Dietary sugar silences a colonization factor in amammalian gut symbiontGuy E. Townsend IIa,b, Weiwei Hana,b, Nathan D. Schwalm IIIa,b, Varsha Raghavana,b, Natasha A. Barrya,b,Andrew L. Goodmana,b, and Eduardo A. Groismana,b,1

aDepartment of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06536; and bYale Microbial Sciences Institute, West Haven, CT 06516

Edited by Jeffrey I. Gordon, Washington University School of Medicine in St. Louis, St. Louis, MO, and approved November 16, 2018 (received for reviewAugust 9, 2018)

The composition of the gut microbiota is largely determined byenvironmental factors including the host diet. Dietary componentsare believed to influence the composition of the gut microbiota byserving as nutrients to a subset of microbes, thereby favoring theirexpansion. However, we now report that dietary fructose andglucose, which are prevalent in the Western diet, specificallysilence a protein that is necessary for gut colonization, but notfor utilization of these sugars, by the human gut commensal Bac-teroides thetaiotaomicron. Silencing by fructose and glucose re-quires the 5′ leader region of the mRNA specifying the protein,designated Roc for regulator of colonization. Incorporation of theroc leader mRNA in front of a heterologous gene was sufficient forfructose and glucose to turn off expression of the correspondingprotein. An engineered strain refractory to Roc silencing by thesesugars outcompeted wild-type B. thetaiotaomicron in mice fed adiet rich in glucose and sucrose (a disaccharide composed of glu-cose and fructose), but not in mice fed a complex polysaccharide-rich diet. Our findings underscore a role for dietary sugars thatescape absorption by the host intestine and reach the microbiota:regulation of gut colonization by beneficial microbes indepen-dently of supplying nutrients to the microbiota.

fructose | gene expression | glucose | leader mRNA | microbiota

The gut microbiota is critical to human health (1). The com-position of the gut microbiota can be modified by diet (2–8).

For example, complex polysaccharides commonly referred to asdietary fiber remain undigested in the small intestine, reach themicrobiota in the distal gut, and promote colonization by bene-ficial microbes associated with lean and healthy individuals (3, 4,7, 9, 10). Accordingly, polysaccharide-rich diets favor expansionof those organisms that can take up and break down dietary fiber(7, 11–13). Conversely, diets rich in simple sugars favor the ex-pansion of organisms that utilize mucosal glycans because simplesugars are believed to be absorbed in the small intestine and,thus, are unavailable to the microbiota in the distal gut (12, 14).The monosaccharide fructose can escape absorption in the

small intestine and reach the microbiota in the distal gut (15, 16),where microbiota-derived products of fructose metabolism enterthe host blood (15). Given the alarming increase in consumptionof fructose and sucrose (a heterodimer of glucose and fructose)by Western populations (17, 18), we wondered how these simplesugars impact colonization by Bacteroides thetaiotaomicron, amember of the gut microbiota associated with lean and healthyindividuals.The BT3172 gene, herein named roc for “regulator of colo-

nization,” is required for B. thetaiotaomicron colonization ofgerm-free mice fed a polysaccharide-rich diet (19). By contrast,the roc mutant exhibits no fitness defect in mice fed a simplesugar diet composed of glucose and sucrose (19). These findingssuggested that Roc promotes gut colonization in a diet-dependentmanner by mediating the utilization of a dietary component(s) pre-sent in the complex polysaccharide-rich diet but absent fromthe simple sugar chow. This is because Roc belongs to a class ofproteins, designated hybrid two-component systems, that activate

transcription of clustered polysaccharide utilization genes in re-sponse to a polysaccharide-derived ligand (19–24). Roc directlycontrols the adjacent polysaccharide utilization genes BT3173 andBT3174 (21), which likely facilitate the utilization of the Roc ligand.However, the mRNA abundance of BT3173 was less than twofoldlower in the roc mutant than in the wild-type strain in bacteriacollected from the ceca of mice fed a polysaccharide-rich diet (19).These results imply that the ceca of mice fed a polysaccharide-richdiet contain very low amounts of the Roc-activating ligand, theidentity of which remains unknown. How, then, does diet controlRoc’s ability to promote gut colonization by B. thetaiotaomicron?We report that dietary glucose and sucrose silence Roc ex-

pression and that Roc is dispensable for utilization of glucose andsucrose-derived fructose. We establish that the mRNA leaderpreceding the roc-coding region is necessary and sufficient for Rocsilencing by fructose and glucose. Furthermore, we engineered astrain that is refractory to silencing by these simple sugars anddemonstrate that the engineered strain outcompetes wild-typeB. thetaiotaomicron in mice fed glucose and sucrose. Our findingsdemonstrate how dietary simple sugars can suppress gut coloniza-tion in a commensal bacterium just by altering the levels of a col-onization factor dispensable for the utilization of such sugars.

