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Antibiotics save lives but can have unwanted effects on our gut microbes, thereby contributing to disease. A mechanistic understanding of how such microbes respond to antibiotics is hence critical. Recently in mSphere , Nilson et al. investigated the metabolite dependence of antibiotic susceptibility in Bacteroides thetaiotaomicron , an abundant and important member of our gut microbiota (R. Nilson, S. Penumutchu, F. S. Pagano, and P. Belenky, mSphere 9:e00103-24, 2024, https://doi.org/10.1128/msphere.00103-24). Their uncovered findings suggest the possibility of potentiating antibiotics with metabolites to reduce post-antibiotic "blooming" of B. thetaiotaomicron and the associated development of gut symbiosis., Competing Interests: The author declares no conflict of interest.

Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron . Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron . Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide., Importance: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource., Competing Interests: The authors declare no conflict of interest.

Understanding how the human gut microbiota contribute to the metabolism of dietary carbohydrates is of great interest, particularly those with ferulic acid (FA) decorations that have manifold health benefits. Here, we report the crystal structure of a decameric feruloyl esterase ( Bt Fae) from Bacteroides thetaiotaomicron in complex with methyl ferulate (MFA), revealing that MFA is situated in a noncatalytic substrate binding pocket adjacent to the catalytic pocket. Molecular docking and mutagenesis studies further demonstrated that the adjacent pocket affects substrate binding in the active site and negatively regulates the Bt Fae activity on both synthetic and natural xylan substrates. Additionally, quantum mechanics (QM) calculations were employed to investigate the catalytic process of Bt Fae from substrate binding to product release, and identified TS_2 in the acylation step is rate-limiting. Collectively, this study unmasks a novel regulatory mechanism of FAE activity, which may contribute to further investigation of FA-conjugated polysaccharides metabolism in the human gut.

The flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions. Here, based on study of enzymes from the phytopathogen Agrobacterium tumefaciens, we reveal a conserved Mn 2+ metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3-keto-glucosides and water addition to the resulting 2-hydroxy-3-keto-glycal product, yielding 3-keto-glucose. Extending our search of GUL-encoded putative lyases to the human gut commensal Bacteroides thetaiotaomicron, we discover a Ca 2+ metallocenter active site in a putative glycoside hydrolase-like protein and demonstrate its catalytic function in the eliminative cleavage of 3-keto-glucosides of opposite (α) anomeric configuration as preferred by the A. tumefaciens enzyme (β). Structural and biochemical comparisons reveal the molecular-mechanistic origin of 3-keto-glucoside lyase stereo-complementarity. Our findings identify a basic set of GUL-encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in the bacterial domain of life., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes., (© 2024 Wiley Periodicals LLC.)

Antibiotic therapy alters bacterial abundance and metabolism in the gut microbiome, leading to dysbiosis and opportunistic infections. Bacteroides thetaiotaomicron ( Bth ) is both a commensal in the gut and an opportunistic pathogen in other body sites. Past work has shown that Bth responds to β-lactam treatment differently depending on the metabolic environment both in vitro and in vivo . Studies of other bacteria show that an increase in respiratory metabolism independent of growth rate promotes susceptibility to bactericidal antibiotics. We propose that Bth enters a protected state linked to an increase in polysaccharide utilization and a decrease in the use of simple sugars. Here, we apply antibiotic susceptibility testing, transcriptomic analysis, and genetic manipulation to characterize this polysaccharide-mediated tolerance (PM tolerance) phenotype. We found that a variety of mono- and disaccharides increased the susceptibility of Bth to several different β-lactams compared to polysaccharides. Transcriptomics indicated a metabolic shift from reductive to oxidative branches of the tricarboxylic acid cycle on polysaccharides. Accordingly, supplementation with intermediates of central carbon metabolism had varying effects on PM tolerance. Transcriptional analysis also showed a decrease in the expression of the electron transport chain (ETC) protein NQR and an increase in the ETC protein NUO, when given fiber versus glucose. Deletion of NQR increased Bth susceptibility while deletion of NUO and a third ETC protein NDH2 had no effect. This work confirms that carbon source utilization modulates antibiotic susceptibility in Bth and that anaerobic respiratory metabolism and the ETC play an essential role.IMPORTANCEAntibiotics are indispensable medications that revolutionized modern medicine. However, their effectiveness is challenged by a large array of resistance and tolerance mechanisms. Treatment with antibiotics also disrupts the gut microbiome which can adversely affect health. Bacteroides are prevalent in the gut microbiome and yet are frequently involved in anaerobic infections. Thus, understanding how antibiotics affect these bacteria is necessary to implement proper treatment. Recent work has investigated the role of metabolism in antibiotic susceptibility in distantly related bacteria such as Escherichia coli . Using antibiotic susceptibility testing, transcriptomics, and genetic manipulation, we demonstrate that polysaccharides reduce β-lactam susceptibility when compared to monosaccharides. This finding underscores the profound impact of metabolic adaptation on the therapeutic efficacy of antibiotics. In the long term, this work indicates that modulation of metabolism could make Bacteroides more susceptible during infections or protect them in the context of the microbiome., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays., (© 2024. The Author(s).)

