Effect of reducing agents on the acidification capacity and the proton motive force of Lactococcus lactis ssp. cremoris resting cells.

International audience; Reducing agents are potential inhibitors of the microbial growth. We have shown recently that dithiothreitol (DTT), NaBH(4) and H(2) can modify the proton motive force of resting cells of Escherichia coli by increasing the membrane protons permeability [Eur. J. Biochem. 262 (1999) 595]. In the present work, the effect of reducing agents on the resting cells of Lactococcus lactis ssp. cremoris, a species widely employed in dairy processes was investigated. DTT did not affect the acidification nor the DeltapH, in contrast to the effect previously reported on E. coli. The DeltaPsi was slightly increased (30 mV) at low pH (pH 4) in the presence of 31 mM DTT or 2.6 mM NaB…

Effect of pHand age of culture on cellular fatty acid composition of Leuconostoc oenos

Z. DRICI-CACHON, J.F. CAVIN AND C. DIVIES. 1996. This study is concerned with the fatty acid composition of three strains of Leuconostoc oenos grown at different pH. The most abundant fatty acids were C18: 1w9, C19: 0 cy(w9,10) and C16:0, followed by C16: 1w9 and C14: 0. The pH considerably modified the fatty acid distribution in Lo107 (an acidophilic strain) and Lo8413 (a moderately acidophilic strain). However, moderate changes occurred for LoATCC 23277 (a less acidophilic strain). At pH 2.9, Lo107 has a remarkably high level of C19: 0 cy-(w9,10) and C19:0 cy(w11,12). Proportions of C18: 1 and C19:0 cyclo acids varied mainly with the pH of the medium and also as a function of growth phase…

Proton-dependent kinetics of citrate uptake in growing cells ofLactococcus lactissubsp.lactisbv.diacetylactis

The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.

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; …

Effect of chelatants on gellan gel rheological properties and setting temperature for immobilization of living bifidobacteria.

The effect of various concentrations of sequestrants (sodium citrate, sodium metaphosphate, and EDTA) was studied on gellan gel (1.5-2.5% (w/v)) setting temperature and rheological properties. Addition of EDTA between 0 and 0.8% (w/v) led to a progressive decrease of setting temperature. Citrate and metaphosphate decreased this parameter when added up to 0.4 or 0.6%, depending on gellan gum concentration, eventually resulting in the absence of gel formation at room temperature for the 1.5% gellan solution containing 0.4% citrate. This effect was accompanied by a significant decrease of gel strength and stiffness and might be attributed to the binding of the divalent cations required for cha…

Molecular characterization of the phenolic acid metabolism in the lactic acid bacteria Lactobacillus plantarum

The lactic acid bacteria Lactobacillus plantarumdisplays substrate-inducible decar- boxylase activities on p-coumaric, caffeic and ferulic acids. Purification of the p-coumaric acid decarboxylase (PDC) was performed. Sequence of the N-terminal part of the PDC led to the cloning of the corresponding pdc gene. Expression of this gene in Escherichia colirevealed that PDC displayed a weak activity on ferulic acid, detectable in vitro in the presence of ammonium sulfate. Transcrip- tional studies of this gene in L. plantarum demonstrated that the pdc transcription is phenolic acid- dependent. A mutant deficient in the PDC activity, designated LPD1, was constructed to study phe- nolic acid altern…

Addition of reducing agent dithiothreitol improves 4-decanolide synthesis by the genus Sporidiobolus.

Two species of the genus Sporidiobolus, S. johnsonii and S. ruinenii, were used to study the effect of the reducing agent, dithiothreitol (DTT), on 4-decanolide production using ricinoleic acid as the substrate. The results indicate that the addition of DTT into the cultures significantly enhanced 4-decanolide biosynthesis by the two species.

Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator.

ABSTRACTPediococcus pentosaceusdisplays a substrate-inducible phenolic acid decarboxylase (PAD) activity onp-coumaric acid. Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of theLactobacillus plantarum pdcgene was used to screen aP. pentosaceusgenomic library in order to clone the corresponding gene of this bacteria. One clone detected with this probe displayed a low PAD activity. Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity withL. plantarumPDC enzyme. This ORF was identified as thepadAgene. A second O…

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…

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 μm long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is respo…

Modes d’invasion et d’expression de Botrytis cinerea, champignon pathogène de la Vigne

Introduction : Le champignon phytopathogene Botrytis cinerea est un polyphage qui cause d'enormes degâts en agriculture. La culture de la vigne represente en France des enjeux economiques importants. La vigne est touchee par la maladie de la pourriture grise due a B. cinerea, conduisant a des pertes de rendement importantes. L'objectif de cette etude est, compte tenu des effets sur le developpement fongique de facteurs environnementaux (Sautour et al., 2001a, b), la caracterisation du caractere pathogene et la comparaison des structures de populations de B. cinerea isolees de differentes regions de France : Bourgogne, Languedoc et Champagne. Materiel et methodes : Les suspensions de conidie…

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pHe) on the intracellular pH (pHi) of wild-type and citrate negative variants (cit−) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pHi of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pHe was decreased from 7 to 4, the pHi decreased from 7 to about 5. The adjunctio…

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.

