts on bacterial adaptation are discussed.
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40. |
Schümann C,
Michlmayr H,
Eder R,
Del Hierro AM,
Kulbe KD,
Mathiesen G,
Nguyen TH,
( 2012 ) Heterologous expression of Oenococcus oeni malolactic enzyme in Lactobacillus plantarum for improved malolactic fermentation. PMID : 22452826 : DOI : 10.1186/2191-0855-2-19 PMC : PMC3366906 Abstract >>
Lactobacillus plantarum is involved in a multitude of food related industrial fermentation processes including the malolactic fermentation (MLF) of wine. This work is the first report on a recombinant L. plantarum strain successfully conducting MLF. The malolactic enzyme (MLE) from Oenococcus oeni was cloned into the lactobacillal expression vector pSIP409 which is based on the sakacin P operon of Lactobacillus sakei and expressed in the host strain L. plantarum WCFS1. Both recombinant and wild-type L. plantarum strains were tested for MLF using a buffered malic acid solution in absence of glucose. Under the conditions with L-malic acid as the only energy source and in presence of Mn2+ and NAD+, the recombinant L. plantarum and the wild-type strain converted 85% (2.5 g/l) and 51% (1.5 g/l), respectively, of L-malic acid in 3.5 days. Furthermore, the recombinant L. plantarum cells converted in a modified wine 15% (0.4 g/l) of initial L-malic acid concentration in 2 days. In conclusion, recombinant L. plantarum cells expressing MLE accelerate the malolactic fermentation.
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41. |
( 1996 ) Nucleotide sequence of plasmid p4028, a cryptic plasmid from Leuconostoc oenos. PMID : 8938055 : DOI : 10.1006/plas.1996.0034 Abstract >>
The Leuconostoc oenos plasmid p4028 was cloned in pBlueScript (SK+), and its complete nucleotide sequence was determined. The analysis of the nucleotide sequence revealed five open reading frames, all of them located on the same strand and grouped in two clusters separated by a short noncoding stretch. A similarity search against the other sequences deposited in the EMBL and GenBank databases showed that p4028 has no significant similarity with any of the sequences checked. Nevertheless, a putative ATP-binding motif was found in ORF2. A more detailed analysis of this ORF suggests that it could encode for a DNA-dependent ATPase.
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42. |
( 1996 ) Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos. PMID : 8938052 : DOI : 10.1006/plas.1996.0031 Abstract >>
Analysis of the nucleotide sequence of Leuconostoc oenos plasmid pOg32 (2544 bp) revealed three open reading frames (ORFs) in one of the DNA strands preceded by putative promoters and ribosome binding sites. ORF1 corresponded to a polypeptide similar to the replication proteins (Rep) of pT181 plasmid family. A nucleotide sequence like the double strand origin (DSO) of the same plasmid family was found inside the presumptive rep gene. ORF2 is capable of encoding a protein resembling the recombination enzyme (Pre) of two lactobacilli plasmids. Immediately upstream of the presumed pre gene, a sequence similar to the consensus RSA regions of those plasmids was encountered. Between ORF3 and ORF1, a sequence comparable to the RSB consensus region of the single strand origin ssoA was detected. The putative RSB region was located at the 3'-end of an imperfect palindrome which may be a single stranded origin. The described features, common to other small plasmids of Gram-positive bacteria, suggest that pOg32 replicates by the rolling circle mechanism. The amino acid sequence encoded by ORF3 was not similar to any known protein.
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43. |
( 1996 ) Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos. PMID : 8978099 : DOI : 10.1111/j.1574-6968.1996.tb08614.x Abstract >>
The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.
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44. |
( 1996 ) Analysis of the beta' subunit of DNA-dependent RNA polymerase does not support the hypothesis inferred from 16S rRNA analysis that Oenococcus oeni (formerly Leuconostoc oenos) is a tachytelic (fast-evolving) bacterium. PMID : 8863429 : DOI : 10.1099/00207713-46-4-1004 DOI : 10.1099/00207713-46-4-1004 Abstract >>
rRNA sequencing has shown that leuconostocs comprise three distinct phylogenetic lineages which have been designated separate genera (viz., the genera Leuconostoc sensu stricto, Oenococcus, and Weissella). In addition, the 16S rRNA line formed by Oenococcus oeni (formerly Leuconostoc oenos) is exceptionally long; this fact, together with variations in the compositions of conserved positions in the 16S rRNA, has led to the hypothesis (D. Yang and C. R. Woese, Syst. Appl. Microbiol. 12:145-149, 1989) that this organism is a fast-evolving bacterium. Previous evidence that the leuconostocs should be divided into three genera and that O. oeni is an example of tachytelic evolution has come solely from rRNA analyses. In this study we seqenced the rpoC gene encoding the beta' subunit of DNA-dependent RNA polymerase of leuconostocs and performed a comparative phylogenetic analysis. The subdivision of the leuconostocs into three distinct lineages was confirmed by the rpoC gene data, but no evidence that indicated that O. oeni is evolving at an extraordinary rate was found. If O. oeni is truly tachytelic, then fast-evolving phenomena would be expected to occur throughout the whole genome, including this independent molecular chronometer.
