A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism

Erica D. Sonnenburg, Justin L. Sonnenburg, Jill K. Manchester, Elizabeth E. Hansen, Herbert C. Chiang, and Jeffrey I. Gordon*

Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108

Contributed by Jeffrey I. Gordon, April 21, 2006 Bacteroides thetaiotaomicron is a prominent member of our nor- Sequencing individual representatives of the major divisions of mal adult intestinal microbial community and a useful model for bacteria as well as whole community DNA is revealing the gene studying the foundations of human–bacterial mutualism in our content of the gut ‘‘microbiome’’ and will allow in silico predic- densely populated distal gut microbiota. A central question is how tions to be made of the microbiota’s metabolic potential (7). members of this microbiota sense nutrients and implement an Germ-free (GF) mice, colonized with one or more sequenced appropriate metabolic response. B. thetaiotaomicron contains a representatives of the human gut microbiota, are providing large number of glycoside hydrolases not represented in our own insights about the transcriptional and metabolic networks ex- proteome, plus a markedly expanded collection of hybrid two- pressed by community members in their habitats (3). component system (HTCS) proteins that incorporate all domains One important question about the operation of this microbial found in classical two-component environmental sensors into one community is how its members respond to changes in their polypeptide. To understand the role of HTCS in nutrient sensing, nutrient environments. Subsumed under this question is the issue we used B. thetaiotaomicron GeneChips to characterize their ex- of how these microbes sense the nutrient landscape of their gut pression in gnotobiotic mice consuming polysaccharide-rich or habitats and adapt to changes in resource availability. The -deficient diets. One HTCS, BT3172, was selected for further anal- answers could help us to attribute niches (professions) to com- ysis because it is induced in vivo by polysaccharides, and its munity members, understand how a microbiota is selected, absence reduces B. thetaiotaomicron fitness in polysaccharide-rich elucidate how community robustness is maintained, and develop diet-fed mice. Functional genomic and biochemical analyses of WT strategies for intentionally manipulating the operations of the and BT3172-deficient strains in vivo and in vitro disclosed that gut microbiota so as to provide additional benefits to human ␣-mannosides induce BT3172 expression, which in turn induces hosts. One benefit could be a new era of personalized nutrition expression of secreted ␣-mannosidases. Yeast two-hybrid screens where diet is matched to the nutrient processing capacity of an revealed that the cytoplasmic portion of BT3172’s sensor domain individual’s microbiota. serves as a scaffold for recruiting glucose-6-phosphate isomerase We have used Bacteroides thetaiotaomicron as a model gut and dehydrogenase. These interactions are a unique feature of symbiont to address some of these issues. In the most compre- BT3172 and specific for the cytoplasmic face of its sensor domain. hensive 16S rRNA enumeration study of the human gut micro- Loss of BT3172 reduces glycolytic pathway activity in vitro and in biota published to date, this Gram-negative obligate anaerobe vivo. Thus, this HTCS functions as a metabolic reaction center, comprised 12% of all Bacteroidetes, and 6% of all colonic coupling nutrient sensing to dynamic regulation of monosaccha- bacteria in three healthy adults (5). The B. thetaiotaomicron ride metabolism. An expanded repertoire of HTCS proteins with genome sequence (8) revealed an unprecedented expansion of diversified sensor domains may be one reason for B. thetaiotaomi- genes dedicated to polysaccharide metabolism: its ‘‘glycobiome’’ cron’s success in our intestinal ecosystem. (genes involved in carbohydrate acquisition and processing) in- cludes an arsenal of 226 glycoside hydrolases and 15 polysaccharide Bacteroides thetaiotaomicron ͉ glycoside hydrolases ͉ gut microbial lyases capable of cleaving many of the glycosidic linkages present in ecology ͉ metabolic regulation ͉ signal transduction plant glycans that are not digestible by humans (3, 8, 9). The