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fofanova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hybrid two-component systems (HTCSs) comprise a major class of transcription regulators of polysaccharide utilization genes in Bacteroides . Distinct from classical two-component systems in which signal transduction is carried out by intermolecular phosphotransfer between a histidine kinase (HK) and a cognate response regulator (RR), HTCSs contain the membrane sensor HK and the RR transcriptional regulator within a single polypeptide chain. Tethering the DNA-binding domain (DBD) of the RR with the dimeric HK domain in an HTCS could potentially promote dimerization of the DBDs and would thus require a mechanism to suppress DNA-binding activity in the absence of stimulus. Analysis of phosphorylation and DNA-binding activities of several HTCSs from Bacteroides thetaiotaomicron revealed a DBD suppression mechanism in which an inhibitory interaction between the DBD and the phosphoryl group-accepting receiver domain (REC) decreases autophosphorylation rates of HTCS-RECs and represses DNA-binding activities in the absence of phosphorylation. Sequence analyses and structure predictions identified a highly conserved sequence motif correlated with a conserved inhibitory domain arrangement of REC and DBD. The presence of the motif, as in most HTCSs, or its absence, in a small subset of HTCSs, is likely predictive of two distinct regulatory mechanisms evolved for different glycans. Substitutions within the conserved motif relieve the inhibitory interaction and result in elevated DNA-binding activities in the absence of phosphorylation. Our data suggest a fundamental regulatory mechanism shared by most HTCSs to suppress DBD activities using a conserved inhibitory interdomain arrangement to overcome the challenge of the fused HK and RR components., Importance: Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides , the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides ., Competing Interests: The authors declare no conflict of interest.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-β-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract., (© 2024. The Author(s).)

The extent to which bacterial lipids produced by the gut microbiota penetrate host tissues is unclear. Here, we combined mass spectrometry approaches to identify lipids produced by the human gut symbiont Bacteroides thetaiotaomicron (B. theta) and spatially track these bacterial lipids in the mouse colon. We characterize 130 B. theta lipids by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using wild-type and mutant B. theta strains to confidently identify lipid structures and their interconnected pathways in vitro. Of these, 103 B. theta lipids can be detected and spatially mapped in a single MALDI mass spectrometry imaging run. We map unlabeled bacterial lipids across colon sections of germ-free and specific-pathogen-free (SPF) mice and mice mono-colonized with wild-type or sphingolipid-deficient (BTMUT) B. theta. We observe co-localization of bacterially derived phosphatidic acid with host tissues in BTMUT mice, consistent with lipid penetration into host tissues. These results indicate limited and selective transfer of bacterial lipids to the host., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A ( BT3337 ), B ( BT3338 ), and C ( BT3339 )], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCE Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events., Competing Interests: The authors declare no conflict of interest.

