Source Tracking and Global Distribution of the Tigecycline Non-Susceptible tet(X).

The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America, and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes. IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors.

A compromised developmental trajectory of the infant gut microbiome and metabolome in atopic eczema.

Evidence is accumulating that the establishment of the gut microbiome in early life influences the development of atopic eczema. In this longitudinal study, we used integrated multi-omics analyses to infer functional mechanisms by which the microbiome modulates atopic eczema risk. We measured the functionality of the gut microbiome and metabolome of 63 infants between ages 3 weeks and 12 months with well-defined eczema cases and controls in a sub-cohort from the Growing Up in Singapore Toward healthy Outcomes (GUSTO) mother-offspring cohort. At 3 weeks, the microbiome and metabolome of allergen-sensitized atopic eczema infants were characterized by an enrichment of Escherichia coli and Klebsiella pneumoniae, associated with increased stool D-glucose concentration and increased gene expression of associated virulence factors. A delayed colonization by beneficial Bacteroides fragilis and subsequent delayed accumulation of butyrate and propionate producers after 3 months was also observed. Here, we describe an aberrant developmental trajectory of the gut microbiome and stool metabolome in allergen sensitized atopic eczema infants. The infographic describes an impaired developmental trajectory of the gut microbiome and metabolome in allergen-sensitized atopic eczema (AE) infants and infer its contribution in modulating allergy risk in the Singaporean mother-offspring GUSTO cohort. The key microbial signature of AE is characterized by (1) an enrichment of Escherichia coli and Klebsiella pneumoniae which are associated with accumulation of pre-glycolysis intermediates (D-glucose) via the trehalose metabolic pathway, increased gene expression of associated virulence factors (invasin, adhesin, flagellin and lipopolysaccharides) by utilizing ATP from oxidative phosphorylation and delayed production of butyrate and propionate, (2) depletion of Bacteroides fragilis which resulted in lower expression of immunostimulatory bacterial cell envelope structure and folate (vitamin B9) biosynthesis pathway, and (3) accompanied depletion of bacterial groups with the ability to derive butyrate and propionate through direct or indirect pathways which collectively resulted in reduced glycolysis, butyrate and propionate biosynthesis.

Divergent responses of bacterial activity, structure, and co-occurrence patterns to long-term unbalanced fertilization without nitrogen, phosphorus, or potassium in a cultivated vertisol.

Unbalanced fertilization lacking nitrogen (N), phosphorus (P), or potassium (K) is a worldwide phenomenon; however, whether they affect bacterial community composition and intraspecific interactions in a similar pattern and how they affect bacterial activity are not systematically compared. Soils under different kinds of unbalanced fertilization in a 21-year field experiment were collected to investigate the variation in dehydrogenase activity (DHA), bacterial community diversity, structure, composition, and possible interactions. Compared to the balanced fertilization of NPK, the DHA from unbalanced fertilization of NP, PK, and NK was 8.70, 11.59, and 14.17% lower, respectively, and from the unfertilized treatment (Nil) was 13.41% lower; however, the Shannon index from NP, PK, and Nil was 4.48-7.21% higher and from NK was 3.95% lower. Based on principal coordinate analyses (PCoA), bacterial community structure was separated by N application or not along PCo1 and was further separated by P application or not along PCo2, indicating a more influence by N deficiency. Moreover, the structure was mainly determined by soil pH, soil organic carbon (SOC), and total phosphorus (TP). The network complexity using co-occurrence analysis followed the order NP > NPK > PK > NK > Nil, indicating a more influence by P deficiency on intraspecific interactions. Structural equation modeling (SEM) revealed that the reduced DHA in NP was mainly regulated by the decreased SOC and increased Shannon index, in PK by the decreased SOC and increased Shannon index and pH, and in NK by the decreased SOC and TP and increased PCo2. The significantly lower abundance of Bacteroidetes and Chitinophagaceae in NK may also contribute to the reduced DHA. Our results imply that N deficiency had the greatest impact on bacterial community structure and composition, P deficiency had the greatest impact on network construction and bacterial activity, and K deficiency has minimal effect. Our results also suggest that main factors regulating the variation in soil functions may vary among different nutrient deficiencies.

Comparisons of Effects on Intestinal Short-Chain Fatty Acid Concentration after Exposure of Two Glycosidase Inhibitors in Mice.

