A high-throughput microbial glycomics platform for prebiotic development

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Modesto , View ORCID Profile Seth G. Kabonick , View ORCID Profile Jennifer E. Lausch , View ORCID Profile Kamalesh Verma , View ORCID Profile Kailyn M. Winokur , View ORCID Profile Jessica E. Gaydos , View ORCID Profile Asia Poudel , View ORCID Profile Gregory Yochum , View ORCID Profile Guy E. Townsend II doi: https://doi.org/10.1101/2025.07.13.664583 Jennifer L. Modesto a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jennifer L. Modesto Seth G. Kabonick a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Seth G. Kabonick Jennifer E. Lausch a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jennifer E. Lausch Kamalesh Verma a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kamalesh Verma Kailyn M. Winokur a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kailyn M. Winokur Jessica E. Gaydos a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jessica E. Gaydos Asia Poudel a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Asia Poudel Gregory Yochum a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA d Department of Surgery, Penn State College of Medicine , Hershey, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gregory Yochum Guy E. Townsend II a Department of Molecular and Precision Medicine, Penn State College of Medicine , Hershey, PA, USA b Penn State One Health Microbiome Center, Pennsylvania State University, State College , PA, USA c Center for Molecular Carcinogenesis and Toxicology, Pennsylvania State University, State College , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Guy E. Townsend II For correspondence: gtownsend{at}pennstatehealth.psu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The mammalian intestine contains diverse carbohydrate pools that govern the gut microbiome composition. Structurally distinct polysaccharides, also called glycans, are differentially consumed by gut microbial subsets and direct their abundance by controlling gene expression and metabolite production. Therefore, identifying gut microbial accessible carbohydrates (MACs) is necessary to develop new prebiotics that beneficially manipulate the gut microbiome. However, no methods exist to efficiently examine MACs in biologically-derived mixtures. Here, we present a high-throughput platform to detect MACs from various plant, animal, and microbial sources using a genome-wide library of engineered Bacteroides thetaiotaomicron ( Bt ) strains that harness their endogenous glycan detection machinery. We demonstrate that this platform exhibits specific and sensitive responses to glycan mixtures and use bacterially-encoded proteins to characterize a previously unknown MAC from yeast. Expanding this technology across gut Bacteroides species will generate a broadly applicable approach to characterize heterogeneous glycan mixtures and identify prebiotic substrates. Main Commensal microbes harvest carbon from structurally diverse MACs, to achieve extraordinary cell densities in the mammalian intestine 1 – 5 . The glycosidic linkages that tether constituent monosaccharides into each MAC are frequently resistant to degradation in the gastrointestinal tract and therefore require substrate-specific enzymes to degrade 6 – 11 . Gut microbes can utilize available MACs by expressing carbohydrate-active enzymes that target specific glycosidic linkages 1 , 8 , 11 , 12 . Characterizing MACs present in biologically-derived mixtures informs how and why the gut microbiota changes due to host dietary choices, genetics, or disease because chemically-distinct glycans support the expansion of defined microbial subsets. Additionally, identifying MACs that can influence microbial abundance and metabolism can promote engineered strain engraftment and gene expression to leverage the gut microbiome as a therapeutic target 13 . Glycan mixtures have traditionally been examined using tandem chromatography and mass spectrometry, arrayed glycan binding aptamers, or lectins that probe for a range of known carbohydrate structures 14 – 16 . Although these approaches can characterize glycan content in various sample types, they are unable to directly detect MACs. Microbes access unique MACs by producing proteins that sequester, transport, degrade, and detect target molecules in their environment. For example, members of the dominant human intestinal phylum, Bacteroidetes , possess glycan sensors encoded within polysaccharide utilization genetic loci (PULs) that direct rapid, dramatic, and predictable increases to corresponding PUL gene transcripts following recognition of their target glycan structures 4 , 6 , 7 , 17 . Ultimately, these sensors direct increased expression of surface glycan binding proteins (SGBPs), outer membrane transporters (SusCD), and glycolytic enzymes for consumption of available MACs. Different Bacteroides species encode vast and partially unique PUL repertoires, which endow each with the ability to access various target glycans that establish distinct intra-intestinal niches. We previously demonstrated that introduction of PUL promoters into a plasmid-borne Bacteroides -optimized lux cassette, p Bolux, converts these bacteria into biosensors of known and unknown target glycans present in biologically-derived mixtures 18 . We reasoned that PUL reporters could reflect the exquisite sensitivity and specificity of their cognate sensor proteins to detect MACs in glycan mixtures and be implemented to isolate and characterize those molecules in combination with corresponding PUL SGBPs and glycolytic enzymes. Here, we generated a genome-wide Bt reporter library and arrayed these strains into a scalable and distributable high-throughput platform that sensitively indicates target glycans from various host-, microbial-, and plant-derived mixtures. We demonstrate that this approach distinguishes structurally distinct MACs according to monosaccharide composition, glycosidic linkages, and polysaccharide degree of polymerization. We use this technology to detect previously unknown PUL target glycans and employ a recombinant SGBP to isolate and characterize an unknown MAC in Baker’s yeast ( Saccharomyces cerevisiae). Collectively, these results establish Bacteroides species as specific and sensitive toolkits to directly detect, isolate, and characterize unknown PUL target glycans that could be developed into prebiotics. Expanding these tools into additional Bacteroides species will support broad, untargeted glycomics applications to examine heterogenous glycan mixtures and overcome current detection limitations. Ultimately, our platform can be used to develop new prebiotics that manipulate gut microbial abundance and metabolism to benefit human health. Results Construction of an arrayed Bt PUL reporter library We established that individual Bt PUL sensors direct predicted PUL transcript increases 19 , 20 because engineered strains expressing constitutively active sensor proteins from PULs 16, 19, 49, 53, and 57, exhibited dramatic increases in corresponding transcript abundances ( Fig. 1a , Supplementary Table 1) 21 , 22 . To examine whether sensor-directed PUL transcription can be faithfully reported by corresponding transcriptional reporters on a genome-wide scale, we generated a complete library of Bt PUL reporter plasmids by introducing putative promoters identified from RNAseq datasets 23 and mapped transcription start sites 24 , 25 from annotated Bt PULs 26 , 27 into p Bolux 18 (Supplementary Table 2). Although Bt possesses 88 PULs, we generated 91 total reporter strains because PULs 14, 52, 74, and 75 possess two divergently transcribed susCD pairs that were assigned unique reporters (denoted a and b) and PUL63 was omitted due to the absence of a susD -like gene ( Fig. 1a , Supplementary Table 1, 2). Following cryopreservation of batch-produced 96-well plates, individual replicates were revived by the addition of rich media and incubated overnight before transfer to a 384-well plate and supplied 4 distinct carbohydrate preparations, one of which was always galactose as a negative control ( Fig. 1b ). Fold changes in growth-adjusted bioluminescence generated over 18 hours ( Fig. 1c ) were calculated from the area under each curve from strains supplied unique glycan preparations and normalized to a strain harboring the promoter-less