End Products Of Glucose Metabolism Research Articles

Blautia parvula sp. nov., isolated from Japanese faecal samples.

Two Gram-positive, anaerobic, non-spore-forming and coccoid or oval-shaped bacterial strains, namely, DN0138T and DN0266, were isolated from faecal samples of healthy Japanese people. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DN0138T clustered with a species of the genus Blautia and was closely related to Blautia producta JCM 1471T, Blautia coccoides JCM 1395T, Blautia hominis KB1T and 'Blautia marasmi' Marseille-P2377, with sequence similarities of 98.6, 98.5, 98.8 and 98.2 %, respectively. The average nucleotide identity values were 85.3 % for B. producta JCM 1471T, 85.0 % for B. coccoides NCTC 11035T, 84.3 % for B. hominis KB1T and 84.3 % for 'B. marasmi' Marseille-P2377. The major end products of glucose metabolism were acetic acid, lactic acid and succinic acid. The genome length of strain DN0138T was 6 247 046 bp with 46.7 mol% G+C content of genome sequence. Based on their phenotypic, cellular fatty acid and phylogenetic characteristics, the three isolates represent a novel species within the genus Blautia, for which the name Blautia parvula sp. nov. is proposed. The type strain is DN0138T (=NBRC 113351T=BCRC 81349T).

1474-P: Testosterone Amplifies Glucose-Stimulated Insulin Secretion from Male ß-Cells via Soluble Adenylate Cyclase

Male mice with elimination of the androgen receptor (AR) in islet β cells (βARKO) exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of islet-derived and exogenous GLP-1 via GLP-1R in mouse and human islets. Using electron microscopy, we observe that DHT increases the size of granules and the frequency of multivesicular granules, suggesting that DHT promotes multivesicular exocytosis. Using live β cell models expressing organelle-specific cAMP sensors and novel allosteric inhibitors in human islets, we show that DHT enhances the ability of GLP-1 to produce cAMP at the plasma membrane and endosomes and enhances islet GSIS via the bicarbonate (HCO3 -) sensor, soluble adenylate cyclase (sAC) . The end-products of glucose metabolism in the TCA cycle are CO2 and H2O, which is followed by rapid conversion of CO2 to HCO3 - and H+ by carbonic anhydrase (CA) . Therefore, CO2 production is translated to HCO3 - and specifically sensed by sAC. Bioenergetics analysis of DHT-stimulated respiration in mouse islets using a Seahorse Analyser revealed that DHT decreases glycolytic acidification and increases mitochondrial acidification (H+ efflux rate) , suggesting that DHT increases mitochondrial production of CO2 followed by conversion to HCO3 - and H+, allowing HCO3 - to stimulate sAC. We discuss experiments of single-cell transcriptomics, proteomics and metabolomics dissecting the impact of DHT on human islets glycolysis and mitochondrial CO2 production. Consistent with DHT increasing production of CO2 followed by conversion to HCO3 - by CA and activation of sAC, in cultured human male islets, the CA inhibitor acetazolamide blunted DHT-enhancement of GSIS. Together these experiments provide novel insights into the role of DHT action via an extranuclear AR in enhancing GSIS. Disclosure M.Qadir: None. D.Hodson: Other Relationship; Celtarys Research. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. W.Xu: None. D.Nasteska: None. M.Bhondeley: None. P.Mota de sa: None. C.R.Evans: None. J.Buck: None. L.Levin: None. C.Burant: None. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

ТАКСОНОМИЧЕСКАЯ И ФУНКЦИОНАЛЬНАЯ ХАРАКТЕРИСТИКА МИКРОБИОТЫ РУБЦА ЛАКТИРУЮЩИХ КОРОВ ПОД ВЛИЯНИЕМ ПРОБИОТИКА ЦЕЛЛОБАКТЕРИНА+

