Fructose and glucose mediates enterotoxin production and anaerobic metabolism of Bacillus cereus ATCC14579(T).

AIMS: To determine the effects of carbohydrates on Bacillus cereus ATCC14579(T) anaerobic metabolism and enterotoxin production in amino acids rich medium. METHODS AND RESULTS: Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose-fructose mixture) was examined in synthetic mMOD medium under continuous cultures (mu = 0.2 h(-1)). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety). CONCLUSIONS: Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment.

Phosphodiesterase type 5 inhibition coupled to strong reinforcement results in two periods of transient retention loss in the young chick.

Previous behavioural studies which have administered phosphodiesterase type-5 (PDE5) inhibitors have consistently demonstrated improved retention. However, when young chicks were trained on a strongly reinforced passive avoidance task 100microM zaprinast caused two periods of transient retention loss. This is opposed to past findings and may suggest an effect on retrieval. It is hypothesised that the level of reinforcement is central to this phenomenon. The molecular corollary of this may be the need to maintain cGMP homeostasis such that strong reinforcement+zaprinast may impair retention through the production of excessive levels of cGMP. This was demonstrated by two challenge studies whereby increasing concentrations of 8-Br-cGMP were administered in the presence of the guanylate cyclase inhibitor ODQ (100microM; ic) resulting in an inverted "U-shaped" retention curve. These findings suggest a more complex role for PDE5 and cGMP in memory processing than previously described and question the role of PDE5 inhibitors as nootropes under all circumstances.

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.

Kinetic analysis of growth and xanthan gum production with Xanthomonas campestris on sucrose, using sequentially consumed nitrogen sources.

A batch fermentation strategy using Xanthomonas campestris ATCC 13951 for xanthan gum production has been established in which all essential medium components are supplied at the onset. This has been achieved using sucrose as sole sugar feedstock. Sequential consumption of nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) was observed to facilitate the further optimisation of the medium. Biomass accumulation was limited by phosphate availability. Xanthan yields of more than 60% (grams of xanthan per gram of sugar) have been obtained with constant acetyl content. However, pyruvyl substitution decreased as the growth rate declined, due to the metabolic constraints specific to phosphate depletion. High rates of carbon conversion into xanthan were observed throughout the culture and the ATP/ADP ratio was not affected by the decline in the specific growth rate.

Molecular physiology of sugar catabolism in Lactococcus lactis IL1403.

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.

Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism.

Two strains of Lactococcus lactis ssp. cremoris, MG 1820 and MG 1363, which differed by the presence or absence of the lactose plasmid, respectively, were cultivated in batch-mode fermentation on lactose as carbon substrate. A correlation between the rate of sugar consumption, the growth rate, and the type of metabolism was observed. The MG 1820 strain grew rapidly on lactose and homolactic fermentation occurred. The major regulating factor was the NADH/NAD(+) ratio proportional to the catabolic flux, which inhibited glyceraldehyde-3-phosphate dehydrogenase activity. This control led to an increase in metabolite concentration upstream of this enzyme, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, and inhibition of pyruvate formate lyase activity, while lactate dehydrogenase was strongly activated by the high coenzyme ratio. The contrary was observed during growth of the MG 1363 strain. Further investigation during growth of L. lactis ssp. lactis NCDO 2118 on galactose as carbon substrate and on various culture media enabling the growth rate to proceed at various rates demonstrated that the relative flux between catabolism and anabolism was the critical regulating parameter rather than the rate of glycolysis itself. In a minimal medium, where anabolism was strongly limited, the rate of sugar consumption was reduced to a low value to avoid carbon and energy waste. Despite this low sugar consumption rate, the catabolic flux was in excess relative to the anabolic capability and the NADH/NAD+ ratio was high, typical of a situation of nonlimiting catabolism leading to a homolactic metabolism.

Competence regulation by oxygen availability and by Nox is not related to specific adjustment of central metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism.

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (Delta malE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.

Metabolism of Lactococcus lactis subsp. cremoris MG 1363 in acid stress conditions.

The metabolism of glucose by Lactococcus lactis subsp. cremoris MG 1363 remains homolactic whatever the pH of the culture medium. The growth rate decreased with the acidification of the medium until a limit pH value of 4.0 for which no growth was observed. In contrast, the specific rate of glucose consumption decreased only for very low pH values, i.e., below 4.5. The efficiency of biomass synthesis relative to the energy supply decreased when the medium pH diminished, as illustrated by Y(ATP) values. This observation was related to the increase in both components of the proton-motive force when the pH decreased. The growth stopped when the internal pH reached a limit value of 5.4 due to organic acid accumulation.

Reductive cleavage of demeton-S-methyl by Corynebacterium glutamicum in cometabolism on more readily metabolizable substrates.

Corynebacterium glutamicum is able to biotransform demeton-S-methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. Among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton-S-methyl biotransformation. The reaction mechanism of demeton-S-methyl biotransformation involves reductive cleavage of an S-C bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.

Growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by Corynebacterium glutamicum.

