Carbohydrate catabolic flexibility in the mammalian intestinal commensal Lactobacillus ruminis revealed by fermentation studies aligned to genome annotations.
ABSTRACT: BACKGROUND: Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates. RESULTS: In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-?-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-?-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-?-D-Gluco-oligosaccharides. CONCLUSIONS: This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.
Project description:We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.
Project description:Lactobacillus ruminis, an autochthonous member of the gastrointestinal microbiota of humans and many animals, is a less characterized but interesting species for many reasons, including its intestinal prevalence and possible positive roles in host-microbe crosstalk. In this study, we isolated a novel L. ruminis strain (GRL 1172) from porcine feces and analyzed its functional characteristics and niche adaptation factors in parallel with those of three other L. ruminis strains (a human isolate, ATCC 25644, and two bovine isolates, ATCC 27780 and ATCC 27781). All the strains adhered to fibronectin, type I collagen, and human colorectal adenocarcinoma cells (HT-29), but poorly to type IV collagen, porcine intestinal epithelial cells (IPEC-1), and human colon adenocarcinoma cells (Caco-2). In competition assays, all the strains were able to inhibit the adhesion of Yersinia enterocolitica and enterotoxigenic Escherichia coli (ETEC, F4+) to fibronectin, type I; collagen, IPEC-1, and Caco-2 cells, and the inhibition rates tended to be higher than in exclusion assays. The culture supernatants of the tested strains inhibited the growth of six selected pathogens to varying extents. The inhibition was solely based on the low pH resulting from acid production during growth. All four L. ruminis strains supported the barrier function maintenance of Caco-2 cells, as shown by the modest increase in trans-epithelial electrical resistance and the prevention of dextran diffusion during co-incubation. However, the strains could not prevent the barrier damage caused by ETEC in the Caco-2 cell model. All the tested strains and their culture supernatants were able to provoke Toll-like receptor (TLR) 2-mediated NF-?B activation and IL-8 production in vitro to varying degrees. The induction of TLR5 signaling revealed that flagella were expressed by all the tested strains, but to different extents. Flagella and pili were observed by electron microscopy on the newly isolated strain GRL 1172.
Project description:Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of ?-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this ?-glucanase is an exo-acting enzyme on ?-(1,3)- and ?-(1,6)-linked glucan substrates and an endo-acting enzyme on ?-(1,4)-linked glucan substrates. Hydrolysis of short ?-(1,3), ?-(1,4), and ?-(1,3)/?-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-?-(1,3)/(1,6) and endo-?-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.
Project description:Commensal bacteria often have an especially rich source of glycan-degrading enzymes which allow them to utilize undigested carbohydrates from the food or the host. The species Ruminococcus gnavus is present in the digestive tract of ?90% of humans and has been implicated in gut-related diseases such as inflammatory bowel diseases (IBD). Here we analysed the ability of two R. gnavus human strains, E1 and ATCC 29149, to utilize host glycans. We showed that although both strains could assimilate mucin monosaccharides, only R. gnavus ATCC 29149 was able to grow on mucin as a sole carbon source. Comparative genomic analysis of the two R. gnavus strains highlighted potential clusters and glycoside hydrolases (GHs) responsible for the breakdown and utilization of mucin-derived glycans. Transcriptomic and functional activity assays confirmed the importance of specific GH33 sialidase, and GH29 and GH95 fucosidases in the mucin utilisation pathway. Notably, we uncovered a novel pathway by which R. gnavus ATCC 29149 utilises sialic acid from sialylated substrates. Our results also demonstrated the ability of R. gnavus ATCC 29149 to produce propanol and propionate as the end products of metabolism when grown on mucin and fucosylated glycans. These new findings provide molecular insights into the strain-specificity of R. gnavus adaptation to the gut environment advancing our understanding of the role of gut commensals in health and disease.
Project description:Inulin-type fructans (ITF) and arabinoxylan oligosaccharides (AXOS) are broken down to different extents by various bifidobacterial strains present in the human colon. To date, phenotypic heterogeneity in the consumption of these complex oligosaccharides at the strain level remains poorly studied. To examine mechanistic variations in ITF and AXOS constituent preferences present in one individual, ITF and AXOS consumption by bifidobacterial strains isolated from the simulator of the human intestinal microbial ecosystem (SHIME) after inoculation with feces from one healthy individual was investigated. Among the 18 strains identified, four species-independent clusters displaying different ITF and AXOS degradation mechanisms and preferences were found. Bifidobacterium bifidum B46 showed limited growth on all substrates, whereas B. longum B24 and B. longum B18 could grow better on short-chain-length fractions of fructooligosaccharides (FOS) than on fructose. B. longum B24 could cleave arabinose substituents of AXOS extracellularly, without using the AXOS-derived xylose backbones, whereas B. longum B18 was able to consume oligosaccharides (up to xylotetraose) preferentially and consumed AXOS to a limited extent. B. adolescentis B72 degraded all fractions of FOS simultaneously, partially degraded inulin, and could use xylose backbones longer than xylotetraose extracellularly. The strain-specific degradation mechanisms were suggested to be complementary and indicated resource partitioning. Specialization in the degradation of complex carbohydrates by bifidobacteria present on the individual level could have in vivo implications for the successful implementation of ITF and AXOS, aiming at bifidogenic and/or butyrogenic effects. Finally, this work shows the importance of taking microbial strain-level differences into account in gut microbiota research.IMPORTANCE It is well known that bifidobacteria degrade undigestible complex polysaccharides, such as ITF and AXOS, in the human colon. However, this process has never been studied for strains coexisting in the same individual. To examine strain-dependent mechanistic variations in ITF and AXOS constituent preferences present in one individual, ITF and AXOS consumption by bifidobacterial strains isolated from the SHIME after inoculation with feces from one healthy individual was investigated. Among the 18 bifidobacterial strains identified, four species-independent clusters displaying different ITF and AXOS degradation mechanisms and preferences were found, indicating that such strains can coexist in the human colon. Such specialization in the degradation of complex carbohydrates by bifidobacteria present on the individual level could have in vivo implications for the successful implementation of ITF and AXOS, aiming at bifidogenic and/or butyrogenic effects.