A temperature-type model for describing the relationship between fungal growth and water activity

Growth of Penicillium chrysogenum, Aspergillus flavus, Cladosporium cladosporioides and Alternaria alternata at their respective optimum temperatures was studied in Potato Dextrose Agar (PDA) medium at different water activities (a(w)) adjusted with glycerol. The growth rate (mu) was expressed as the increase in colony radius per unit of time. This paper extends the model that showed the relationship between temperature and bacterial growth rate developed by Rosso et al. [J. Theor. Biol. 162 (1993) 447] to describe the influence of a(w) on fungal development. An excellent correlation between the experimental data and the model predictions was obtained, the regression coefficients (r2) were …

Citrate lyases of lactic acid bacteria

Citrate Iyase is a key enzyme of the citrate metabolism which is involved in flavor and texture of many fermented milk products. Citrate Iyase which catalyses the cleavage of citrate into oxaloacetate and acetate is a multienzyme complex composed of three proteins: an acyl carrier protein (ACP); a citrate, acetate-ACP transferase; and a citryl-S-ACP Iyase. The citrate Iyase is active only when the thioester residue of the prosthetic group bound to ACP is acetylated. In the presence of citrate, the transferase mediates the formation of citryl-S-acyl carrier protein by acyl exchange and liberation of acetate. Then the Iyase subunit cleaves the citryl-S-ACP with libe- ration of oxaloacetate an…

Genetic organization of the citCDEF locus and identification of mae and clyR genes from Leuconostoc mesenteroides.

ABSTRACT In this paper, we describe two open reading frames coding for a NAD-dependent malic enzyme ( mae ) and a putative regulatory protein ( clyR ) found in the upstream region of citCDEFG of Leuconostoc mesenteroides subsp. cremoris 195. The transcriptional analysis of the citrate lyase locus revealed one polycistronic mRNA covering the mae and citCDEF genes. This transcript was detected only on RNA prepared from cells grown in the presence of citrate. Primer extension experiments suggest that clyR and the citrate lyase operon are expressed from a bidirectional A-T-rich promoter region located between mae and clyR.

Mechanism of the Citrate Transporters in Carbohydrate and Citrate Cometabolism in Lactococcus and Leuconostoc Species

ABSTRACT Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178–2185, 1996). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis . The cloned gene was exp…

Wine Production by Immobilised Cell Systems

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.

Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization

By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the…

Gene Cloning, Transcriptional Analysis, Purification, and Characterization of Phenolic Acid Decarboxylase from Bacillus subtilis

Phenolic acids, also called substituted cinnamic acids, are important lignin-related aromatic acids and natural constituents of plant cell walls. These acids (particularly ferulic, p-coumaric, and caffeic acids) bind the complex lignin polymer to the hemicellulose and cellulose in plants (1) or are generally esterified with tartaric acid (for example, in grape must, wine, and cider) and can be released as free acids during wine making by some cinnamoyl esterase activities (9). Most often, free phenolic acids are metabolized by different microorganisms into 4-vinyl derivatives and then are eventually reduced into 4-ethyl derivatives (5, 6). Some of these volatile phenols, particularly vinyl …

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster

ABSTRACT A citrate lyase (EC 4.1.3.6 ) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris . This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD , citE , and citF , encoding, respectively, the three ci…

A method for screening diacetyl and acetoin-producing bacteria on agar plates

A simple method for screening bacteria for diacetyl and acetoin production was developed. This method is based on the ability of diacetyl and acetoin to form a red insoluble complex with α-naphthol in the presence of creatine. Addition of carboxymethyl-cellulose containing calcium citrate in the medium allowed discrimination between citrate utilizing and non-utilizing bacteria.

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…

Medium for Screening Leuconostoc oenos Strains Defective in Malolactic Fermentation

A new sensitive medium was developed to screen and isolate mutagenic Leuconostoc oenos strains defective in malolactic fermentation. The essential components of the medium included fructose (22 mM), l -malic acid (74.6 mM), bromocresol green (as pH indicator), and cellulose powder. The wild-type colonies turned blue, but defective malolactic colonies gave an acid reaction and remained yellow-green.

