Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization.

Primary bile acids (BAs) are a collection of host-synthesized metabolites that shape physiology and metabolism. BAs transit the gastrointestinal tract and are subjected to a variety of chemical transformations encoded by indigenous bacteria. The resulting microbiota-derived BA pool is a mediator of host-microbiota interactions. Bacterial bile salt hydrolases (BSHs) cleave the conjugated glycine or taurine from BAs, an essential upstream step for the production of deconjugated and secondary BAs. Probiotic lactobacilli harbor a considerable number and diversity of BSHs; however, their contribution to Lactobacillus fitness and colonization remains poorly understood. Here, we define and compare the functions of multiple BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri Our genetic and biochemical characterization of lactobacilli BSHs lend to a model of Lactobacillus adaptation to the gut. These findings deviate from previous notions that BSHs generally promote colonization and detoxify bile. Rather, we show that BSH enzymatic preferences and the intrinsic chemical features of various BAs determine the toxicity of these molecules during Lactobacillus growth. BSHs were able to alter the Lactobacillus transcriptome in a BA-dependent manner. Finally, BSHs were able to dictate differences in bacterial competition in vitro and in vivo, defining their impact on BSH-encoding bacteria within the greater gastrointestinal tract ecosystem. This work emphasizes the importance of considering the enzymatic preferences of BSHs alongside the conjugated/deconjugated BA-bacterial interaction. These results deepen our understanding of the BA-microbiome axis and provide a framework to engineer lactobacilli with improved bile resistance and use probiotics as BA-altering therapeutics.

The microbial-derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

Clostridioides difficile is a Gram-positive, spore-forming anaerobe that causes clinical diseases ranging from diarrhea and pseudomembranous colitis to toxic megacolon and death. C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial-derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration against C. difficile R20291 and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Additionally, we tested the cytotoxicity of these bile acids through Caco-2 cell apoptosis and viability assays to gauge their effects on the host. Here, we show that the epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA, while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. IMPORTANCE In the search for a novel therapeutic that targets Clostridioides difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that LCA epimers isolithocholate (iLCA) and LCA epimers isoallolithocholate (iaLCA) specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression, and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Multiple TonB homologs are important for carbohydrate utilization by Bacteroides thetaiotaomicron.

IMPORTANCE: The human gut microbiota, including Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source, and fermentation products such as short-chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, the formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for the development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Surface glycan-binding proteins are essential for cereal beta-glucan utilization by the human gut symbiont Bacteroides ovatus.

The human gut microbiota, which underpins nutrition and systemic health, is compositionally sensitive to the availability of complex carbohydrates in the diet. The Bacteroidetes comprise a dominant phylum in the human gut microbiota whose members thrive on dietary and endogenous glycans by employing a diversity of highly specific, multi-gene polysaccharide utilization loci (PUL), which encode a variety of carbohydrases, transporters, and sensor/regulators. PULs invariably also encode surface glycan-binding proteins (SGBPs) that play a central role in saccharide capture at the outer membrane. Here, we present combined biophysical, structural, and in vivo characterization of the two SGBPs encoded by the Bacteroides ovatus mixed-linkage ß-glucan utilization locus (MLGUL), thereby elucidating their key roles in the metabolism of this ubiquitous dietary cereal polysaccharide. In particular, molecular insight gained through several crystallographic complexes of SGBP-A and SGBP-B with oligosaccharides reveals that unique shape complementarity of binding platforms underpins specificity for the kinked MLG backbone vis-à-vis linear ß-glucans. Reverse-genetic analysis revealed that both the presence and binding ability of the SusD homolog BoSGBPMLG-A are essential for growth on MLG, whereas the divergent, multi-domain BoSGBPMLG-B is dispensable but may assist in oligosaccharide scavenging from the environment. The synthesis of these data illuminates the critical role SGBPs play in concert with other MLGUL components, reveals new structure-function relationships among SGBPs, and provides fundamental knowledge to inform future (meta)genomic, biochemical, and microbiological analyses of the human gut microbiota.