ResultsRoc Is Required for Gut Colonization but Not for Expression of ItsRegulated Genes in Mice Fed a Polysaccharide-Rich Diet. We de-termined that a strain deleted for the roc gene was dramaticallyoutcompeted by wild-type B. thetaiotaomicron in germ-free mice

Significance

Diet is known to alter the gut microbiota composition bysupplying nutrients that promote the expansion of particularmicroorganisms. However, we demonstrate that fructose, acommon dietary additive in the Western world, decreases theabundance of a regulator of gut colonization in the human gutcommensal Bacteroides thetaiotaomicron. Rendering the levelsof this colonization factor refractory to silencing by fructoseconfers an advantage in mice fed a fructose-rich diet. We pro-vide a singular example of the host diet controlling the amountsof a bacterial protein necessary for murine gut colonization by abeneficial gut commensal bacterium, but dispensable for growthon such dietary components.

Author contributions: G.E.T., A.L.G., and E.A.G. designed research; G.E.T., W.H., N.D.S.,V.R., and N.A.B. performed research; G.E.T. and W.H. contributed new reagents/analytictools; G.E.T., W.H., N.D.S., V.R., and E.A.G. analyzed data; and G.E.T. and E.A.G. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: eduardo.groisman@yale.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1813780115/-/DCSupplemental.

Published online December 17, 2018.

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fed a polysaccharide-rich diet (SI Appendix, Fig. S1). However,roc-dependent activation of target genes was not observed inbacteria collected from the intestinal lumen of mice fed apolysaccharide-rich diet (SI Appendix, Fig. S2). Our findings,which are in full agreement with previous results (19), argue thatRoc contributes to gut colonization additionally to or indepen-dently of regulating genes in the polysaccharide utilization locus(PUL) containing the roc gene.

Fructose and Glucose Decrease the Steady-State Levels of the RocProtein. We examined Roc amounts in bacteria grown in thelaboratory in the presence of various monosaccharides com-prising the polysaccharide-rich or simple sugar diets. Both glu-cose and fructose reduced Roc amounts to a similar extent whensupplied as sole carbon sources in laboratory media (Fig. 1A).This dramatic reduction in Roc amounts appears specific tothese two monosaccharides because Roc was readily detectablein media containing arabinose, galactose, mannose, N-acetyl-glucosamine, rhamnose, or xylose (Fig. 1 A and B).Fructose and glucose exert a dominant effect when supplied

along with sugars that do not silence Roc. That is to say, fructoseand glucose decreased Roc amounts within 1 h of addition tocultures growing in galactose-containing media (Fig. 1C and SIAppendix, Fig. S3A), and the decrease was more pronounced by 3 h(Fig. 1C and SI Appendix, Fig. S3A). By contrast, no changes inRoc abundance were observed when additional galactose wassupplied to the cultures growing in galactose-containing media(Fig. 1C and SI Appendix, Fig. S3A). Shifting bacteria fromfructose- or glucose-containing media to media lacking a carbonsource triggered a rapid accumulation of Roc, resulting in a 17.2-and 20-fold increase by 60 min, respectively (Fig. 1 D and E andSI Appendix, Fig. S3 B and C). Fructose and glucose appear toact specifically on Roc because they did not alter the amounts of

BT3334 and BT1635, which are Roc sequelogs (i.e., they exhibitsequence similarity) (SI Appendix, Fig. S4).To determine if fructose could exert this effect as a component

of a complex polysaccharide, we examined Roc levels duringgrowth on the fructose homopolymers, fructo-oligosaccharides(fos) and levan. Roc levels were not silenced by fos, but thecombination of monomeric fructose with fos reduced Roc levelsto those in cells grown in fructose alone (Fig. 1F). Conversely,levan silenced Roc to levels similar to monomeric fructose alone(Fig. 1F), most likely due to the extracellular liberation of freefructose from levan by the secreted endo-levanase encoded byBT1760 in B. thetaiotaomicron (23).Cumulatively, these data demonstrate that monomeric glucose

and fructose similarly reduce Roc abundance even in the pres-ence of other carbohydrates. Moreover, the data provide apossible explanation for the wild-type strain not exhibiting acompetitive advantage over the roc mutant in mice fed glucoseand sucrose: by dramatically decreasing Roc abundance, these sugarsrender wild-type B. thetaiotaomicron effectively Roc-deficient.

Fructose and Glucose Reduce Roc Abundance at the PosttranscriptionInitiation Level. We determined that transcription of the roc genestarts from an identical position (Fig. 2A) and yields similaramounts of nascent transcript in bacteria grown in either glucoseor galactose (SI Appendix, Fig. S5), sugars that either silence Rocor do not silence Roc, respectively (Fig. 1A). Identical tran-scription start sites were also observed under both conditions forthe BT3334 gene (Fig. 2B and SI Appendix, Fig. S5), whichspecifies a protein of the same family as Roc. The roc mRNAamounts changed less than 4-fold in bacteria grown in eightdifferent carbohydrates (SI Appendix, Fig. S6), which is much lessthan the 20-fold change in Roc protein abundance (Fig. 1A).

Fig. 1. Roc amounts are dramatically reduced by glucose and fructose. (A) Western blot analysis of crude extracts from B. thetaiotaomicron (GT593) grown tomidexponential phase in minimal media containing arabinose (ara), fructose (fru), galactose (gal), glucose (glu), mannose (man), N-acetylglucosamine (nag),rhamnose (rha), or xylose (xyl). Blot was probed with anti-HA and anti-DnaK antibodies. (B) Fold difference in Roc amounts relative to bacteria grown infructose calculated from Western blot quantification (n = 7 biological replicates; error bars represent SEM; P values derived from two-tailed Student’s t test;n.s. indicates P values ≥ 0.05; **P < 0.01; and ***P < 0.001). (C) Western blot analysis of crude extracts from B. thetaiotaomicron (GT593) grown in 0.25%galactose (0 h) and following addition of either 0.25% fructose or 0.25% galactose (1–3 h). Blot was probed with anti-HA and anti-GroEL antibodies. (D)Western blot analysis of crude extracts from B. thetaiotaomicron (GT593) grown in fructose (−5 min) and switched to media lacking a carbon source for 15and 60 min. Blot was probed with anti-HA and anti-GroEL antibodies. (E) Roc amounts were calculated from quantified Western blots in B. thetaiotaomicron(GT593) grown in fructose to midexponential phase and subsequently shifted to carbon-source—deficient media (n = 6 biological replicates; error barsrepresent SEM; P values derived from two-tailed Student’s t test; n.s. indicates P values ≥ 0.05; **P < 0.01; and ***P < 0.001). (F) Western blot analysis of crudeextracts from B. thetaiotaomicron (GT593) grown to midexponential phase in minimal media containing fructose (fru), fructose oligosaccharides (fos) withand without fructose, and levan (lev) with and without fructose. The blot was probed with anti-HA and anti-GroEL antibodies.

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That glucose and fructose control Roc abundance after theinitiation of transcription is supported by two additional exper-imental results: first, the roc promoter failed to confer fructose-or glucose-dependent silencing to a heterologous gene (Fig. 2 Cand D and SI Appendix, Fig. S7). And second, glucose reducedRoc protein amounts even when the roc gene was transcribedfrom a heterologous promoter (Fig. 2 E and F and SI Appendix,Fig. S8).

The roc mRNA Leader Is Necessary and Sufficient for Roc Silencing byFructose and Glucose. To explore whether the 5′ leader region ofthe roc mRNA (Fig. 2A) is solely responsible for regulation ofRoc amounts by glucose, we engineered two strains: one in whichthe 26-nt leader of the BT3334 gene (Fig. 2B) was replaced bythe 54-nt roc leader (Fig. 2 C and D and SI Appendix, Fig. S7);and another, in which the 54-nt roc leader region was replaced bythe 26-nt leader from the BT3334 gene (Fig. 2 E and F and SIAppendix, Fig. S8) (20).The strain with the roc leader preceding the BT3334-coding