The human colon hosts hundreds of commensal bacterial species, many of which ferment complex dietary carbohydrates. To transform these fibers into metabolically accessible compounds, microbes often express a series of dedicated enzymes homologous to the starch utilization system (Sus) encoded in polysaccharide utilization loci (PULs). The genome of Bacteroides thetaiotaomicron ( Bt ), a common member of the human gut microbiota, encodes nearly 100 PULs, conferring a strong metabolic versatility. While the structures and functions of individual enzymes within the PULs have been investigated, little is known about how polysaccharide complexity impacts the function of Sus-like systems. We here show that the activity of Sus-like systems depends on polysaccharide size, ultimately impacting bacterial growth. We demonstrate the effect of size-dependent metabolism in the context of dextran metabolism driven by the specific utilization system PUL48. We find that as the molecular weight of dextran increases, Bt growth rate decreases and lag time increases. At the enzymatic level, the dextranase BT3087, a glycoside hydrolase (GH) belonging to the GH family 66, is the main GH for dextran utilization, and BT3087 and BT3088 contribute to Bt dextran metabolism in a size-dependent manner. Finally, we show that the polysaccharide size-dependent metabolism of Bt impacts its metabolic output in a way that modulates the composition of a producer-consumer community it forms with Bacteroides fragilis . Altogether, our results expose an overlooked aspect of Bt metabolism that can impact the composition and diversity of microbiota., Importance: Polysaccharides are complex molecules that are commonly found in our diet. While humans lack the ability to degrade many polysaccharides, their intestinal microbiota contain bacterial commensals that are versatile polysaccharide utilizers. The gut commensal Bacteroides thetaiotaomicron dedicates roughly 20% of their genomes to the expression of polysaccharide utilization loci for the broad range utilization of polysaccharides. Although it is known that different polysaccharide utilization loci are dedicated to the degradation of specific polysaccharides with unique glycosidic linkages and monosaccharide compositions, it is often overlooked that specific polysaccharides may also exist in various molecular weights. These different physical attributes may impact their processability by starch utilization system-like systems, leading to differing growth rates and nutrient-sharing properties at the community level. Therefore, understanding how molecular weight impacts utilization by gut microbe may lead to the potential design of novel precision prebiotics., Competing Interests: The authors declare no conflict of interest.

The genus Bacteroides , a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila . These peptides facilitate efficient protein transport across cellular membranes in Bacteroides , a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Sulfate- and sulfite-reducing bacteria (SRB) are a group of strict anaerobes found within the human gut. Bilophila wadsworthia , a sulfite-reducing bacterium which produces hydrogen sulfide (H 2 S) from taurine and isethionate respiration, is a common member of the healthy commensal human gut microbiota but has been implicated in several disease states including inflammatory bowel disease and colorectal cancer. Bacteroides thetaiotaomicron , one of the most prominent gut bacteria, has sulfatases which release sulfate, serving as a potential substrate for sulfate-reducing bacteria. Here, we showed that when B. thetaiotaomicron and B. wadsworthia were in co-culture, there was a significant increase in B. thetaiotaomicron 's growth and in H 2 S production by B. wadsworthia . Differential gene expression analysis revealed increased expression of B. wadsworthia 's dsrMKJOP complex in co-culture, which delivers electrons for sulfite reduction to H 2 S. This was accompanied by a decreased expression of genes associated with taurine, sulfolactate, and thiosulfate respiration, indicating that B. thetaiotaomicron may provide an alternative source of sulfite to B. wadsworthia . We hypothesized adenosine 5'-phosphosulfate (APS) to be this intermediate. Indeed, B. wadsworthia was able to grow using APS or sulfite as electron acceptors. Endometabolomic and transcriptomic analyses revealed decreased production of indole by B. thetaiotaomicron in co-culture with B. wadsworthia due to enhanced tryptophan utilization by B. wadsworthia . The results of this microbe-microbe interaction could have significant pro-inflammatory effects in the human gut environment.