Today rations for dairy cows are designed to provide the highest growth rate and productivity in a short period of time. However, such intensive livestock farming affects, first of all, the health of animals, since metabolic pathways inherent in ruminants are disrupted. The use of 16S metagenomics approaches makes it possible to assess the genetic and metabolic diversity of the bovine microbiome, which allows identifying factors that can contribute to an increase in productivity and an improvement in the health of the host. In the feeding trial, dairy cows were fed with dietary probiotic Cellobacterin+ based on the Enterococcus faecium 1-35 strain (the winter-spring period of 2018, JSC PZ Plamya, Gatchinsky District, Leningrad Province). Two groups of ten Holsteinized black-and-white dairy cows (Bos taurus taurus) of the 2nd and 3rd lactation with an average annual milk yield of 7000-7500 kg were used. The basal diet was 10 kg compound feed, 2 kg yellow corn, 0.5 kg sunflower cake, 0.5 kg rapeseed cake, 1 kg hay, 25 kg grass silage, 1 kg beet molasses, and 0.2 kg MINVIT®-3 (Russia). In the morning, the test cows were fed with dietary Cellobacterin+ (OOO BIOTROF, St. Petersburg) at 40 g per cow. Cicatricial contents (10-50 g) were collected from three cows of each group at the end of the experiment. Fasting blood was taken for biochemical analysis from the tail vein with vacutainers. The blood was analyzed for total protein, total bilirubin, glucose, calcium, phosphorus, urea, reserve alkalinity, ketone bodies. The mass fraction of fat in milk was analyzed according to GOST 5867-90, protein according to GOST 23327-98, and the number of somatic cells according to GOST R 54761-2011. Total DNA from the studied samples was extracted using the Genomic DNA Purification Kit (Fermentas, Inc., Lithuania) according to the attached instructions. Amplification for subsequent NGS sequencing was run (a Veriti Thermal Cycler, Life Technologies, Inc., USA) using the eubacterial primers (IDT) 343F (5´-CTCCTACGGRRSGCAGCAG-3´) and 806R (5´-GGACTANVGGGTWTCTAAT-3´) flanking the V1V3 region of the 16S rRNA gene. Metagenomic sequencing (a MiSeq system, Illumina, Inc., USA) was performed with a MiSeq Reagent Kit v3 (Illumina, Inc., USA). Chimeric sequences were excluded from analysis using the USEARCH 7.0 program (http://drive5.com/usearch/). The processing of the obtained reads using the bioinformatics platform CLC Bio GW 7.0 (Qiagen, the Netherlands) included overlapping, quality filtering (QV > 15), and primer trimming. The taxonomic affiliation of microorganisms to genus was determined using the RDP Classifier program (http://rdp.cme.msu.edu/). Mathematical and statistical processing of the results was carried out using the software packages Microsoft Office Excel 2003, R-Studio (Version 1.1.453) (https://rstudio.com). The mean values (M) and standard errors of the means (±SEM) were calculated. The results were deemed significant at p < 0.05. Analysis of microbial β-diversity of the samples by the principal component method was performed according to the Weighted UniFrac PCoA Emperor method using the QIIME software package. Reconstruction and prediction of the functional content of the metagenome, gene families, and enzymes was performed using the PICRUSt2 software package (v.2.3.0). MetaCyc database (https://metacyc.org/) was used to analyze metabolic pathways and enzymes. Feeding the probiotic had a significant effect (p = 0.049) on an increase in milk yield, as well as on a decrease (p = 0.003) in the somatic cell number in milk by 38,000/ml per cow. The NGS sequencing provided a complete taxonomic and functional characterization of the cicatricial microbiota, including uncultivated representatives. Significant differences were found between the groups for 13 bacterial genera. In particular, in the rumen of cows treated with the probiotic Cellobacterin+, compared to the control group, a lower proportion of the order Clostridia were found, namely the bacteria of the genera Anaerofilum sp. (2.3 times lower, p ≤ 0.05) and Anaerostipes sp. (1.8 times lower, p ≤ 0.05) that produce lactate in the rumen as the end product of glucose metabolism. A decrease occurred in the abundance of the genera Campylobacter, Gemella, Mycoplasma, Shewanella (p ≤ 0.05), and Fusobacterium (including F. necrophorum) (p ≤ 0.001) among which pathogens are often found. Changes in the taxonomic structure of rumen microbiota as influenced by the probiotic were also associated with metabolic changes. The predicted functional potential of seven metabolic pathways was enhanced in cows fed Cellobacterin+ compared to the control animals. Thus, when fed Cellobacterin+, there was a 3.5-fold increase (p ≤ 0.05) in the predicted level of microbiome metabolic capabilities associated with the synthesis of glyoxylate from allantoin, and 2.3-fold increase (p ≤ 0.05) in the biosynthesis of propionate from L-glutamate. These findings allow us to suggest an important role of the biological product Cellobacterin+ for maintaining the homeostasis of metabolic processes.

