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The differential expression of five genes related to adaptation to wine conditions in Oenococcus oeni was evaluated by studying transcriptional changes in wine-like media and in real wine during malolactic fermentation. Two strains isolated from high ethanol wine and another, less tolerant, strain were used. Expression of the selected genes (mleA, citE, hsp18, clpP and gsh) was studied by means of RT-qPCR. A higher level of gene expression was found for the more ethanol-tolerant strains, especially for citE, hsp18 and clpP. Thus, these three genes might be good markers for the more stress-resistant strains of O. oeni. In most cases, the results of expression in wine-like medium with 14 % ethanol had a similar trend to those in real wine, suggesting that the wine-like medium can be a good model for studying the stress response in O. oeni. Different expression between strains was well correlated with their ability to perform malolactic fermentation. In this way, the identified gene markers can be used in the selection of new strains as starters for malolactic fermentation.

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.

The goal of this study was to analyze how the profiles of membrane proteins of Oenococcus oeni change under particular stress conditions of wine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis protein profiles of membrane fraction showed that a 40-kDa protein was overexpressed in the presence of SO2. The sequence of its N-terminal fragment showed a significant identity with glyceraldehyde-3-phosphate dehydrogenases (GAPDHs), but the protein showed no GAPDH activity. This sequence was compared with those of other GAPDHs with ClustalW alignment, and it was found to be somewhat similar to that of the cell-wall and membrane proteins of other lactic acid bacteria.

A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lysogeny with mitomycin C as inducer. As a result of this test, 45% of the strains proved to be lysogenic, suggesting that lysogeny is widespread among bacteria isolated from wines during malolactic fermentation. The sensitivity of bacteria to phages was very different, depending on the strain. All the lysogenic strains were resistant to infection by the temperate phage they released. Some phages infected none of the strains. Phages of Oenoc. oeni had a classical morphology, an isometric head, and a long striated tail. With the broadest host strain as an indicator, phages were detected in wines after malolactic fermentation.

Agrobacterium MC501 and the mixed culture composed of Agrobacterium MC501, Xanthomonas MC701, Corynebacterium sp. MC401, and Corynebacterium sp. MC402, all previously isolated from a coal mine area by an enrichment culture with dibenzothiophene (DBT), were used to study DBT sulfone desulfurization. Both cultures were able to use DBT sulfone as a sole source of sulfur for growth. This compound was metabolized to 2-hydroxybiphenyl (2PP) and sulfate. Agrobacterium MC501 and the mixed culture could also utilize a wide range of organic and inorganic sulfur compounds as aources of sulfur such as DBT, thianthrene, diphenylsulfide, thiophene-2-carboxylate, dibutilsulfide, methionine, cysteine, sulfate, and sulfite. Based on these results, the above-mentioned strains can be used to characterize and study coal desulfurization.

Four out of 187 strains, from enrichment cultures of dibenzothiophene (DBT), grew on DBT or thiophene 2-carboxylate as S sources. The four isolates, presumptively identified as Agrobacterium sp., Xanthomonas sp. and Corynebacterium spp., individually and together desulphurized DBT, producing 2-hydroxybiphenyl and sulphate.

The microbial degradation of dibenzothiophene (DBT) and other organosulphur compounds such as thiophene-2-carboxylate (T2C) is of interest for the potential desulphurization of coal. The feasibility of degradation of DBT and T2C by Pseudomonas putida and other bacteria was analysed. Pseudomonas putida oxidized sulphur from DBT in the presence of yeast extract, but it did not when DBT was the sole source of carbon.

The growth and oxygen consumption of a variety of thermophilic, acidophilic bacteria in the presence of thiophene-2-carboxylate (T2C) and dibenzothiophene (DBT) have been determined. T2C was extremely toxic to the acidophiles in comparison with neutrophiles, but appeared to be degraded by a heterotrophicSulfolobus- like thermophile. DBT proved to be unstable at high temperatures, even in the absence of bacteria, and was not a substrate for the thermophiles.

Aims: The goal of this study was to develop a reproducible method for molecular typing strains of Oenococcus oeni, and also to apply it in the study of population dynamics of these strains during malolactic fermentation of wine. Methods and Results: A new method of multiplex randomly amplified polymorphic DNA (RAPD)-PCR has been developed, based on the combination of one random 10-mer and one specific 23-mer oligonucleotide in a single PCR. This method generates unique and discriminant DNA profiles for strains of O. oeni. The strains of this species were also clearly distinguished from other species of lactic acid bacteria. The method was applied to study the dynamics of O. oeni strains during malolactic fermentation, in three vintages in the same cellar. Conclusions: A fast and reliable method for typing strains of O. oeni has been designed and optimized. It improves the reproducibility and rapidity of conventional RAPD-PCR, and it has been validated monitoring the population dynamics during malolactic fermentation. Significance and Impact of the Study: This method will be a good tool to study the population dynamics of bacteria during malolactic fermentation and to evaluate the performance of new malolactic starter cultures and their dominance over the native microbiota.

Aims: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. Methods and Results: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. Conclusions: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. Significance and Impact of the Study: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.

This study shows that the growth of Oenococcus oeni CECT 4100 in a synthetic medium is affected by phenolic compounds in different ways, depending on their type and concentration. Generally they have no effects at low concentrations, but hydroxycinnamic acids are inhibitory at high concentrations. Malolactic fermentation was stimulated in the presence of catechin and quercetin, but increasingly delayed with increasing amounts of p-coumaric acid. Gallic acid appeared to delay or inhibit the formation of acetic acid from citric acid. This could lead to a better control of malolactic fermentation and suppress the increase in volatile acidity, which is undesirable in the wine-making process.

Phenol-degrading strain QT 31 ofPseudomonas sp. was selected among other phenol-resistant bacteria from activated sludges of wastewater treatment plant of an oil refinery. Its capacity of degradation was studied at different periods of adaptation, reaching a phenol biodegradation rate of 28 mg/l phenol per hour, from minimal, medium with 1000 mg/l phenol, after adaptation for 20 days.

A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested.

A Technicon AutoAnalyzer has been adapted to analyse lysine of fermentation broths from lysine-producer mutants, with a photometric method with ninhydrin. Linearity and accuracy have been tested, with errors lower than 5.5%. The great advantadge is its speed, analysing one sample every 5 minutes, automatically.

A strain of Pseudomonas has been studied for its nickel accumulation capacity. Most assays were carried out exposing cells to the metal in a resting state. Results indicate an extracellular, metabolically independent adsorption that is decreased by the presence of acetate. A possible exchange of nickel for magnesium from the outer membrane is suggested. Nickel tolerance of this strain in minimal medium is limited.

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking.