Atsushi Yokota

Bile Acid Is a Host Factor That Regulates the Composition of the Cecal Microbiota in Rats

BACKGROUND & AIMS Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. METHODS Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. RESULTS Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. CONCLUSIONS Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.

Mechanism of Growth Inhibition by Free Bile Acids in Lactobacilli and Bifidobacteria

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.

Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.

Is bile acid a determinant of the gut microbiota on a high-fat diet?

Recently, we discovered that bile acid, a main component of bile, is a host factor that regulates the composition of the cecal microbiota in rats. Because bile secretion increases on a high-fat diet and bile acids generally have strong antimicrobial activity, we speculated that bile acids would be a determinant of the gut microbiota in response to a high-fat diet. The observed changes in the rat cecal microbiota triggered by cholic acid (the most abundant bile acid in human biliary bile) administration resemble those found in animals fed high-fat diets. Here, we discuss the rationale for this hypothesis by evaluating reported diet-induced gut microbiota alterations based on the postulate that bile acids worked as an underlying determinant. The identification of host factors determining the gut microbiota greatly contributes to understanding the causal relationships between changes in the gut microbiota and disease development, which remain to be elucidated.

Production of inulin fructotransferase (depolymerizing) by Arthrobacter sp. H65-7 and preparation of DFA III from inulin by the enzyme

Abstract A bacterial strain H65-7 isolated from soil as an inulin-assimilating microorganism produces inulin fructotransferase (inulase II) which converts inulin into di- d -fructofuranose-1,2′:2,3′-dianhydride (DFA III). This strain was classified as Arthrobacter sp. The inulase II production was induced by inulin, and markedly enhanced by the addition of yeast extract. Under optimal conditions, the enzyme activity in the culture supernatant reached 90 units/ml after cultivation for 18 h. The optimum pH and temperature for the enzyme reaction were 5.5 and 60°C, respectively. The enzyme was stable within a pH range of 4.5 to 9.0 and at up to 75°C. Using this crude enzyme, 300 mg/ml of inulin was converted into 237 mg/ml of DFA III after incubation for 4 h.

Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid aas the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32–40 h at pH6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.

Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

ABSTRACT To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

Population dynamics of Bifidobacterium species in human feces during raffinose administration monitored by fluorescence in situ hybridization-flow cytometry.

ABSTRACT The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.

Purification and Properties of an Inulin Fructotransferase (Depolymerizing) from Arthrobacter sp. H65-7

Abstract Arthrobacter sp. H65-7 produced inulin fructotransferase (depolymerizing) which converts inulin into di- d -fructofuranose 1, 2′ : 2, 3′ dianhydride (DFA III) and small amounts of oligosaccharides. The enzyme was purified 8-fold with a yield of 13% from a culture supernatant by ammonium sulfate precipitation followed by DEAE-Toyopearl 650 M column chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 49,000 by SDS-polyacrylamide gel electrophoresis, and 100,000 by gel filtration. The isoelectric point of the enzyme was determined to be pH 4.7. The optimal pH and temperature for the enzyme reaction were 5.5, and 60°C, respectively. The enzyme was stable with a pH range of 4.5 to 9.0, and at up to 70°C. After exhaustive digestion of inulin by this enzyme, nystose and 1-F-fructofuranosyl-nystose were produced in addition to DFA III.

Endophytes as Producers of Xylanase

One hundred and sixty-nine endophytic fungi and 81 endophytic bacteria were isolated from 14 plants in total. Among them, 155 fungi (91.7%) and 52 bacteria (64%) were found to produce xylanase. The inside part of plants is a novel and good source for isolating xylanase producers in comparison with soil.