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…

Comparison of α-acetolactate synthase and α-acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp.

Cell-free extracts of Leuconostoc and Lactococcus species were tested for their alpha-acetolactate synthase and alpha-acetolactate decarboxylase activities. In Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc lactis, the Km of alpha-acetolactate synthase for pyruvate was close to 10 mM whereas it was 30 mM in Lactococcus lactis subsp. lactis biovar. diacetylactis. The Km of alpha-acetolactate decarboxylase for alpha-acetolactic acid was very low (0.3 mM) in Leuconostoc species in comparison to Lactococcus lactis subsp. lactis biovar. diacetylactis (60 mM). In the latter bacterium, alpha-acetolactate decarboxylase showed a sigmoidal de…

Citrate permease gene expression inLactococcus lactissubsp.lactisstrains IL1403 and MG1363

Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis. Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.

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 …

Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PREVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIES. 1996. The conversion from citrate positive (Cit+) to citrate negative (Cit-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit+ phenotype was observed. The Cit- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit+ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit- mutants encoded for a citrate…

Purification and characterization of the catabolic ?-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris

The α-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris was purified to homogeneity in SDS-PAGE. The enzyme is a trimer of 3×55,000 Da. It was unstable but could be preserved by addition of pyruvate and thiamine pyrophosphate in the buffer. The enzyme exhibits Michaelis-Menten kinetics, and Km for pyruvate is 10 mM. Three intermediates in glucose metabolism (ATP, 3-phosphoglycerate, and phosphoenolpyruvate) exhibit a noncompetitive inhibition towards the enzyme. This enzyme does not require any divalent metal ion for activity. The α-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris is not inhibited by the branched-chain amino acids (valine, leucine, and…

Membrane fluidity of stressed cells of Oenococcus oeni

International audience; The determination of membrane fluidity in whole cells of Oenococcus oeni was achieved by membrane probe 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy measurements. The results demonstrated instantaneous fluidity variations with cells directly stressed during the measure. Heat (42°C) or acid (pH 3.2) shocks decreased the anisotropy values (fluidising effects), whereas an ethanol shock (10% ethanol, v/v) increased the membrane rigidity. The velocities of fluidity variation with non-adapted or adapted cells (incubation in inhibitory growth conditions) were compared. The adaptation of the cells to acid conditions had no effect on the membrane fluidity variation a…

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, Xg (growth + acidification) and Xng (acidification), and one inactive, Xi. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fe…

Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum.

ABSTRACTLactobacillus plantarumdisplays a substrate-induciblepadAgene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator ofpadAwas located in thepadAlocus based on its 52% identity with PadR, thepadAgene transcriptional regulator ofPediococcus pentosaceus(L. Barthelmebs, B. Lecomte, C. Diviès, and J.-F. Cavin, J. Bacteriol.182:6724-6731, 2000). Deletion of theL. plantarum padRgene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently orientedpadA. ThepadRgene is cotranscribed with a downstream open reading frame (ORF1), the product of which m…

Kinetics of citrate uptake in growing cells ofLeuconostocspp.

Citrate uptake was studied in growing cells of Leuconostoc mesenteroides subsp. mesenteroides. A Michaelis-Menten pattern with the dianionic form of citrate as the limiting substrate has been proposed. It was validated for different fermentations varying the initial citrate concentrations and the pH medium. This latter did not modify the rate of the process which was clearly confirmed using experiments with resting cells. The model was used to compare the kinetics of citrate consumption between several strains of Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc mesenteroides subsp. cremoris and Leuconostoc lactis.

Theme 4: Immobilized Cell Technology in Wine Production

AbstractIn spite of its traditional nature, wine making is largely concerned with the progress of biotechnology. High cell density reactors have potential for enology: improved performance of alcoholic and malolactic fermentations, smaller scale fermentation facilities, adaptation to continuous processes. Among the immobilization techniques, cell entrapment in alginate beads seems to be an impressive one. Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as “Methode champenoise” and sparkling wine are among the industrial applications. Knowledge of kinetics and physiology in microorganisms in heterogeneous media has expanded in the last few years. The use of…

Prediction of conidial germination of Penicillium chrysogenum as influenced by temperature, water activity and pH.

