Galacto- and Fructo-oligosaccharides Utilized for Growth by Cocultures of Bifidobacterial Species Characteristic of the Infant Gut.

Bifidobacterial species are common inhabitants of the gut of human infants during the period when milk is a major component of the diet. Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum subspecies longum, and B. longum subspecies infantis have been detected frequently in infant feces, but B. longum subsp. infantis may be disadvantaged numerically in the gut of infants in westernized countries. This may be due to the different durations of breast milk feeding in different countries. Supplementation of the infant diet or replacement of breast milk using formula feeds is common in Western countries. Formula milks often contain galacto- and/or fructo-oligosaccharides (GOS and FOS, respectively) as additives to augment the concentration of oligosaccharides in ruminant milks, but the ability of B. longum subsp. infantis to utilize these potential growth substrates when they are in competition with other bifidobacterial species is unknown. We compared the growth and oligosaccharide utilization of GOS and FOS by bifidobacterial species in pure culture and coculture. Short-chain GOS and FOS (degrees of polymerization [DP] 2 and 3) were favored growth substrates for strains of B. bifidum and B. longum subsp. longum, whereas both B. breve and B. longum subsp. infantis had the ability to utilize both short- and longer-chain GOS and FOS (DP 2 to 6). B. breve was nevertheless numerically dominant over B. longum subsp. infantis in cocultures. This was probably related to the slower use of GOS of DP 3 by B. longum subsp. infantis, indicating that the kinetics of substrate utilization is an important ecological factor in the assemblage of gut communities.IMPORTANCE The kinds of bacteria that form the collection of microbes (the microbiota) in the gut of human infants may influence health and well-being. Knowledge of how the composition of the infant diet influences the assemblage of the bacterial collection is therefore important because dietary interventions may offer opportunities to alter the microbiota with the aim of improving health. Bifidobacterium longum subspecies infantis is a well-known bacterial species, but under modern child-rearing conditions it may be disadvantaged in the gut. Modern formula milks often contain particular oligosaccharide additives that are generally considered to support bifidobacterial growth. However, studies of the ability of various bifidobacterial species to grow together in the presence of these oligosaccharides have not been conducted. These kinds of studies are essential for developing concepts of microbial ecology related to the influence of human nutrition on the development of the gut microbiota.

Sharing a ß-Glucan Meal: Transcriptomic Eavesdropping on a Bacteroides ovatus-Subdoligranulum variabile-Hungatella hathewayi Consortium.

Whole-transcriptome analysis was used to investigate the molecular interplay between three bacterial species that are members of the human gut microbiota. Bacteroides ovatus, Subdoligranulum variabile, and Hungatella hathewayi formed associations in cocultures fed barley ß-glucan, a constituent of dietary fiber. B. ovatus depolymerized ß-glucan and released, but did not utilize, 3-O-ß-cellobiosyl-d-glucose (DP3) and 3-O-ß-cellotriosyl-d-glucose (DP4). These oligosaccharides provided growth substrates for S. variabile and H. hathewayi with a preference for DP4 in the case of the latter species. There was increased transcription of a B. ovatus mixed-linkage-ß-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the ß-glucan utilization locus did not occur in continuous culture. Evidence for interactions relating to provision of cobalamin, alterations to signaling, and modulation of the "stringent response" (an adaptation to nutrient deprivation) were detected. Overall, we established a bacterial consortium based on barley ß-glucan in vitro, which can be used to investigate aspects of the functional blueprint of the human gut microbiota.IMPORTANCE The microbial community, mostly composed of bacterial species, residing in the human gut degrades and ferments polysaccharides derived from plants (dietary fiber) that would not otherwise be digested. In this way, the collective metabolic actions of community members extract additional energy from the human diet. While the variety of bacteria present in the microbial community is well known, the formation of bacterial consortia, and the consequent interactions that result in the digestion of dietary polysaccharides, has not been studied extensively. The importance of our work was the establishment, under laboratory conditions, of a consortium of gut bacteria that formed around a dietary constituent commonly present in cereals. This enabled the metabolic interplay between the bacterial species to be studied. This kind of knowledge is required to construct an interactive, metabolic blueprint of the microbial community that inhabits the human gut.