region produced significantly less BT3334 protein when grown infructose or glucose than cultures provided xylose or galactose(Fig. 2 C and D) and increased BT3334 levels following a switchto media lacking a carbon source (SI Appendix, Fig. S7). Bycontrast, the strain with the BT3334 leader preceding the roc-coding region produced similar Roc amounts regardless of thecarbohydrate provided (Fig. 2 E and F) and failed to increaseRoc amounts following a switch from glucose to no-carbonconditions (SI Appendix, Fig. S8). As discussed above, glucoseand fructose silence Roc (Fig. 1A) but not BT3334 (SI Appen-dix, Fig. S4 A and B) in wild-type B. thetaiotaomicron. Thesedata establish that the 5′ leader region of the roc transcript is

necessary and sufficient to control Roc abundance in responseto glucose.In agreement with the notion that the roc mRNA leader con-

trols the response to glucose, the nucleotide sequence of the rocleader exhibits little shared identity with the nucleotides precedingthe coding region of BT1635 despite this gene specifying a proteinexhibiting 74% amino acid identity to Roc but is not silenced byfructose or glucose (SI Appendix, Fig. S4 C and D).

An 11-nt Region in the roc mRNA Leader Is Critical for Silencing byFructose and Glucose. To identify positions within the roc 5′ leader(Fig. 3A) necessary for Roc silencing by fructose and glucose, weconstructed a library of plasmids harboring randomly generatedmutations in the roc 5′ leader region preceding a roc genespecifying a C-terminally HA-tagged Roc protein. The plasmidlibrary was then screened in a roc-deficient B. thetaiotaomicronstrain using an implementation of a classic approach for thisanaerobic microbe: directly immunoblotting colonies recoveredfrom glucose-containing solid media using anti-HA antibodies(SI Appendix, Fig. S9).Roc levels were 16.7- to 35.7-fold higher in strains with plas-

mids harboring single-nucleotide substitutions at four positionsbetween the putative ribosome binding site (RBS) (25) and thestart codon of roc than in the parental plasmid with the wild-typeroc leader (Fig. 3 B and C). The four positions are critical forrepression by glucose and fructose (as opposed to de-repressingRoc production under all conditions) because the mutationsmodestly altered Roc abundance (i.e., a 1.8- to 2.7-fold increase)when bacteria were grown in galactose (Fig. 3 B and C).Comparison of the putative leader sequences from roc

sequelogs in 18 other Bacteroides species reveals that the 11 nt

Fig. 2. The 5′ roc mRNA leader is necessary and sufficient for repression by glucose. (A) Nucleotide sequence of the roc 5′ mRNA leader (blue) up to the startcodon (underlined) with the putative RBS shaded in black. (B) Nucleotide sequence of the BT3334 5′ mRNA leader (red) up to the start codon (underlined)with the putative RBS shaded in black. (C) Western blot analysis of crude extracts from B. thetaiotaomicron strains deleted for both the BT3334 and roc genesand complemented with a single-copy plasmid harboring the BT3334-coding region preceded by its native promoter and leader (GT534), the roc promoterand its native leader (GT640), or its native promoter but the roc leader (GT663), grown as indicated in the figure. (D) Fold difference in BT3334 levels inB. thetaiotaomicron strains described in C grown in galactose (gal), glucose (glu), or xylose (xyl) relative to the same strains grown in fructose (fru) calculatedfrom quantified Western blots (n = 6 biological replicates; error bars represent SEM; P values derived from two-tailed Student’s t test). (E) Western blotanalysis of crude extracts from B. thetaiotaomicron strains deleted for both the BT3334 and roc genes and complemented with a single-copy plasmid har-boring the roc-coding region preceded by its native promoter and leader (GT530), the BT3334 promoter and its native leader (GT670), or its native promoterand the BT3334 leader (GT663) grown as indicated in the figure. (F) Fold difference in Roc amounts in B. thetaiotaomicron strains described in C grown ingalactose (gal), glucose (glu), or xylose (xyl) relative to the same strains grown in fructose (fru) calculated from quantified Western blots (n = 6 biologicalreplicates, error bars represent SEM; P values derived from two-tailed Student’s t test; n.s. indicates P values ≥ 0.05, *P < 0.05; **P < 0.01; and ***P < 0.001).