M. SAUTOUR, A. ROUGET, P. DANTIGNY, C. DIVIES AND M. BENSOUSSAN. 2001 Aims: Conidial germination of Penicillium chrysogenum was carried out under operating conditions compatible with a pastries manufacturing process. Methods and Results: A range, limited by two experimental values, was defined for each environmental factor tested: temperature (15 or 25°C), water activity (0·75 or 0·85) and pH (3·5 or 5·5). A closed device was made, which maintained an equilibrium between water activity of the culture medium and atmospheric relative humidity during 25 days, to follow spore germination. The combined effects of temperature, water activity and pH on spore germination were studied by applying fa…

Plasmid-Mediated Resistance to Antimicrobial Agents among Listeriae

The resistance to 14 antiseptic-disinfectant and dye compounds of 208 strains of Listeria (132 L. monocytogenes, 63 L. innocua, 8 L. seeligeri, 1 L. ivanovii, 1 L. welshimeri, and 3 Listeria spp.) was tested by the agar-dilution procedure. The Listeria strains were isolated from different varieties of foods, environments of cheese dairies, humans, and wild birds. A total of 14 (6.7%) Listeria strains (12 L. monocytogenes and 2 L. innocua) were resistant to benzalkonium chloride, hexamidine diisethionate, and ethidium bromide. This multiple resistance was observed more frequently from strains of Listeria spp. detected on carcasses of poultry (47%) than strains isolated from human listeriosis…

Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential.

International audience; The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane prot…

Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli.

ABSTRACT Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (−100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (−320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzy…

Immobilised Cell Technology in Winery and Fruit Wine Production

Winemaking is largely concerned with the progress of biotechnology and especially with the use of high cell density reactors. Entrapment is the most widely method to immobilise cells; several matrix can be used (alginate, carrageenan, agar) with different geometry (beads, fibres, plates). Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as the "Methode champenoise" and sparkling wines are among the industrial applications. Whereas prospects for this technology appear encouraging, further research is needed to optimise reaction variables, improve the long-term stability of the reactors, and understand more about secondary metabolite production by yeasts unde…

Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris

International audience; The biodiversity of growth and energetics in Leuconostoc sp. has been studied in MRS lactose medium with and without citrate. On lactose alone, Ln. lactis has a growth rate double that of Ln. cremoris and Ln. mesenteroides. The pH is a more critical parameter for Ln. mesenteroides than for Ln. lactis or Ln. cremoris; without pH control Ln. mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln. lactis and Ln. cremoris. In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and …

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides.

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in D-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation.

Cloning of branched chain amino acid biosynthesis genes and assays of alpha-acetolactate synthase activities in Leuconostoc mesenteroides subsp. cremoris.

Abstract A genomic library from Leuconostoc mesenteroides subsp. cremoris (Lmc) in Escherichia coli was screened for α-acetolactate synthase (ALS) activity using a phenotypic test detecting the production of acetolactate or related C 4 derivatives (diacetyl, acetoin or 2,3-butanediol) in the culture. Four recombinant E. coli clones, with plasmids containing overlapping DNA fragments and displaying anabolic ALS activity, were selected. This activity is encoded by an ilvB gene belonging to a putative operon which contains genes highly similar to the genes of the branched chain amino acid (BCAA) operon of Lactococcus lactis subsp. lactis. This putative BCAA operon is not functional as the ilvA…

Expression in Escherichia coli of Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities and Method for Screening Recombinant E. coli Strains Expressing These Enzymes

ABSTRACT Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum , Pediococcus pentosaceus , Bacillus subtilis , and Bacillus pumilus were expressed in Escherichia coli , and their activities on p -coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved …

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 (

Modeling of continuous Ph-stat stirred tank reactor withLactococcus lactisssp.lactisbv.diacetylactisimmobilized in calcium alginate gel beads

A dynamic diffusion-reaction-growth model is proposed for the study of lactic fermentation, the bioconversion of citric acid, and cell release in an immobilized cell reactor [pH-stat continuous stirred tank-reactor (CSTR)]. The model correctly simulates the onset of fermentation and colonization of the gel, followed by the steady state. External diffusion is nonlimiting and internal diffusion is limited by high cell densities at the periphery of the gel beads. Lactose-citrate cometabolism in the gel is related to the distribution of active included biomass within the gel and to gradients of substrates (lactose, citrate) and products (lactate, pH) in the beads. The utilization of lactose is …

Resistance of Immobilized Lactic Acid Bacteria to the Inhibitory Effect of Quaternary Ammonium Sanitizers

Abstract The inhibitory effect of quaternary ammonium sanitizers (QAS) on the fermentation activity of lactic acid bacteria was investigated. Benzalkonium chloride and didecyl dimethyl ammonium chloride show the same inhibitory effect. In MRS agar medium and MRS liquid medium their minimum inhibitory concentration was below 2 μg/mL. Interfering molecules in milk increased this concentration of inhibitory QAS above 8 μg/mL. Both growth and acidification were decreased but by immobilizing cells in calcium alginate gel beads this inhibitory effect could be reduced. The longer the beads were used for successive acidification and were colonized by lactic acid bacteria, the better was the acidifi…