Histone Lactylation: A New Role for Glucose Metabolism

Lactate is an end product of glucose metabolism, which serves metabolic and nonmetabolic functions. A new study by Zhang etal. establishes a novel function for lactate whereby it is utilized in a new histone modification, histone lysine lactylation, to regulate gene expression in macrophages.

Isolation and whole genome sequencing of a Ruminococcus-like bacterium, associated with irritable bowel syndrome

In our previous studies on the intestinal microbiota in irritable bowel syndrome (IBS), we identified a bacterial phylotype with higher abundance in patients suffering from diarrhea than in healthy controls. In the present work, we have isolated in pure culture strain RT94, belonging to this phylotype, determined its whole genome sequence and performed an extensive genomic analysis and phenotypical testing. This revealed strain RT94 to be a strict anaerobe apparently belonging to a novel species with only 94% similarity in the 16S rRNA gene sequence to the closest relatives Ruminococcus torques and Ruminococcus lactaris. The G + C content of strain RT94 is 45.2 mol% and the major long-chain cellular fatty acids are C16:0, C18:0 and C14:0. The isolate is metabolically versatile but not a mucus or cellulose utilizer. It produces acetate, ethanol, succinate, lactate and formate, but very little butyrate, as end products of glucose metabolism. The mechanisms underlying the association of strain RT94 with diarrhea-type IBS are discussed.

Fusibacter fontis sp. nov., a sulfur-reducing, anaerobic bacterium isolated from a mesothermic Tunisian spring.

Strain KhalAKB1T, a mesophilic, anaerobic, rod-shaped bacterium, was isolated from water collected from a mesothermic Tunisian spring. Cells were Gram-staining-positive rods, occurring singly or in pairs and motile by one lateral flagellum. Strain KhalAKB1T grew at 15-45 °C (optimum 30 °C), at pH 5.5-8.5 (optimum pH 7.0) and in the presence of 0-35 g NaCl l- 1 (optimum 1 g NaCl l- 1). It fermented yeast extract and a wide range of carbohydrates including cellobiose, d-glucose, d-ribose, sucrose, d-xylose, maltose, d-galactose and starch as electron donors. Acetate, ethanol, CO2 and H2 were end products of glucose metabolism. It reduced elemental sulfur, but not sulfate, thiosulfate or sulfite, into sulfide. The DNA G+C content was 37.6 mol%. The predominant cellular fatty acids were C14 : 0 and C16 : 0. Phylogenetic analysis of the 16S rRNA gene sequence suggested Fusibacter bizertensis as the closest relative of this isolate (identity of 97.2 % to the type strain). Based on phenotypic, phylogenetic and genotypic taxonomic characteristics, strain KhalAKB1T is proposed to be assigned to a novel species within the genus Fusibacter, order Clostridiales, Fusibacter fontis sp. nov. The type strain is KhalAKB1T ( = DSM 28450T = JCM 19912T).

Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei

Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

High environmental temperature increases glucose requirement in the developing chicken embryo.

Environmental conditions during the perinatal period influence metabolic and developmental processes in mammals and avian species, which could impact pre- and postnatal survival and development. The current study investigated the effect of eggshell temperature (EST) on glucose metabolism in broiler chicken embryos. Broiler eggs were incubated at a high (38.9°C) or normal (37.8°C) EST from day 10.5 of incubation onward and were injected with a bolus of [U-13C]glucose in the chorio-allantoic fluid at day 17.5 of incubation. After [U-13C]glucose administration, 13C enrichment was determined in intermediate pools and end-products of glucose metabolism. Oxidation of labeled glucose occurred for approximately 3 days after injection. Glucose oxidation was higher in the high than in the normal EST treatment from day 17.6 until 17.8 of incubation. The overall recovery of 13CO2 tended to be 4.7% higher in the high than in the normal EST treatment. An increase in EST (38.9°C vs 37.8°C) increased 13C enrichment in plasma lactate at day 17.8 of incubation and 13C in hepatic glycogen at day 18.8 of incubation. Furthermore, high compared to normal EST resulted in a lower yolk-free body mass at day 20.9 (−2.74 g) and 21.7 (−3.81 g) of incubation, a lower hepatic glycogen concentration at day 18.2 (−4.37 mg/g) and 18.8 (−4.59 mg/g) of incubation, and a higher plasma uric acid concentration (+2.8 mg/mL/+43%) at day 21.6 of incubation. These results indicate that the glucose oxidation pattern is relatively slow, but the intensity increased consistently with an increase in developmental stage of the embryo. High environmental temperatures in the perinatal period of chicken embryos increased glucose oxidation and decreased hepatic glycogen prior to the hatching process. This may limit glucose availability for successful hatching and could impact body development, probably by increased gluconeogenesis from glucogenic amino acids to allow anaerobic glycolysis.

Lactobacillus porcinae sp. nov., isolated from traditional Vietnamese nem chua

A species diversity study of lactic acid bacteria occurring in traditional Vietnamese nem chua yielded an isolate, LMG 26767(T), that could not be assigned to a species with a validly published name. The isolate was initially investigated by 16S rRNA gene sequence analysis, which revealed that it belonged to the genus Lactobacillus, with Lactobacillus manihotivorans and Lactobacillus camelliae as the closest relatives (98.9 % and 96.9 % gene sequence similarity to the type strains, respectively). Comparative (GTG)5-PCR genomic fingerprinting confirmed the unique taxonomic status of the novel strain. DNA-DNA hybridization experiments, DNA G+C content determination, sequence analysis of the phenylalanyl-tRNA synthase (pheS) gene, and physiological and biochemical characterization demonstrated that strain LMG 26767(T) represents a novel species, for which the name Lactobacillus porcinae sp. nov. is proposed; the type strain is LMG 26767(T) ( = CCUG 62266(T)). Biochemically, L. porcinae can be distinguished from L. manihotivorans and L. camelliae by its carbohydrate fermentation profile, absence of growth at 45 °C, and production of d- and l-lactate as end products of glucose metabolism.

Succinatimonas hippei gen. nov., sp. nov., isolated from human faeces

A novel strictly anaerobic, non-spore-forming, non-motile, non-flagellated, rod-shaped, Gram-negative bacterium (YIT 12066T) was isolated from human faeces. The isolate was negative for catalase, oxidase, urease, hydrolysis of aesculin and gelatin, nitrate reduction and indole production. The major end products of glucose metabolism were succinate and acetate. The major cellular fatty acids (>10%) were C14:0, C18:1omega7c, C18:1omega9c, C16:1omega7c and C16:0. The G+C content of the DNA was 40.3 mol%. 16S rRNA gene sequence analysis showed that strain YIT 12066T was most closely related to members of the family Succinivibrionaceae, with sequence similarity of 92-87%. However, some phenotypic characteristics such as cellular morphology and the major fatty acid profile of strain YIT 12066T were markedly different from those of other members of the family Succinivibrionaceae. On the basis of both phylogenetic and phenotypic evidence, it is suggested that strain YIT 12066T represents a novel species in a new genus, for which the name Succinatimonas hippei gen. nov., sp. nov. is proposed; the type strain of Succinatimonas hippei is YIT 12066T (=DSM 22608T =JCM 16073T).

