Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni

47 ref.; International audience

RVVS 2013

Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos

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; …

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis

International audience; Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxe…

Truncated internalin A and asymptomatic Listeria monocytogenes carriage: in vivo investigation by allelic exchange

ABSTRACT Allelic exchange of the region coding for the C terminus of InlA between one epidemic (with an 80-kDa InlA) and one asymptomatic (with a 47-kDa InlA) carriage Listeria monocytogenes strain confirmed the need for this region for internalin entry in vitro. Interestingly, restoration of internalin A functionality did not result in full virulence in chicken embryo assays.

Etude biochimiques et physiologiques d'un "heat shock protein" de faible poids moléculaire d'une bactérie lactique

National audience

Acid tolerance inLeuconostoc oenos. Isolation and characterization of an acid-resistant mutant

The acid tolerance ofLeuconostoc oenos was examined in cells surviving at pH 2.6, which is lower than the acid limit of growth (about pH 3.0). Acid-adapted cells survived better than non-adapted cells. Tolerance to acid stress was found to be dependent upon the adaptive pH. Acid resistance was increased by an order of magnitude for cultures adapted to a pH of about 2.9. Inhibiting protein synthesis with chloramphenicol prior to acid shock revealed that acid adaptation may involve two separate systems, one of which appears to be independent of protein synthesis. The acid-resistant mutant LoV8413, isolated during a long-term survival screen at pH 2.6, was found to be able to grow in acidic me…

Le rôle du système agr chez Listeria monocytogènes

National audience; La capacité des bactéries à développer un biofilm constitue une des principales difficultés quant à la maîtrise de la contamination : ces structures en augmentent les risques et rendent notamment les micro-organismes plus résistants aux désinfectants. Des études ont pu démontrer le rôle du système de communication agr dans la formation de biofilms de L. monocytogenes, agent pathogène particulièrement recherché en industrie agro-alimentaire.

Assessment of the roles of LuxS, Autoinducer 2 and its precursor S-Ribosyl Homocysteine, in cell attachment during biofilm formation by Listeria monocytogenes EGD-e

No abstract

Cloning and sequencing of the gene encoding α-acetolactate decarboxylase fromLeuconostoc oenos

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.

An improved protocol for electroporation ofOenococcus oeniATCC BAA-1163 using ethanol as immediate membrane fluidizing agent

Aims:  To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain. Methods and Results:  The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 103 per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm−1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions:  An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EP…

Exopolysaccharide produced by Weissella confusa: Chemical characterisation, rheology and bioactivity

Abstract The purpose of this study was to characterise the production of exopolysaccharide (EPS) by indigenous lactic acid bacteria (LAB) isolated from traditional Algerian dairy products and to evaluate their possible use in agri-foods. Among the collection of isolated strains, the strain Weissella confusa (W4) was selected for its ability to produce EPS once exposed to a sucrose culture medium. EPS produced were first isolated with a standardised method and further characterised in terms of molecular size, antioxidant activity, and rheological properties. Its direct implication in the texture and syneresis of acid milk gel was evaluated offering interesting industrial applications for its…

Insights into genome plasticity of the wine-making bacterium Oenococcus oeni strain ATCC BAA-1163 by decryption of its whole genome.

International audience; Studying genomes of O. oeni strains having opposite oenological aptitudes is important for understanding why this lactic acid bacterium involved in malolactic fermentation is so well adapted to wine. Here, the genome of a strain ATCC BAA-1163, is described and compared with the recently reported genome of the better wine-adapted strain PSU-1. The BAA-1163 genome (8X) was obtained by shotgun sequencing and Phrap assembling. Compact and 62% AT-rich, it consists of a circular 1,792,103-bp chromosome and a 3,948-bp plasmid. It was analysed through a CAAT-Box annotation platform and manually inspected. A total of 51 RNA genes were detected, including two rRNA operons (the…

Influence des conditions environnementales (statique versus dynamique) sur le développement en biofilm de Listeria monocytogenes EGD-e. Developpement en biofilm et expression d'Agr de Listeria monocytogenes

National audience

Indigenous Listeria spp. in biowastes and during composting

International audience

Oligopeptide assimilation and transport by Oenococcus oeni

International audience; Aims: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. Methods and Results: Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolys…

Induction of stress proteins inLeuconostoc oenos to perform direct inoculation of wine

The enhancement or induction of the protein synthesis was clearly observed in cells ofL. oenos labeled with35S for five proteins during heat shock at 42°C and acid shock at pH 3. Furthermore, no stress protein was induced after exposure ofL. oenos to ethanol shock 10% (v/v). Moreover, survival ofL. oenos in wine and ability to perform alolactic fermentation was improved after direct inoculation when cells were pretreated at 42°C.