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45. |
( 1996 ) Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. PMID : 8919788 : PMC : PMC167893 Abstract >>
Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank. Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments. Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure. The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present. The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells. The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species. The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core. Energy-dependent L-[14C]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector. Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.
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46. |
( 1996 ) Genetic organization of the mle locus and identification of a mleR-like gene from Leuconostoc oenos. PMID : 8953720 : PMC : PMC168275 Abstract >>
Characterization of the mle locus harboring the malolactic enzyme gene mleA and malate permease gene mleP from Leuconostoc oenos was completed in this study by mRNA analysis. Northern (RNA) blot experiments revealed a 2.6-kb transcript, suggesting an operon structure harboring mleA and mleP genes. Primer extension analysis showed that the mle operon has a single transcription start site located 17 nucleotides upstream of the ATG translation start site for the mleA gene. We found sequences, TTGACT and TATGAT (which are separated by 18 bp), that are closely related to the gram-positive and Escherichia coli consensus promoter sequences. Upstream of the mleA gene, an 894-bp open reading frame that transcribed divergently from the mle operon was found. Sequence analysis and expression in E. coli minicells suggest that this open reading frame encodes a polypeptide with an apparent molecular mass of 34 kDa belonging to the LysR-type regulatory protein family. Protein comparisons showed the highest level of identity with the MleR regulatory protein from Lactococcus lactis, which is involved in the expression of the malolactic genes in the presence of L-malate. However, the MleR-like protein of L. oenos seems different from the protein of Lactococcus lactis, since no regulation of the malolactic enzyme by L-malate was effective under our experimental conditions.
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47. |
( 1993 ) Sequence analysis of Leuconostoc oenos DNA: organization of pLo13, a cryptic plasmid. PMID : 8302929 : DOI : 10.1006/plas.1993.1053 Abstract >>
A Leuconostoc oenos plasmid, pLo13, was studied to analyze its genetic organization and to define its functions. The nucleotide sequence (3948 bp) revealed three major open reading frames. Features commonly found in plasmids that replicate via a rolling-circle mechanism were identified within pLo13: first, a sequence coding for a protein with an amino acid sequence homologous to the plasmid recombination enzymes (Pre), but for which a specific target site similar to those previously described was not found; second, a sequence probably encoding a replication protein (Rep). The putative pLo13 Rep protein amino acid sequence is divergent from the pC194-pUB110 family Rep proteins. However, the consensus sequence of the Rep protein active site was found, as well as the Rep protein consensus target site. No sequence similar to the previously described minus-origins (SSOs) are present in pLo13; nevertheless, a 200-bp sequence rich in imperfect palindromes may be involved in the minus-strand replication. These overall differences are in agreement with the previously reported important phylogenetic distance existing between Ln. oenos and other lactic acid bacteria.
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48. |
( 1997 ) Molecular characterization of the gene encoding an 18-kilodalton small heat shock protein associated with the membrane of Leuconostoc oenos. PMID : 9023938 : PMC : PMC168350 Abstract >>
In Leuconostoc oenos, different stresses such as heat, ethanol, and acid shocks dramatically induce the expression of an 18-kDa small heat shock protein called Lo 18. The corresponding gene (hsp18) was cloned from a genomic library of L. oenos constructed in Escherichia coli. A 2.3-kb DNA fragment carrying the hsp18 gene was sequenced. The hsp18 gene encodes a polypeptide of 148 amino acids with a calculated molecular mass of 16,938 Da. The Lo18 protein has a significant identity with small heat shock proteins of the alpha-crystallin family. The transcriptional start site was determined by primer extension. This experiment allowed us to identify the promoter region exhibiting high similarity to consensus promoter sequences of gram-positive bacteria, as well as E. coli. Northern blot analysis showed that hsp18 consists of a unique transcription unit of 0.6 kb. Moreover, hsp18 expression seemed to be controlled at the transcriptional level. This small heat shock protein was found to be peripherally associated with the membrane of L. oenos.