Substrate Use Prioritization by a Coculture of Five Species of Gut Bacteria Fed Mixtures of Arabinoxylan, Xyloglucan, ß-Glucan, and Pectin.

Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, ß-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order ß-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and ß-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology.IMPORTANCE This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 µm long was observed.

Preferential use of plant glycans for growth by Bacteroides ovatus.

B. ovatus is a member of the human gut microbiota with a broad capability to degrade complex glycans. Here we show that B. ovatus degrades plant polysaccharides in a preferential order, and that glycan structural complexity plays a role in determining the prioritisation of polysaccharide usage.

Bifidobacterium bifidum ATCC 15696 and Bifidobacterium breve 24b Metabolic Interaction Based on 2'-O-Fucosyl-Lactose Studied in Steady-State Cultures in a Freter-Style Chemostat.

Infants fed breast milk harbor a gut microbiota in which bifidobacteria are generally predominant. The metabolic interactions of bifidobacterial species need investigation because they may offer insight into the colonization of the gut in early life. Bifidobacterium bifidum ATCC 15696 hydrolyzes 2'-O-fucosyl-lactose (2FL; a major fucosylated human milk oligosaccharide) but does not use fucose released into the culture medium. However, fucose is a growth substrate for Bifidobacterium breve 24b, and both strains utilize lactose for growth. The provision of fucose and lactose by B. bifidum (the donor) allowing the growth of B. breve (the beneficiary) conforms to the concept of syntrophy, but both strains will compete for lactose to multiply. To determine the metabolic impact of this syntrophic/competitive relationship on the donor, the transcriptomes of B. bifidum were determined and compared in steady-state monoculture and coculture using transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR). B. bifidum genes upregulated in coculture included those encoding alpha-l-fucosidase and carbohydrate transporters and those involved in energy production and conversion. B. bifidum abundance was the same in coculture as in monoculture, but B. breve dominated the coculture numerically. Cocultures during steady-state growth in 2FL medium produced mostly acetate with little lactate (acetate:lactate molar ratio, 8:1) compared to that in monobatch cultures containing lactose (2:1), which reflected the maintenance of steady-state cells in log-phase growth. Darwinian competition is an implicit feature of bacterial communities, but syntrophy is a phenomenon putatively based on cooperation. Our results suggest that the regulation of syntrophy, in addition to competition, may shape bacterial communities.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the infant bowel) using in vitro experimentation with bacterial cultures maintained under controlled growth and environmental conditions. We studied the growth of bifidobacteria whose nutrition centered on the hydrolysis of a human milk oligosaccharide. The results revealed responses relating to metabolism occurring in a Bifidobacterium bifidum strain when it provided nutrients that allowed the growth of Bifidobacterium breve, and so discovered biochemical features of these bifidobacteria in relation to metabolic interaction in the shared environment. These kinds of experiments are essential in developing concepts of bifidobacterial ecology that relate to the development of the gut microbiota in early life.

Polysaccharide compositions of collenchyma cell walls from celery (Apium graveolens L.) petioles.

