Biodiversity trends in Europe: development and testing of a species trend indicator for evaluating progress towards the 2010 target.

This paper presents a trial of a species population trend indicator for evaluating progress towards the 2010 biodiversity target in Europe, using existing data. The indicator integrates trends on different species (groups), and can be aggregated across habitats and countries. Thus, the indicator can deliver both headline messages for high-level decision-making and detailed information for in-depth analysis, using data from different sources, collected with different methods. International non-governmental organizations mobilized data on over 2800 historical trends in national populations of birds, butterflies and mammals, for a total of 273 species. These were combined by habitat and biogeographical region to generate a pilot pan-European scale indicator. The trial indicator suggests a decline of species populations in nearly all habitats, the largest being in farmland, where species populations declined by an average of 23% between 1970 and 2000. The indicator is potentially useful for monitoring progress towards 2010 biodiversity targets, but constraints include: the limited sensitivity of the historical data, which leads to conservative estimates of species decline; a potential danger of ambiguity because increases in opportunistic species can mask the loss of other species; and failure to account for pre-1970 population declines. We recommend mobilizing additional existing data, particularly for plants and fishes, and elaborating further the criteria for compiling representative sets of species. For a frequent, reliable update of the indicator, sound, sensitive and harmonized biodiversity monitoring programmes are needed in all pan-European countries.

Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the lactobacillus reuteri wild-type strain and by mutant strains

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or 1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted alpha-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2-->6)-beta-D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.

Purification and characterization of thermophilin T, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0040.

ACA-DC 0040 produced an antimicrobial agent, which was named thermophilin T, active against several lactic acid bacteria strains of different species and food spoilage bacteria, such as Clostridium sporogenes C22/10 and Cl. tyrobutyricum NCDO-1754. The crude antimicrobial compound is sensitive to proteolytic enzymes and alpha-amylase, heat-stable (100 degrees C for 30 min), resistant to pH exposure at pH 1-12 and demonstrates a bactericidal mode of action against the sensitive strain Lactococcus cremoris CNRZ-117. The production of bacteriocin was optimized approximately 10-fold in an aerobic fermenter held at constant pH 5.8 and 6.2. Ultrafiltration experiments with culture supernatant fluids containing the bacteriocin, and further estimation of molecular weight with gel filtration chromatography, revealed that bacteriocin in the native form has a molecular weight in excess of 300 kDa. SDS-gel electrophoresis of partially purified thermophilin T showed that bacteriocin activity was associated with a protein band of approximately 2.5 kDa molecular mass.

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test.

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the hdcA genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc aenos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. aenos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdcA gene products of Gram-positive bacteria. The amplification products of the hdcA genes from Lac. buchneri and Leuct. aenos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdcA gene. In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46.

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80 degrees C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2.4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.

Occurrence and formation of biologically active amines in foods.

Attention is given to the toxicology of biogenic amines and their occurrence and formation in foods, with special emphasis on fermented foods. The role of the associated flora, the starter cultures used and their interaction is discussed.

Comparison of the effects of fluoride and the ionophore nigericin on acid production by Streptococcus mutans and the resultant in vitro enamel demineralization.

In an in vitro demineralization experiment with a plaque of S. mutans C180-2 over enamel, the effect of 0.5 mmol fluoride/L was compared with that of 10 nmol nigericin/L. Since the effects of both substances on the acid production in a dense cell suspension were of the same magnitude, and since nigericin did not affect the enamel solubility, the present demineralization experiment may differentiate between the effect of fluoride on bacterial metabolism alone and the combined effect of fluoride on both the bacterial metabolism and enamel solubility. In our experiments, the effect on bacterial metabolism accounted for 75% of the caries-inhibiting effect of fluoride. Microradiographic data showed subsurface lesions in the control and nigericin group and a slight surface-softening in the fluoride group. In addition, the effects of fluoride and nigericin on the cellular internal pH during glycolysis were studied. Analysis of these data suggested that glycolysis was inhibited when the intracellular pH reached about pH 5.2, which supports the view that acidification of the cell cytoplasm is a mechanism by which fluoride inhibits glycolysis.

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.

Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture.

The lactate concentration gradient and the components of the electrochemical proton gradient (delta micro H+) were determined in cells of Streptococcus cremoris growing in batch culture. The membrane potential (delta psi) and the pH gradient (delta pH) were determined from the accumulation of the lipophilic cation tetraphenylphosphonium and the weak acid benzoate, respectively. During growth the external pH decreased from 6.8 to 5.3 due to the production of lactate. Delta pH increased from 0 to -35 mV, inside alkaline (at an external pH of 5.7), and fell to zero directly after growth stopped. Delta psi was nearly constant at -90 mV during growth and also dissipated within 40 min after termination of growth. The internal lactate concentration decreased from 200 mM at the beginning of growth (at pH 6.8) to 30 mM at the end of growth (at pH 5.3); the external lactate concentration increased from 8 to 30 mM due to the fermentation of lactose. Thus, the lactate gradient decreased from 80 mV to zero as growth proceeded and the external pH decreased. From the data obtained on delta psi, delta pH, and the lactate concentration gradient, the H+/lactate stoichiometry (n) was calculated. The value of n varied with the external pH from 1.9 (at pH 6.8) to 0.9 (at pH values below 6). This implies that especially at high pH values the carrier-mediated efflux of lactate supplies a significant quantity of metabolic energy to S. cremoris cells. At pH 6.8 this energy gain was almost two ATP equivalents per molecule of lactose consumed if the H+/ATP stoichiometry equals 2. These results supply strong experimental evidence for the energy recycling model postulated by Michels et al.

Generation of an electrochemical proton gradient by lactate efflux in membrane vesicles of Escherichia coli.

The 'energy-recycling model' [Michels et al. (1979) FEMS Microbiol. Lett. 5, 357-364] postulates the generation of an electrochemical gradient across the bacterial cytoplasmic membrane by carrier-mediated efflux of metabolic endproducts in symport with protons. Experimental evidence for this model is presented. In membrane vesicles from Escherichia coli ML 308-255 L-lactate translocation (both uptake and efflux) is carrier-mediated. The H+/L-lactate stoichiometry varies, depending on the external pH, between 1 and 2. This change in stoichiometry is most likely the result of a protonation of the lactate carrier protein. This process has a pK of 6.75. L-Lactate efflux from membrane vesicles, loaded with 50 mM potassium L-lactate, results at an external pH of 6.6 in an 11-fold accumulation of proline inside the vesicles. This accumulation is completely inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The uptake of proline is not the result of a potassium or an osmotic gradient. At an external pH of 6.6 efflux of L-lactate from the vesicles leads to the generation of an electrical potential across the membrane of -55 mV, as is demonstrated from the accumulation of the lipophilic cation tetraphenylphosphonium.