Project description:Carbohydrates are complex macromolecules in biological metabolism. Enzymatic synthesis of carbohydrates is recognized as a powerful tool to overcome the problems associated with large scale synthesis of carbohydrates. Novel enzymes with significant transglycosylation ability are still in great demand in glycobiology studies. Here we report a novel glycoside hydrolase family 16 "elongating" β-transglycosylase from Paecilomyces thermophila (PtBgt16A), which efficiently catalyzes the synthesis of higher polymeric oligosaccharides using β-1,3/1,4-oligosaccharides as donor/acceptor substrates. Further structural information reveals that PtBgt16A has a binding pocket around the -1 subsite. The catalytic mechanism of PtBgt16A is partly similar to an exo-glycoside hydrolase, which cleaves the substrate from the non-reducing end one by one. However, PtBgt16A releases the reducing end product and uses the remainder glucosyl as a transglycosylation donor. This catalytic mechanism has similarity with the catalytic mode of amylosucrase, which catalyzes the transglycosylation products gradually extend by one glucose unit. PtBgt16A thus has the potential to be a tool enzyme for the enzymatic synthesis of new β-oligosaccharides and glycoconjugates.
Project description:Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.
Project description:BACKGROUND: The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade. RESULTS: The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group. CONCLUSIONS: The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli.
Project description:Cellobiose 2-epimerase epimerizes and isomerizes ?-1,4- and ?-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6?Å resolution and belonged to the monoclinic space group P2?, with unit-cell parameters a=63.9, b=85.1, c=79.8?Å, ?=110.8°. With a Matthews coefficient VM of 2.18?Å3?Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85?Å resolution and belonged to the orthorhombic space group P2?2?2?, with unit-cell parameters a=55.9, b=80.0, c=93.7?Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26?Å3?Da(-1) and a solvent content of 45.6%.
Project description:Foodborne pathogens are a major source of morbidity and mortality worldwide. For this cause, exploring various effective ways of suppressing their spread is at the forefront of many research projects. The current study aims to investigate the <i>in vitro</i> organic acid production of <i>S. thermophilus</i> KLDS 3.1003 and <i>L. bulgaricus</i> KLDS 1.0207 strains, their <i>in vivo</i> suppression of and immuno-modulatory effects against <i>E. coli</i> ATCC 25922 and <i>S. aureus</i> ATCC 25923 pathogens. First, lactic and acetic acid production using three carbon sources - 1% glucose (control), 1% sucrose, and 1% fructo-oligosaccharides (FOS) - was determined by HPLC. For the <i>in vivo</i> section, a total of 40 BALB/c mice were purchased and divided into 10 treatment groups (control and nine treatments). Animals were given 1 week to acclimatize and then fed treatment diets for 14 days. Afterward, hematological (RBC, WBC, HB, PLT, Neutrophils, Eosinophils, Lymphocytes, and Monocytes) and histopathological analyses were carried out. All analyses were done in triplicate. Results show that lactic and acetic acid productions for both strains increased with supplementation and were highest after 1% FOS addition. Regardless of carbon source, <i>L. bulgaricus</i> KLDS 1.0207 produced higher (<i>P</i> < 0.05) amounts of lactic and acetic acids than <i>S. thermophilus</i> KLDS 3.1003. Also, generally better hematological parameters in probiotic groups than the control (<i>P</i> < 0.05) were observed. In some instances, mice in probiotic treatment groups had better immunity levels (lymphocytes, monocytes, neutrophils, eosinophils) than those in the control and pathogen groups. Histopathological studies showed that no anomalies were associated with <i>S. thermophilus</i> KLDS 3.1003 and <i>L. bulgaricus</i> KLDS 1.0207 administration. In conclusion, <i>S. thermophilus</i> KLDS 3.1003 and <i>L. bulgaricus</i> KLDS 1.0207 strains are not only probiotic candidates but can have therapeutic applications.