Acarbose and voglibose are the most widely used diabetes drugs as glycosidase inhibitors. In this study, the use of these two inhibitors significantly increased the content of starch in large intestine, and altered the concentration of short-chain fatty acids (SCFAs) by affecting the intestinal microbiota. However, there are some differences in the intestinal microbiome of the two groups of mice, mainly in bacteria such as Bacteroidaceae bacteroides and Desulfovibrionaceae desulfovibrio. The productions of acetate and propionate in caecum in voglibose group were significantly higher than those in acarbose group and two kinds of glycosidase inhibitors were close in the production of butyrate in caecum. The Tax4Fun analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) data indicated that different productions of acetate and propionate between acarbose group and voglibose group may be related to 2-oxoisovalerate dehydrogenase and pyruvate oxidase. In addition, in-vitro experiments suggested that voglibose had less effect on epithelial cells than acarbose after direct stimulation. According to the recent researches of SCFAs produced by intestinal microbiota, our comparative study shown higher concentration of these beneficial fatty acids in the lumen of voglibose-treated mice, which implied a lower level of inflammation.

Intermittent Fasting Confers Protection in CNS Autoimmunity by Altering the Gut Microbiota.

Multiple sclerosis (MS) is more common in western countries with diet being a potential contributing factor. Here we show that intermittent fasting (IF) ameliorated clinical course and pathology of the MS model, experimental autoimmune encephalomyelitis (EAE). IF led to increased gut bacteria richness, enrichment of the Lactobacillaceae, Bacteroidaceae, and Prevotellaceae families and enhanced antioxidative microbial metabolic pathways. IF altered T cells in the gut with a reduction of IL-17 producing T cells and an increase in regulatory T cells. Fecal microbiome transplantation from mice on IF ameliorated EAE in immunized recipient mice on a normal diet, suggesting that IF effects are at least partially mediated by the gut flora. In a pilot clinical trial in MS patients, intermittent energy restriction altered blood adipokines and the gut flora resembling protective changes observed in mice. In conclusion, IF has potent immunomodulatory effects that are at least partially mediated by the gut microbiome.

Understanding the prebiotic potential of different dietary fibers using an in vitro continuous adult fermentation model (PolyFermS).

Consumption of fermentable dietary fibers (DFs), which can induce growth and/or activity of specific beneficial populations, is suggested a promising strategy to modulate the gut microbiota and restore health in microbiota-linked diseases. Until today, inulin and fructo-oligosaccharides (FOS) are the best studied DFs, while little is known about the gut microbiota-modulating effects of ß-glucan, α-galactooligosaccharide (α-GOS) and xylo-oligosaccharide (XOS). Here, we used three continuous in vitro fermentation PolyFermS model to study the modulating effect of these DFs on two distinct human adult proximal colon microbiota, independently from the host. Supplementation of DFs, equivalent to a 9 g daily intake, induced a consistent metabolic response depending on the donor microbiota. Irrespective to the DF supplemented, the Bacteroidaceae-Ruminococcaceae dominated microbiota produced more butyrate (up to 96%), while the Prevotellaceae-Ruminococcaceae dominated microbiota produced more propionate (up to 40%). Changes in abundance of specific bacterial taxa upon DF supplementation explained the observed changes in short-chain fatty acid profiles. Our data suggest that the metabolic profile of SCFA profile may be the most suitable and robust read-out to characterize microbiota-modulating effects of a DF and highlights importance to understand the inter-individual response to a prebiotic treatment for mechanistic understanding and human application.

The Starch Utilization System Assembles around Stationary Starch-Binding Proteins.

Bacteroides thetaiotaomicron (Bt) is a prominent member of the human gut microbiota with an extensive capacity for glycan harvest. This bacterium expresses a five-protein complex in the outer membrane, called the starch utilization system (Sus), which binds, degrades, and imports starch into the cell. Sus is a model system for the many glycan-targeting polysaccharide utilization loci found in Bt and other members of the Bacteroidetes phylum. Our previous work has shown that SusG, a lipidated amylase in the outer membrane, explores the entire cell surface but diffuses more slowly as it interacts with starch. Here, we use a combination of single-molecule tracking, super-resolution imaging, reverse genetics, and proteomics to show that SusE and SusF, two proteins that bind starch, are immobile on the cell surface even when other members of the system are knocked out and under multiple different growth conditions. This observation suggests a new paradigm for protein complex formation: binding proteins form immobile complexes that transiently associate with a mobile enzyme partner.

Shifting the balance of fermentation products between hydrogen and volatile fatty acids: microbial community structure and function.

Fermentation is a key process in many anaerobic environments. Varying the concentration of electron donor fed to a fermenting community is known to shift the distribution of products between hydrogen, fatty acids and alcohols. Work to date has focused mainly on the fermentation of glucose, and how the microbial community structure is affected has not been explored. We fed ethanol, lactate, glucose, sucrose or molasses at 100 me- eq. L-1, 200 me- eq. L-1 or 400 me- eq. L-1 to batch-fed cultures with fermenting, methanogenic communities. In communities fed high concentrations of electron donor, the fraction of electrons channeled to methane decreased, from 34% to 6%, while the fraction of electrons channeled to short chain fatty acids increased, from 52% to 82%, averaged across all electron donors. Ethanol-fed cultures did not produce propionate, but did show an increase in electrons directed to acetate as initial ethanol concentration increased. In glucose, sucrose, molasses and lactate-fed cultures, propionate accumulation co-occurred with known propionate producing organisms. Overall, microbial communities were determined by the substrate provided, rather than its initial concentration, indicating that a change in community function, rather than community structure, is responsible for shifts in the fermentation products produced.

