Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Proteomics analysis of Bifidobacterium longum NCC2705 growing on glucose, fructose, mannose, xylose, ribose, and galactose

To investigate the molecular mechanisms underlying carbohydrate uptake and connected metabolic pathways of Bifidobacterium longum NCC2705, the proteomic profiles of bacteria grown on different carbon sources including glucose, fructose, mannose, xylose, ribose, and galactose were analyzed. Our results show that all sugars tested were catabolized via the bifid shunt. Sixty-eight proteins that exhibited changes in abundance of threefold or greater were identified by MS. A striking observation was the differential expression of proteins related to the pyruvate metabolism. Further analysis of acetic acid and lactic acid in the culture supernatants by HPLC at the end of fermentation showed that more lactic acid was produced during growth on fructose, ribose, xylose, galactose and more acetic acid was produced during the fermentation of glucose and mannose. Growth experiments revealed that B. longum NCC2705 preferentially used fructose, ribose, xylose, and galactose with higher growth rates over glucose and mannose. Furthermore, five proteins (GroEL, Eno, Tal, Pgm, and BL0033) exhibited clear phosphorylation modifications at serine and/or tyrosine residues. BL0033, a component of an ATP-binding cassette (ABC) transporter, was significantly more abundant in bacteria grown on fructose and, to a lesser extent, ribose and xylose. RT-PCR analysis revealed that all genes of the ABC transporter are induced in the presence of these sugars suggesting that BL0033, BL0034, BL0035, and BL0036 constitute an ABC transporter with fructose as preferred substrate.     

Sugar transport systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.     

Arabinogalactan utilization in continuous cultures of Bifidobacterium longum: effect of co-culture with Bacteroides thetaiotaomicron

Studies showed that the plant cell wall polysaccharide arabinogalactan supported growth of Bifidobacterium longum in batch culture. Galactose was also utilized, but not arabinose, the other major constituent sugar of the polymer. Enzymes required for hydrolysis of arabinogalactan ('arabinogalactanase', alpha-arabinopyranosidase, beta-galactosidase) were inducible and cell-associated in B. longum, and their expression was repressed by glucose. Considerable amounts of alpha-arabinopyranosidase and beta-galactosidase were synthesized during growth on arabinogalactan, but only low levels of arabinogalactanase were detected. B. longum only grew on arabinogalactan in continuous culture under putative carbon-excess conditions. In C-limited chemostats, the bifidobacterium could not establish unless Bacteroides thetaiotaomicron was present in co-culture. The relationship between the two organisms was not simply commensal; at low specific growth rates, bacteroides cell population densities were approximately 30% lower than those recorded in axenic culture, indicating the existence of competitive interactions with the bifidobacterium. In contrast, at high specific growth rates, a mutualistic association was observed, in that Bact. thetaiotaomicron was maintained in the chemostats at high dilution rates if bifidobacteria were also present. Measurements of residual carbohydrate in spent culture fluid from C-limited chemostats indicated that a large part of the arabinogalactan molecule could not be broken down by either B. longum or Bact. thetaiotaomicron alone, or in co-culture. Formate and acetate were the major fermentation products of B. longum cultured in the presence of high concentrations of arabinogalactan, confirming that these bacteria were growing under energy-limited conditions.     

Oxygen- and glucose-dependent expression of Trhxt1, a putative glucose transporter gene of Trichoderma reesei

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.     

Molecular and genetical analysis of the fructose-glucose transport system in the cyanobacterium Synechocystis PCC6803

Complementation for glucose transport capacity of deficient mutants from Synechocystis PCC6803 allowed the cloning of the corresponding gene, glcP. The protein predicted from one open reading frame (ORF) in the DNA sequence was 468 residues long. It showed 46-60% amino acid sequence homology and similarity in size and predicted structure (including twelve probable membrane-spanning regions) with a group of non-phosphorylating sugar transporters from mammals, yeasts and Escherichia coli. A second ORF, 64 base pairs downstream from glcP, was detected. Its function, dispensable under auto- and heterotrophic conditions, could not be determined. Genetic analysis of mutants confirmed that the resistance to fructose, acquired simultaneously with the deficiency in glucose transport, resulted from mutations in the glcP gene, whose approximate location could be determined.