Charles Diviès

Knockout of the p-Coumarate Decarboxylase Gene from Lactobacillus plantarum Reveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

ABSTRACT Lactobacillus plantarum NC8 contains a pdcgene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomalpdc 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 withp-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 withp-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates.

Two human faeces carriage isolates of Listeria monocytogenes (H1 and H2) were compared to reference strains (ScottA and LO28) with regard to their lethality in 14-day-old chick embryos, their haemolytic and phospholipase (phosphatidylcholine-phospholipase C and phosphatidylinositol-phospholipase C) activities and their invasiveness towards Caco-2 cells. Experimental infection of chick embryos allowed discrimination of the strains into those exhibiting high virulence (ScottA and H2), those exhibiting slightly attenuated virulence (LO28) and those exhibiting low virulence (H1). A similar percentage mortality and time to death for embryos was observed when they were infected with H2 as was seen with infection by the reference strain ScottA. Therefore, human carriage strain H2 was considered potentially pathogenic. In contrast to H2 and ScottA, H1 exhibited low virulence. Using the tissue-culture cell-line model, it was found that carriage strain H1 was unable to enter Caco-2 cells efficiently, even though it was similar to the virulent strains in terms of the enzymic activities involved in pathogenicity. Detection of the internalins InlA and InlB, involved in the internalization of L. monocytogenes in the host cells, by immunoblot indicated that a truncated form of InlA was produced by H1. Taken together, these data provide a starting point for the study of the behaviour of two types of human faeces carriage strains and their characterization.

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).

A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase

In Leuconostoc oenos, a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42°C), acid (pH 3) or ethanolic (12% (v/v)) shocks. Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30u200ah) during this growth phase. Significant identity was found between the N‐terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells.

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.

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.

Measurement of the intracellular pH in Escherichia coli with the internally conjugated fluorescent probe 5- (and 6-)carboxyfluorescein succinimidyl ester

The 5- (and 6-)carboxyfluorescein succinimidyl ester (cFDASE) is a pH-dependent fluorescent probe which conjugates with the aliphatic amine functions of the cells. Because of this property, the fluorescence efflux rate is low, 10 to 40% after 30 min of fermentable carbon source incubation. Thus, this technique allows a continuous high-time measurement (>30 min) without correction of the signal, but also a dynamic study phenomenon of few seconds. Due to the advantages presented by this technique, the method was adapted for Escherichia coli. The internal pH measurement obtained under various conditions are in accuracy with those from radiolabelled technique and from other fluorescence probe, with the advantages mentioned previously.

Membrane Potential-generating Transport of Citrate and Malate Catalyzed by CitP of Leuconostoc mesenteroides

Citrate uptake in Leuconostoc mesenteroides subsp. mesenteroides 19D is catalyzed by a secondary citrate carrier (CitP). The kinetics and mechanism of CitP were investigated in membrane vesicles of L. mesenteroides. The transporter is induced by the presence of citrate in the medium and transports both citrate and malate. In spite of sequence homology to the Na-dependent citrate carrier of Klebsiella pneumoniae, CitP is not Na- dependent, nor is CitP Mg-dependent. The pH gradient (ΔpH) is a driving force for citrate and malate uptake into the membrane vesicles, whereas the membrane potential (Δ) counteracts transport. An inverted membrane potential (inside positive) generated by thiocyanide diffusion can drive citrate and malate uptake in membrane vesicles. Analysis of the forces involved showed that a single unit of negative charge is translocated during transport. Kinetic analysis of citrate counterflow at different pH values indicated that CitP transports the dianionic form of citrate (Hcit) with an affinity constant of 20 μM. It is concluded that CitP catalyzes Hcit/H symport. Translocation of negative charge into the cell during citrate metabolism results in the generation of a membrane potential that contributes to the protonmotive force across the cytoplasmic membrane, i.e. citrate metabolism in L. mesenteroides generates metabolic energy. Efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate co-metabolism, is observed, suggesting that under physiological conditions, CitP may function as an electrogenic precursor/product exchanger rather than a symporter. The mechanism and energetic consequences of citrate uptake are similar to malate uptake in lactic acid bacteria.

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 free-cells for normal and two-layer beads respectively. This result was reproducible for several consecutive runs. Also, the problems linked to storage acidification (souring, wheying-off) appeared later and living lactic acid bacteria were maintained in the product.