Alistipes indistinctus sp. nov. and Odoribacter laneus sp. nov., common members of the human intestinal microbiota isolated from faeces

Two anaerobic, non-spore-forming, non-motile, Gram-negative-staining bacteria, strains YIT 12060(T) and YIT 12061(T), were isolated from human faeces. Cells of strain YIT 12060(T) were coccoid to rod-shaped with round ends, positive for catalase, negative for indole and oxidase production, produced succinic and acetic acids as end products of glucose metabolism in peptone/yeast extract/glucose medium and had a DNA G+C content of 55.2 mol%. The main respiratory quinones were MK-10 (40%) and MK-11 (57%). Fatty acid analysis demonstrated the presence of a high concentration of iso-C(15 : 0) (56%). Following 16S rRNA gene sequence analysis, this strain was found to be most closely related to species of the genus Alistipes, with 90.9-92.6% gene sequence similarities to type strains of this species. Phylogenetic analysis and biochemical data supported the affiliation of strain YIT 12060(T) to the genus Alistipes of the family 'Rikenellaceae'. Strain YIT 12060(T) therefore represents a novel species of the genus Alistipes for which the name Alistipes indistinctus sp. nov. is proposed; the type strain is YIT 12060(T) (=DSM 22520(T)=JCM 16068(T)). Cells of the other isolate, strain YIT 12061(T), were pleomorphic rods that were asaccharolytic, catalase- and oxidase-negative, positive for gelatin hydrolysis and indole production, produced small amounts of succinic, acetic and iso-valeric acids as end products of metabolism in peptone/yeast extract medium and had a DNA G+C content of approximately 42.4 mol%. On the basis of 16S rRNA gene sequence similarity values, this strain was shown to belong to the family 'Porphyromonadaceae' and related to the type strains of Odoribacter splanchnicus (89.6%) and Odoribacter denticanis (86.2%); similarity values with strains of recognized species within the family 'Porphyromonadaceae' were less than 84 %. Biochemical data supported the affiliation of strain YIT 12061(T) to the genus Odoribacter. Strain YIT 12061(T) therefore represents a novel species for which the name Odoribacter laneus sp. nov. is proposed; the type strain is YIT 12061(T) (=DSM 22474(T)=JCM 16069(T)).

Paraprevotella clara gen. nov., sp. nov. and Paraprevotella xylaniphila sp. nov., members of the family 'Prevotellaceae' isolated from human faeces

Two anaerobic, non-spore-forming, pleomorphic, Gram-negative rods, designated YIT 11840T and YIT 11841T, were isolated from human faeces. The organisms were catalase-negative, produced succinic and acetic acids as end products of glucose metabolism and had DNA G+C contents of approximately 48-49 mol%. Although the phenotypic characteristics of these two strains were very similar, analysis of their 16S rRNA gene sequences showed that they are only distantly related (93.8%), indicating that they represent two different species. A comparative sequence analysis revealed that these two species are members of the family 'Prevotellaceae' but are phylogenetically distant (<88% sequence similarity) from the known genera belonging to this family, including Prevotella, Hallela and Xylanibacter. On the basis of the phylogenetic analysis and physiological tests, strains YIT 11840T and YIT 11841T represent two novel species of a new genus, for which the names Paraprevotella clara gen. nov., sp. nov. (type strain YIT 11840T=JCM 14859T=DSM 19731T), the type species, and Paraprevotella xylaniphila sp. nov. (type strain YIT 11841T=JCM 14860T=DSM 19681T) are proposed.