The organophosphorous pesticide, demeton-S-methyl was transformed by Corynebacterium glutamicum in co-metabolism with more readily degradable substrates. Glucose, acetate and fructose were tested as growth substrates, and the highest demeton-S-methyl biotransformation average rate (0.78 mg l(-1) h(-1)) and maximum instantaneous rate (1.4 mg l(-1) h(-1)) were achieved on fructose. This higher efficiency seems to be linked to the atypical behavior of C. glutamicum grown on fructose, characterized by a prolonged period of accelerating growth instead of a constant growth rate observed on glucose or acetate. More precisely, for growth rates in the 0.1-0.4 h(-1) range, a direct coupling between the specific demeton-S-methyl consumption rate and the growth rate was demonstrated on fructose during batch-, steady state continuous- or continuous cultures with a controlled transient growth rate (accelerostat technology). The demeton-S-methyl biotransformation was more favoured during an acceleration phase of the growth rate.

Phenol degradation by Ralstonia eutropha: colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations.

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.

Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts.

Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human mu-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%-90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water.

Codependency: predictors and psychometric issues.

This study examined the relationships between codependency and age, gender, self-confidence, autonomy, and succorance, which is the quality of soliciting emotional support from others. The study also tested the validity of the Spann-Fischer Codependency Scale (Fischer, Spann, & Crawford, 1991). Ninety-five undergraduates completed a demographic sheet, the Adjective Check List (Gough & Heilbrun, 1983), the Spann-Fischer Codependency Scale, the Co-Dependents Anonymous Checklist (Whitfield, 1991), and a questionnaire developed for this study based on the work of Hemfelt, Minirth, and Meier (1989). As predicted, codependency was negatively related to self-confidence and positively related to succorance. However, contrary to expectation, a negative relationship between codependency and autonomy was not found. In addition, low self-confidence was the strongest predictor of codependency. Finally, all three measures of codependency were strongly related, attesting to the convergent validity of the Spann-Fischer Codependency Scale. Future studies should further investigate the role of emotional autonomy and codependency and should begin to utilize an experimental approach, making predictions regarding the behavior of codependent and noncodependent persons in experimental situations.

Electrospray ionization mass spectrometric analysis of blood for differentiation of species.

The National Fish and Wildlife Forensic Laboratory is responsible for the determination of species of birds, reptiles, and mammals from the United States, as well as international species falling under the protection of CITES treaties. We have recently found electrospray ionization mass spectrometry to be an effective means of rapidly analyzing blood samples for species identification. Nearly 1000 individuals were analyzed which comprised 62 species represented by birds, mammals, and reptiles. Whole blood and dried blood samples were analyzed without purification to provide simultaneous molecular weights from the alpha- and beta-proteins present in each sample's hemoglobin. The combination of the two molecular weights for the hemoglobin proteins (i.e., alpha/beta-pairs) was used as species determining markers. In all, 133 distinctive alpha/beta-pairs were observed from the individuals analyzed. Despite the variability in the hemoglobins evaluated, 86% of these alpha/beta-pairs were found to be diagnostic for a particular species to the exclusion of all other species studied. No other single protein system studied by a single analytical technique can so effectively resolve species from a wide range of taxa as can the hemoglobin system when analyzed by electrospray ionization mass spectrometry.

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Metabolic analysis of glutamate production by Corynebacterium glutamicum.

The dynamic behavior of the metabolism of Corynebacterium glutamicum during L-glutamic acid fermentation, was evaluated by quantitative analysis of the evolution of intracellular metabolites and key enzyme concentrations. Glutamate production was induced by an increase of the temperature and a final concentration of 80 g/l was attained. During the production phase, various other compounds, notably lactate, trehalose, and DHA were secreted to the medium. Intracellular metabolites analysis showed important variations of glycolytic intermediates and NADH, NAD coenzymes levels throughout the production phase. Two phenomena occur during the production phase which potentially provoke a decrease in the glutamate yield: Both the intracellular concentrations of glycolytic intermediates and the NADH/NAD ratio increase significantly during the period in which the overall metabolic rates decline. This correlates with the decrease in glutamate yield due in part to the production of lactate and also to the period of the fermentation in which growth no longer occurred.

Novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol.

The use of a membrane bioreactor with cell retention to achieve high biomass concentrations has been examined for phenol degradation by the bacteria Alcaligenes eutrophus. This process is particularly interesting for toxic substrates as the hydraulic dilution rate and the growth rate are independently controlled. In the case of a transitory excess of phenol, this potentially toxic situation can be overcome by modifying the substrate concentration or the dilution rate without any loss of cells. The injection of a gas phase at the filter inlet increased both the permeate flow rate (by a factor of 1. 75) and the oxygen transfer capacity (by a factor of 1.5). This has enabled the cell concentration to reach a maximal value of 60 g L-1 with a hydraulic dilution rate of 0.5 h-1 and a phenol feed concentration of 8 g L-1. The volumetric productivity of this process corresponds to a phenol degradation rate approaching 100 kg m-3 day-1. The on-line measurement of the characteristic yellow color of 2-hydroxymuconate semialdehyde, a metabolic intermediate of the phenol degradation pathway, in the permeate provides an interesting basis for process control of phenol supply into the reactor since the color intensity correlates directly to the specific rate of phenol degradation.

Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose.

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO2 during growth on fructose. These differences in carbon-metabolite accumulation are indicative of a different pattern of carbon-flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (> 50% of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon-isotope distribution patterns of the glutamate pool after growth on 1-13C- or 6-13C-enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80% of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD+ ratio susceptible to inhibit both glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.