Project description:Bacteroidetes have multiple polysaccharide utilization loci (PUL) which are specific for the decomposition of polysaccharides. The marine Bacteroidetes strain Flavimarina sp. Hel_I_48 encodes two separate PULs which target xylose-containing polysaccharides. We elucidated the specificity of these two PULs by correlating proteome data with biochemical activities of the encoded carbohydrate active enzymes. Proteomics revealed that one PUL targets glucuronoxylans whereas the other PUL targets arabinoxylans.
Project description:Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria (MHB), using largely uncharacterized mechanisms. In Zobellia galactanivorans, we discovered and characterized the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, providing a model system for carrageenan utilization by MHB. We further demonstrate that carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and several distal genes. The genetic structure of the carrageenan utilization system is well conserved in marine Bacteroidetes, but modified in other MHB phyla. The core system is completed by additional functions which can be assumed by non-orthologous genes in different species. This complex genetic structure is due to multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.
Project description:The marine Flavobacterium Formosa agariphila KMM 3901T is able to use a broad range of different carbohydrates as growth substrates. This is reflected in the strain’s repertoire of 13 polysaccharide utilization loci (PUL) in total. One PUL – termed as PUL H – is responsible for ulvan degradation, which is a widely distributed, algal-derived polysaccharide. The PUL comprises almost 40 genes, coding for transporters, lyases, glycoside hydrolases or sulfatases, among others. These proteins catalyse the breakdown of ulvan or the uptake of degradation products. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed that corresponding PUL encoded proteins were substrate specific up-regulated in ulvan-cultivated cells. The sulphated polysaccharide ulvan also induced the specific expression of proteins necessary for subsequent monosaccharide degradation. Compared to a control (fructose-cultivated cells), expression of PUL H additionally responded to rhamnose, a basic component of ulvan, indicating that this monosaccharide might signal ulvan availability in the environment. Our proteome analyses proofed a substrate specific expression of proteins involved in ulvan utilization and allowed us to deduce a comprehensive degradation pathway for this complex marine polysaccharide.
Project description:The adult human gut microbial community is typically dominated by two bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from E. rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the former possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole genome transcriptional profiling of both organisms in their distal gut (cecal) habitat as well as host responses, high resolution proteomic analysis of cecal contents, and biochemical assays of carbohydrate metabolism. B. thetaiotaomicron adapts to E. rectale by upregulating expression of a variety of polysaccharide utilization loci (PULs) encoding numerous glycoside hydrolase gene families, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is utilized by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of major gut bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability. Experiment Overall Design: four biological replicates per group. NMRI mice, males (12-14 weeks old).
Project description:The adult human gut microbial community is typically dominated by two bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from E. rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the former possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole genome transcriptional profiling of both organisms in their distal gut (cecal) habitat as well as host responses, high resolution proteomic analysis of cecal contents, and biochemical assays of carbohydrate metabolism. B. thetaiotaomicron adapts to E. rectale by upregulating expression of a variety of polysaccharide utilization loci (PULs) encoding numerous glycoside hydrolase gene families, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is utilized by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of major gut bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability. The interactions between E. rectale and B. thetaiotaomicron were characterized by performing whole genome transcriptional profiling of each species after colonization of gnotobiotic mice with each organism alone, or in combination. E. rectale was also profiled during in vitro growth.
Project description:The phylum Bacteroidetes is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host, and a potential role in a wide range of disease syndromes. The predominance of Bacteroidetes and the genus Bacteroides in the distal gut is due in large part to the expansion of paralogous gene clusters, termed Polysaccharide Utilization Loci (PULs), dedicated to the uptake and catabolism of host derived and dietary polysaccharides. It is generally thought that the diversity of PULs is key to Bacteroides successful competition for nutrients in the gut environment. The nutritive value of the available polysaccharides varies greatly and thus their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that control expression in response to the presence of specific glycan substrates. However the existence of additional regulatory mechanisms has been predicted to explain phenomena such as the hierarchical control, catabolite repression, and the fine tuning of gene expression to match catabolic activity. Using Bacteroides fragilis as a model organism, this report describes a previously unknown layer of regulatory control in which cis-encoded antisense small RNAs (sRNA) act as repressors of the PULs’ catabolic genes. Nearly 30% of B. fragilis PULs are subject to this type of sRNA control and these PULs tend to be more closely linked to the utilization of host-derived glycans than dietary polysaccharides. The findings described here indicate the presence of a global control mechanism that underlies the known regulatory circuits which modulate PUL expression in response to substrate availability, and hence provide novel insight into regulation of the gut Bacteroidetes physiology.