Dialister succinatiphilus sp. nov. and Barnesiella intestinihominis sp. nov., isolated from human faeces

Two anaerobic, non-spore-forming, bacteria (YIT 11850(T) and YIT 11860(T)) that stained Gram-negative, were isolated from human faeces. Cells of strain YIT 11850(T) were coccobacilli, asaccharolytic and largely unreactive, with only traces of lactate and propionate as metabolic end products; however, strain YIT 11850(T) was able to decarboxylate succinate to propionate. The DNA G+C content of strain YIT 11850(T) was 51.9 mol%. Following 16S rRNA gene sequence analysis, this strain was found to be most closely related to Dialister propionicifaciens, with 95.1 % sequence similarity between the two taxa. Biochemical data supported the affiliation of strain YIT 11850(T) to the genus Dialister. Strain YIT 11850(T) therefore represents a novel species for which the name Dialister succinatiphilus sp. nov. is proposed; the type strain is YIT 11850(T) (=DSM 21274(T)=JCM 15077(T)). Cells of the other isolate, strain YIT 11860(T), were non-motile, rod-shaped, positive for aesculin hydrolysis, negative for indole production, produced succinic and acetic acids as end products of glucose metabolism and possessed a DNA G+C content of 45.5 mol%. On the basis of 16S rRNA gene sequence similarity values, this strain was shown to belong to the family 'Porphyromonadaceae' related to Barnesiella viscericola (96.0 %); similarity values with species within the family 'Porphyromonadaceae' with validly published names were less than 86 %. Biochemical data supported the affiliation of strain YIT 11860(T) to the genus Barnesiella. Strain YIT 11860(T) therefore represents a novel species for which the name Barnesiella intestinihominis sp. nov. is proposed; the type strain is YIT 11860(T) (=DSM 21032(T)=JCM 15079(T)).

Knock-downs of Iron-Sulfur Cluster Assembly Proteins IscS and IscU Down-regulate the Active Mitochondrion of Procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.

C/ebpδ Null Mouse as a Model for the Double Knock-out of slc5a8 and slc5a12 in Kidney

slc5a8 and slc5a12 represent the high affinity and low affinity Na+/lactate co-transporters, respectively, in the kidney. Here we show that these transporters are expressed in the apical membrane of the proximal tubular cells in mouse kidney, indicating that these transporters are likely to mediate the first step in the renal reabsorption of lactate. Interestingly, the renal expression of both transporters is almost completely ablated in mice homozygous for the deletion of the transcription factor c/ebpdelta. This effect is tissue-specific since the expression of the transporters is not affected in non-renal tissues. The functional role of C/EBPdelta in the expression of SLC5A8 and SLC5A12 is demonstrable in HEK293 cells in reporter assays using gene-specific promoters. The ablation of the transporters in the kidney is accompanied by a marked increase in urinary excretion of lactate as well as a decrease in blood levels of lactate in c/ebpdelta-/- mice. These data provide evidence for an obligatory role for slc5a8 and slc5a12 in the renal absorption of lactate. In addition, we show that urinary excretion of urate is significantly elevated in c/ebpdelta-/- mice even though the expression of URAT1, the transporter responsible for the apical membrane uptake of urate in renal proximal tubule, is not altered. These data provide in vivo evidence for the functional coupling between lactate reabsorption and urate reabsorption in the kidney. Thus, the fortuitous double knock-out of slc5a8 and slc5a12 in kidney in c/ebpdelta-/- mice reveals the physiologic role of these transporters in the renal handling of lactate and urate.

