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ABSTRACT Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques—a known mucin degrader that has been implicated in inflammatory bowel diseases (IBDs)—degrades mucin glycoproteins or their component O-linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong α-L-fucosidase, sialidase and β1,4-galactosidase activities. There was a lack of detectable sulfatase and weak β1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron. This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which might contribute to its association with IBD.IMPORTANCEAn important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade the various components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate species associated with bacterial mucus damage and subsequent disease susceptibility. Ruminococcus torques is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in R. torques and found that it shares mucin degradation products with another species of gut bacteria, Bacteroides thetaiotaomicron. Our findings underscore the importance of understanding mucin degradation mechanisms in different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately affect mucus damage and could therefore be key players in effects that result from reductions in mucus integrity.

Summary: Parkinson’s disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Epithelial cells that line the gut secrete complex glycoproteins that form a mucus layer to protect the gut wall from enteric pathogens. Here, the authors provide a comprehensive characterisation of endo-acting glycoside hydrolases expressed by mucin-degrading members of the microbiome that are able to cleave the O-glycan chains of a range of different animal and human mucins.

ABSTRACT Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multilocus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors. IMPORTANCE Nonharmful bacteria are the primary microbial symbionts that inhabit the human gastrointestinal tract. These bacteria play many beneficial roles and in some cases can modify disease states, making it important to understand which nutrients sustain specific lineages. This knowledge will in turn lead to strategies to intentionally manipulate the gut microbial ecosystem. We designed a scalable, high-throughput platform for measuring the ability of gut bacteria to utilize polysaccharides, of which many are derived from dietary fiber sources that can be manipulated easily. Our results provide paths to expand phenotypic surveys of more diverse gut bacteria to understand their functions and also to leverage dietary fibers to alter the physiology of the gut microbial community.

Summary: Reproducible in vivo models are necessary to address functional aspects of the gut microbiome in various diseases. Here, we present a gnotobiotic mouse model that allows for the investigation of specific microbial functions within the microbiome. We describe how to culture 14 different well-characterized human gut species and how to verify their proper colonization in germ-free mice. This protocol can be modified to add or remove certain species of interest to investigate microbial mechanistic details in various disease models.For complete details on the use and execution of this protocol, please refer to Desai et al. (2016).

Commensal butyrate-producing bacteria belonging to the Firmicutes

How dietary β-mannans are utilized by gut Gram-positive bacteria is unclear. Here, the authors uncover the enzymatic pathway for β-mannan metabolism in Roseburia intestinalis and show that these polysaccharides promote beneficial gut bacteria, highlighting a potential for β-mannan-based therapeutic interventions.

The change of dietary habits in Western societies, including reduced consumption of fiber, is linked to alterations in gut microbial ecology. Nevertheless, mechanistic connections between diet-induced microbiota changes that affect colonization resistance and enteric pathogen susceptibility are still emerging. We sought to investigate how a diet devoid of soluble plant fibers impacts the structure and function of a conventional gut microbiota in specific-pathogen-free (SPF) mice and how such changes alter susceptibility to a rodent enteric pathogen. We show that absence of dietary fiber intake leads to shifts in the abundances of specific taxa, microbiome-mediated erosion of the colonic mucus barrier, a reduction of intestinal barrier-promoting short-chain fatty acids, and increases in markers of mucosal barrier integrity disruption. Importantly, our results highlight that these low-fiber diet-induced changes in the gut microbial ecology collectively contribute to a lethal colitis by the mucosal pathogen Citrobacter rodentium, which is used as a mouse model for enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Our study indicates that modern, low-fiber Western-style diets might make individuals more prone to infection by enteric pathogens via the disruption of mucosal barrier integrity by diet-driven changes in the gut microbiota, illustrating possible implications for EPEC and EHEC infections.

ABSTRACT The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL. IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

Summary: There is increasing evidence that gut microbiome perturbations, also known as dysbiosis, can influence colorectal cancer development. To understand the mechanisms by which the gut microbiome modulates cancer susceptibility, we examine two wild-type mouse colonies with distinct gut microbial communities that develop significantly different tumor numbers using a mouse model of inflammation-associated tumorigenesis. We demonstrate that adaptive immune cells contribute to the different tumor susceptibilities associated with the two microbial communities. Mice that develop more tumors have increased colon lamina propria CD8+ IFNγ+ T cells before tumorigenesis but reduced CD8+ IFNγ+ T cells in tumors and adjacent tissues compared with mice that develop fewer tumors. Notably, intratumoral T cells in mice that develop more tumors exhibit increased exhaustion. Thus, these studies suggest that microbial dysbiosis can contribute to colon tumor susceptibility by hyperstimulating CD8 T cells to promote chronic inflammation and early T cell exhaustion, which can reduce anti-tumor immunity. : The gut microbiome is capable of modulating intestinal inflammation and tumorigenesis. Yu et al. demonstrate that dysbiosis can lead to increased susceptibility to inflammation-associated colon tumorigenesis via the induction of pro-inflammatory CD8+ IFNγ+ T cells, which can lead to increased T cell exhaustion within the tumor microenvironment. Keywords: microbiome, colitis, colon cancer, CD8 T cells, T cell exhaustion, inflammation

ABSTRACT Dietary fiber provides a variety of microbiota-mediated benefits ranging from anti-inflammatory metabolites to pathogen colonization resistance. A healthy gut microbiota protects against Clostridioides difficile colonization. Manipulation of these microbes through diet may increase colonization resistance to improve clinical outcomes. The primary objective of this study was to identify how the dietary fiber xanthan gum affects the microbiota and C. difficile colonization. We added 5% xanthan gum to the diet of C57BL/6 mice and examined its effect on the microbiota through 16S rRNA gene amplicon sequencing and short-chain fatty acid analysis. Following either cefoperazone or an antibiotic cocktail administration, we challenged mice with C. difficile and measured colonization by monitoring the CFU. Xanthan gum administration is associated with increases in fiber-degrading taxa and short-chain fatty acid concentrations. However, by maintaining both the diversity and absolute abundance of the microbiota during antibiotic treatment, the protective effects of xanthan gum administration on the microbiota were more prominent than the enrichment of these fiber-degrading taxa. As a result, mice that were on the xanthan gum diet experienced limited to no C. difficile colonization. Xanthan gum administration alters mouse susceptibility to C. difficile colonization by maintaining the microbiota during antibiotic treatment. While antibiotic-xanthan gum interactions are not well understood, xanthan gum has previously been used to bind drugs and alter their pharmacokinetics. Thus, xanthan gum may alter the activity of the oral antibiotics used to make the microbiota susceptible. Future research should further characterize how this and other common dietary fibers interact with drugs. IMPORTANCE A healthy gut bacterial community benefits the host by breaking down dietary nutrients and protecting against pathogens. Clostridioides difficile capitalizes on the absence of this community to cause diarrhea and inflammation. Thus, a major clinical goal is to find ways to increase resistance to C. difficile colonization by either supplementing with bacteria that promote resistance or a diet to enrich for those already present in the gut. In this study, we describe an interaction between xanthan gum, a human dietary additive, and the microbiota resulting in an altered gut environment that is protective against C. difficile colonization.

Polysaccharides are the primary structural cell wall and energy storage molecules of seaweed. Here, the authors show how the geographically restricted dietary polysaccharide agarose is selectively utilized by the human intestinal bacterium Bacteroides uniformis, providing insight into how carbohydrate metabolism evolves within the human microbiome.

Summary: Microbial utilization of complex polysaccharides is a major driving force in shaping the composition of the human gut microbiota. There is a growing appreciation that finely tuned polysaccharide utilization loci enable ubiquitous gut Bacteroidetes to thrive on the plethora of complex polysaccharides that constitute “dietary fiber.” Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs) are a key family of plant cell wall polysaccharides with recognized health benefits but whose mechanism of utilization has remained unclear. Here, we provide molecular insight into the function of an archetypal MLG utilization locus (MLGUL) through a combination of biochemistry, enzymology, structural biology, and microbiology. Comparative genomics coupled with growth studies demonstrated further that syntenic MLGULs serve as genetic markers for MLG catabolism across commensal gut bacteria. In turn, we surveyed human gut metagenomes to reveal that MLGULs are ubiquitous in human populations globally, which underscores the importance of gut microbial metabolism of MLG as a common cereal polysaccharide. : Mixed-linkage β(1,3)/β(1,4)-glucan (MLG) is an important complex dietary polysaccharide (dietary fiber), the degradation of which in the human gut depends on the resident microbiota. Tamura et al. outline the molecular mechanism of MLG utilization by Bacteroides ovatus and reveal that the majority of surveyed humans possess MLG-utilizing Bacteroidetes. Keywords: microbiota, dietary fiber, complex carbohydrates, polysaccharide, polysaccharide utilization locus, Bacteroidetes, mixed-linkage glucan, barley beta-glucan, oat beta-glucan, carbohydrate-active enzymes

Delivering fibers more distally could be important to prevent or treat colonic diseases such as cancer and ulcerative colitis. Here, we hypothesized that fermentation of fast-fermenting soluble fibers by the colonic microbiota is delayed when they are presented in a mixture due to hierarchical utilization of fibers. A series of in vitro fermentation studies was performed using fecal microbiota obtained from three healthy donors using single dietary fibers [arabinoxylan, chondroitin sulfate (CS), galactomannan (GM), polygalacturonic acid (PGA), xyloglucan (XG)] and a mixture containing an equal amount of each. Substrate disappearance analysis, as measured by GC–MS, revealed that CS, PGA, and XG utilization was delayed when present in the mixture. 16S rRNA sequencing showed certain fibers consistently increased specific genera in the microbiota of all donor groups. Mixing different types of fermentable dietary fibers might be a logical strategy for delivering fibers into more distal regions of the colon.