p Bolux plasmid and cultures supplied galactose alone ( Fig. 1d ). Download figure Open in new tab Fig. 1. Construction of a genome-wide Bt PUL reporter library array. a, Cartoon depicting 88 Bt PULs denoted by individual genes (innermost black circles). Dots emanating inwards represent significantly increased PUL transcripts measured by RNA-seq from strains engineered to produce constitutively active sensor proteins from PUL16 (red), PUL19 (orange), PUL49 (green), PUL53 (cyan), or PUL57 (purple) and color-corresponding heatmaps are positioned centrally. The outermost symbols represent distinct classes of known or predicted PUL sensor proteins (filled square: ECF, extracytoplasmic sigma factor; filled triangle: HTCS, hybrid two-component system; filled circle: SusR, starch utilization system regular; open circle: other classes) and corresponding glycan structures that activate them emanate radially. b, schematic depicting PUL reporter construction and glycan analysis using the arrayed Bt library. c-d, Bioluminescence ( c ) from all 91 Bt reporters over 18 hours following introduction of hyaluronic acid and normalized by identical strains supplied galactose and corresponding ( d ) AUC normalized by responses from a strain containing p Bolux . All significantly increased reporters have corresponding colors and insignificant increases are displayed in grayscale. N=3, error is SEM. e, Heatmap of AUC over 18 hours from all reporters supplied mixtures containing galactose and various purified polysaccharides. N≥2. All statistics were calculated using 2-way ANOVA with Fisher’s LSD test and *** indicates P -values < 0.001, and ** < 0.01. Bt PUL reporters distinguish between glycosidic linkages and monosaccharide composition To examine the Bt PUL reporter array functionality, we examined changes in bioluminescence from all strains following the introduction of various purified mono- and poly-saccharide preparations. As expected, the monosaccharides, fructose and ribose, increased bioluminescence from their respective PUL reporters, P- PUL22 and P- PUL37 , which agree with previous findings ( Extended Data Fig. 1a ) 18 . However, ribose also increased PUL22 transcripts 28 and corresponding bioluminescence from P- PUL22 ( Extended Data Fig. 1a, b ). Ribose is likely contaminated with fructose because a Bt strain lacking sensor PUL22 ( BT1754 ) harboring P- PUL22 exhibited no bioluminescence increases when supplied ribose, whereas this strain harboring P- PUL37 exhibited similar responses to wild-type Bt ( Extended Data Fig. 1c ). In contrast, a strain lacking sensor PUL37 ( BT2802 ), which is unable to increase PUL37 transcripts or utilize ribose as a sole carbon source 28 , did not display increased activity when harboring P- PUL37 ( Extended Data Fig. 1c ). Similarly, P- PUL22 also exhibited increased bioluminescence when supplied mannose ( Extended Data Fig. 1a ), that required sensor PUL22 ( Extended Data Fig. 1b ), suggesting that the mannose preparation examined here was also contaminated with fructose. These results indicate that the Sensor PUL22 , which binds fructose with high affinity 7 , is responsible for increasing PUL22 expression when supplied ribose or mannose. Ultimately, these data highlight the specificity of PUL reporters for their target ligands and reflect their extraordinary sensitivity in mixtures. Download figure Open in new tab Extended Data Fig. 1. Monosaccharide-responsive PUL reporters exhibit extraordinary sensitivity. a, A heatmap displaying normalized bioluminescence responses to monosaccharides. N≥2. b-c , Normalized biolumi-nescence from wild-type (black) or corresponding sensor -deficient (red) Bt strains harboring ( b ) P- PUL22 or ( c ) P- PUL37 supplied the indicated monosaccharides. N=8, error is SEM. P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values 0.05. We established that Bt PUL reporters can distinguish between MACs based on glycosidic linkages, monosaccharide composition, and degree of polymerization (d.o.p.). Distinct strains exhibited increased bioluminescence according to the administration of various purified plant, animal, and microbially-derived MACs consumed via known PULs 4 , 6 , 7 , 11 , 28 , 29 . Strikingly, P- PUL66 , P- PUL48 , and P- PUL56 were specifically activated by amylose, dextran, and pustulan, respectively, which are distinct glucose homopolymers differing by their α1,4-, α1,6-, and β1,6-glycosidic linkages ( Fig. 1e ). Each PUL is regulated by their respective SusR-like sensor proteins, encoded by BT3703 (Sensor PUL66 ) 30 , BT3091 (Sensor PUL48 ) 31 , and BT3309 (Sensor PUL56 ) 32 , to increase target PUL transcription and utilize these MACs as growth substrates. Sensor PUL66 and Sensor PUL56 differentiate between glycosidic linkage because their corresponding PUL reporters only exhibit increased bioluminescence when supplied α1,4- or β1,6-linked glucose disaccharides ( Fig. 2a ). However, P- PUL48 can also distinguish between d.o.p. because bioluminescence from this strain only increased when supplied isomaltooligosaccharides greater than 3 α1,6-linked glucose units and requires at least 6 residues to reach activity similar to dextran ( Fig. 2b ). These data demonstrate that SusR-like PUL sensors detect distinct glycosidic linkages and d.o.p.. Download figure Open in new tab Fig. 2. Bt PUL reporters distinguish between monosaccharide composition, d.o.p., and glycosidic linkage. a, A heatmap displaying normalized AUC following the introduction of various oligosaccharides to the Bt PUL reporter array. n=2. b, Normalized AUC from P- PUL48 supplied isomaltooligosaccharides with increasing d.o.p. N=6, error is SEM. c, Normalized AUC from wild-type (black) or sensor- PUL 58 -deficient ( ΔBT2160 , red) strains harboring P- PUL58 supplied the indicated di- and tri-saccharides. N≥3, error is SEM. d, Normalized AUC from wild-type (black) or ΔBT2160 (red) strains harboring P- PUL46 supplied the indicated cello-oligosaccha-rides. N=4, error is SEM. For panels b-d , P -values were computed using 2-way ANOVA. *** indicates values 0.05. Further examination of purified oligosaccharides yielded several unexpected outcomes. First, monomeric glucose and various glucose-disaccharides, including maltose, isomaltose, trehalose, and β-gentibiose increased bioluminescence from P- PUL58 ( Fig. 2a ). Similarly, supplying glucose-fructose heterodimers, turanose and palatinose, stimulated greater than 10-fold increases from this strain, whereas sucrose did not, suggesting that this PUL reporter responds to glucose- and fructose-containing disaccharides subsets. We hypothesized that P- PUL58 could be governed by the SusR-like sensor, encoded by BT2160 , that is an important Bt fitness determinant during growth in trehalose and palatinose 33 . Accordingly, Δsensor PUL 58 harboring P- PUL58 did not exhibit increased bioluminescence in any condition, indicating that this sensor facilitates PUL58 transcription in response to various disaccharides ( Fig. 2c ). Curiously, this sensor also responds to glucosinolates derived from cruciferous vegetables and is required for their conversion into isothiocyanates by increasing transcription of the linked genes BT2159-56 34 . A deeper examination of how and why this distally encoded sensor protein responds to these signals is necessary to understand this regulatory relationship. Unexpectedly, P- PUL46 detected a Bt -inaccessible carbohydrate 35 because introduction of the β1,4-linked glucose disaccharide, cellobiose, increased bioluminescence from this strain ( Fig. 2a, d ). This response was d.o.p.-specific because there was no change when supplied cellulose-derived tetra- or hexa-saccharides and this response did not require sensor PUL 58 ( Fig. 2d ). Because Bt cannot utilize cellulose or its polysaccharide breakdown products including cellobiose 35 , these results suggest that cellobiose is a signal in the intestine. Bt PUL reporters detect discreet carbohydrate structures The Bt PUL array can also distinguish between various microbial-, animal-, and plant glycans covalently tethered into polymeric complexes. For example, Bt utilization of yeast cell wall α-mannans ( Extended Data Fig. 2a ) requires three PULs: 36, 68, and 69 36 , 37 , which increase activity from the reporter strains, P- PUL36 , P- PUL68, and P- PUL69 by the addition of purified α-mannan ( Fig. 1e ). The corresponding sensor proteins, BT2628 (Sensor PUL36 ) and BT3786 (Sensor PUL68 ) facilitate mannan-responsive PUL36/68 transcription because bioluminescence from strains harboring either P- PUL36 or P- PUL68 is not apparent in a strain lacking both sensors ( Δsensors PUL36/68 ; Extended Data Fig. 2b ), consistent with previous transcript measurements 36 . Alternatively, PUL69 transcription is facilitated by Sensor PUL69 , encoded by BT3853 36 , which is required for increased bioluminescence from P- PUL69 when supplied α-mannan ( Extended Data Fig. 2b ). Sensors PUL36/68/69 are collectively required for mannan utilization as a sole carbon source ( Extended Data Fig. 2c ) but detect discreet carbohydrate structures because the branched α1,3-[α1,6] trisaccharide increases P- PUL36/68 bioluminescence ( Extended Data Fig. 2d, e ). Conversely, P- PUL69 did not respond to any di- or tri-saccharide examined, indicating it responds to a different oligo-mannan structure ( Extended Data Fig. 2f ). These data highlight that PUL sensor proteins direct cognate PUL expression following recognition of distinct glycan moieties assembled as larger polymers. Download figure Open in new tab Extended Data Fig. 2. Bt mannan PUL reporters detect structurally distinct manno-oligosaccharides. a, Cartoon representation of yeast cell wall glycan structures containing mannoproteins decorated with α-mannan tethered to β 1,6-glucan by a GPI-anchor remnant glycan. b , Normalized bioluminescence from wild-type , sensor PUL36/68 -, or sensor- PUL69 -deficient Bt strains harboring P- PUL36, P-PUL68 , or P- PUL69 supplied α-mannan and normalized by responses from isogenic strains harboring p Bolux . N=8, error is SEM. c, Growth of strains described in ( b ) supplied α-mannan as a sole carbon source. N=8, error is SEM. d-f, strains harboring ( d ) P- PUL36 , ( e ) P- PUL68 , or ( f ) P- PUL69 were supplied the indicated manno-di-, tri-saccharides, or poly-saccharides. n=4, error is SD. For panels b,d-f, P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values 0.05. Bt possess 3 PULs that respond to glycosaminoglycans (GAGs), which are intestinally abundant heteropolymers comprised of amine sugars and uronic acids, including previously characterized PULs 57 6 , 10 and 85 38 , and the uncharacterized PUL20 ( Fig. 3a ). Strains harboring P- PUL57 exhibited increased bioluminescence when supplied chondroitin sulfate (CS) or dermatan sulfate (DS; Fig. 1e ), which are variably sulfated linear polymers of repeating, disaccharide units comprised of glucuronic acid (GluA) and β1,3-linked N-acetylgalactosamine (GalNAc). P- PUL57 also responds to hyaluronic acid 18 , an unsulfated GAG whose disaccharide unit is comprised of gluA and β1,3-linked N-acetylglucosamine (GlcNAc) because the Sensor PUL57 protein, BT3334, cannot distinguish between either CS- or HA-derived disaccharide subunits ( Fig. 3b ) 6 . Introduction of CS, DS, and HA also increased bioluminescence from P- PUL20 ( Fig. 1e ), consistent with previous transcriptional measurements 3 , suggesting that all GAGs activate an additional PUL. However, Sensor PUL57 is not responsible for increasing bioluminescence from P- PUL20 because: 1.) CS- and HA-derived disaccharides increased activity from P- PUL57 but not P- PUL20 ( Fig. 3b ), 2.) heterologous expression of a constitutively active Sensor PUL57 (BT3334*) increased PUL57 but not PUL20 transcripts ( Fig. 1a ), and 3.) BT3334* increased bioluminescence from strains harboring P- PUL57 but not P- PUL20 ( Fig. 3c ). Furthermore, a distinct GAG, heparin, also increased bioluminescence from only P- PUL20 and P- PUL85 ( Fig. 1e ), whereas the composite disaccharide comprised of alternating N-acetylglucosamine (GlcNAc) and uronic acid increased activity from only P- PUL85 ( Fig. 3b ). We propose that PUL20 responds to the common GAG tetrasaccharide linker, GlcA-[β1-3]-Gal-[β1-3]-Galβ-[1-4]-Xyl2P-β-Ser ( Fig. 1a ), because CS from shark cartilage stimulates increased bioluminescence from P- PUL20 and P- PUL57 but no other contaminating glycans following co-incubation with SGBP PUL57 ( Fig. 3d ) 18 , indicating that the PUL20-target glycan is covalently tethered to CS. Download figure Open in new tab Fig. 3. PUL 20 responds to a unique GAG-tethered glycan. a, cartoon representing PUL20 activated by GAGs. b , Normalized bioluminescence from Bt strains harboring P- PUL20, P- PUL38, P- PUL47, P- PUL57, or P- PUL85 supplied unsaturated disaccharides (di0S) derived from CS (blue), HA (red), or heparin (purple) and normalized by responses from a strain harboring p Bolux . N=6. c, Normalized bioluminescence from a strain expressing BT3334* harboring the reporters described in ( b ) and normalized by identical strains harboring the empty vector cultured in galactose as a sole carbon source. N=4. d, Normalized bioluminescence from Bt strains harboring P- PUL20, P- PUL38, P- PUL47, P- PUL57, or P- PUL85 supplied SGBP PUL57 co-purified material following incubation with shark CS and normalized by responses from a strain harboring p Bolux . N=6. For panels b-d , error is SEM and P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values 0.05. Plant cell wall glycans called pectins and hemicelluloses, are frequently assembled into complex polymeric structures ( Extended Data Fig. 3a ) that are targeted by distinct Bt PULs 1 , 4 , 9 , 11 . This is exemplified by P- PUL7 , which exhibited increased bioluminescence when supplied arabinan ( Fig. 1e ). However, arabinan also elicited increases from strains harboring P- PUL5 , P- PUL13 , P- PUL22 , P- PUL47 , P- PUL48 , P- PUL65 , P- PUL77 , and P- PUL86 , indicating this preparation also contains arabinogalactan (PUL5), rhamnogalacturonan II (PULs 13 & 21), fructose (PUL22), dextran (PUL48), rhamnogalacturonan I (PUL77), and pectic galactan (PUL86) ( Extended Data Fig. 3b ). Reporter activity faithfully reflects sensor-dependent activity because the Sensor PUL7 (BT0366) 39 , which recognizes arabino-oligomers of at least 6 α1,5-linked arabinose residues 4 , was only required to stimulate P- PUL7 when supplied arabinan ( Extended Data Fig. 3b ). Consistent with this notion, purified oligo-galacturonate increased bioluminescence from its corresponding reporter, P- PUL75a&b ( Fig. 2a ), whereas highly polymeric PGA also increased activity from P- PUL7 , P- PUL22 , P- PUL47 , P- PUL48 , and P- PUL66 , and P- PUL86 ( Fig. 1e ), demonstrating that PUL reporters respond predictably to complex MACs comprised of multiple ligands 1 , 4 , 7 , 9 , 11 . Finally, the ubiquitous food additives carrageenan, xantham gum, locust bean gum, karaya gum, and gum arabic are complex heteropolymers of largely Bt -inaccessible carbohydrates 9 , 35 but contain small amounts of detectable structures because each elicits increased bioluminescence from at least one PUL reporter ( Fig. 1e ). For example, P- PUL5 is dramatically activated by purified arabinogalactan and exhibits significant increases when supplied karaya gum and gum arabic 9 ( Fig. 1e ). Collectively, these data demonstrate the exquisite specificity of Bt PUL reporters for complex multi-valent MACs, facilitated by the cognate sensor proteins. Download figure Open in new tab Extended Data Fig. 3. Bt PUL reporters recognize discreet glycan moieties from plant pectins. a, Cartoon representation of plant pectin structures including arabinan (PUL7), rhamnogalacturonan I (RGI, PUL77), and pectic galactan (PUL86). b , Normalized bioluminescence from wild-type or Δsensor PUL7 Bt strains harboring the indicated PUL reporters supplied arabinan and normalized by responses from a strain harboring p Bolux . n=8, error is SEM. P -values were calculated using 2-way ANOVA with Fisher’s LSD test and * indicates values <0.05, *** 0.05. The Bt PUL reporter array can faithfully survey glycan mixtures Bt PUL reporters can simultaneously examine MACs present in heterogeneous glycan mixtures because P- PUL5 , P- PUL22 , P- PUL57 , and P- PUL48 exhibited increased bioluminescence when supplied an equal mixture of purified arabinogalactan (AG, PUL5), levan (PUL22), hyaluronan (HA, PUL57), and dextran (PUL48) ( Fig. 1e ). Additionally, this mixture increased activity from P- PUL20 and P- PUL47 , which were both increased by HA ( Fig. 1c-e ), displaying faithful reporting of lab-prepared mixtures. To characterize biologically derived mixtures, we