“ Bacteroides goldsteinii sp. nov.” Isolated from Clinical Specimens of Human Intestinal Origin

Phenotypic and phylogenetic studies were performed on an unknown gram-negative, strictly anaerobic, rod-shaped bacterium isolated from human clinical specimens. This organism was indole negative, resistant to 20% bile, produced acetic and a lesser amount of succinic acids as the major end products of glucose metabolism, and possessed a G+C content of approximately 43 mol%. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of the Cytophaga-Flavobacter-Bacteroides phylum of gram-negative bacteria and formed a close association (with an average sequence similarity of 93.6%) with the second subcluster of the Porphyromonas cluster in the Bacteroides subgroup. Phylogenetically and phenotypically it resembled Bacteroides merdae; however, a 16S rRNA gene sequence divergence of approximately 5.5% between the unknown bacterium and B. merdae, as well as distinguishable biochemical characteristics, demonstrate that the unknown bacterium is genotypically and phenotypically distinct and represents a previously unknown subline within the Porphyromonas phylogenetic cluster. Furthermore, a DNA-DNA reassociation value of 17.8% between isolates WAL 12034(T) (the type strain of this novel taxon) and ATCC 43184(T) (B. merdae type strain) also documented the separateness of the unknown species and B. merdae. Based on the phenotypic and phylogenetic findings, a new species, "Bacteroides goldsteinii sp. nov," is proposed. The G+C content of the DNA is 43 mol% for Bacteroides. The type strain of "B. goldsteinii" is WAL 12034(T) (= CCUG 48944(T) = ATCC BAA-1180(T)).

A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.

New Functions for Parts of the Krebs Cycle in Procyclic Trypanosoma brucei, a Cycle Not Operating as a Cycle

We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

Isolation from oil reservoirs of novel thermophilic anaerobes phylogenetically related to Thermoanaerobacter subterraneus: reassignment of T. subterraneus, Thermoanaerobacter yonseiensis, Thermoanaerobacter tengcongensis and Carboxydibrachium pacificum to Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov. as four novel subspecies.

Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively. Both strains, together with Thermoanaerobacter yonseiensis KB-1(T) (=DSM 13777(T)), Thermoanaerobacter tengcongensis MB4(T) (=DSM 15242(T)) and Carboxydibrachium pacificum JM(T) (=DSM 12653(T)), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858(T) (=DSM 13054(T)), which was isolated recently from an oilfield reservoir in south-west France. Marked phenotypic differences exist between the three oilfield isolates (T. subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used. Differences were also observed in the DNA G+C contents of all organisms. Similarly to T. subterraneus, strains SL9 and OCA1, and also T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism [0.8-1.0 mol L-alanine produced (mol glucose consumed)(-1)] and reduced thiosulfate, but not sulfate, to sulfide. Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T. subterraneus, strain OCA1 and strain SL9), T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp. subterraneus subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. yonseiensis subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. tengcongensis subsp. nov., comb. nov. and Caldanaerobacter subterraneus subsp. pacificus subsp. nov., comb. nov.

Anaerofustis stercorihominis gen. nov., sp. nov., from human feces

Phenotypic and phylogenetic studies were performed on an unidentified Gram-positive, strictly anaerobic, non-spore-forming, rod-shaped bacterium isolated from human feces. The organism was catalase-negative, resistant to 20% bile, produced acetic and butyric acids as end products of glucose metabolism, and possessed a G+C content of approximately 70 mol%. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of the Clostridium sub-phylum of the Gram-positive bacteria, and formed a loose association with rRNA cluster XV. Sequence divergence values of 12% or greater were observed between the unidentified bacterium and all other recognized species within this and related rRNA clusters. Treeing analysis showed the unknown anaerobe formed a deep line branching near to the base of rRNA cluster XV and phylogenetically represents a hitherto unknown taxon, distinct from Acetobacterium, Eubacterium sensu stricto, Pseudoramibacter and other related organisms. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from feces be classified in a new genus Anaerofustis, as Anaerofustis stercorihominis sp. nov. The type strain of Anaerofustis stercorihominis is ATCC BAA-858 T=CCUG 47767 T.