B. thetaiotaomicron genome also contains a sizeable he adult human gut is home to tens of trillions of microbes. collection of genes involved in environmental sensing and signal TThis community is composed primarily of members of the transduction. This collection includes 54 sensor kinases and 29 domain Bacteria but also contains representatives from Archaea response regulators (8) that form ‘‘classical’’ two-component and Eukarya (1). Most bacteria live in the colon where, among systems, plus a family of 32 unique proteins that incorporate all their other functions, they extract energy from dietary polysac- of the domains found in classical two-component systems into a charides that would otherwise be lost because our human single polypeptide. Classical two-component systems transduce genome does not encode the requisite glycoside hydrolases and signals from the cell’s outside environment (pH, ions, and polysaccharide lyases needed for cleaving their glycosidic link- ages (2, 3). Intestinal absorption of the short-chain fatty acid end products of microbial polysaccharide fermentation accounts for Conflict of interest statement: No conflicts declared. up to 10% of our daily harvest of calories (4). Two divisions of Freely available online through the PNAS open access option. bacteria, the Bacteroidetes which include the saccharolytic Bac- Abbreviations: GF, germ-free; HTCS, hybrid two-component system; MM, minimal medium; teroides, and the Firmicutes, account for Ͼ99% of all phyloge- MM-G, MM with 0.5% glucose; PR, polysaccharide-rich; CPS, capsular polysaccharide syn- thesis; TYG, tryptone͞yeast extract͞glucose; GS, 35% glucose͞35% sucrose. netic types (phylotypes) identified in the colon (5). Data deposition: The GeneChip data sets reported in this paper have been deposited Several recent developments have set the stage for defining in the Gene Expression Omnibus database, www.ncbi.nlm.nih.gov͞geo͞ (accession the structure and operations of this community. Comprehensive nos. GSM40917–GSM40926, GSM40886–GSM40888, GSM40897–GSM40899, GSM40902– 16S rRNA sequence-based enumerations combined with new GSM40906, and GSE4703). computational methods are allowing community membership to *To whom correspondence should be addressed. E-mail: [email protected]. be defined and compared between healthy individuals (5, 6). © 2006 by The National Academy of Sciences of the USA

8834–8839 ͉ PNAS ͉ June 6, 2006 ͉ vol. 103 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603249103 Downloaded by guest on September 24, 2021 metabolites) to its genome via a multistep phosphorelay. The Our GeneChip analysis revealed that 18 of the 32 HTCS family prototypic two-component system consists of (i) a membrane- members have significantly different levels of expression among localized sensor protein containing histidine kinase and phos- the three growth conditions tested [P Ͻ 0.01; empirical false phoacceptor domains (annotated as HATPase࿝c and HisKA in discovery rate, 0%]. These differentially regulated HTCS genes the Pfam database; http:͞͞pfam.wustl.edu͞), and (ii) a cytoplas- cluster into four groups: (i) highest expression in MM-G (two mic response regulator protein containing a response regulator HTCS); (ii) highest expression in the ceca of mice on the GS diet receiver domain (Response࿝reg) and a signal output domain (three); (iii) highest expression in the ceca of animals on the PR (10). The majority of response regulators are transcription diet (ten); and (iv) highest expression in mice on both diets factors that contain one of three types of DNA-binding domains compared with MM-G (three) (Fig. 1A). The distal human gut, like (trans࿝reg࿝c, GerE, or HTH࿝8). B. thetaiotaomicron’s 32 ‘‘hybrid the ceca of our gnotobiotic mice, normally provides an environment two-component system’’ (HTCS) proteins are composed of an rich in polysaccharides for B. thetaiotaomicron. Therefore, we chose N-terminal periplasmic sensor that is interrupted by up to five BT3172 for further analysis because it represents the group of predicted transmembrane segments, plus four conserved cyto- HTCS genes that are most highly expressed in the ceca of mice on plasmic domains [HisKA, HATPase࿝c, Response࿝reg, plus a the PR diet (10-fold higher levels than in MM-G at mid-log phase; helix–turn–helix