Quantification of HSP27 and HSP70 Molecular Chaperone Activities

Stress-inducible heat-shock proteins (HSPs, like HSP70 and HSP27) are molecular chaperones that -protect cells from stress damage by keeping cellular proteins in a folding competent state and preventing them from irreversible aggregation. HSP27 and HSP70 chaperone activities are useful indicators to test chemical products and physical stress impact on protein denaturation, to select HSP inhibitors, or to -determine the implication of the chaperone function in other HSP activities, such as apoptosis. We have developed two simple and fast chaperone activity tests for HSP27 and HSP70 that we initially set up to test the effect of potential HSP inhibitors obtained after screening of chemical an…

Differential expression of the agr locus of Listeria monocytogenes EGD-e

International audience

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

 ; Aims: Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. Methods and Results: A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenanc…

Genetic organization of the mle locus and identification of a mleR-like gene from Leuconostoc oenos

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 t…

Biotechnical applications of small heat shock proteins from bacteria.

The stress responses of most bacteria are thought to involve the upregulation of small heat shock proteins. We describe here some of the most pertinent aspects of small heat shock proteins, to highlight their potential for use in various applications. Bacterial species have between one and 13 genes encoding small heat shock proteins, the precise number depending on the species considered. Major efforts have recently been made to characterize the protein protection and membrane stabilization mechanisms involving small heat shock proteins in bacteria. These proteins seem to be involved in the acquisition of cellular heat tolerance. They could therefore potentially be used to maintain cell via…

The biofilm mode of life boosts the anti-inflammatory properties ofLactobacillus

Summary The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or plankto…

Agr system of Listeria monocytogenes EGD-e: role in adherence and differential expression pattern.

ABSTRACT In this study, we investigated the agrBDCA operon in the pathogenic bacterium Listeria monocytogenes EGD-e. In-frame deletion of agrA and agrD resulted in an altered adherence and biofilm formation on abiotic surfaces, suggesting the involvement of the agr system of L. monocytogenes during the early stages of biofilm formation. Real-time PCR experiments indicated that the transcript levels of agrBDCA depended on the stage of biofilm development, since the levels were lower after the initial attachment period than during biofilm growth, whereas transcription during planktonic growth was not growth phase dependent. The mRNA quantification data also suggested that the agr system was a…

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase

International audience; Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The express…

Antibacterial properties and reduction of MRSA biofilm with a dressing combining polyabsorbent fibres and a silver matrix

Objective: This study was designed to evaluate the antibacterial activity of a wound dressing which combines polyacrylate fibres and a silver lipido-colloid matrix (UrgoClean Ag, silver polyabsorbent dressing), against biofilm of methicillin-resistant Staphylococcus aureus (MRSA). Method: Samples of silver polyabsorbent dressing and the neutral form of this dressing (UrgoClean) were applied to biofilms of MRSA formed on a collagen I-coated surface, cultured for 24 hours. Different exposure times were tested (1, 2, 4 and 7 days) without dressing change. The biofilm reduction was quantified by using culture methods and by confocal laser scanning microscopy experiments. Results: The applicatio…

Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production

Aims:  The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. Methods and Results:  Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in th…

Stress Responses of Oenococcus oeni

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium responsible for malolactic fermention in wine. The stress responses of O. oeni have been studied at both the molecular and physiological levels. Genes encoding stress proteins mainly belong to the CtsR regulon. Other regulation mechanisms seem to coexist in O. oeni and may correspond to posttranscriptional regulation. Maintenance of the cell membrane integrity under stress conditions seems to be a prerequisite for survival in wine. The active cell response to protect membrane function under stress conditions requires changes in fatty acid composition and involves stress proteins. Various solute transporters and energy…

Acid sensitivity of neomycin-resistant mutants ofOenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity

Mutants of Oenococcus oeni were isolated as spontaneous neomycin-resistant mutants. Three of these mutants harbored a significantly reduced ATPase activity that represented 50% of that of the wild-type strain. Their growth rates were also impaired at pH 5.3 (46-86% of the wild-type level). However, the profiles of sugar consumption appeared identical to those of the parental strain. At pH 3.2, all the mutant strains failed to grow and a drastic decrease in viability was observed after an acid shock. Surprisingly, all the isolated mutants were devoid of malolactic activity. These results suggest that the ATPase and malolactic activities of O. oeni are linked to each other and play a crucial …

Listeria monocytogenes EGD-e biofilms: no mushrooms but a network of knitted chains.

ABSTRACT Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and…

Protective role of glutathione addition against wine-related stress in Oenococcus oeni

FIliació URV: SIInclòs a la memòria: SI Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were ab…

Inactivation of a small heat shock protein affects cell morphology and membrane fluidity in Lactobacillus plantarum WCFS1.