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positioned between the putative RBS (25) and start codon arerelatively well conserved, while the remainder of the leader hasdiverged (SI Appendix, Fig. S10). Furthermore, two of the fourpositions required to control Roc levels in response to glucoseand fructose are conserved among all of the analyzed sequencesupstream of Roc sequelogs of at least 83% amino acid identity.These data suggest that roc homologs are similarly regulated inother Bacteroides species.

Fructose and Glucose Are Present in the Cecum of Mice Fed a Glucose/Sucrose Diet. We determined that fructose abundance in the ce-cum of mice fed a glucose/sucrose chow was 2.6-fold higher thanin animals fed a polysaccharide-rich chow (Fig. 4A). By contrast,glucose was not detected in the ceca of mice fed either diet (Fig.4B), which likely reflects efficient glucose absorption by the gutepithelium (15). However, when cecal extracts were treated withinvertase, an enzyme that liberates glucose assembled in oligo-and polysaccharides, we detected 2.7-fold more glucose in mice fedthe glucose/sucrose diet relative to those fed the polysaccharide-rich diet (Fig. 4B). Given that sucrose hydrolysis is the sole sourceof fructose in the cecum of glucose/sucrose-fed mice, and thatglucose is detected only after invertase addition, our results arguethat sucrose is present in the distal gut and that sucrose hydrolysisexposes the microbiota to both fructose and glucose.

Rendering Roc Expression Refractory to Silencing by Fructose andGlucose Confers a Colonization Advantage to Mice Fed a Glucose/Sucrose Diet. If a glucose/sucrose diet decreases B. thetaiotaomi-cron fitness in the gut by reducing Roc abundance, a strainproducing Roc in the presence of fructose or glucose (SI Ap-pendix, Fig. S11) should exhibit a fitness advantage over both astrain lacking the roc gene and wild-type B. thetaiotaomicron. Ashypothesized, the engineered strain with the BT3334 leaderpreceding the roc coding region achieved five times greaterabundance than wild-type B. thetaiotaomicron and the roc-

deficient mutant in mice fed a glucose/sucrose diet by day 15(Fig. 4C).The colonization advantage exhibited by the engineered strain

is specific to the gut of mice fed a glucose/sucrose diet because:first, the abundance of the engineered strain hardly changed inmice fed a polysaccharide-rich diet over 15 d (Fig. 4D), whereasthe abundance of wild-type B. thetaiotaomicron and of the roc mu-tant increased and decreased, respectively (Fig. 4D), in agreementwith previous results (SI Appendix, Fig. S1) (19). And second, theengineered strain grew similarly to wild-type B. thetaiotaomicronin laboratory media with fructose or glucose as the sole carbonsource (SI Appendix, Fig. S12). These data demonstrate that theroc mRNA leader is necessary for B. thetaiotaomicron’s responseto dietary sources of fructose, as it is for the response to fructoseand glucose in laboratory media (Fig. 1).

DiscussionOur findings reveal how common dietary additives endemic toWestern human populations can impact gut colonization by themicrobiota independently of their ability to serve as nutrients(Fig. 4 and SI Appendix, Fig. S12). We establish that fructose andglucose silence a critical colonization factor, called Roc, in awidely distributed gut commensal bacterium B. thetaiotaomicron(Fig. 1). Furthermore, we demonstrate that fructo-oligosaccharides,which are used as prebiotics in humans (26), fail to silence Roc(Fig. 1F), indicating that the presence of monomeric fructose isrequired for this effect. Additionally, we determine that a short5′ mRNA leader is necessary and sufficient for the silencing effectsthat fructose and glucose exert on the amounts of the associatedcoding region (Figs. 2 and 3). The identified mRNA leader maybe used to engineer gut microbes to alter their behaviors in responseto dietary components.Roc is a transcriptional regulator that controls expression

of genes in the PUL that also harbors the roc gene (21). How-ever, Roc appears to promote gut colonization independently

Fig. 3. Mutations in the roc leader alleviate Roc repression by fructose and glucose. (A) Nucleotide sequence of the roc 5′ leader with four positions requiredfor repression by glucose. (B) Western blot analysis of crude extracts from B. thetaiotaomicron harboring plasmids encoding the wild-type roc sequence or theindicated single-nucleotide substitutions in the roc 5′ leader following growth in fructose, galactose, or glucose. (C) Fold difference in Roc amounts inB. thetaiotaomicron mutant strains described in B grown in fructose, galactose, or glucose relative to the wild-type strain grown in the same condition,calculated from quantified Western blots (n = 6 biological replicates; error bars represent SEM; P values derived from two-tailed Student’s t test; *P < 0.05;**P < 0.01; and ***P < 0.001).