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49. |
Le Jeune C,
Lonvaud-Funel A,
ten Brink B,
Hofstra H,
van der Vossen JM,
( 1995 ) Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test. PMID : 7730207 : Abstract >>
On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the hdcA genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc aenos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. aenos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdcA gene products of Gram-positive bacteria. The amplification products of the hdcA genes from Lac. buchneri and Leuct. aenos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdcA gene. In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.
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( 2013 ) Structural and biochemical characterisation of a NAD?-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications. PMID : 23385476 : DOI : 10.1007/s00253-013-4725-0 Abstract >>
Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni(2+)-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni(2+), activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD(+)/NADH, whereas no activity has been observed with NADP(+)/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn(2+). The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni(2+)-dependent activity towards 1,3-propanediol.
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( N/A ) Malolactic enzyme from Oenococcus oeni: heterologous expression in Escherichia coli and biochemical characterization. PMID : 23196745 : DOI : 10.4161/bioe.22988 PMC : PMC3669155 Abstract >>
Malolactic enzymes (MLE) are known to directly convert L-malic acid into L-lactic acid with a catalytical requirement of nicotinamide adenine dinucleotide (NAD (+)) and Mn (2+) ; however, the reaction mechanism is still unclear. To study a MLE, the structural gene from Oenococcus oeni strain DSM 20255 was heterologously expressed in Escherichia coli, yielding 22.9 kU l (-1) fermentation broth. After affinity chromatography and removal of apparently inactive protein by precipitation, purified recombinant MLE had a specific activity of 280 U mg (-1) protein with a recovery of approximately 61%. The enzyme appears to be a homodimer with a molecular mass of 128 kDa consisting of two 64 kDa subunits. Characterization of the recombinant enzyme showed optimum activity at pH 6.0 and 45�XC, and Km, Vmax and kcat values of 4.9 mM, 427 U mg (-1) and 456 sec (-1) for L-malic acid, 91.4 ?M, 295 U mg (-1) and 315 sec (-1) for NAD (+) and 4.6 ?M, 229 U mg (-1) and 244 sec (-1) for Mn (2+) , respectively. The recombinant MLE retained 95% of its activity after 3 mo at room temperature and 7 mo at 4�XC. When using pyruvic acid as substrate, the enzyme showed the conversion of pyruvic acid with detectable L-lactate dehydrogenase (L-LDH) activity and oxidation of NADH. This interesting observation might explain that MLE catalyzes a redox reaction and hence, the requirements for NAD (+) and Mn (2+) during the conversion of L-malic to L-lactic acid.
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( 2012 ) Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni. PMID : 23053071 : DOI : 10.1007/s00253-012-4409-1 Abstract >>
The study of gene expression and accurate quantitation of target genes in any organism depends on correct normalisation. Due to the increase in studies on Oenococcus oeni gene expression, there is a clear need for alternative reference genes in order to reliably measure expression levels. In this manuscript, we propose the approach of using multiple reference genes to provide a more robust basis for establishing a reference gene set. The identification and evaluation of a panel of nine reference genes, including the commonly used ldhD, for real-time PCR normalisation was performed in O. oeni. Expression levels of these reference genes were then measured by real-time qPCR in an independent set of O. oeni samples (n = 30). The nine genes were ranked according to their stability of gene expression measure (M) using geNorm to identify the most consistently expressed reference genes. This approach resulted in the identification of multiple reference genes that may be used for a screening and more robust normalisation of target gene expression measured by real-time RT-qPCR. Expression of esterase genes was then measured in these O. oeni samples in the presence of known esterase substrates. The results give an indication of how these genes may be involved in ester synthesis and hydrolysis in O. oeni.
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( 2012 ) Identification of pOENI-1 and related plasmids in Oenococcus oeni strains performing the malolactic fermentation in wine. PMID : 23139835 : DOI : 10.1371/journal.pone.0049082 PMC : PMC3489775 Abstract >>
Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.
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( 1998 ) Histidine carboxylase of Leuconostoc oenos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene. PMID : 9633629 : Abstract >>
Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc oenos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively alpha and beta. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. oenos 9204 HDC was synthesized as a precursor proHDC pi 6 (Mr 205,000). A cleavage between Ser-81 and Ser-82 generated the alpha (Mr 25,380) and beta (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (alpha beta)6. At the optimal pH of 4.8, the HDC activity exhibited a simple Michaelis-Menten kinetic (K(m) = 0.33 mmol l-1, Vmax = 17.8 mumol CO2 min-1 mg-1), while at pH 7.6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor (Ki = 32 mmol l-1). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.
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