A small heat shock gene of Lactobacillus plantarum strain WCFS1 was deleted using a Cre-lox based system. Compared to the wild type, the ∆hsp 18.55 mutant strain displayed a similar growth rate when cultivated either under optimal temperature or under different stress conditions such as heat, low pH and salt stress. However, a longer lag phase was observed when the ∆hsp 18.55 mutant strain was cultivated under short intense heat stress (50 °C). This suggests that the hsp 18.55 gene of L. plantarum may be involved in recovery of L. plantarum stressed cells in the early stage of high temperature stress. In addition, morphology of the mutant cells, investigated by scanning electron microscopy,…

Chemical Transfers Occurring Through Oenococcus oeni Biofilm in Different Enological Conditions

International audience; Chardonnay wine malolactic fermentations were carried out to evaluate the chemical transfers occurring at the wood/wine interface in the presence of two different bacterial lifestyles. To do this, Oenococcus oeni was inoculated into must and wine in its planktonic and biofilm lifestyles, whether adhering or not to oak chips, leading to three distinct enological conditions: (i) post-alcoholic fermentation inoculation in wine in the absence of oak chips, (ii) post-alcoholic fermentation inoculation in wine in the presence of oak chips, and (iii) co-inoculation of both Saccharomyces cerevisiae and O. oeni directly in Chardonnay musts in the presence of oak chips. Classi…

A small HSP, Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium

International audience; The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytopl…

Peptidases specific for proline-containing peptides and their unusual peptide-dependent regulation in Oenococcus oeni

International audience; Growth of the lactic acid bacterium (LAB) Oenococcus oeni, which is involved in malolactic fermentation during the winemaking process, is stimulated by peptides originating from yeast. In this study, we investigated the impact of peptides on O. oeni growth, peptidase activity and the expression of genes encoding the studied peptidases. Low levels of PepN activity and very high levels of PepI activity were observed in O. oeni, whereas levels of PepX activity were intermediate. The level of biosynthesis of these O. oeni peptidases was shown to depend on peptides present in the culture medium. These results were confirmed by transcriptional analyses of putative pep gene…

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties

IF 3.682; International audience; Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobaci…

Distinct amino acids of  the Oenococcus oeni small heat shock protein Lo18 are essential for damaged protein protection and membrane stabilization

The small heat shock protein (smHsp) Lo18 from lactic acid bacteria Oenococcus oeni reduces in vitro thermal aggregation of proteins and modulates the membrane fluidity of native liposomes. An absence of information relating to the way in which the smHsp demonstrates a stabilizing effect for both proteins and membranes prompted this study. We expressed three Lo18 proteins with amino acid substitutions in Escherichia coli to investigate their ability to prevent E. coli protein aggregation and their capacity to stabilize E. coli whole-cell membranes. Our results showed that the alanine 123 to serine substitution induces a decrease in chaperone activity in denaturated proteins, and that the ty…

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation

International audience; Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were gr…

Resveratrol-induced xenophagy promotes intracellular bacteria clearance in intestinal epithelial cells and macrophages

International audience; Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be bene ficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebra f…

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

International audience; Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Delta pep4, which is deleted for the proteinase A gene. Fermentation with the mutant Delta pep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Delta pep4 strain took 10 days longer than the control. This diffe…

CtsR is the master regulator of stress response gene expression in Oenococcus oeni.

ABSTRACT Although many stress response genes have been characterized in Oenococcus oeni , little is known about the regulation of stress response in this malolactic bacterium. The expression of eubacterial stress genes is controlled both positively and negatively at the transcriptional level. Overall, negative regulation of heat shock genes appears to be more widespread among gram-positive bacteria. We recently identified an ortholog of the ctsR gene in O. oeni . In Bacillus subtilis , CtsR negatively regulates expression of the clp genes, which belong to the class III family of heat shock genes. The ctsR gene of O. oeni is cotranscribed with the downstream clpC gene. Sequence analysis of t…

Discovering lactic acid bacteria by genomics

International audience; This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the pu…

Interactions in dual species biofilms between Listeria monocytogenes EGD-e and several strains of Staphylococcus aureus.