Vancomycin (VAN) treatment in Clostridioides difficile infection (CDI) suffers from a relatively high rate of recurrence, with a variety of reasons behind this, including biofilm-induced recurrent infections. C. difficile can form monophyletic or symbiotic biofilms with other microbes in the gut, and these biofilms protect C. difficile from being killed by antibiotics. In this study, we analyzed the ecological relationship between Bacteroides thetaiotaomicron and C. difficile and their formation of symbiotic biofilm in the VAN environment. The production of symbiotic biofilm formed by C. difficile and B. thetaiotaomicron was higher than that of C. difficile and B. thetaiotaomicron alone in the VAN environment. In symbiotic biofilms, C. difficile was characterized by increased production of the toxin protein TcdA and TcdB, up-regulation of the expression levels of the virulence genes tcdA and tcdB , enhanced bacterial cell swimming motility and c-di-GMP content, and increased adhesion to Caco-2 cells. The scanning electron microscope (SEM) combined with confocal laser scanning microscopy (CLSM) results indicated that the symbiotic biofilm was elevated in thickness, dense, and had an increased amount of mixed bacteria, while the fluorescence in situ hybridization (FISH) probe and plate colony counting results further indicated that the symbiotic biofilm had a significant increase in the amount of C. difficile cells, and was able to better tolerate the killing of the simulated intestinal fluid. Taken together, C. difficile and B. thetaiotaomicron become collaborative in the VAN environment, and targeted deletion or attenuation of host gut B. thetaiotaomicron content may improve the actual efficacy of VAN in CDI treatment.

Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanism of OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodology for screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors declare no conflict of interest.

Achyranthes bidentata (A. bidentata) is a famous traditional Chinese medicine (TGM) for treatment osteoporosis. Polysaccharides, a major factor for shaping the gut microbiota, are the primary ingredients of A. bidentata. However, bioactivity of A. bidentata polysaccharide on human gut microbiota (HGM) remains unknown. Here, a homogeneous pectic polysaccharide A23-1 with average molecular weight of 93.085 kDa was extracted and purified from A. bidentata. And A23-1 was compsed of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 7.26: 0.76: 5.12: 2.54: 23.51: 60.81. GC-MS, partial acid hydrolysis and NMR results indicated the backbone of A23-1 was composed of 1, 2, 4-Rhap and 1, 4-GlapA, while the branches were composed of galactose, arabinose, glucose and glucuronic acid. Further, A23-1 was found to be degraded into monosaccharides and fragments. Taking Bacteroides thetaiotaomicron (BT) as a model, we suggested three polysaccharide utilization loci (PULs) might be involved in the A23-1 degradation. Degraded products generated by BO might not support the growth of probiotics. Besides, acetate and propionate as the main end products were generated by Bacteroides spp. and probiotics utilizing A23-1. These findings suggested A23-1 was possible one of food sources of human gut Bacteroides spp., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

The α-amylase, SusG, is a principal component of the Bacteroides thetaiotaomicron (Bt) starch utilization system (Sus) used to metabolize complex starch molecules in the human gastrointestinal (GI) tract. We previously reported the non-microbicidal growth inhibition of Bt by the acarbose-mediated arrest of the Sus as a potential therapeutic strategy. Herein, we report a computational approach using density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation to explore the interactive mechanism between acarbose and SusG at the atomic level in an effort to understand how acarbose shuts down the Bt Sus. The docking analysis reveals that acarbose binds orthosterically to SusG with a binding affinity of -8.3 kcal/mol. The MD simulation provides evidence of conformational variability of acarbose at the active site of SusG and also suggests that acarbose interacts with the main catalytic residues via a general acid-base double-displacement catalytic mechanism. These results suggest that small molecule competitive inhibition against the SusG protein could impact the entire Bt Sus and eliminate or reduce the system's ability to metabolize starch. This computational strategy could serve as a potential avenue for structure-based drug design to discover other small molecules capable of inhibiting the Sus of Bt with high potency, thus providing a holistic approach for selective modulation of the GI microbiota., (© 2023 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