Inositol-phosphodihydroceramides in the periodontal pathogen Tannerella forsythia: Structural analysis and incorporation of exogenous myo-inositol.

BACKGROUND: Unique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed. METHODS: Lipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI-MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium. RESULTS: T. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2. CONCLUSION: The study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol. GENERAL SIGNIFICANCE: The effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets.

Structural basis for the divergence of substrate specificity and biological function within HAD phosphatases in lipopolysaccharide and sialic acid biosynthesis.

The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, and are separated by diffuse active-site sequence boundaries. We infer a high tolerance of the KDO8PP catalytic platform to amino acid replacements that in turn influence substrate specificity changes and thereby facilitate the divergence in biological function.

Multiple infections with Cardinium and two strains of Wolbachia in the spider mite Tetranychus phaselus Ehara: revealing new forces driving the spread of Wolbachia.

Cytoplasmic incompatibility (CI) has been proposed as a major mechanism by which certain strains of Wolbachia to invade and persist in host populations. However, mechanisms that underlie the invasion and persistence of non-CI strains are less well understood. Here, we established a spider mite Tetranychus phaselus population multiply infected by Cardinium as well as two distinct lineages of Wolbachia, designated wCon and wOri, to study the forces driving the spread of the non-CI strain of Wolbachia wOri. Interestingly, we found that wOri provided a longevity advantage to its female hosts under ideal conditions, making wOri stay longer in this population, and then being transmitted to more offspring. Furthermore, the lifespan of uninfected females was reduced when mated with multiple-infected males. As a result, the uninfected population is attenuated by the multiple-infected males. Thus, we infer that the host age effects of multiple infection may represent sufficient forces driving the spread of wOri through the host population.

Production of secoisolariciresinol from defatted flaxseed by bacterial biotransformation.

AIMS: Secoisolariciresinol (SECO) is increasingly recognized for potential clinical application because of its preventive effects against breast and colon cancers, atherosclerosis and diabetes, and its production through biotransformation has been attempted. However, previously reported bacteria all required stringent anaerobic culture conditions, precluding large-scale production. Here, we report the isolation and characterization of bacteria that produce SECO under less stringent anaerobic culture conditions. METHODS AND RESULTS: Using defatted flaxseed as raw material, we isolated a facultative anaerobic bacterium from human faeces that hydrolysed secoisolariciresinol diglucoside-3-hydroxy-3-methyl glutaric acid (SDG-HMGA) oligomers in flaxseed to produce SECO. Both conventional assays and 16S rRNA gene sequence analysis demonstrated its close relatedness with Bacteroides uniformis. The transformation efficiency of SDG in defatted flaxseed to SECO was more than 80% by this bacterial strain. We investigated factors that might influence fermentation, such as redox potential and pH, for large-scale fermentation of defatted flaxseed to produce SECO. CONCLUSIONS: The method to produce SECO through biotransformation of defatted flaxseed with this bacterial strain is highly efficient and economic. SIGNIFICANCE AND IMPACT OF THE STUDY: This bacterial strain can transform SDG to SECO under less stringent anaerobic culture conditions, which will greatly facilitate industry-scale production of SECO.

Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a simulator of the human intestinal microbial ecosystem.

In this study, the prebiotic potential of arabinoxylan oligosaccharides (AXOS) was compared with inulin in two simulators of the human intestinal microbial ecosystem. Microbial breakdown of both oligosaccharides and short-chain fatty acid production was colon compartment specific, with ascending and transverse colon being the predominant site of inulin and AXOS degradation, respectively. Lactate levels (+5.5 mM) increased in the ascending colon during AXOS supplementation, while propionate levels (+5.1 mM) increased in the transverse colon. The concomitant decrease in lactate in the transverse colon suggests that propionate was partially formed over the acrylate pathway. Furthermore, AXOS supplementation strongly decreased butyrate in the ascending colon, this in parallel with a decrease in Roseburia spp. and Bacteroides/Prevotella/Porphyromonas (-1.4 and -2.0 log CFU) levels. Inulin treatment had moderate effects on lactate, propionate and butyrate levels. Denaturing gradient gel electrophoresis analysis revealed that inulin changed microbial metabolism by modulating the microbial community composition. In contrast, AXOS primarily affected microbial metabolism by 'switching on' AXOS-degrading enzymes (xylanase, arabinofuranosidase and xylosidase), without significantly affecting microbial community composition. Our results demonstrate that AXOS has a higher potency than inulin to shift part of the sugar fermentation toward the distal colon parts. Furthermore, due to its stronger propionate-stimulating effect, AXOS is a candidate prebiotic capable of lowering cholesterol and beneficially affecting fat metabolism of the host.