BACKGROUND: Collenchyma serves as a mechanical support tissue for many herbaceous plants. Previous work based on solid-state NMR and immunomicroscopy suggested collenchyma cell walls (CWs) may have similar polysaccharide compositions to those commonly found in eudicotyledon parenchyma walls, but no detailed chemical analysis was available. In this study, compositions and structures of cell wall polysaccharides of peripheral collenchyma from celery petioles were investigated. RESULTS: This is the first detailed investigation of the cell wall composition of collenchyma from any plant. Celery petioles were found to elongate throughout their length during early growth, but as they matured elongation was increasingly confined to the upper region, until elongation ceased. Mature, fully elongated, petioles were divided into three equal segments, upper, middle and lower, and peripheral collenchyma strands isolated from each. Cell walls (CWs) were prepared from the strands, which also yielded a HEPES buffer soluble fraction. The CWs were sequentially extracted with CDTA, Na2CO3, 1 M KOH and 4 M KOH. Monosaccharide compositions of the CWs showed that pectin was the most abundant polysaccharide [with homogalacturonan (HG) more abundant than rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II)], followed by cellulose, and other polysaccharides, mainly xyloglucans, with smaller amounts of heteroxylans and heteromannans. CWs from different segments had similar compositions, but those from the upper segments had slightly more pectin than those from the lower two segments. Further, the pectin in the CWs of the upper segment had a higher degree of methyl esterification than the other segments. In addition to the anticipated water-soluble pectins, the HEPES-soluble fractions surprisingly contained large amounts of heteroxylans. The CDTA and Na2CO3 fractions were rich in HG and RG-I, the 1 M KOH fraction had abundant heteroxylans, the 4 M KOH fraction was rich in xyloglucan and heteromannans, and cellulose was predominant in the final residue. The structures of the xyloglucans, heteroxylans and heteromannans were deduced from the linkage analysis and were similar to those present in most eudicotyledon parenchyma CWs. Cross polarization with magic angle spinning (CP/MAS) NMR spectroscopy showed no apparent difference in the rigid and semi-rigid polysaccharides in the CWs of the three segments. Single-pulse excitation with magic-angle spinning (SPE/MAS) NMR spectroscopy, which detects highly mobile polysaccharides, showed the presence of arabinan, the detailed structure of which varied among the cell walls from the three segments. CONCLUSIONS: Celery collenchyma CWs have similar polysaccharide compositions to most eudicotyledon parenchyma CWs. However, celery collenchyma CWs have much higher XG content than celery parenchyma CWs. The degree of methyl esterification of pectin and the structures of the arabinan side chains of RG-I show some variation in the collenchyma CWs from the different segments. Unexpectedly, the HEPES-soluble fraction contained a large amount of heteroxylans.

Monoglobus pectinilyticus gen. nov., sp. nov., a pectinolytic bacterium isolated from human faeces.

A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae, but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7 % sequence similarity). Strain 14T shared ~99 % sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6 µm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae. The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae, Which Forms Biofilms Composed of a Novel Exopolysaccharide.

Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 µm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α-d-rhamnan with side chains of terminal α-d-Fucf and an α-d-1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds.

RNA-stable-isotope probing shows utilization of carbon from inulin by specific bacterial populations in the rat large bowel.

Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope ((13)C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [(13)C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect (13)C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the (13)C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA-stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut.

Discovery of lipids from B. longum subsp. infantis using whole cell MALDI analysis.

Bifidobacteria are dominant members of the microbial community in the intestinal tract of infants, and studies have shown that glycolipids extracted from the cell surface of these bacteria elicit beneficial immune responses. Accordingly, the identification and structural characterization of glycolipids from the cell wall of bifidobacteria is the first step in correlating glycolipid structure with biological activity. Using whole cell MALDI as a screening tool, we herein present for the first time the identification and structural elucidation of the major polar lipids from Bifidobacterium longum subs. infantis. The lipids identified include an unprecedented plasmenyl cyclophosphatidic acid and a mixed acetal glycolipid, with the latter subsequently being isolated and found to suppress the innate immune response.

In vitro fermentation of prebiotic oligosaccharides by Bifidobacterium lactis HN019 and Lactobacillus spp.