Project description:The phylum Bacteroidetes is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host, and a potential role in a wide range of disease syndromes1-3. The predominance of Bacteroidetes and the genus Bacteroides in the distal gut is due in large part to the expansion of paralogous gene clusters, termed Polysaccharide Utilization Loci (PULs), dedicated to the uptake and catabolism of host derived and dietary polysaccharides4,5. It is generally thought that the diversity of PULs is key to Bacteroides successful competition for nutrients in the gut environment6. The nutritive value of the available polysaccharides varies greatly and thus their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that control expression in response to the presence of specific glycan substrates. However the existence of additional regulatory mechanisms has been predicted to explain phenomena such as the hierarchical control, catabolite repression, and the fine tuning of gene expression to match catabolic activity7-9. Using Bacteroides fragilis as a model organism, this report describes a previously unknown layer of regulatory control in which cis-encoded antisense small RNAs (sRNA) act as repressors of the PULs’ catabolic genes. Nearly 30% of B. fragilis PULs are subject to this type of sRNA control and these PULs tend to be more closely linked to the utilization of host-derived glycans than dietary polysaccharides. The findings described here indicate the presence of a global control mechanism that underlies the known regulatory circuits which modulate PUL expression in response to substrate availability, and hence provide novel insight into regulation of the gut Bacteroidetes physiology.
Project description:The phylum Bacteroidetes is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host, and a potential role in a wide range of disease syndromes1-3. The predominance of Bacteroidetes and the genus Bacteroides in the distal gut is due in large part to the expansion of paralogous gene clusters, termed Polysaccharide Utilization Loci (PULs), dedicated to the uptake and catabolism of host derived and dietary polysaccharides4,5. It is generally thought that the diversity of PULs is key to Bacteroides successful competition for nutrients in the gut environment6. The nutritive value of the available polysaccharides varies greatly and thus their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that control expression in response to the presence of specific glycan substrates. However the existence of additional regulatory mechanisms has been predicted to explain phenomena such as the hierarchical control, catabolite repression, and the fine tuning of gene expression to match catabolic activity7-9. Using Bacteroides fragilis as a model organism, this report describes a previously unknown layer of regulatory control in which cis-encoded antisense small RNAs (sRNA) act as repressors of the PULsâ catabolic genes. Nearly 30% of B. fragilis PULs are subject to this type of sRNA control and these PULs tend to be more closely linked to the utilization of host-derived glycans than dietary polysaccharides. The findings described here indicate the presence of a global control mechanism that underlies the known regulatory circuits which modulate PUL expression in response to substrate availability, and hence provide novel insight into regulation of the gut Bacteroidetes physiology. This is a 4 chip study with 8 technical replicates on each chip. This was an in vitro, exploratory study to determine if mutation or overexpression of a sRNA associated with the Don locus would affect gene expression. In vitro cultures were grown in defined media with mucin glycans as the sole carbon source. The two chips representing growth of the wild type strain (638R) on mucin glycans were also used in a related study GSE53883 (GSM1303101 and GSM1303102).
Project description:The adult human gut microbial community is typically dominated by two bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from E. rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the former possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole genome transcriptional profiling of both organisms in their distal gut (cecal) habitat as well as host responses, high resolution proteomic analysis of cecal contents, and biochemical assays of carbohydrate metabolism. B. thetaiotaomicron adapts to E. rectale by upregulating expression of a variety of polysaccharide utilization loci (PULs) encoding numerous glycoside hydrolase gene families, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is utilized by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of major gut bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.