The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas.

AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated. METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents. Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV. The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7. CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc. mesenteroides physiology. The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc. mesenteroides in the fermentation process.

Comparison of the effects of temperature and water activity on growth rate of food spoilage moulds.

The influence of temperature (T) and water activity (aw) on the growth rate (mu) of seven moulds (Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Mucor racemosus, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma harzianum) was assessed in suboptimal conditions. Firstly, the dependence of fungal growth on temperature, at aw 0.99, was modelled through an approach described previously for bacteria. A dimensionless growth rate variable: mu(dimalpha)=mu/mu (optalpha) depended on the following normalised temperature: T(dim)=(T-T(min))/(T(opt)- T(min)) according to a power function: mu(dimalpha)=[T(dim)]alpha, where alpha was an exponent to be estimated. Secondly, the same approach was used to describe the influence of aw on fungal growth, at the respective optimum temperatures for each mould. Similarly, mu(dimbeta)=mu/mu(optbeta) depended on the following normalised water activity: a(wdim)=(aw-a(wmin))/(a(wopt)-a(wmin)) according to a power function: mu(dimbeta)=[a(wdim)](beta). Results show: (i) for each mould, the alpha-value is significantly less than the beta-value, confirming that water activity has a greater influence than temperature on fungal development; (ii) the alpha-values and the beta-values depend on the mould; (iii) the alpha-value is less than 1 for the mesophilic mould A. flavus, whereas the other moulds are characterised by higher alpha-values ranging from 1.10 to 1.54; (iv) the mesophilic A. flavus exhibits a low beta-value, 1.50, compared to the hydrophilic T. harzianum, beta=2.44, while beta-values are within the range (1.71-2.37) for the other moulds.

Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum.

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.

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

The small heat shock protein (smHSP) family has been extensively studied in eukaryotic cells. SmHSP assemble into large multimeric structures and possess chaperone activity that can prevent protein aggregation in vitro. Few studies on prokaryotic smHSP are actually available and no smHSP from lactic acid bacteria has been characterized at a biochemical level to date. Here we report on the Lo18 membrane-associated smHSP from the lactic acid bacterium Oenococcus oeni. Using size exclusion chromatography, nondenaturing pore-exclusion PAGE and in vitro and in vivo cross-linking experiments, the multimeric structure of Lol8 from O. oeni or expressed in Escherichia coli was investigated. In vitro, Lo18 is able to form a trimer and a higher oligomer which could be a dodecamer. Experiments strongly suggest that the same structures exist in vivo. First, Lo18 prevented thermal aggregation of citrate synthase and lactate dehydrogenase even at 60degreesC. These findings showed that the prokaryotic smHSP Lo18 can function as a molecular chaperone in vitro. Second, Lo18 did not protect lactate dehydrogenase from thermal inactivation and did not assist in enzymatic activity restoration after thermal aggregation, suggesting that other components may be needed for the refolding of the enzyme in an active conformation. Third, we showed that membrane association of Lo18 depends on the temperature upshift. Moreover, expression of this smHSP was induced by administration of a membrane fluidiser, the benzyl alcohol, suggesting that Lo18 expression could be regulated by the level of membrane fluidity.

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 greater than 0.990, with the exception of that for A. flavus (r2 = 0.982). In addition, the use of such a model allows predictions of the cardinal water activities: a(wmin), a(wopt) and a(wmax). The estimation of the minimum water activity (a(wmin)) was in accordance with data literature for all the moulds considered here, but seemed to be slightly underestimated for P. chrysogenum and A. flavus when compared to our experimental values. The estimations of the optimal water activity (a(wopt)) and the optimal growth rate (muopt) were in excellent agreement to the experimental results for the four moulds. Through this example, it is suggested that the same approach for modelling can be used for various microorganisms (e.g. bacteria and moulds), and different environmental parameters (e.g. temperature and water activity).

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. CONCLUSION: It was demonstrated that the three genes were needed for acquisition of this acid tolerance phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: Heterologous expression of Oenococcus genes could be used to confer acidophilic behaviour on strains of biotechnological interest.

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

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 degrees 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 factorial design methodology. CONCLUSIONS: Higher rates of spore germination were associated with a high level of water activity. The incubation temperature also had a positive effect. A significant positive interaction between water activity and temperature was observed. Under these specific experimental conditions, pH did not have a significant effect on conidial germination. SIGNIFICANCE AND IMPACT OF THE STUDY: A model describing the behaviour of fungal conidia is proposed.

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.

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 in determining PAD substrate specificity and catalytic capacity. In order to test phenolic acid toxicity, the levels of growth of recombinant E. coli displaying and not displaying PAD activity were compared on medium supplemented with different concentrations of phenolic acids and with differing pHs. Though these acids already have a slight inhibitory effect on E. coli, vinyl phenol derivatives, created during decarboxylation of phenolic acids, were much more inhibitory to the E. coli control strain. To take advantage of this property, a solid medium with the appropriate pH and phenolic acid concentration was developed; in this medium the recombinant E. coli strains expressing PAD activity form colonies approximately five times smaller than those formed by strains devoid of PAD activity.

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

Pediococcus pentosaceus displays a substrate-inducible phenolic acid decarboxylase (PAD) activity on p-coumaric acid. Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of the Lactobacillus plantarum pdc gene was used to screen a P. pentosaceus genomic 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 with L. plantarum PDC enzyme. This ORF was identified as the padA gene. A second ORF was located just downstream of the padA gene and displayed 37% identity with the product of the Bacillus subtilis yfiO gene. Subcloning, transcriptional analysis, and expression studies with Escherichia coli of these two genes under the padA gene promoter, demonstrated that the genes are organized in an autoregulated bicistronic operonic structure and that the gene located upstream of the padA gene encodes the transcriptional repressor of the padA gene. Transcription of this pad operon in P. pentosaceus is acid phenol dependent.

Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism.

Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol. min(-1). mg(-1) in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

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

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 expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

Membrane fluidity of stressed cells of Oenococcus oeni.

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 degrees 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 after an acid shock. In contrast, the rates of membrane fluidity variation of adapted cells were 5- and 3-fold lower after a heat shock and an ethanol shock, respectively. These results suggest the positive effect of an adaptation on the membrane response and can help to explain the mechanisms of stress tolerance in Oenococcus oeni.

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

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 growth failure (pH 5.0, < or =-100 mV).

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

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 enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.

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.

The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase.

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42 degrees C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5' end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli.

Acid sensitivity of neomycin-resistant mutants of Oenococcus 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 role in the mechanism of resistance to an acid stress.

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

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.

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

A genomic library from Leuconostoc mesenteroides subsp. cremoris (Lmc) in Escherichia coli was screened for alpha-acetolactate synthase (ALS) activity using a phenotypic test detecting the production of acetolactate or related C4 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 gene is interrupted by a single mutation and the strain is auxotrophic for the three BCAAs. Only a very low anabolic ALS activity was present in cell-free extracts of Lmc and no transcript from the ilvB gene could be detected. Instability of ilvB expression in E. coli was the consequence of a frequent IS5 insertion sequence in this gene. Despite the detection of a high catabolic ALS activity in Lmc, no catabolic ALS activity gene could be found in the BCAA gene locus, indicating the presence of a catabolic als gene in the Lmc chromosome that could be absent or not expressed in the screened library.

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

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 proton permeability without any modification of ATPase activity. We propose that low ORP values de-energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.