The utilisation of various prebiotic oligosaccharides by probiotic strains of Bifidobacterium lactis, Lactobacillus rhamnosus and Lactobacillus acidophilus was investigated in order to determine the synbiotic potential of various prebiotic/probiotic combinations. Analysis by HPLC and high-performance anion-exchange chromatography of the cell-free medium taken during growth of the three probiotic bacteria showed differences in the consumption of the various oligosaccharides. Analysis of galactooligosaccharides showed that both L. rhamnosus and B. lactis consumed mostly mono- and di-saccharide, while L. acidophilus consumed oligosaccharides up to trisaccharide. Both B. lactis and L. acidophilus utilised fructooligosaccharides and inulin, but showed different patterns of oligosaccharide consumption. Only L. rhamnosus grew on ß-glucan oligosaccharides and preferentially consumed the trisaccharide. The results indicate the synbiotic potential of the various probiotic/prebiotic combinations, particularly L. acidophilus/galactooligosaccharides, L. acidophilus/fructooligosaccharides or inulin and L. rhamnosus/ß-glucan oligosaccharides.

Does harvesting age matter? Changes in structure and rheology of a shear-thickening polysaccharide from Cyathea medullaris as a function of age.

A shear-thickening polysaccharide from the New Zealand Black tree fern (Cyathea medullaris, commonly known as mamaku) extracted from different age fronds (stage 1: young, stage 2: fully grown and stage 3: old) was characterised in terms of structure and rheological properties. Constituent sugar analysis and 1H and 13C NMR revealed a repeating backbone of -4)-ß-D-GlcpA-(1 â†’ 2)-α-D-Manp-(1→, for all mamaku polysaccharide (MP) samples from different age fronds without any alterations in molecular structure. However, the molecular weight (Mw) was reduced with increasing age, from ~4.1 × 106 to ~2.1 × 106 Da from stage 1 to stage 3, respectively. This decrease in Mw (and size) consequently reduced the shear viscosity (ηs-Stage 1 > Î·s-Stage 2 > Î·s-Stage 3). However, the extent of shear-thickening and uniaxial extensional viscosity of MP stage 2 was greater than MP stage 1, which was attributed to a greater intermolecular interaction occurring in the former. Shear-thickening behaviour was not observed in MP stage 3.

Ulvans are not equal - Linkage and substitution patterns in ulvan polysaccharides differ with Ulva morphology.

Ulva are hardy green seaweeds that contain the sulfated polysaccharide ulvan and grow in two distinct morphologies: foliose and tubular. The authors hypothesise that ulvan from tubular species are more structurally complex than ulvans from foliose species. Herein, using standardised methods, the glycosyl linkage positions and sulfate ester substitutions of constituent monosaccharides of ulvan isolated from foliose (U. lacinulata and U. stenophylloides) and tubular (U. prolifera and U. ralfsii) species of Ulva were investigated. Comparison of native ulvans with 80 and 100 °C desulfated counterparts indicated that 4-linked rhamnose is predominantly 3-O-sulfated in all four ulvans. Ulvans from the foliose species predominantly contained →3,4)-Rhap-(1→, →4)-GlcAp-(1→ and →4)-IdoAp-(1→, collectively accounting for 67 to 81 mol% of the total linkages. In contrast, these same linkages in ulvans from the tubular species only collectively accounted for 29 to 36 mol%. Instead, ulvan from tubular species contained a combination of →2,3,4)-Rhap-(1→, terminal Rhap-(1→, →4)-GlcAp-(1→, →4)-Xylp-(1→, and/or →4)-Galp-(1→ in high proportions; some of the latter three residues were also likely O-2 sulfated. The results presented here suggest that ulvan from foliose species are predominantly unbranched polysaccharides composed of repeat disaccharides while ulvans from tubular species contain a greater diversity of branch and sulfate substitution locations.

Whole-transcriptome shotgun sequencing (RNA-seq) screen reveals upregulation of cellobiose and motility operons of Lactobacillus ruminis L5 during growth on tetrasaccharides derived from barley ß-glucan.

Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley ß-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-ß-cellotriosyl-d-glucose [LDP4] or 4-O-ß-laminaribiosyl-d-cellobiose [CDP4]) present in ß-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3-O-ß-cellotriosyl-d-glucose released by ß-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis.

Novel 4-O-ß-d-xylopyranosyl-3,6-anhydro-l-galactopyranosyl disaccharide units in a polysaccharide from the red alga Pyrophyllon subtumens.