Dysfunction in host immune responses and pathologic alterations in the gut microbiota, referred to as dysbiosis, can both contribute to the development of inflammatory bowel disease (IBD). However, it remains unclear how specific changes in host immunity or the microbiota cause disease. We previously demonstrated that the loss of the innate immune receptor NLRP6 in mice resulted in impaired production of interleukin-18 (IL-18) and increased susceptibility to epithelial-induced injury. Here, we show that NLRP6 is important for suppressing the development of spontaneous colitis in the Il10−/− mice model of IBD and that NLRP6 deficiency results in the enrichment of Akkermansia muciniphila. A. muciniphila was sufficient for promoting intestinal inflammation in both specific-pathogen-free and germ-free Il10−/− mice. Our results demonstrate that A. muciniphila can act as a pathobiont to promote colitis in a genetically susceptible host and that NLRP6 is a key regulator of its abundance.

ABSTRACT Woody biomass is a sustainable and virtually unlimited source of hemicellulosic polysaccharides. The predominant hemicelluloses in softwood and hardwood are galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), respectively. Based on the structure similarity with common dietary fibers, GGM and AGX may be postulated to have prebiotic properties, conferring a health benefit on the host through specific modulation of the gut microbiota. In this study, we evaluated the prebiotic potential of acetylated GGM (AcGGM) and highly acetylated AGX (AcAGX) obtained from Norwegian lignocellulosic feedstocks in vitro. In pure culture, both substrates selectively promoted the growth of Bifidobacterium, Lactobacillus, and Bacteroides species in a manner consistent with the presence of genetic loci for the utilization of β-manno-oligosaccharides/β-mannans and xylo-oligosaccharides/xylans. The prebiotic potential of AcGGM and AcAGX was further assessed in a pH-controlled batch culture fermentation system inoculated with healthy adult human feces. Results were compared with those obtained with a commercial fructo-oligosaccharide (FOS) mixture. Similarly to FOS, both substrates significantly increased (P

OBJECTIVES/GOALS: This study seeks to culture and characterize mucus-degrading microbes from the microbiome of inflammatory bowel disease (IBD) patients. METHODS/STUDY POPULATION: Stool will be collected from IBD patients and healthy first-degree relatives, then enriched for mucin-degrading microbes through growth on porcine rectal mucin. Dilution plating in both liquid and solid culture formats will be employed to isolate strains capable of growth on mucin. Cultures that are positive for mucin degradation will be identified with 16S rRNA sequencing; unique isolates will be genome sequenced and transcriptionally profiled on simple monosaccharides and mucin in order to identify putative mucin-degrading genes. The abundance of novel enzymes, pathways, and microbes will be compared in healthy and IBD patient populations using existing datasets in the literature. RESULTS/ANTICIPATED RESULTS: We expect to isolate previously uncultured mucin-degrading microbes, which will likely include new strains and possibly new species of bacteria. Through the transcriptomic characterization of mucin-degrading pathways, we will expand the lexicon of known mucin-degrading enzymes and pathways used by bacteria in the human colon. We expect mucin-degrading microbes to be more abundant and active in IBD patients compared to healthy controls. DISCUSSION/SIGNIFICANCE OF IMPACT: There is no cure for IBD and treatment relies heavily on suppressing a patient’s immune system. This research seeks to understand the contribution of the gut microbiota in the pathogenesis of IBD, which may lead to future therapeutic targets.

ABSTRACT Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be introduced into the gut microbe Bacteroides thetaiotaomicron to identify genes based on activity screening. Our results support the continuing development of genetically tractable systems to obtain information about gene function.

ABSTRACT When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet.