International audience; Six environmental isolates of Staphylococcus aureus and one collection strain were investigated for their ability to form monospecies biofilms and dual species biofilms with Listeria monocytogenes EGD-e on stainless steel coupons. All isolates were able to grow as biofilms but their ability to form monospecies biofilms differed. The population of L. monocytogenes EGD-e in dual species biofilms was not affected by the presence of S. aureus isolates except for strain CIP 53.156. The effect of L. monocytogenes EGD-e on the population of S. aureus was strain dependent: S. aureus population either increased or decreased or was not affected in the presence of L. monocytoge…

Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum

Abstract Lactobacillus plantarum cells displayed substrate-inducible decarboxylase activities on p-coumaric and ferulic acids of 0.6 and 0.01 μmol min−1 mg−1, respectively. Activity in uninduced cells or corresponding cell extracts was undetectable (

Evaluation of the Sensory Profiles of Texas High Plains Tempranillo and Cabernet Sauvignon Wines

A trained panel was asked to evaluate the sensory profiles of Tempranillo and Cabernet Sauvignon varietal wines from the Texas High Plains American Viticultural Area. The panelists evaluated 20 different wines using a modified conventional descriptive analysis and a modified citation frequency-based method. Our study revealed a significant correlation between both methods in the distribution of wines. The two varietal wines could not be significantly distinguished by their average sensory profiles, although butter, caramel and lavender were significantly stronger in Tempranillo and black currant in Cabernet Sauvignon. Both average sensory profiles were mainly driven by their oak-related cha…

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

Aims:  To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain-specific primers. Strains were further grouped using a multiplex RAPD-PCR analysis. Then, six strains were inoculated in two wine-like media with two differe…

Techniques d'études de la formation des biofilms. Retour d'expérience issu du domaine "agro-alimentaire" et du domaine "environnemental"

No abstract

Increase of sulfite tolerance in Oenococcus oeni by means of acidic adaptation

International audience; Sulfite is an antimicrobial agent used at the beginning of winemaking to avoid development of undesirable microorganisms. However, Oenococcus oeni, which is mainly responsible for the malolactic fermentation, has to grow in wine and therefore has to be resistant to sulfite. This study showed that acid-adapted cells of O. oeni survived better than non-adapted cells in the presence of a high sulfite concentration (30 mg l-1). Addition of a sub-lethal concentration of sulfite (15 mg l31) during the adaptation step in acidic medium increases the sulfite tolerance. Moreover, sulfite appeared to be able to induce a heat shocklike response. Our results suggest that pH homeo…

A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters.

The lactic acid bacterium Oenococcus oeni, mainly responsible for malolactic fermentation (MLF), is used in new winery process as starter culture for direct inoculation. The difficulty to master MLF according to the wine led us to search a new approach to select effective O. oeni strains. Biochemical and molecular tests were performed in order to characterize three strains of O. oeni selected for malolactic starter elaboration. Malolactic and ATPase activities that appeared as a great interest in MLF were measured and the expression of a small heat shock protein Lo18 was evaluated by immunoblotting and real-time PCR. These results were correlated with the performances of strains in two red …

Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then w…

Sensitivity to acetic acid, ability to colonize abiotic surfaces and virulence potential of Listeria monocytogenes EGD-e after incubation on parsley leaves.

International audience; Abstract Aim: To investigate how the survival of Listeria monocytogenes on parsley leaves may affect its ability to sustain process-related harsh conditions and its virulence. Methods and Results: Parsley seedlings were spot inoculated with stationary phase cells of L. monocytogenes EGD-e and incubated for 15 days. Each day, bacterial cells were harvested and enumerated, and their ability to survive acetic acid challenge (90 min, pH 4.0), to colonize abiotic surfaces and to grow as biofilms was assessed. After a 3-log decrease over the first 48 h, the population stabilized to about 10(6) CFU g(-1) until the sixth day. After the sixth day, L. monocytogenes was no long…

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°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…

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5 degrees C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100-110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-k…

Absence of malolactic activity is a characteristic of H+-ATPase-deficient mutants of the lactic acid bacterium Oenococcus oeni.

ABSTRACT The lack of malolactic activity in H + -ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni . Results obtained in this study show that th…

Modulation of autophagy by probiotic bacteria: selecting and engineering strains able to stimulate autophagy in intestinal epithelial cells

National audience

Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts

ABSTRACT For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes , Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli . Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetabl…

Improved acid tolerance of a recombinant strain of Escherichia coli expressing genes from the acidophilic bacterium Oenococcus oeni.

Aims:Oenococcus oeni is a lactic acid bacterium used in wine fermentation. Two open reading frames (orfB and orfC) were identified in the upstream region of the hsp18 gene, encoding the small heat-shock protein Lo18. Expression of these genes in conditions of acid stress was studied in Escherichia coli. Methods and Results: Sequence analysis showed that orfB encodes a putative transcriptional regulator of the LysR family. The protein encoded by orfC shares homologies with multi-drug resistance systems. Heterologous expression of orfB, orfC and hsp18 genes in Escherichia coli significantly enhanced the viability of the host strain under acidic conditions. Conclusions: It was demonstrated tha…

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

ABSTRACT Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Δ ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR - clpC operon and hsp1 .

Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives.

Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation. A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality. Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation. The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure. Furthermore, yeast that ferment must before or toget…