TenA thiamin-degrading enzymes are commonly found in prokaryotes, plants, fungi and algae and are involved in the thiamin salvage pathway. The gut symbiont Bacteroides thetaiotaomicron (Bt) produces a TenA protein (BtTenA) which is packaged into its extracellular vesicles. An alignment of BtTenA protein sequence with proteins from different databases using the basic local alignment search tool (BLAST) and the generation of a phylogenetic tree revealed that BtTenA is related to TenA-like proteins not only found in a small number of intestinal bacterial species but also in some aquatic bacteria, aquatic invertebrates, and freshwater fish. This is, to our knowledge, the first report describing the presence of TenA-encoding genes in the genome of members of the animal kingdom. By searching metagenomic databases of diverse host-associated microbial communities, we found that BtTenA homologues were mostly represented in biofilms present on the surface of macroalgae found in Australian coral reefs. We also confirmed the ability of a recombinant BtTenA to degrade thiamin. Our study shows that BttenA-like genes which encode a novel sub-class of TenA proteins are sparingly distributed across two kingdoms of life, a feature of accessory genes known for their ability to spread between species through horizontal gene transfer., (© 2023. The Author(s).)

Extracellular vesicles are produced in all three domains of life, and their biogenesis has common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. Bacteroides thetaiotaomicron ( Bt ), a prominent member of the human intestinal microbiota, produces significant amounts of outer membrane vesicles (OMVs) which have been proposed to play key physiological roles. Here, we employed a dual marker system, consisting of outer membrane- and OMV-specific markers fused to fluorescent proteins to visualize OMV biogenesis by time-lapse microscopy. Furthermore, we performed comparative proteomic analyses to show that, in Bt , the OMV cargo is adapted for the optimal utilization of different polysaccharides. We also show that a negatively charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. Our results demonstrate that OMV production is the result of a highly regulated process in Bt .

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut 1 . Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Glutamate decarboxylase catalyzes the conversion of glutamate to γ-aminobutyric acid, which plays a vital role in the gut-brain axis. Herein, a novel glutamate decarboxylase from Bacteroides thetaiotaomicron (BTGAD) was heterologously expressed. BTGAD possessed high catalytic efficiency at 60℃ and pH 3.6. As pH response, N-terminal sequence (NTS), C-terminal sequence (CTS), and β-hairpin in BTGAD coordinately regulated its activity under different pH. NTS folded into a loop under acidic pH, and the truncation of NTS severely reduced its activity to 4.2%. While CTS occupied the active site under neutral pH and became disordered to release the inhibition effect under acidic conditions. The β-hairpin, located near the active site, swung and formed open and closed conformations, which acted as an activity switch. This study provides the molecular basis for the coordinated regulation mechanism of BTGAD and lays a theoretical foundation for understanding the metabolism of dietary glutamate and its interaction relationships with the gut-brain axis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bacteroides thetaiotaomicron is a gram negative bacterium within the human gut microbiome that metabolizes a wide range of dietary and mucosal polysaccharides. Here, we analyze the proteome response of B. thetaiotaomicron cultivated on two different carbon sources, glucose and sucrose. Two quantitative LC-MS based proteomics approaches, encompassing label free quantification and isobaric labeling by tandem mass tags were applied. The results obtained by both workflows were compared with respect to the number of identified and quantified proteins, peptides supporting identification and quantification, sequence coverage, and reproducibility. A total of 1719 and 1696 proteins, respectively, were quantified, covering 35 % of the predicted B. thetaiotaomicron proteome. The data show that B. thetaiotaomicron widely maintains its intracellular proteome upon change of the carbohydrates and that major changes are observed solely in the machinery necessary to make use of the carbon sources provided. With respect to the central role of carbohydrates on gut health these data contribute to the understanding of how different carbohydrates contribute to shape bacterial community in the gut microbiome. All proteomics raw data have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033704., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

Why oxygen ceases the growth of strictly anaerobic bacteria is a longstanding question, yet the answer remains unclear. Studies have confirmed that the dehydratase-fumarase containing an iron-sulfur cluster ([4Fe-4S]) is inactivated upon exposure to oxygen in the intestinal obligate anaerobe, Bacteroides thetaiotaomicron (B. thetaiotaomicron); this blocks fumarate respiration, which is the essential energy-producing pathway in anaerobes. Here, we substituted the [4Fe-4S]-dependent fumarase in B. thetaiotaomicron with an iron-free isozyme from E. coli (Ec-FumC). Results show that Ec-FumC successfully performed the catalytic function of fumarase in B. thetaiotaomicron, as the fum-mutant strain that expressed Ec-FumC exhibited succinate-producing ability under anaerobic growth conditions. Ec-FumC is oxygen-resistant and remains active to produce fumarate upon aeration; however, B. thetaiotaomicron mutant that expressed Ec-FumC did not convert fumarate to succinate during air exposure. Biochemical assays of inverted membrane vesicles from wild-type B. thetaiotaomicron confirmed that the electron flux from NADH to fumarate was less efficient in the presence of air as compared to that without oxygen. Our findings suggest that the anaerobic fumarate respiration might be paralyzed due to electron dissipations upon aeration of the obligate anaerobe., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis., (© 2022. The Author(s).)

Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbially-derived gut metabolites are important contributors to host phenotypes, many of which may link microbiome composition to metabolic disease. However, relatively few metabolites with known bioactivity have been traced from specific microbes to host tissues. Here, we use a labeling strategy to characterize and trace bacterial sphingolipids from the gut symbiont Bacteroides thetaiotaomicron to mouse colons and livers. We find that bacterial sphingolipid synthesis rescues excess lipid accumulation in a mouse model of hepatic steatosis and observe the transit of a previously uncharacterized bacterial sphingolipid to the liver. The addition of this sphingolipid to hepatocytes improves respiration in response to fatty-acid overload, suggesting that sphingolipid transfer to the liver could potentially contribute to microbiota-mediated liver function. This work establishes a role for bacterial sphingolipids in modulating hepatic phenotypes and defines a workflow that permits the characterization of other microbial metabolites with undefined functions in host health., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii ( Rb ) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron ( Bt ). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆ sus Bt ) can grow within the halo. Furthermore, when these ∆ sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides . These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.

Members of the gut-dwelling Bacteroides genus have remarkable abilities in degrading a diverse set of fiber polysaccharide structures, most of which are found in the mammalian diet. As part of their metabolism, they convert these fibers to organic acids that can in turn provide energy to their host. While many studies have identified and characterized the genes and corresponding proteins involved in polysaccharide degradation, relatively little is known about Bacteroides genes involved in downstream metabolic pathways. Bacteroides thetaiotaomicron is one of the most studied species from the genus and is representative of this group in producing multiple organic acids as part of its metabolism. We focused here on several organic acid synthesis pathways in B. thetaiotaomicron, including those involved in formate, lactate, propionate, and acetate production. We identified potential genes involved in each pathway and characterized these through gene deletions coupled to growth assays and organic acid quantification. In addition, we developed and employed a Golden Gate-compatible plasmid system to simplify alteration of native gene expression levels. Our work both validates and contradicts previous bioinformatic gene annotations, and we develop a model on which to base future efforts. A clearer understanding of Bacteroides metabolic pathways can inform and facilitate efforts to employ these bacteria for improved human health or other utilization strategies. IMPORTANCE Both humans and animals host a large community of bacteria and other microorganisms in their gastrointestinal tracts. This community breaks down dietary fiber and produces organic acids that are used as an energy source by the body and can also help the host resist infection by various pathogens. While the Bacteroides genus is one of the most common in the gut microbiota, it is only distantly related to bacteria with well-characterized metabolic pathways and it is therefore unclear whether research insights on organic acid production in those species can also be directly applied to the Bacteroides. By investigating multiple genetic pathways for organic acid production in Bacteroides thetaiotaomicron, we provide a basis for deeper understanding of these pathways. The work further enables greater understanding of Bacteroides-host relationships, as well as inter-species relationships in the microbiota, which are of importance for both human and animal gut health.

The mammalian intestinal tract contains trillions of bacteria. However, the genetic factors that allow gut symbiotic bacteria to occupy intestinal niches remain poorly understood. Here, we identified genetic determinants required for Bacteroides thetaiotaomicron colonization in the gut using transposon sequencing analysis. Transposon insertion in BT2391, which encodes a hybrid two-component system, increased the competitive fitness of B. thetaiotaomicron. The BT2391 mutant showed a growth advantage in a mucin-dependent manner and had an increased ability to adhere to mucus-producing cell lines. The increased competitive advantage of the BT2391 mutant was dependent on the BT2392-2395 locus containing susCD homologs. Deletion of BT2391 led to changes in the expression levels of B. thetaiotaomicron genes during gut colonization. However, colonization of the BT2391 mutant promoted DSS colitis in low-fiber diet-fed mice. These results indicate that BT2391 contributes to a sustainable symbiotic relationship by maintaining a balance between mucosal colonization and gut homeostasis., (© 2022. The Microbiological Society of Korea.)

