Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118.

alpha-Acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable. The purification was carried out from E. coli in which the enzyme was expressed 36-fold higher. The specific activity was 24-fold enhanced after purification. The main characteristics of alpha-acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine. The enzyme is composed of six identical subunits of 26,500 Da.

Purification, properties and DNA sequence of the D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. cremoris.

The complete sequence of the D-lactate dehydrogenase (D-ldh) gene from Leuconostoc mesenteroides subsp. cremoris, cloned in Escherichia coli, were determined. The deduced amino acid sequence showed homologies with all members of the D-specific-2-hydroxyacid dehydrogenase family. Furthermore, the essential residues detected so far as being involved in catalysis were also conserved. Purification of the enzyme revealed physico-chemical properties corresponding to those predicted from the sequence. The active enzyme was a dimer of 40-kDa subunits. The Km values for pyruvate, lactate, NADH and NAD were 0.3, 19, 0.03 and 0.16 mM, indicating that the enzyme reduced pyruvate in vivo. Besides the D-LDH activity, L. mesenteroides subsp. cremoris also displayed HicDH enzymatic activity, catalysing the reduction of pyruvate analogs. The purified D-LDH displayed low HicDH-type activity; therefore, differences in specificity profiles between the crude extract and the purified enzyme suggested the occurrence of a specific HicDH.

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.

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc 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.

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.

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 chain association during gelation by chelatants. With the aim of lowering the gel setting temperature during the cell entrapment process while maintaining high mechanical properties, a gel made of 2.5% gellan gum and 0.2% sodium citrate was used to entrap Bifidobacterium longum ATCC 15707. Ions and pH of the inoculum during the immobilization step influenced the long-term mechanical stability of the gel beads during continuous fermentation in a stirred tank reactor. High stability as well as high biocatalyst activity was obtained when a washed cell suspension was used as the inoculum. Gellan gel produced by dissolving gellan gum in a sodium citrate solution may be a promising entrapment matrix for temperature-sensitive cells such as mesophilic lactic acid bacteria and eukaryotic cells.

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).

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.

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.

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 cases or carriers (11.5%), red meats (10%), cheeses (5.4%), wild birds (0.9%), and environments of cheese dairies (0%). Among resistant strains, 10 groups of strains (71.5%) were differentiated by serogroup, phage typing, and sensitivity or resistance to cadmium. Extrachromosomal DNA was found in all resistant strains and was transferred at a high frequency among Listeria spp. (8.7 x 10(-6) to 1 x 10(-3) transconjugant CFUs per one donor CFU). These resistances were also transferable between L. monocytogenes and Staphylococcus aureus with similar transfer frequencies (7.8 x 10(-6) to 1 x 10(-4) and between strains of Staphylococcus aureus with similar transfer frequencies from 8 x 10(-7) to 3.3 x 10(-6). These results suggest that emergence of this multiple resistance in Listeria spp. could be due to acquisition of a replicon originating in staphylocci.

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.

[Remarks on ethanol oxidation by an "Acetobacter xylinum" microbial electrode (author's transl)]. / Remarques sur l'oxydation de l'éthanol par une "électrode microbienne" d'Acetobacter xylinum

A "microbial electrode" for ethanol assay has been designed using combination of an oxygen probe and cellulosic pellicle of Acetobacter xylinum. Assay is feasible with an ethanol concentration below 0.4 mM on a pH range of 2,5-7. The formation of acetic acid leds to a competitive inhibition of ethanol oxidation as observed with free cells. Pellicle stability at room temperature is good over a ten hours period. At 4 degrees C, film preservation is quite satisfactory over a ten days storage period. The author compares the ethanol oxidation kinetics observed, using both cellulosic pellicles and free cells of A. xylinum.

[Use of labeling with 3H6-thymidine in the study of the multiplication of lactobacillus casei immobilized in a polyacrylamide gel]. / Application du marquage par la 3H6-thymidine à l'étude de la multiplication de Lactobacillus casei inclus dans un gel de polyacrylamide.

When 3H6-thymidine labelled bacteria (1.2 desintegration per cell/day) are immobilized in a polyacrylamide gel lattice, the decimal reduction time for their malic acid decarboxylase activity is extended to 38 days. Thus, the growth of bacteria in the gel lattice is very poor. The author has evaluated both the DNA turnover rate during the exponential growth phase for the free cells and the DNA specific activity of the population of immobilized cells. The viability of bacteria isolated from the gel lattice decreases with time, as does their thermal stability for malic acid decarboxylase activity. This activity is partially restored after 65 days. With respect to the protein, deoxyribose and ribose cell constituents, the protein/deoxyribose, protein/ribose and deoxyribose/ribose ratios fluctuats considerably with time. These fluctuations are more marked than those observed for the free cells during their different growth phases.

[Study on L-malic acid catabolism by "Lactobacillus casei" cells immobilized into polyacrylamide gel lattice (author's transl)]. / Etude du catabolisme de l'acide L-malique par Lactobacillus casei emprisonné dans un gel de polyacrylamide

Study on L-malic acid catabolism by Lactobacillus casei cells immobilized into polyacrylamide gel lattice has shown that the pH profil of malic acid decarboxylase activity does not differ significantly from that of free cells. The apparent enzymatic constant value as determined by the Warburg manometric method is 5 X 10(-3) M for free cells and 1.25 X 10(-2) M for immobilized cells. Malic acid decarboxylase activity can be preserved in immobilized cells over a 12 month period when the reaction vessel is fed continuously with the growth medium. It is noteworthy that the reaction vessel is similar to a constant activity reactor whose apparent enzymatic constant value, of 6,25 X 10(-3) M, does not differ significantly from that of free cells. Labelling of the cells with 14C-glycin has shown that the immobilized cells released 14 per cent of the radioactivity over a 65 hour period, indicating that the cells are possibly in a non proliferating state. After 9 months of working, a large fraction of viable bacterial cells can be isolated again. The use of such a reactor for producing metabolites is proposed.

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 that transcribed divergently from the mle operon was found. Sequence analysis and expression in E. coli minicells suggest that this open reading frame encodes a polypeptide with an apparent molecular mass of 34 kDa belonging to the LysR-type regulatory protein family. Protein comparisons showed the highest level of identity with the MleR regulatory protein from Lactococcus lactis, which is involved in the expression of the malolactic genes in the presence of L-malate. However, the MleR-like protein of L. oenos seems different from the protein of Lactococcus lactis, since no regulation of the malolactic enzyme by L-malate was effective under our experimental conditions.

Development and Use of a Screening Procedure for Production of (alpha)-Acetolactate by Lactococcus lactis subsp. lactis biovar diacetylactis Strains.

A method was developed to screen and isolate mutagenized Lactococcus lactis subsp. lactis biovar diacetylactis strains accumulating (alpha)-acetolactate. This compound is accumulated by (alpha)-acetolactate decarboxylase-deficient strains and undergoes spontaneous degradation into diacetyl on agar plates. The diacetyl produced is detected by a colorimetric reaction yielding a red halo around the colonies.

Modeling of continuous Ph-stat stirred tank reactor with Lactococcus lactis ssp. lactis bv. diacetylactis immobilized 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 limited by reaction, whereas that of citrate is limited by diffusion. Cell release from gel to the liquid medium occurs in the external spherical cap of the beads. In this peripheral zone viability is maintained at around 90%. (c) 1995 John Wiley & Sons Inc.

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.