measured reporter responses to PMOGs, a mixture of porcine mucosal-derived O-glycans that increases many Bt PUL transcripts 3 , 18 , 40 . When supplied PMOG, we detected increased activity in GAG-, and O-glycan-specific PUL reporters, consistent with previous transcript measurements 3 , 18 , 40 indicating that the reporter strains can faithfully examine biologically-derived mixtures ( Fig. 4a ). To prepare other host-, diet-, and microbially-derived mixtures, we adapted the oxidative-release of natural glycans (ORNG) method that can liberate glycans from a wide variety of biological materials 41 . We determined that a preparation of porcine mucosal glycans using ORNG (PMG), yielded similar reporter profiles as PMOG ( Fig. 4a ), but did display a 2-fold decrease in bioluminescence from a strain harboring P- PUL14b and a 2-fold increase in signal from P- PUL48 , indicating glycan subsets are differentially extracted or detected when this approach is applied ( Extended Data Fig. 4a ). Download figure Open in new tab Extended Data Fig. 4. Extraction- and species-specific reporter responses. a-c, Log2 fold differences between reporter responses from relevant strains supplied ( a ) porcine gastric mucin prepared using ORNG (PMG) or β-elimination (PMOG), ( b ) fresh human (HIG) and boar (BCG), and ( c ) human milk oligosaccharides (HMO) prepared using ORNG or enzymatic treatment (ProK). N≥2, error is SD. Download figure Open in new tab Fig. 4. Arrayed Bt PUL reporter strains differentiate MAC sources. a, Heatmap representing Bt PUL array responses to the indicated biologically-derived mixtures clustered by reporter activation similarity. n≥2. b, PCoA of Bt reporter responses to different MACs from ( a ) using the Jaccard similarity index and Ward D2 distance calculations. c, Distribution of unique and shared predicted PULs across the indicated Bacteroides species. To determine how gut microbial responses differ between animal and human mucosal material, we compared human-derived intestinal glycan mixtures collected from various patient intestinal samples recovered following surgery (HIG) and porcine cecal mixtures from freshly slaughtered boars (BCG). HIG elicited similar patterns of reporter activity as PMOG and fresh porcine cecal glycans, suggesting that mammalian hosts largely supply gut microbes with similar growth substrates ( Fig. 4a ). For example, HIG and BCG elicited similar bioluminescence from P- PUL14 b, P- PUL20 , P- PUL38 , P- PUL66 , and P- PUL72 ( Extended Data Fig. 4b ). However, the following distinctions were readily apparent: on one hand, human material elicited reduced bioluminescence from the CS/HA reporter P- PUL57 and heparin reporter P- PUL85 , but on the other hand they elicited increased responses from P- PUL76 , P-PUL81, and P-PUL86 ( Extended Data Fig. 4b ). Furthermore, human milk glycans (HMG) elicited similar responses to HIG, however, signals were improved when HMG were prepared by defatting and proteinase K treatment ( Extended Data Fig. 4c ), suggesting that ORNG can result in glycan peeling from some source materials 42 . We used ORNG to prepare various animal, plant, and fungal material that are abundant components of the human diet ( Fig. 4a ) and examined Bt reporter responses. As expected, plant-, animal-, and microbially-derived material occupied distinct space following PCoA analysis, indicating that MAC distribution reflects source phylogeny at the phylum level ( Fig. 4b ). For example, dietary plant products emanating from both mono- and di-cot species distinctly increased bioluminescence from strains harboring P- PUL5 (arabinogalactan), P- PUL7 (arabinan), P- PUL13 and P- PUL21 (RGII), P- PUL22 (fructan), P- PUL66 (starch/glycogen), P- PUL75 (PGA), and P- PUL86 (RGI), which is consistent with common plant fibers. Conversely, mammalian derived mixtures stimulated P- PUL14b (N-glycans), P- PUL20 (GAG associated), P- PUL38 (unknown mucin O -glycan), P- PUL57 (CS/HA), P- PUL66 (glycogen), and P- PUL76 (unknown mucin O -glycan). Finally, fungal products consistently increased activity from strains containing P- PUL29 (unknown), P- PUL36/68 (α-mannan), P- PUL56 (β-glucan), P- PUL66 (starch/glycogen), and P- PUL69 (α-mannan). Accordingly, processed foods containing both plant and fungal products, including breads and beer, contained reporter increases consistent with both grains and yeast ( Fig. 4a ). Unexpectedly, several PUL reporters did not exhibit bioluminescence increases when supplied any purified carbohydrate or mixture examined in this study, suggesting either these target glycans are not present in the mixtures prepared for this work or the corresponding reporter plasmid may not contain the appropriate regulatory sequence to facilitate increased bioluminescence. However, we determined that the latter is an unlikely scenario because qPCR analysis for susC transcripts from PULs whose corresponding reporters failed to increase bioluminescence were not increased when supplied various mixtures ( Extended Data Fig. 5a ). Furthermore, expression of constitutively active sensor proteins from PULs 16, 19, 49, 53, and 57, which increased target PUL transcription ( Fig. 1a ) 21 , 22 , stimulated bioluminescence increases when these strains contained the corresponding PUL reporter ( Fig. 3c , Extended Data Fig. 5b ). A notable exception is PUL2, which targets an unknown glycan, exhibits 180-fold increased transcripts when supplied yeast extract and red-wine derived glycan mixtures ( Extended Data Fig. 5a ), but whose corresponding reporter did not exhibit increased bioluminescence despite engineering an additional plasmid that included an internal transcription start site 25 , 40 ( Extended Data Fig. 5c ). Thus, the majority of PULs can be converted into glycan biosensors with a small minority that may require additional optimization to use p Bolux . Finally, establishing the functionality of various PUL reporters with unknown target glycans ( Extended Data Fig. 5b ) that are not activated across the common foods examined in this study, indicate that future investigations should focus on other glycan sources, including co-resident microbes and fermented products. Download figure Open in new tab Extended Data Fig. 5. PUL expression is faithfully reported. a, Heatmap depicting the indicated susC transcript measurements from Bt cultures supplied the indicated glycan mixtures. N=3. b, Bioluminescence from strains expressing the indicated constitutively active PUL sensor and either p Bolux (black) or the corresponding PUL reporter (red) normalized by strains harboring an empty vector. N=4, error is SEM. c, Cartoon of PUL2 (top) and normalized bioluminescence (bottom) from 2 different PUL reporters containing distinct TSS combinations supplied grape juice or yeast extract glycans (YEG). N=4, error is SEM. For panels b,c , P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values 0.05. Unique PULs across p Bolux -compatible Bacteroides species This study demonstrates how Bt PUL reporters can be readily constructed and deployed to detect MAC content in various glycan preparations. However, other Bacteroides species possess unique PUL repertoires that putatively confer distinct glycan accessibility, indicating that they recognize a partially unique subset of carbohydrate structures ( Fig. 4c, Supplementary Table 3). For example, B. fragilis PULs facilitate its growth in host-derived glycan preparations including those distinct from Bt target glycans 22 , whereas B. ovatus 43 and B. uniformis 44 possess PUL repertoires that target Bt -inaccessible hemicelluloses. We previously demonstrated that p Bolux can report promoter-dependent transcription in B. ovatus 18 and B. fragilis 45 , and this is consistent in B. uniformis and B. cellulosilyticus because introduction of their corresponding rpoD promoters in this plasmid increase bioluminescence from all five species ( Extended Data Fig. 6 ). Therefore, construction of future PUL reporter libraries in these species will expand the breadth of glycan detection capabilities to comprehensively characterize MAC content in host-, microbial-, and dietary-components. Download figure Open in new tab Extended Data Fig. 6. Various Bacteroides species can be converted into glycan biosensors. Normalized bioluminescence values from Bt , B. fragilis ( Bf ), B. uniformis ( Bu ), B. ovatus ( Bo ), and