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Although the polysaccharide utilization loci (PULs) activated by pectin have been defined, due to the complex of side-chain structure, the degradative mechanisms still remain vague. Thus, we hypothesize that there may have other specific PULs to target pectin. Here, we characterize loci-encoded proteins expressed by Bacteroides thetaiotaomicron (BT) that are involved in the pectin capturing, importation, de-branching and degradation into monosaccharides. Totally, four PULs contain ten enzymes and four glycan binding proteins which including a novel surface enzyme and a surface glycan binding protein are identified. Notably, PUL2 and PUL3 have not been reported so far. Further, we show that the degradation products support the growth of other Bacteroides spp. and probiotics. In addition, genes involved in this process are conservative in other Bacteroides spp. Our results further highlight the contribution of Bacteroides spp. to metabolism the pectic network., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes . Here, we report the identification of RBPs that are broadly distributed across the Bacteroidetes , with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron Δ rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The Δ rbpB mutant had reduced expression of BT1871 , encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes . IMPORTANCE The human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides , that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates, and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors., Competing Interests: Declaration of interests R.J.X. is a consultant to Novartis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Capsular polysaccharides (CPSs) protect bacteria from host and environmental factors. Many bacteria can express different CPSs and these CPSs are phase variable. For example, Bacteroides thetaiotaomicron (B. theta ) is a prominent member of the human gut microbiome and expresses eight different capsular polysaccharides. Bacteria, including B. theta , have been shown to change their CPSs to adapt to various niches such as immune, bacteriophage, and antibiotic perturbations. However, there are limited tools to study CPSs and fundamental questions regarding phase variance, including if gut bacteria can express more than one capsule at the same time, remain unanswered. To better understand the roles of different CPSs, we generated a B. theta CPS1-specific antibody and a flow cytometry assay to detect CPS expression in individual bacteria in the gut microbiota. Using these novel tools, we report for the first time that bacteria can simultaneously express multiple CPSs. We also observed that nutrients such as glucose and salts had no effect on CPS expression. The ability to express multiple CPSs at the same time may provide bacteria with an adaptive advantage to thrive amid changing host and environmental conditions, especially in the intestine.

Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of polysaccharide utilization loci (PULs), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic β(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), Bacteroides uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct β-glucans. Ligand complexes provide definitive insight into β(1,3)-glucan selectivity in the HGM, including structural features enabling dual β(1,3)-glucan/mixed-linkage β(1,3)/β(1,4)-glucan-binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the trimodular BtSGBP-B and BuSGBP-B revealed a tandem repeat of carbohydrate-binding module-like domains connected by long linkers. In contrast, BfSGBP-B comprises a bimodular architecture with a distinct β-barrel domain at the C terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) is a form of nonspecific chronic intestinal inflammation associated with gut microbiome dysbiosis. Modulating the composition of the intestinal flora may be a viable means of alleviating such inflammatory pathology. Bacteroides thetaiotaomicron (B. thetaiotaomicron) is a symbiotic intestinal microbe that has been associated with IBD, although the mechanistic basis for this association remains to be clarified. In this present study, we determined that B. thetaiotaomicron can alleviate colonic inflammation through mechanisms associated with the modulation of tryptophan metabolism and T cell subsets within inflamed intestinal tissues. Specifically, we found that B. thetaiotaomicron promotes the preferential differentiation of anti-inflammatory Treg/Th2 cells while suppressing the relative differentiation of pro-inflammatory Th1/Th17 cells, thereby decreasing inflammation within the colon. At a molecular level, B. thetaiotaomicron treatment was linked to altered CpG methylation within the Foxp3 promoter that was associated with enhanced Treg cell functionality. In a murine dextran sulfate sodium (DSS) colitis model system, B. thetaiotaomicron increased the levels of the aryl hydrocarbon receptor (AHR) ligands indole metabolites-indole acetic acid (IAA) and indole propionic acid (IPA), thereby increasing AHR activation that is related to changes of transcription factor expression profiles within T cells. In summary, our data suggest that B. thetaiotaomicron can activate AHR and modulate CD4 + T cell differentiation profiles in a murine DSS colitis model system, suggesting that this bacterium may be of therapeutic relevance for the treatment of IBD., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way, the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin., (© 2020 John Wiley & Sons Ltd.)