Thalli of the endemic epiphytic New Zealand red seaweed Pyrophyllon subtumens are known to contain a high level of xylose and a notable amount of arabinose but the extracted polysaccharide has not been characterised. The linkage/substitution of individual sugars within the water-soluble polysaccharide extract and various derivatives were determined by chemical and spectroscopic methods. No 3-linked sugars nor any d-galactose were found, which excluded agar-, carrageenan- or mixed 3-linked/4-linked ß-d-xylan-type polysaccharides found in many other red macroalgae. Instead, the polysaccharide backbone contained predominantly 4-linked ß-d-xylopyranosyl, 4-linked 3,6-anhydro-l-galactopyranosyl and 4-linked l-galactopyranosyl units. Some of each type of sugar were sulfated at various positions. Some xylosyl units were substituted at the 2- or 3-position with l-arabinosyl units. The polysaccharide is complex and likely contains a range of structures. However, partial sequencing was successfully used to recover and identify a novel disaccharide 4-O-d-xylopyranosyl-3,6-anhdydro-l-galactopyranose, which indicates a unique →4)-ß-d-Xylp-(1 â†’ 4)-3,6-anhydro-l-Galp-(1 â†’ repeat unit in the polysaccharide.

Characterizing kiwifruit carbohydrate utilization in vitro and its consequences for human faecal microbiota.

It is well accepted that our gut bacteria have coevolved with us in relation to our genetics, diet and lifestyle and are integrated metabolically with us to affect our gut health adversely or beneficially. "Who is there" may vary quite widely between individuals, as might "how they do it", but "what they make" may be less variable. Many different individual species of bacteria can perform the same saccharolytic functions and so the availability of substrate (host or diet-derived) along with the degradative enzymes they possess may be key drivers of gut ecology. In this case study, we discuss detailed microbial ecology and metabolism analysis for three individuals following 48 h of in vitro faecal fermentation, using green kiwifruit as the substrate. In parallel, we have analyzed the chemical changes to the kiwifruit carbohydrates present in the fermenta to close the circle on substrate usage/degradative enzymes possessed/microbes present/microbial byproducts produced. In the absence of host carbohydrate, we see that kiwifruit carbohydrates were differentially utilized to drive microbial diversity, yet resulted in similar byproduct production. The starting ecology of each individual influenced the quantitative and qualitative microbial changes; but not necessarily the metabolic byproduct production. Thus, we propose that it is the consistent functional changes that are relevant for assessment of gut health benefits of any food. We recommend that in this era of large scale genotype/-omics studies that hypothesis-driven, bottom-up research is best placed to interpret metagenomic data in parallel with functional, phenotypic data.

Synthesis and biological activity of the lipoteichoic acid anchor from Streptococcus sp. DSM 8747.

In this study, the role of lipoteichoic acid (LTA) anchors in the activation of the innate immune response was investigated through the chemical synthesis of a series of LTA derivatives and the determination of their ability to induce NO production in bone marrow-derived macrophages (BMM). To this end, an efficient synthesis of the sn-3-O-(α-D-galactofuranosyl)-1,2-di-O-acylglycerol LTA core was developed, which was then used as a key structure to produce both phosphate and glycerylphosphate-funtionalised LTA anchors, as well as galactofuranosyldiglycerides with different fatty acid chain lengths. With a series of LTA anchors in hand, we then determined the effect of these glycolipids on the innate immune response by exploring their capacity to activate macrophages. Here, we report that several of the LTA-derivatives were able to induce NO production by BMMs. In general, the unnatural (sn-1) core glycolipid anchors showed lower levels of activity than the corresponding natural (sn-3) analogues, and the activity of the glycolipids also appears to be dependent on the length of lipid present, with an optimum lipid length of C20 for the sn-3 derivatives. Interestingly, a triacylated anchor and the 6-O-phosphorylated anchor, showed only modest activity, while the 6-O-glycerophosphorylated derivative was unable to induce NO production. Taken as a whole, our results highlight the subtle effects that glycolipid length can have on the ability to activate BMMs.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.