SusE facilitates starch uptake independent of starch binding in B. thetaiotaomicron.

The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model system for nutrient acquisition by gut Bacteroidetes, a dominant phylum of gut bacteria. The Sus includes SusCDEFG, which assemble on the cell surface to capture, degrade and import starch. While SusD is an essential starch-binding protein, the precise role(s) of the partially homologous starch-binding proteins SusE and SusF has remained elusive. We previously reported that a non-binding version of SusD (SusD*) supports growth on starch when other members of the multi-protein complex are present. Here we demonstrate that SusE supports SusD* growth on maltooligosaccharides, and determine the domains of SusE essential for this function. Furthermore, we demonstrate that SusE does not need to bind starch to support growth in the presence of SusD*, suggesting that the assembly of SusCDE is most important for maltooligosaccharide uptake in this context. However, starch binding by proteins SusDEF directs the uptake of maltooligosaccharides of specific lengths, suggesting that these proteins equip the cell to scavenge a range of starch fragments. These data demonstrate that the assembly of core Sus proteins SusCDE is secondary to their glycan binding roles, but glycan binding by Sus proteins may fine tune the selection of glycans from the environment.

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.

Updates to Clostridium difficile Spore Germination.

Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringensC. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes.

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-uptake system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex.

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.

Loss of Bacteroides thetaiotaomicron bile acid-altering enzymes impacts bacterial fitness and the global metabolic transcriptome.

IMPORTANCE: Recent work on bile salt hydrolases (BSHs) in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it are not well understood. In this study, we set out to define if and how Bacteroides thetaiotaomicron (B. theta) uses its BSHs and hydroxysteroid dehydrogenase to modify bile acids to provide a fitness advantage for itself in vitro and in vivo. Genes encoding bile acid-altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci. This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut.

Evolution in fast forward: a potential role for mutators in accelerating Staphylococcus aureus pathoadaptation.

Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.

Loss of Bacteroides thetaiotaomicron bile acid altering enzymes impact bacterial fitness and the global metabolic transcriptome.

Bacteroides thetaiotaomicron (B. theta) is a Gram-negative gut bacterium that encodes enzymes that alter the bile acid pool in the gut. Primary bile acids are synthesized by the host liver and are modified by gut bacteria. B. theta encodes two bile salt hydrolases (BSHs), as well as a hydroxysteroid dehydrogenase (HSDH). We hypothesize that B. theta modifies the bile acid pool in the gut to provide a fitness advantage for itself. To investigate each gene's role, different combinations of genes encoding bile acid altering enzymes (bshA, bshB, and hsdhA) were knocked out by allelic exchange, including a triple KO. Bacterial growth and membrane integrity assays were done in the presence and absence of bile acids. To explore if B. theta's response to nutrient limitation changes due to the presence of bile acid altering enzymes, RNASeq analysis of WT and triple KO strains in the presence and absence of bile acids was done. WT B. theta is more sensitive to deconjugated bile acids (CA, CDCA, and DCA) compared to the triple KO, which also decreased membrane integrity. The presence of bshB is detrimental to growth in conjugated forms of CDCA and DCA. RNA-Seq analysis also showed bile acid exposure impacts multiple metabolic pathways in B. theta, but DCA significantly increases expression of many genes in carbohydrate metabolism, specifically those in polysaccharide utilization loci or PULs, in nutrient limited conditions. This study suggests that bile acids B. theta encounters in the gut may signal the bacteria to increase or decrease its utilization of carbohydrates. Further study looking at the interactions between bacteria, bile acids, and the host may inform rationally designed probiotics and diets to ameliorate inflammation and disease. Importance: Recent work on BSHs in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it is not well understood. In this study we set out to define if and how B. theta uses its BSHs and HSDH to modify bile acids to provide a fitness advantage for itself in vitro and in vivo. Genes encoding bile acid altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci (PULs). This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut. This work will aid in our understanding of how to rationally manipulate the bile acid pool and the microbiota to exploit carbohydrate metabolism in the context of inflammation and other GI diseases.

The microbial derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration (MIC) against C. difficile R20291, and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Here we show that epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile . LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. Importance: In the search for a novel therapeutic that targets C. difficile , bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that iLCA and iaLCA specifically are potent inhibitors of C. difficile , affecting key virulence factors including growth, toxin expression and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Multiple TonB Homologs are Important for Carbohydrate Utilization by Bacteroides thetaiotaomicron.

The human gut microbiota is able to degrade otherwise undigestible polysaccharides, largely through the activity of the Bacteroides. Uptake of polysaccharides into Bacteroides is controlled by TonB-dependent transporters (TBDT) whose transport is energized by an inner membrane complex composed of the proteins TonB, ExbB, and ExbD. Bacteroides thetaiotaomicron (B. theta) encodes 11 TonB homologs which are predicted to be able to contact TBDTs to facilitate transport. However, it is not clear which TonBs are important for polysaccharide uptake. Using strains in which each of the 11 predicted tonB genes are deleted, we show that TonB4 (BT2059) is important but not essential for proper growth on starch. In the absence of TonB4, we observed an increase in abundance of TonB6 (BT2762) in the membrane of B. theta, suggesting functional redundancy of these TonB proteins. Growth of the single deletion strains on pectin galactan, chondroitin sulfate, arabinan, and levan suggests a similar functional redundancy of the TonB proteins. A search for highly homologous proteins across other Bacteroides species and recent work in B. fragilis suggests that TonB4 is widely conserved and may play a common role in polysaccharide uptake. However, proteins similar to TonB6 are found only in B. theta and closely related species suggesting that the functional redundancy of TonB4 and TonB6 may be limited across the Bacteroides. This study extends our understanding of the protein network required for polysaccharide utilization in B. theta and highlights differences in TonB complexes across Bacteroides species.

Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota.

Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.

Complete Genome Sequence of Lactobacillus johnsonii NCK2677, Isolated from Mice.

We report the closed genome sequence of a Lactobacillus johnsonii strain (NCK2677) that was isolated from a cefoperazone-treated mouse model designed for the study of Clostridioides difficile infection. Illumina and Nanopore sequencing reads were assembled into a circular 1,951,416-bp chromosome with a G+C content of 34.7%, containing 1,865 genes.

A Cell-Surface GH9 Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus.

Dietary fiber is an important food source for members of the human gut microbiome. Members of the dominant Bacteroidetes phylum capture diverse polysaccharides via the action of multiple cell surface proteins encoded within polysaccharide utilization loci (PUL). The independent activities of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) for the harvest of various glycans have been studied in detail, but how these proteins work together to coordinate uptake is poorly understood. Here, we combine genetic and biochemical approaches to discern the interplay between the BoGH9 endoglucanase and the xyloglucan-binding proteins SGBP-A and SGBP-B from the Bacteroides ovatus xyloglucan utilization locus (XyGUL). The expression of BoGH9, a weakly active xyloglucanase in isolation, is required in a strain that expresses a non-binding version of SGBP-A (SGBP-A*). The crystal structure of the BoGH9 enzyme suggests the molecular basis for its robust activity on mixed-linkage ß-glucan compared to xyloglucan. However, catalytically inactive site-directed mutants of BoGH9 fail to complement the deletion of the active BoGH9 in a SGBP-A* strain. We also find that SGBP-B is needed in an SGBP-A* background to support growth on xyloglucan, but that the non-binding SGBP-B* protein acts in a dominant negative manner to inhibit growth on xyloglucan. We postulate a model whereby the SGBP-A, SGBP-B, and BoGH9 work together at the cell surface, likely within a discrete complex, and that xyloglucan binding by SGBP-B and BoGH9 may facilitate the orientation of the xyloglucan for transfer across the outer membrane.