B. cellulosilyticus ( Bc ) type strains harboring either p Bolux (black) or a plasmid containing the corresponding rpoD promoter (red). N=8, error is SEM. P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values < 0.001. Isolation and characterization of a Saccharomyces cerevisiae glycan consumed by Bt To explore the utility of PULs as glycomics tools for prebiotic development, we characterized an unknown glycan detected in a biologically derived mixture. We selected PUL71 ( Fig. 5a ) because yeast extract glycans (YEG) elicited 32-fold increased bioluminescence from strains harboring P- PUL71 , which was also stimulated by preparations from yeast containing products like red wine and animal feeds, but not breads or beers ( Fig. 4a ). Furthermore, P- PUL71 activity was increased in yeast-derived mannan prebiotic mannooligosaccharides (Bio MOS) but absent in host- and most diet-derived mixtures ( Fig. 4a ). Furthermore, this glycan is an excellent prebiotic candidate because a strain lacking PUL71 ( ΔBT3957-BT3965 ) exhibited a 5-fold fitness defect relative to wild-type Bt in mice supplied a 0.5% YEG solution in place of water ( Fig. 5b ). PUL71 includes a unique glycan sensor protein class, encoded by BT3957 , that possesses a periplasmic glycan binding domain and a cytoplasmic DNA-binding domain similar to HTCSs ( Extended Data Fig. 7a ). We established that the putative sensor, BT3957, controls PUL71 transcription because a sensor PUL 71 -deficient strain ( Δsensor PUL71 ) harboring P- PUL71 failed to exhibit increased activity in response to YEG ( Extended Data Fig. 7b ). Conversely, sensor PUL36/68 - or sensor PUL69 -deficient strains harboring P- PUL71 exhibited similar bioluminescence as wild-type Bt supplied YEG ( Extended Data Fig. 7b ). PUL71 responds to a glycan distinct from other well characterized yeast-derived glycans including α-mannan, which activates P- PUL36/68/69 ( Extended Data Fig. 2b ) 36 and β-1,6-glucan, which stimulates P- PUL56 ( Fig. 2a ) 32 , because bioluminescence from P- PUL71 was unchanged when supplied these purified glycans ( Fig. 1e ) and Δsensor PUL71 exhibited similar activity to wild-type Bt harboring P- PUL36 or P- PUL56 when supplied YEG ( Extended Data Fig. 7b ), indicating that Sensor PUL71 recognizes a unique yeast-derived glycan that increases PUL71 expression. Download figure Open in new tab Extended Data Fig. 7. PUL reporters demonstrate a unique assemblage of distinct yeast glycans. a, Alpha-fold structure of Sensor PUL71 (BT3957) with labels indicating the periplasmic (PP) carbohydrate-binding module (CBM), the transmembrane (TM) region in the inner membrane (IM), and the cytoplasmic (CP) receiver (Rec) and helix-turn-helix (HTH) domains. b, Normalized AUC from wild-type Bt or the indicated sensor-deficient strains harboring P- PUL36 , P- PUL56 , P- PUL69 , or P- PUL71 supplied YEG. c&e, Normalized AUC from YEG-activated reporters supplied co-purified material following incubation of YEG with recombinant ( c ) SGBP PUL71 or ( e ) SGBP PUL56 . N=6, error is SEM. d&g, Growth of wild-type Bt or the indicated sensor-deficient strains supplied co-purified material following incubation of YEG with recombinant ( d ) SGBP PUL71 or ( g ) SGBP PUL56 . N=3, error is SEM. f, Growth of wild-type Bt or Δsensor PUL 56 supplied β-gentibiose. N=8, error is SEM. h, HPAEC-PAD analysis of YEG treated with GH20 PUL56 . i, Normalized bioluminescence from a strain harboring P- PUL71 supplied various di- and oligo-mannosaccha-rides or YEG. N=4, error is SEM. For panels b , c , e , and i , P -values were calculated using 2-way ANOVA with Fisher’s LSD test. * indicates values < 0.05, ** < 0.01, *** 0.05. Download figure Open in new tab Fig. 5. PUL71 consumes an unknown yeast cell wall glycan. a, Cartoon schematic of PUL71, which responds to yeast glycans. b, Fitness index of a PUL71 -deficient strain relative to wild-type Bt in mice supplied a 0.5% YEG solution compared to those supplied water. N=5 and error is SEM. P -values were calculated using 2-way ANOVA with Bonferroni correction. *** indicates values 0.05. c, Normalized bioluminescence of YEG-induced reporter strains supplied SGBP PUL71 -isolated glycans from GH30 PUL56 -treated YEG. N=6, error is SEM. P -values were calculated using 2-way ANOVA with Fisher’s LSD test. *** indicates values 0.05. d&e, Growth of wild-type and the indicated sensor-deficient Bt strains supplied ( d ) SGBP PUL71 -co-purifying material following incubation with GH30 PUL56 -treated YEG or ( e ) untreated YEG. N=6, error is SEM. To characterize the PUL71 target glycan, we co-incubated YEG with recombinant hexahistidine-tagged SGBP PUL71 (BT3961) and recovered protein-bound glycans using metal affinity chromatography 18 . The recovered glycans were enriched for the PUL71 target over other MACs present in YEG because the co-purifying material significantly increased activity from P- PUL71, but P- PUL7 (α-arabinan), P- PUL22 (fructans), P- PUL29 (unknown), P- PUL48 (dextran), P- PUL66 (glycogen), and P- PUL72 (high mannose N-glycan 36 ) were no longer detectable ( Extended Data Fig. 7c ). However, P- PUL36/68/69 and P- PUL56 also exhibited increased activity indicating that α-mannan and β-glucan co-purified with the PUL71 target ( Extended Data Fig. 7c ). Significantly activated PULs contributed to accessing the SGBP PUL71 co-purifying material because the corresponding sensor-deficient strains exhibited reduced growth on this material as a sole carbon source, with Δsensor PUL71 exhibiting the largest reduction ( Extended Data Fig. 7d ). We hypothesized that the BT3961-purified material contained covalently tethered carbohydrates comprised of the PUL71-, PUL36/68/69-, and PUL56-target glycans because the yeast cell-wall is an interconnected matrix comprised of chitin, β1,3-, and β1,6-glucans decorated with mannoproteins through a GPI-anchor remnant glycan ( Extended Data Fig. 2a ). Consistent with this notion, material co-purifying with the SGBP PUL56 , BT3313, incubated with YEG, also increased bioluminescence from strains harboring P- PUL56 , P- PUL36/68 , and P- PUL71 , although bioluminescence from P- PUL69 was not significantly increased possibly because the corresponding ligand was below the limit of detection ( Extended Data Fig. 7e ). Accordingly, only Δsensor PUL56 , which is required for growth on β1,6-glucan ( Extended Data Fig. 7f ), exhibited substantially reduced growth when supplied SGBP PUL56 -isolated material ( Extended Data Fig. 7g ), indicating the other target glycans are present in relatively low abundances. Collectively, these data demonstrate that the PUL56 and PUL71 SGBPs isolate their corresponding target glycans when assembled into polymeric structures. To determine how the PUL71-target glycan is tethered to β1,6-glucan and α-mannan, we treated YEG with the PUL56-encoded β1,6-glucosidase (BT3312), referred to here as GH20 PUL56 , which liberated glucose and β-gentibiose from YEG ( Extended Data Fig. 7h ). We determined that GH PUL56 treatment removed β1,6-glucan, but not α-mannan, from the PUL71 target because SGBP PUL71 -isolated material no longer increased bioluminescence from P- PUL56 but exhibited significant increases from P- PUL36/68/69 ( Fig. 5c ). Accordingly, Δsensor PUL71 and Δsensors PUL36/68/69 each exhibited reduced growth on this material as a sole carbon source and growth was completely abolished in a strain lacking all four sensors ( Δsensors PUL36/68/69/71 ) ( Fig. 5d ), whereas these strains grew on YEG with reduced growth maxima ( Fig. 5e ). P-PUL71 likely responds to a distinct manno-oligosaccharide because introduction of various purified substrates were unable to increase bioluminescence ( Extended Data Fig. 7i ). Furthermore, compositional analysis demonstrated that SGBP PUL71 -isolated material from untreated YEG contained glucose ( Extended Data Fig. 8a ), whereas this monosaccharide was no longer detectable when YEG was pre-treated with GH20 PUL56 ( Extended Data Fig. 8b ). Consistent with this finding, HPAEC-PAD analysis revealed that SGBP PUL56 -isolated glycans from untreated YEG contained mannose ( Extended Data Fig. 8c ), further suggesting that yeast β1,6-glucan and α-mannan are tethered in the absence of mannoproteins, which are eliminated by ORNG extraction. These data indicate that yeast α-mannan, β-glucan, and PUL71-target glycan are covalently