ABSTRACT Many symbiotic gut bacteria possess the ability to degrade multiple polysaccharides, thereby providing nutritional advantages to their hosts. Like microorganisms adapted to other complex nutrient environments, gut symbionts give different metabolic priorities to substrates present in mixtures. We investigated the responses of Bacteroides thetaiotaomicron, a common human intestinal bacterium that metabolizes more than a dozen different polysaccharides, including the O-linked glycans that are abundant in secreted mucin. Experiments in which mucin glycans were presented simultaneously with other carbohydrates show that degradation of these host carbohydrates is consistently repressed in the presence of alternative substrates, even by B. thetaiotaomicron previously acclimated to growth in pure mucin glycans. Experiments with media containing systematically varied carbohydrate cues and genetic mutants reveal that transcriptional repression of genes involved in mucin glycan metabolism is imposed by simple sugars and, in one example that was tested, is mediated through a small intergenic region in a transcript-autonomous fashion. Repression of mucin glycan-responsive gene clusters in two other human gut bacteria, Bacteroides massiliensis and Bacteroides fragilis, exhibited variable and sometimes reciprocal responses compared to those of B. thetaiotaomicron, revealing that these symbionts vary in their preference for mucin glycans and that these differences occur at the level of controlling individual gene clusters. Our results reveal that sensing and metabolic triaging of glycans are complex processes that vary among species, underscoring the idea that these phenomena are likely to be hidden drivers of microbiota community dynamics and may dictate which microorganisms preferentially commit to various niches in a constantly changing nutritional environment. IMPORTANCE Human intestinal microorganisms impact many aspects of health and disease, including digestion and the propensity to develop disorders such as inflammation and colon cancer. Complex carbohydrates are a major component of the intestinal habitat, and numerous species have evolved and refined strategies to compete for these coveted nutrients. Our findings reveal that individual bacteria exhibit different preferences for carbohydrates emanating from host diet and mucosal secretions and that some of these prioritization strategies are opposite to one another. Thus, we reveal new aspects of how individual bacteria, some with otherwise similar metabolic potential, partition to “preferred niches” in the complex gut ecosystem, which has important and immediate implications for understanding and predicting the behavioral dynamics of this community.

ABSTRACT Gut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the model Bacteroides thetaiotaomicron starch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on the B. thetaiotaomicron cell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to control Bacteroidetes in the intestinal tract to enhance human health and treat disease. IMPORTANCE In this study, we used nanometer-scale superresolution imaging to reveal dynamic interactions between the proteins involved in starch processing by the prominent human gut symbiont Bacteroides thetaiotaomicron in real time in live cells. These results represent the first working model of starch utilization system (Sus) complex assembly and function during glycan catabolism and are likely to describe aspects of how other Sus-like systems function in human gut Bacteroidetes. Our results provide unique mechanistic insights into a glycan catabolism strategy that is prevalent within the human gut microbial community. Proper understanding of this conserved nutrient uptake mechanism is essential for the development of dietary or pharmaceutical therapies to control intestinal tract microbial populations, to enhance human health, and to treat disease.

ABSTRACT To compete for the dynamic stream of nutrients flowing into their ecosystem, colonic bacteria must respond rapidly to new resources and then catabolize them efficiently once they are detected. The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model for nutrient acquisition by symbiotic gut bacteria, which harbor thousands of related Sus-like systems. Structural investigation of the four Sus outer membrane proteins (SusD, -E, -F, and -G) revealed that they contain a total of eight starch-binding sites that we demonstrated, using genetic and biochemical approaches, to play distinct roles in starch metabolism in vitro and in vivo in gnotobiotic mice. SusD, whose homologs are abundant in the human microbiome, is critical for the initial sensing of available starch, allowing sus transcriptional activation at much lower concentrations than without this function. In contrast, seven additional binding sites across SusE, -F, and -G are dispensable for sus activation. However, they optimize the rate of growth on starch in a manner dependent on the expression of the bacterial polysaccharide capsule, suggesting that they have evolved to offset the diffusion barrier created by this structure. These findings demonstrate how proteins with similar biochemical behavior can serve orthogonal functions during different stages of cellular adaptation to nutrients. Finally, we demonstrated in gnotobiotic mice fed a starch-rich diet that the Sus binding sites confer a competitive advantage to B. thetaiotaomicron in vivo in a manner that is dependent on other colonizing microbes. This study reveals how numerically dominant families of carbohydrate-binding proteins in the human microbiome fulfill separate and sometimes cooperative roles to optimize gut commensal bacteria for nutrient acquisition. IMPORTANCE Our intestinal tract harbors trillions of symbiotic microbes. A critical function contributed by this microbial community is the ability to degrade most of the complex carbohydrates in our diet, which not only change from meal to meal but also cannot be digested by our own bodies. A numerically abundant group of gut bacteria called the Bacteroidetes plays a prominent role in carbohydrate digestion in humans and other animals. Currently, the mechanisms that allow this bacterial group to rapidly respond to available carbohydrates and then digest them efficiently are unclear. Here, we present novel functions for four carbohydrate-binding proteins present in one member of the Bacteroidetes, revealing that these proteins serve unique and separable roles in either initial nutrient sensing or subsequent digestion. Because the protein families investigated are numerous in other gut bacteria colonizing nearly all humans and animals, our findings are fundamentally important to understanding how symbiotic microbes assist human digestion.

Nature Communications 6 Article number: 7481 (2015); Published 26 June 2015; Updated 5 February 2016 The financial support for the work described in this Article was not fully acknowledged. The Acknowledgements should have included the following: This work was supported in part by a grant to H.J.G.,B.