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…

Combined action of redox potential and pH on heat resistance and growth recovery of sublethally heat-damaged Escherichia coli

International audience; The combined effect of redox potential (RP) (from -200 to 500 mV) and pH (from 5.0 to 7.0) on the heat resistance and growth recovery after heat treatment of Escherichia coli was tested. The effect of RP on heat resistance was very different depending on the pH. At pH 6.0, there was no significant difference, whereas at pH 5.0 and 7.0 maximum resistance was found in oxidizing conditions while it fell in reducing ones. In sub-lethally heat-damaged cells, low reducing and acid conditions allowed growth ability to be rapidly regained, but a decrease in the redox potential and pH brought about a longer lag phase and a slower exponential growth rate, and even led to growt…

Métabolisme sucre-citrate chez Leuconostoc mesenteroides

Sugar citrate cometabolism in Leuconostoc mesenteroides. Bacteria from the genus Leuconostocplay roles in the dairy industry. The most important functions of this bacteria are their ability to produce CO 2 and flavour compounds through lactose heterofermentation and citrate uti- lization. Although the biotechnological role of the citrate metabolism is very important and widely appreciated, little is known about the genetic properties of Leuconostoc spp. In our laboratory, we cloned the genes responsible for citrate metabolism ( clyR mae citCDEFGOPcluster), for D-lactate dehydrogenase (ldhD) and for phosphotransacetylase ( pta). In addition we have planned to con- struct new vectors and we h…

Cream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beads

This investigation was directed towards the development of a process which produces a fermented cream of greatly reduced cell number.Lactococcus lactis subsp.Lactis andLactococcus lactis subsp.lactis biovardiacetylactis were entrapped separately in normal or two-layer Ca-alginate gel beads. Pasteurized cream (31% fat content) was inoculated with free-cells and with normal or two-layer beads. When 8% of the total volume was occupied by the gel, there was 300–800 times more inoculum in this system and the fermentation time was considerably reduced (5h against 18h). When pH 5.0 was reached, the residual free-cell count was 150 and 1800 times less than for a classical inoculation method with fr…

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l−1 lactose and 2 g·l−1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was t…

Action de la chlorhexidine sur l'expression de la virulence de

Abstract Candida albicans is an opportunistic yeast. Its pathogenicity is linked to the susceptibility of the host surface as well as to particular factors of the strain: adhesion, filamentous growth and secretion of proteolytic enzymes. Chlorhexidine digluconate is an antiseptic with fungicidal properties. The action of the antiseptic on the growth of the yeast shows a minimal inhibitory concentration (MIC) at 50 μg·mL−1 and a minimal fungicidal concentration (MFC) at 100 μg·mL−1. The consequences of antiseptic treatment are studied using two indicators of pathogenicity: filamentation and the secretion of acid proteinase. Concerning the morphological indicator, a complete inhibition of fil…

Effects of oxidoreduction potential combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum.

International audience; The effects of oxidoreduction potential (Eh) combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum were studied. The culture medium was set at pH 5, and two different Eh values were adjusted using nitrogen (Eh = +350 mV) or hydrogen (Eh = -300 mV) gas. In reducing condition, the growth was slowed and the acidification delayed at 37 degrees C, but not at 10 degrees C. A synergistic inhibitory effect of reducing Eh, acetic acid and NaCl was observed, mainly for delaying the lag phase before acidification. These results may be explained by changes in ATPase activity, membrane fluidity and surface…

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…

Enterococcus asini sp. nov. isolated from the caecum of donkeys (Equus asinus)

Several Gram-positive, non-spore-forming and non-motile bacteria consisting of pairs or chains of cocci were isolated during an investigation of the bacterial flora of the caecum of donkeys. Physiological and metabolic data indicated that the strains belong to the genus Enterococcus; phenotypic traits of these organisms were not consistent with any of the currently known Enterococcus species. 16S rRNA gene sequence analysis placed these strains in the genus Enterococcus. Their closest relatives are Enterococcus avium, Enterococcus faecium and Enterococcus pseudoavium with a sequence similarity of 97.4%. This group of strains can be differentiated from the other Enterococcus spp. by their ph…

Knockout of thep-Coumarate Decarboxylase Gene fromLactobacillus plantarumReveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

ABSTRACTLactobacillus plantarumNC8 contains apdcgene coding forp-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomalpdcgene was replaced with the deletedpdcgene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolizep-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown thatL. plantarumhas a second acid phenol decarboxylase enzyme, better induced with ferulic acid than withp-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when gl…