assembled into a complex resistant to oxidation, and that the PUL71 target glycan decorates yeast-derived α-mannan prepared using ORNG ( Fig. 4a ) but not alkaline extraction, which was used to generate the α-mannan standard ( Fig. 1e ). PUL71 includes the mannosidases BT3962 and BT3965, which respectively convert α1,2- and α1,4-mannobioses into mannose ( Extended Data Fig. 9a, b ), and a putative mannosidase BT3963, which was unable to hydrolyze any mannobiose examined here ( Extended Data Fig. 9c -f), in agreement with previous results 46 . Collectively, these data suggest that the PUL71-target glycan is an unknown yeast α-mannan decoration comprised of unique glycosidic linkages that can function as a prebiotic. Download figure Open in new tab Extended Data Fig. 8. Monosaccharide composition analysis of SBGP-isolated glycans. a-b, HPAEC-PAD analysis of monosaccharides released from glycans that co-purified with SGBP PUL71 ( a ) before or ( b ) after YEG treatment with GH20 PUL56 . c, HPAEC-PAD analysis of monosaccharides released from glycans that co-purified with SGBP PUL56 following incubation with YEG. Download figure Open in new tab Extended Data Fig. 9. PUL71 GH specificity indicates novel glycan structure. a-f, HPAEC-PAD analysis of reactions containing ( a ) α1,2-mannobiose alone (black) or incubated with BT3962 (blue), ( b ) α1,4-mannobiose alone (black) or incubated with BT3965 (pink), or ( c ) α1,2-mannobiose, ( d ) α1,3-mannobiose, ( e ) α 1,4-mannobiose, or ( f ) α1,6-mannobiose alone (black) or incubated with BT3963 (purple). Discussion We have developed a microbial glycomics platform that harnesses Bacteroides PUL sensors to detect, isolate, and characterize MACs from biologically derived mixtures ( Fig. 1 ). We demonstrated that a genome-wide Bt PUL-reporter library can indicate the monosaccharide composition, glycosidic linkages and d.o.p. of purified Bt accessible-( Fig. 1e , 2b) and - inaccessible carbohydrates ( Fig. 2d ). Furthermore, we demonstrated this toolkit can efficiently characterize MAC content in semi-purified preparations ( Fig. 1e , Extended Data Figs. 1 , 2 ) and various mixtures derived from the host mucosa and dietary material ( Fig. 1e and Fig. 4a ). We establish that reporter responses largely correspond to the biological source of each mixture ( Fig. 4b ), indicating that MAC content reflects phylogeny. The exquisite specificity and sensitivity of Bacteroides reporter strains highlight their utility as glycan biosensors that could be implemented in product quality control to detect carbohydrate contaminants ( Extended Data Fig. 1b, c ) and co-purifying moieties ( Fig. 3 ). Furthermore, this approach could be applied to broader glycomics applications including surveillance of different carbohydrate structures from patient samples, plant cultivars, and microbial isolates ( Fig. 4a ). Finally, Bt PUL reporters can directly identify novel MACs that could be translated into prebiotics to manipulate the gut microbiome composition and products for therapeutic purposes ( Fig. 5 ). Therefore, PUL reporters offer a powerful new toolkit that can be expanded using additional Bacteroides species that possess unique PUL repertoires ( Fig. 4c , Extended Data Fig. 6 ). Materials and Methods Bacterial growth conditions All bacteria were cultured as described previously 47 . Briefly, Bacteroides strains (described in Supplementary Table 5) were cultured on brain-heart infusion agar (MilliporeSigma) containing 5% horse blood (Hardy) under anaerobic conditions (85% N 2 , 12.5% CO 2 , 2.5% H 2 ). Liquid cultures were inoculated from a single colony into TYG media and incubated under anaerobic conditions before sub-culture into Bacteroides minimal media containing 100 mM KH 2 PO 4 (pH=7.2), 15 mM NaCl, 8.5 mM (NH 4 ) 2 SO 4 , 0.5 μg/ml of L-cysteine, 1.9 μM hematin, 200 μM L-histidine, 100 μM MgCl 2 , 1.4 μM FeSO 4 , 50 μM CaCl 2 , 1 μg/ml of vitamin K 3 , 5 ng/ml of vitamin B 12 and individual carbon sources described in Supplementary Table 4. All bacterial strains included the following antibiotics where appropriate: 100 μg/mL ampicillin, 200 μg/mL gentamicin, 2 μg/mL tetracycline, or 25 μg/mL erythromycin (MilliporeSigma). RNA-seq Strains engineered to express constitutively active PUL sensors or corresponding vector control were cultured in triplicate in minimal media containing galactose as the sole carbon source to mid-exponential phase (OD 600 = 0.45 - 0.7) before collection by centrifugation, frozen on dry ice, and stored at −80°C. Cell pellets were treated with RNAprotect Bacteria Reagent (Qiagen) and RNA was harvested using RNeasy Mini Kit with on-column DNase digestion (Qiagen) according to the manufacturer’s instructions. Purified RNA was treated with Turbo DNase (Invitrogen) according to the manufacturer’s instructions and re-purified using RNeasy Mini Kit with on-column DNase digestion. RNA-sequencing was performed by SeqCoast Genomics using a Nextseq 2000 (Illumina) following ribosomal RNA depletion using the Ribo-Zero Plus Microbiome rRNA Depletion Kit (Illumina) to generate 12 million 150bp paired-end reads per sample. Reads were aligned to the B. thetaiotaomicron genome (strain VPI-5482; GenBank accession number NC_004663 ) in Galaxy using Bowtie2 (v2.5.3). The mapped reads were quantified using featureCounts (v2.0.8) and differential gene expression was measured using DEseq2 (v2.11.40.8) and values are listed in Supplementary Table 1. Raw files are available on the NCBI Sequence Read Archive, PRJNA1298319. Reporter construction Reporter plasmids were constructed by amplifying PUL promoter fragments (Supplementary Table 2) using oligonucleotides (Supplementary Table 6) as previously described 18 . Briefly, PUL promoters amplified using Q5 Master Mix (NEB) were combined with pBolux 18 linearized with BamHI-HF and SpeI-HF (NEB) and NEBuilder Master Mix (NEB). Reactions were incubated for 1 hour at 50°C before dialyzed on a 0.025 µM dialysis membrane against water and subsequent electroporation into competent E. coli S17-1. All plasmids were verified by Sanger sequencing. Engineering Bt mutants Indicated Bt genomic deletions were generated using pEXCHANGE- tdk plasmids (Supplementary Table 5) harboring flanking sequences, using amplicon-specific primers (Supplementary Table 6), as previously described 48 . In short, pEXCHANGE constructs were introduced via di-parental mating and chromosomal integration was validated by PCR. Parent strains were counter-selected on solid media containing 200ng/mL 5-fluoro-2-deoxyuridine (DOT Scientific). All strains were validated using linear amplicon sequencing. Bt reporter array production All 91 reporter Bt strains described were cultured in TYG to stationary phase in a distinct position of a 96 deep-well plate (Supplementary Table 2). Each strain was combined with glycerol to a 10% final concentration, and 5 µL of the resulting mixture was dispensed into a sterile 96-well plate using a Mini-96 (Integra), covered with adhesive sterile foil, and placed on dry ice before ultimate storage at −80°C. Bt reporter assays One replicate array plate was thawed and combined with pre-reduced TYG and cultured overnight at 37°C under anaerobic conditions. The following day, each strain was diluted 50-fold into minimal media containing 0.5% galactose and cultured to mid-logarithmic phase before centrifugation at 2,204 x g to pellet cells, resuspension into 2x minimal media containing 0.2% galactose and distribution into a sterile 384-well white, clear bottom microplate (Corning) containing equal amounts of 0.8% carbon. Absorbance and bioluminescence were measured using an Infinite M200 Plex (Tecan) every 15 minutes over 18 hours. Biologically derived glycan mixture preparation Solid material was homogenized in 6% bleach (PureBright) and allowed to stir for 30 minutes at room temperature before addition of 1:100 volumes of formic acid were combined and stirred for 5 additional minutes at room temperature. Reactions were centrifuged at 7200 x g for 10 minutes and the resulting supernatant was combined with bleach to a final concentration of 0.2% and stirred for 16 hours at room temperature before addition of equal volumes of formic acid and stirring for an additional 5 minutes. Reactions were centrifuged at 7200 x g for 10 minutes and the resulting supernatant was sterilized using a 0.22 µm vacuum