Survival and persistence of human and ruminant-specific faecal Bacteroidales in freshwater microcosms.

Amplification of host-specific markers from Bacteroidales faecal anaerobes can rapidly identify the source of faecal pollution. It is necessary to understand persistence and survival of these markers and marker cells, both to interpret quantitative source-tracking data, and to use such data to predict pathogen occurrence. We measured marker persistence and cell survival of two human (HF134, HF183) and two ruminant (CF128, CF193) faecal Bacteroidales markers, compared with Escherichia coli and enterococci. Freshwater microcosms were inoculated with fresh cattle or human faeces and incubated at 13 degrees C in natural light or darkness. Marker persistence was measured by polymerase chain reaction (PCR) and quantitative PCR. Survival of marker cells was measured by real-time quantitative PCR. There was no difference in persistence between the two human-specific Bacteroidales DNA markers in the light and dark microcosms. Cell survival profiles of the two human markers were also similar; both were significantly affected by light. Ruminant markers persisted and survived longer than human markers (14 versus 6 days respectively). CF193 decreased more rapidly than CF128, and light significantly affected CF128 but not CF193. These results support use of host-specific faecal Bacteroidales markers as indicators of recent faecal pollution, but suggest that caution is needed in interpreting quantitative results to indicate proportional contribution of different sources, as individual markers differ in their survival, persistence and response to environmental variables. The survival and persistence profiles for Bacteroidales markers are consistent with survival profiles for several faecal pathogens.

Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide.

Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 microM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.

Roles of mucosal bacteria and succinic acid in colitis caused by dextran sulfate sodium in mice.

Intestines of mice with colitis caused by dextran sulfate sodium (DSS) contain more Bacteroidaceae cells than untreated controls. We investigated the roles of intestinal bacteria and succinic acid, a by-product of Bacteroidaceae metabolism, in this model of colitis. CBA/J mice were given 3% DSS in water for 14 days. After mice were anesthetized and killed, concentrations of organic acids in stools from the cecum and colon were measured. The resected rectum and colon were washed with sterile saline; some specimens were incubated with imipenem in saline for 1 h to kill bacteria on the surfaces and others were not. Their homogenates were cultured anaerobically and aerobically. Separately, 1 mL of 20 mM succinic acid was infused into the rectum of mice, whose anal verge was glued. Animals were anesthetized and killed the next day. The rectum and colon were examined histologically. Concentrations of succinate were higher everywhere in the colon of mice with colitis than in controls. Mice with colitis had more Bacteroidaceae cells, especially B. caccae, than controls. Mice given succinate enemas had focal erosions of the mucosa and edema of the submucosa. Succinic acid, produced abundantly by members of the family Bacteroidaceae, especially B. caccae, may be the ulcerogenic agent in DSS colitis.

In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny.

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K(m) of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1.

Ecology, metabolism, and genetics of ruminal selenomonads.

Selenomonas ruminantium is one of the more prominent and functionally diverse bacteria present in the rumen and can survive under a wide range of nutritional fluctuations. Selenomonas is not a degrader of complex polysaccharides associated with dietary plant cell wall components, but is important in the utilization of soluble carbohydrates released from initial hydrolysis of these polymers by other ruminal bacteria. Selenomonads have multiple carbon flow routes for carbohydrate catabolism and ATP generation, and subspecies differ in their ability to use lactate. Some soluble carbohydrates (glucose, sucrose) appear to be transported via the phosphoenolpyruvate phosphotransferase system, while arabinose and xylose are transported by proton symport. High cell yields and the presence of electron transport components in Selenomonas strains has been documented repeatedly and this may partially account for the energy partitioning observed between energy consumed for growth and maintenance functions. Most strains can utilize ammonia, protein, and/or amino acids as a nitrogen source. Some strains can hydrolyze urea and/or reduce nitrate and use the ammonia for the biosynthesis of amino acids. Experimental evidence suggests that ammonia assimilatory enzymes in some strains may possess unique properties with respect to other presumably similar bacteria. Little is known about the genetics of ruminal selenomonads. Plasmid DNA has been isolated from some strains, but it is unknown what physiological functions may be encoded on these extrachromosomal elements. Due to the predominance of S. ruminantium in the rumen, it is an ideal candidate for genetic manipulation. Once the genetics of this bacterium are better understood, it may be possible to amplify its role in the rumen.

Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces.

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H2, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39-42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.