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.

Alterations to the gut microbiota can cause an amplification of the inflammatory response to intestinal pathogens. We assessed the effect of Bacteroides thetaiotaomicron and Lactobacillus johnsonii on the elimination of Candida species and whether restoration of these two anaerobic bacteria could attenuate the development of colitis in mice. In this study, L. johnsonii and B. thetaiotaomicron interacted directly with Candida species and induced a degradation of the fungal cell wall, mediated via chitinase-like and mannosidase-like activities, which promoted the inhibition of Candida species growth. In the DSS-induced colitis model, oral administration of L. johnsonii and B. thetaiotaomicron to mice reduced the overgrowth of Escherichia coli, Enterococcus faecalis and Candida glabrata populations and resulted in a significant reduction in inflammatory parameters. L. johnsonii and B. thetaiotaomicron decreased pro-inflammatory mediators and enhanced the anti-inflammatory cytokine response with high TLR9 expression and chitinase-like protein-1 activation, which promoted the elimination of C. glabrata from the gut. Overall, these findings provide evidence that L. johnsonii and B. thetaiotaomicron decrease the development of colitis mediated by TLR9 and promote the elimination of C. glabrata from the gut via chitinase-like and mannosidase-like activities.

Unique morphologies can enable bacteria to survive in their native environment. Furthermore, many bacteria change their cell shape to adapt to different environmental conditions. For instance, some bacteria increase their surface area under carbon or nitrogen starvation. Bacteriodes thetaiotaomicron is an abundant human gut species; it efficiently degrades a number of carbohydrates and also supports the growth of other bacteria by breaking down complex polysaccharides. The gut provides a variable environment as nutrient availability is subject to the diet and health of the host, yet how gut bacteria adapt and change their morphologies under different nutrient conditions has not been studied. Here, for the first time, we report an elongated B. thetaiotaomicron morphology under sugar-limited conditions using live-cell imaging; this elongated morphology is enhanced in the presence of sodium bicarbonate. Similarly, we also observed that sodium bicarbonate produces an elongated-length phenotype in another Gram-negative gut bacterium, Escherichia coli . The increase in cell length might provide an adaptive advantage for cells to survive under nutrient-limited conditions.

When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a β-barrel subdomain and an α-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The β-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bacteroides thetaiotaomicron (B. thetaiotaomicron), which resides in the human intestinal tract, has a number of carbohydrate enzymes, including glycoside hydrolase (GH) family 97. Only a few GH 97 enzymes have been characterized to date. In this study, a novel α-galactosidase (Bt_3294) was cloned from B. thetaiotaomicron, expressed in Escherichia coli, and purified using affinity chromatography. This novel enzyme showed optimal activity at 60 °C and pH 7.0. Enzyme activity was reduced by 94.4% and 95.7% in the presence of 5 mM Ca 2+ and Fe 2+ , respectively. It is interesting that Bt_3294 specifically hydrolyzed shorter α-galactosyl oligosaccharides, such as melibiose and raffinose. The D-values of Bt_3294 at 40 °C and 50 °C were about 107 and 6 min, respectively. After immobilization of Bt_3294, the D-values at 40 °C and 50 °C were about 37.6 and 29.7 times higher than those of the free enzyme, respectively. As a practical application, the immobilized Bt_3294 was used to hydrolyze raffinose family oligosaccharides (RFOs) in soy milk, decreasing the RFOs by 98.9%., (Copyright © 2020 Elsevier B.V. All rights reserved.)