filter (Millipore). The resulting material was dialyzed with ultrapure H 2 O using 1KDa cutoff dialysis membrane for 3 days before lyophilization. Human material collection The Pennsylvania State University College of Medicine Institutional Review Board approved this study (IRB Protocol No. HY98-057EP-A). Prior to colectomy, patients gave informed consent to have surgically resected tissue collected and banked into the Carlino Family Inflammatory Bowel and Colorectal Disease Biobank. Diagnoses were confirmed using colonoscopy and biopsy. Surgical specimens were immediately transferred from the operating room to the surgical pathology lab, where the tissue was examined by a pathologist and surgeon. The samples were then transported to the research laboratory on ice for further processing. Mucosa was scraped from approximately 1 inch tissue sections and stored at −80°C until samples were weighed, pooled, and subjected to glycan extraction. Protein expression and purification SGBPs and GHs were recombinantly expressed as previously described 47 . Briefly, inserts encoding each enzyme were amplified from B. thetaiotaomicron VPI-5482 genomic DNA and cloned into pT7-7-N6H4A 18 linearized with NotI-HF and HindIII-HF using NEBuilder Hi-Fi Master Mix (NEB). The resulting plasmids were introduced into E. coli S17-1 and verified by Sanger sequencing. Plasmids were introduced into BL21 and plated on selective media. A single colony was inoculated into Luria Bertani broth (BD) and cultured to mid-logarithmic phase before 50 µM IPTG addition and subsequent incubation for 4 hours at 30°C. Pelleted cells were lysed in Lysis Buffer (20 mM Tris (pH, 8) and 100 mM sodium chloride) and N-terminally hexa-histidine-tagged proteins were recovered following co-incubation with Ni 2+ -NTA resin (Thermo) and elution with Lysis Buffer containing 25 mM Histidine. Recovered proteins were buffer exchanged in 10 mM Tris (pH, 7.4) containing 10% glycerol using appropriate molecular weight cut-off centrifugal concentrators (MilliporeSigma). Protein concentrations were determined using a BCA protein quantification kit (Thermo). SGBP-mediated glycan isolation Glycans were recovered from mixtures using recombinant SGBPs as previously described 18 . Briefly, an Ec strain BL21 cell lysate expressing N-terminal hexa-his-tagged SGBP was combined with 0.5% glycan and 1.0 mL Ni 2+ -NTA resin (Thermo) and incubated overnight at 4°C with rocking. The mixture was packed into a 3 mL gravity flow column and washed with 20 mM Tris (pH=8) and 100mM Sodium Chloride 8. Protein-glycan complexes were eluted with 25 mM histidine (MilliporeSigma), treated with proteinase K before incubation at 65°C for 2 hours. The reaction was combined with 3x volume of 100% ethanol incubated overnight at 4°C with rocking and centrifuged for 15 minutes at 21,291 x g . The pelleted material was dried under filtered air, resuspended in water and combined before lyophilization. qPCR Stationary phase cultures were diluted 50-fold into pre-reduced minimal media containing the 0.5% galactose and incubated to mid-logarithmic phase (OD 600 = 0.45 - 0.65). 1.0 mL of culture was pelleted by centrifugation, immediately placed on dry ice, and stored at −80°C Remaining cultures were pelleted by centrifugation at 7,200 x g for 3 minutes and resuspended in pre-reduced 2X minimal media containing no carbon. Equal amounts of resuspended culture and carbon were combined for a final concentration of 0.5% carbon and incubated for 60-minutes before 1.0 mL of culture was pelleted by centrifugation, immediately placed on dry ice, and stored at −80°C. RNA was isolated using the RNeasy Mini Kit (QIAGEN), quantified by absorbance, and 1 µg was converted to cDNA following addition of the Superscript IV VILO Master Mix with ezDNAse (Invitrogen) per the manufacturer’s directions. Transcript abundances were measured as previously described 47 using a QuantStudio5 (ThermoFisher) and PowerUp SYBR Green Master Mix (ThermoFisher) with amplicon specific primers (Supplementary Table 3). The 16S rRNA, rrs, was used as the reference gene for all qPCR experiments. In vivo competitive fitness of B. thetaiotaomicron strains All animal experiments were performed in accordance with protocols approved by Penn State Institutional Animal Care and Use Committee. 8-12 week old, mixed-gender, germ-free C57/BL6 mice were maintained in flexible plastic gnotobiotic isolators with a 12-hour light/dark cycle and provided a standard, autoclaved mouse chow (LabDiet, 5021) ad libitum 47 . Mice were randomly distributed into 2 groups, which were supplied either autoclaved drinking water or 0.5% yeast extract glycans, 2 days prior to gavage with 10 8 CFU of each indicated strains suspended in 200 μL of phosphate-buffered saline. Input (day 0) abundance of each strain was determined by counting colony forming units following plating on solid media. Fresh fecal pellets were collected at the indicated days and genomic DNA was extracted as described previously 23 . The abundance of each strain was measured by qPCR, using barcode-specific primers (Supplemental Table 2) as described previously 47 . Monosaccharide Composition Analysis Trifluoroacetic acid was added to the indicated glycan preparations to a final concentration of 2N and heated to 100°C for 4 hours. Reactions were cooled to room temperature, dried under filtered air, and washed twice with 50% isopropanol before being dried to completion. Reactions were resuspended in 0.2 mL of deionized water and analyzed by HPAEC-PAD. CAZyme activity YEG was combined with 200 nM GH20 PUL56 to a final concentration of 1.0% in 20 mM Tris (pH=8), incubated for 4 hours at 37 °C, and analyzed by HPAEC-PAD. Mannobioses were combined with 200 nM of each PUL71 GH in 20 mM Tris (pH=8) and 1 µM calcium chloride. Software and statistics Data collection and curation was done in Microsoft Excel. All data was plotted in GraphPad Prism, except for heatmaps and PCoA, which were generated using ggplot2 in RStudio. Statistical analyses were calculated in Prism. For all experiments, N denotes individual biological replicates across at least two independent experiments. Resource Availability All PUL reporter plasmids are available on addgene.com and a complete Bt reporter array will be supplied upon request. All other Bt and Ec strains used in this work are available upon request. Supplementary Information Supplementary Table 1. RNA-seq analysis of Bt strains expressing constitutively active PUL sensors. Supplementary Table 2. Bt PUL reporter plasmids. Supplementary Table 3. PUL similarity across Bacteroides species. Supplementary Table 4. Carbohydrates examined in this study. Supplementary Table 5. Strains and plasmids used in this study. Supplementary Table 6. Oligonucleotides used in this study. Acknowledgements We thank Walter Koltun, Leonard Harris, Troy Ott, Wesley Raup-Konsavage, and Hannah Valensi for assistance producing glycan extracts. We thank Biswa Chourdry and Richard Helm for assistance with carbohydrate compositional assistance. Additionally, we thank Jordan Bisanz for assistance with animal experiments and useful discussions. This project is funded, in-part by National Institutes of Health Grants GM147178 and DK132711 to G.E.T. and a grant with the Pennsylvania Department of Health using Tobacco CURE Funds. The PA Department of Health specifically disclaims responsibility for any analyses, interpretations or conclusions. This work was supported by the Carlino fund to The Penn State Colorectal Disease Biobank. Funder Information Declared National Institute of General Medical Sciences, https://ror.org/04q48ey07 , GM147178 National Institute of Diabetes and Digestive and Kidney Diseases , DK132711 Footnotes Data analysis curation and error corrections. References 1. ↵ Luis , A.S. et al. Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides . Nat Microbiol 3 , 210 – 219 ( 2018 ). OpenUrl PubMed 2. Marcobal , A. et al. Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways . Cell Host Microbe 10 , 507 – 514 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 3. ↵ Martens , E.C. , Chiang , H.C. & Gordon , J.I . Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont . Cell Host Microbe 4 , 447 – 457 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Martens , E.C. et al. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts . PLoS Biol 9 , e1001221 ( 2011 ). OpenUrl CrossRef PubMed 5. ↵ Salyers , A.A. , Vercellotti , J.R. , West , S.E. & Wilkins , T.D . Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon . Appl Environ Microbiol 33 , 319 – 322 ( 1977 ). OpenUrl Abstract / FREE Full Text 6. ↵ Raghavan , V. , Lowe , E.C. , Townsend , G.E. , 2nd . , Bolam , D.N. & Groisman , E.A. Tuning transcription of nutrient utilization genes to catabolic rate promotes growth in a gut bacterium . Mol Microbiol 93 , 1010 – 1025 ( 2014 ). OpenUrl CrossRef PubMed 7. ↵ Sonnenburg , E.D. et al. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations . Cell 141 , 1241 – 1252 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ Bakshani , C.R. et al. Carbohydrate-active enzymes from Akkermansia muciniphila break down mucin O-glycans to completion . Nat Microbiol 10 , 585 – 598 ( 2025 ). OpenUrl PubMed 9. ↵ Cartmell , A. et al. A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation . Nat Microbiol 3 , 1314 – 1326 ( 2018 ). OpenUrl PubMed 10. ↵ Ndeh , D. et al. Metabolism of multiple glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus . Nat Commun 11 , 646 ( 2020 ). OpenUrl CrossRef PubMed 11. ↵ Ndeh , D. et al. Complex pectin metabolism by gut bacteria reveals novel catalytic functions . Nature 544 , 65 – 70 ( 2017 ). OpenUrl CrossRef PubMed 12. ↵ McNulty , N.P. et al. Effects of diet on resource utilization by a model human gut microbiota containing Bacteroides cellulosilyticus WH2, a symbiont with an extensive glycobiome . PLoS Biol 11 , e1001637 ( 2013 ). OpenUrl CrossRef PubMed 13. ↵ Whitaker , W.R. et al. Controlled colonization of the human gut with a genetically engineered microbial therapeutic . Science 389 , 303 – 308 ( 2025 ). OpenUrl PubMed 14. ↵ Paulson , J.C. , Blixt , O. & Collins , B.E . Sweet spots in functional glycomics . Nat Chem Biol 2 , 238 – 248 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 15. Heimburg-Molinaro , J. , Mehta , A.Y. , Tilton , C.A. & Cummings , R.D . Insights Into Glycobiology and the Protein-Glycan Interactome Using Glycan Microarray Technologies . Mol Cell Proteomics 23 , 100844 ( 2024 ). OpenUrl PubMed 16. ↵ Ruhaak , L.R. , Xu , G. , Li , Q. , Goonatilleke , E. & Lebrilla , C.B . Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses . Chem Rev 118 , 7886 – 7930 ( 2018 ). OpenUrl CrossRef PubMed 17. ↵ Raghavan , V. & Groisman , E.A . Species-specific dynamic responses of gut bacteria to a mammalian glycan . J Bacteriol 197 , 1538 – 1548 ( 2015 ). OpenUrl Abstract / FREE Full Text 18. ↵ Modesto , J.L. , Pearce , V.H. & Townsend , G.E ., 2nd Harnessing gut microbes for glycan detection and quantification . Nat Commun 14 , 275 ( 2023 ). OpenUrl CrossRef PubMed 19. ↵ Glowacki , R.W.P. & Martens , E.C . If you eat it, or secrete it, they will grow: the expanding list of nutrients utilized by human gut bacteria . J Bacteriol 203 ( 2020 ). 20. ↵ Schwalm , N.D. , 3rd . & Groisman , E.A. Navigating the Gut Buffet: Control of Polysaccharide Utilization in Bacteroides spp . Trends Microbiol 25 , 1005 – 1015 ( 2017 ). OpenUrl CrossRef PubMed 21. ↵ Townsend , G.E. , 2nd . , Raghavan , V. , Zwir , I. & Groisman , E.A. Intramolecular arrangement of sensor and regulator overcomes relaxed specificity in hybrid two-component systems . Proc Natl Acad Sci U S A 110 , E161 – 169 ( 2013 ). OpenUrl Abstract / FREE Full Text 22. ↵ Cao , Y. , Rocha , E.R. & Smith , C.J . Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections . Proc Natl Acad Sci U S A 111 , 12901 – 12906 ( 2014 ). OpenUrl Abstract / FREE Full Text 23. ↵ Townsend , G.E. , 2nd . et al. A Master Regulator of Bacteroides thetaiotaomicron Gut Colonization Controls Carbohydrate Utilization and an Alternative Protein Synthesis Factor . mBio 11 ( 2020 ). 24. ↵ Ryan , D. , et al. An integrated transcriptomics-functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines . bioRxiv ( 2023 ). 25. ↵ Ryan , D. , Jenniches , L. , Reichardt , S. , Barquist , L. & Westermann , A.J . A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron . Nat Commun 11 , 3557 ( 2020 ). OpenUrl CrossRef PubMed 26. ↵ Terrapon , N. et al. PULDB: the expanded database of Polysaccharide Utilization Loci . Nucleic Acids Res 46 , D677 – D683 ( 2018 ). OpenUrl CrossRef PubMed 27. ↵ Terrapon , N. , Lombard , V. , Gilbert , H.J. & Henrissat , B . Automatic prediction of polysaccharide utilization loci in Bacteroidetes species . Bioinformatics 31 , 647 – 655 ( 2015 ). OpenUrl CrossRef PubMed 28. ↵ Glowacki , R.W.P. et al. A Ribose-Scavenging System Confers Colonization Fitness on the Human Gut Symbiont Bacteroides thetaiotaomicron in a Diet-Specific Manner . Cell Host Microbe 27 , 79 – 92 e79 ( 2020 ). OpenUrl CrossRef PubMed 29. ↵ Reeves , A.R. , D’Elia , J.N. , Frias , J. & Salyers , A.A . A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch . J Bacteriol 178 , 823 – 830 ( 1996 ). OpenUrl Abstract / FREE Full Text 30. ↵ D’Elia , J.N. & Salyers , A.A . Effect of regulatory protein levels on utilization of starch by Bacteroides thetaiotaomicron . J Bacteriol 178 , 7180 – 7186 ( 1996 ). OpenUrl Abstract / FREE Full Text 31. ↵ Jones , D.R. et al. Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron . Sci Rep 9 , 17400 ( 2019 ). OpenUrl CrossRef PubMed 32. ↵ Temple , M.J. et al. A Bacteroidetes locus dedicated to fungal 1,6-beta-glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6-beta-glucanase . J Biol Chem 292 , 10639 – 10650 ( 2017 ). OpenUrl Abstract / FREE Full Text 33. ↵ Liu , H. et al. Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments . Cell Rep 34 , 108789 ( 2021 ). OpenUrl CrossRef PubMed 34. ↵ Liou , C.S. et al. A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont . Cell 180 , 717 – 728 e719 ( 2020 ). OpenUrl CrossRef PubMed 35. ↵ Pudlo , N.A. , et al. Phenotypic and Genomic Diversification in Complex Carbohydrate-Degrading Human Gut Bacteria . mSystems 7 , e0094721 ( 2022 ). OpenUrl CrossRef PubMed 36. ↵ Cuskin , F. et al. Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism . Nature 517 , 165 – 169 ( 2015 ). OpenUrl CrossRef PubMed 37. ↵ Abbott , D.W. , Martens , E.C. , Gilbert , H.J. , Cuskin , F. & Lowe , E.C . Coevolution of yeast mannan digestion: Convergence of the civilized human diet, distal gut microbiome, and host immunity . Gut Microbes 6 , 334 – 339 ( 2015 ). OpenUrl CrossRef PubMed 38. ↵ Lowe , E.C. , Basle , A. , Czjzek , M. , Firbank , S.J. & Bolam , D.N . A scissor blade-like closing mechanism implicated in transmembrane signaling in a Bacteroides hybrid two-component system . Proc Natl Acad Sci U S A 109 , 7298 – 7303 ( 2012 ). OpenUrl Abstract / FREE Full Text 39. ↵ Schwalm , N.D. , 3rd . , Townsend , G.E. , 2nd . & Groisman , E.A. Multiple Signals Govern Utilization of a Polysaccharide in the Gut Bacterium Bacteroides thetaiotaomicron . mBio 7 ( 2016 ). 40. ↵ Ryan , D. et al. An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity . Nat Microbiol 9 , 1130 – 1144 ( 2024 ). OpenUrl PubMed 41. ↵ Song , X. et al. Oxidative release of natural glycans for functional glycomics . Nat Methods 13 , 528 – 534 ( 2016 ). OpenUrl CrossRef PubMed 42. ↵ Vos , G.M. et al. Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents . Anal Chem 95 , 8825 – 8833 ( 2023 ). OpenUrl 43. ↵ Wu , M. et al. Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides . Science 350 , aac5992 ( 2015 ). OpenUrl Abstract / FREE Full Text 44. ↵ Feng , J. et al. Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions . Cell Host Microbe 30 , 200 – 215 e212 ( 2022 ). OpenUrl CrossRef PubMed 45. ↵ Kabonick , S.G. et al. Hierarchical glycolytic pathways control the carbohydrate utilization regulator in human gut Bacteroides . Nat Commun 16 , 4488 ( 2025 ). OpenUrl PubMed 46. ↵ Zhu , Y. et al. Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont . Nat Chem Biol 6 , 125 – 132 ( 2010 ). OpenUrl CrossRef PubMed 47. ↵ Pearce , V.H. , Groisman , E.A. & Townsend , G.E. , 2nd . Dietary sugars silence the master regulator of carbohydrate utilization in human gut Bacteroides species . Gut Microbes 15 , 2221484 ( 2023 ). OpenUrl CrossRef PubMed 48. ↵ Koropatkin , N.M. , Martens , E.C. , Gordon , J.I. & Smith , T.J . Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices . Structure 16 , 1105 – 1115 ( 2008 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted August 02, 2025. 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