Bacteroides thetaiotaomicron: a dynamic, niche-adapted human symbiont

The coevolution of humans with their intestinal microflora has resulted in cooperative relationships that have shaped the biology and the genomes of these symbiotic partners. Bacteroides thetaiotaomicron is one such bacterial symbiont that is a dominant member of the intestinal microbiota of humans and other mammals. The recent report of the genome sequence of B. thetaiotaomicron is the first reported for an abundant Gram-negative organism of the human colonic microbiota and, as such, provides the first glimpse on a genomic scale of the genetic arsenal used by a Gram-negative symbiont to dominate in this ecosystem. The genome has revealed large expansions of many paralogous groups of genes that encode products essential to the organism's ability to successfully compete in this environment. Most noteable is the organism's abundant machinery for utilizing a large variety of complex polysaccharides as a source of carbon and energy. The proteome also reveals the organism's extensive ability to adapt and regulate expression of its genes in response to the changing ecosystem. These factors, as well as others highlighted below, suggest an incredibly flexible and adaptable organism that is exquisitely equipped to dominate in its challenging and competitive niche.     

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

1. Download : Download high-res image (203KB)
2. Download : Download full-size image

     

Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices

The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique alpha-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.     

An anaerobic bacterium,Bacteroides thetaiotaomicron,uses a consortium of enzymes to scavenge hydrogen peroxide

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H₂O₂ that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H₂O₂ in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H₂O₂, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H₂O₂ formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H₂O₂ was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.     

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute‐binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane‐associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute‐binding proteins display a range of glycan‐binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6‐branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch‐degrading Clostridium cluster XIVa organisms in the human gut.     

Mannose foraging by Bacteroides thetaiotaomicron: structure and specificity of the beta-mannosidase, BtMan2A

The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. Although it is likely that these enzymes enable the organism to utilize dietary and host glycans as major nutrient sources, the biochemical properties of these GH2 glycoside hydrolases are currently unclear. Here we report the biochemical properties and crystal structure of the GH2 B. thetaiotaomicron enzyme BtMan2A. Kinetic analysis demonstrates that BtMan2A is a beta-mannosidase in which substrate binding energy is provided principally by the glycone binding site, whereas aglycone recognition is highly plastic. The three-dimensional structure, determined to a resolution of 1.7 A, reveals a five-domain structure that is globally similar to the Escherichia coli LacZ beta-galactosidase. The catalytic center is housed mainly within a (beta/alpha)8 barrel although the N-terminal domain also contributes to the active site topology. The nature of the substrate-binding residues is quite distinct from other GH2 enzymes of known structure, instead they are similar to other clan GH-A enzymes specific for manno-configured substrates. Mutagenesis studies, informed by the crystal structure, identified a WDW motif in the N-terminal domain that makes a significant contribution to catalytic activity. The observation that this motif is invariant in GH2 mannosidases points to a generic role for these residues in this enzyme class. The identification of GH-A clan and GH2 specific residues in the active site of BtMan2A explains why this enzyme is able to harness substrate binding at the proximal glycone binding site more efficiently than mannan-hydrolyzing glycoside hydrolases in related enzyme families. The catalytic properties of BtMan2A are consistent with the flexible nutrient acquisition displayed by the colonic bacterium.     

Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron.

Previous studies of starch utilization by the gram-negative anaerobe Bacteroides thetaiotaomicron have demonstrated that the starch-degrading enzymes are cell associated rather than extracellular, indicating that the first step in starch utilization is binding of the polysaccharide to the bacterial surface. Five transposon-generated mutants of B. thetaiotaomicron which were defective in starch binding (Ms-1 through Ms-5) had been isolated, but initial attempts to identify membrane proteins missing in these mutants were not successful. We report here the use of an immunological approach to identify four maltose-inducible membrane proteins, which were missing in one or more of the starch-binding mutants of B. thetaiotaomicron. Three of the maltose-inducible proteins were outer membrane proteins (115, 65, and 43 kDa), and one was a cytoplasmic membrane protein (80 kDa). The genes encoding these proteins were shown to be clustered in an 8.5-kbp segment of the B. thetaiotaomicron chromosome. Two other loci defined by transposon insertions, which appeared to contain regulatory genes, were located within 7 kbp of the cluster of membrane protein genes. The 115-kDa outer membrane protein was essential for utilization of maltoheptaose (G7), whereas loss of the other proteins affected growth on starch but not on G7. Not all of the proteins missing in the mutants were maltose regulated. We also detected two constitutively produced proteins (32 and 50 kDa) that were less prominent in all of the mutants than in the wild type. Both of these were outer membrane proteins.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.