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of its predicted role in mediating dietary carbohydrate utilization(19) (Fig. 4 C and D). The absence of Roc-dependent targetgene activation in total cecal contents (SI Appendix, Fig. S2)may reflect a spatial and/or temporal distribution of the Rocligand, as suggested for another Bacteroides species (27, 28).Alternatively, Roc may promote gut colonization by othermeans, such as the proposed interactions with glucose 6-phosphateisomerase and glucose 6-phosphate dehydrogenase (19). Al-though Roc interacting with these metabolic enzymes is an in-triguing hypothesis, it is not clear how the cytoplasmic glucose6-phosphate isomerase and glucose 6-phosphate dehydrogenaseproteins would interact with Roc domains predicted to reside inthe periplasm (22).We have demonstrated that the roc leader is necessary and

sufficient to confer glucose and fructose to a heterologous gene(Fig. 2 C and D) and that the roc promoter is dispensable for thisregulation (Fig. 2 E and F). Furthermore, we identified four

nucleotide positions in the short roc mRNA leader locatedbetween the putative ribosome-binding site (25) and start codonthat are necessary for control of the downstream roc-coding re-gion by glucose and fructose (Fig. 3) and conserved in leaderspreceding roc homologs (SI Appendix, Fig. S10). Additionally,roc transcript levels vary less than 4-fold during growth in eightdifferent carbon sources, while protein levels vary almost 20-fold(SI Appendix, Fig. S7). Similarly, the roc mRNA is present atsimilar levels, and transcription is initiated from an identicalposition in cells grown in glucose and galactose. By contrast,13.7-fold more Roc protein is present in cells grown in galactosecompared with glucose. The difference in Roc levels under thesetwo conditions cannot be explained by roc mRNA stability, as roctranscript levels are indistinguishable in the presence of eitherglucose or galactose (SI Appendix, Fig. S13). These findings dem-onstrate that control of Roc occurs independently of transcriptioninitiation or transcript stability and suggest that this regulation isexerted at the level of translation initiation or elongation.We propose that a regulatory protein and/or small RNA acts

on the roc leader to control Roc synthesis. This putative regu-lator is not likely encoded within the Roc-controlled PUL (21),as fructose silences Roc levels similarly in wild-type B. thetaio-taomicron and a strain lacking the region corresponding to theBT3174–BT3180 genes (SI Appendix, Fig. S14 A and B). Fur-thermore, the roc leader is unlikely to operate as a glucose- andfructose-responding riboswitch due to its short length and theabsence of regions that can adopt alternative structures thatcharacterize riboswitches (29). The proposed regulator is likelyto target the conserved portion of the roc mRNA leader betweenthe predicted ribosome-binding site and start codon (SI Appen-dix, Fig. S10).Finally, we demonstrated how colony blotting can be used to

investigate the expression of any protein that can be epitope-tagged (SI Appendix, Fig. S9) in an organism with a limitednumber of reporter genes. Previous attempts to couple geneexpression and reporter function in Bacteroides species have re-quired the exposure to oxygen (30), the application of toxic re-agents (31), or extremely high expression levels (32). We havedeveloped a reporter to genetically dissect regulatory interac-tions that can be adapted to any B. thetaiotaomicron protein ofinterest assuming it can be tagged with an HA epitope. Thisapproach enabled us to screen a library of strains harboringrandom mutations to identify and recover mutants of interest forfurther characterization.

Materials and MethodsMaterials and methods including culturing conditions, strain construction,qPCR, Western blot analysis, mouse experiments, colony blotting, and sugarquantification are detailed in SI Appendix. Experiments using germ-freemice were approved by the Institutional Animal Care and Use Committeeof Yale University.

ACKNOWLEDGMENTS. We thank Hubert Salvail, Bentley Lim, Liza-MarieValle, and Diane Lazo for technical advice and support and Jennifer Aronsonfor comments on the manuscript. This research was supported with fundsfrom Yale University and NIH Grant GM123798 (to E.A.G.).

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