putative DNA-binding domain (HTH࿝AraC) 2-fold higher compared with GS diet-treated animals; P Ͻ 0.01; that is distinct from the HTH࿝8 domain common to classical Fig. 1A). response regulators]. The sensor domains of HTCS proteins are A mixture of 108 colony forming units of WT B. thetaiotaomi- less highly conserved among family members than their intra- cron and 108 colony forming units of an isogenic mutant con- cellular signaling domains (19% vs. 32% average amino acid taining a BT3172-null allele (BT3172::pGERM; erythromycin sequence identity, respectively), suggesting that HTCS proteins resistant) was introduced by gavage into 10 12-week-old GF have diversified to respond to distinct signals but have conserved NMRI male recipients. Five animals were maintained on a PR their method of intracellular signal transduction. diet. The second group had been switched from PR to GS chow B. thetaiotaomicron has more HTCS proteins than any other 14 days before colonization. Both groups of gnotobiotic mice sequenced prokaryote: since their initial identification in the B. were killed 10 days after gavage, and the number of viable WT thetaiotaomicron genome, HTCS family members have been vs. mutant cells in their cecal contents was defined by selective identified in other Bacteroidetes, Proteobacteria, and Chlo- plating. In the PR diet-fed group, WT B. thetaiotaomicron roflexi (ref. 11 and Table 1, which is published as supporting achieved a statistically significant 40-fold higher density than the information on the PNAS web site). Intriguingly, 17 of B. mutant (P Ͻ 0.01 using Student’s t test), whereas in the GS-fed thetaiotaomicron’s HTCS genes are located adjacent to genes group, both strains colonized the cecum at equivalent levels (Fig. encoding paralogs of SusC͞SusD polysaccharide-binding pro- 1B). Colonization of mice consuming one or the other diets with teins, or glycoside hydrolases (8). Based on these findings, we the WT or the mutant strain alone resulted in equivalent have hypothesized that evolution of a large collection of HTCS numbers of colony forming units (P Ͼ 0.2 among treatment paralogs may be a salient feature of the adaptation of B. groups). Control experiments confirmed that there was no thetaiotaomicron (and other Bacteroidetes) to their gut habitats detectable reversion of the mutant to WT in vivo under either (addresses) and niches (12). Below, we present evidence that nutrient condition (Ͻ10Ϫ6) as defined by an erythromycin- supports this hypothesis. resistant phenotype, and that the fitness defect observed with BT3172::pGERM was not due to the insertion of the suicide Results and Discussion vector (pGERM) itself [co-colonizations with WT B. thetaio- BT3172 Expression Improves the Fitness of B. thetaiotaomicron in the taomicron and an isogenic mutant strain lacking an HTCS whose Ceca of Mice Fed a Polysaccharide-Rich (PR) Diet. We used custom expression is similar in mice fed PR and GS diets Affymetrix GeneChips containing probesets that recognize (BT4178::pGERM) produced equivalent levels of colonization 98.7% of B. thetaiotaomicron’s 4,779 known or predicted chro- of both strains in the ceca of PR diet-treated animals (data not mosomal protein-coding genes to characterize HTCS gene ex- shown)]. Based on these results, we concluded that BT3172 plays a pression in three nutrient environments: (i) during growth from role in successful B. thetaiotaomicron colonization of its cecal early log to stationary phase in a batch culture fermenter habitat when organisms are competing for dietary polysaccharides. containing a minimal medium (MM) with 0.5% glucose (MM-G) as the sole fermentable carbon source; (ii) in the ceca Loss of BT3172 Affects ␣-Mannosidase Gene Expression. To obtain a of adult male GF mice belonging to the NMRI inbred line that comprehensive view of the impact of BT3172 deficiency on the were fed a standard autoclaved PR rodent chow diet and B. thetaiotaomicron transcriptome, we performed GeneChip colonized with B. thetaiotaomicron for 10 days (a period that en- profiling of WT and BT3172::pGERM harvested from the ceca compasses 2–3 rounds of turnover of the gut epithelium and its of GF mice that had been colonized for 10 days with either strain overlying mucus layer); and (iii) in the ceca of GF mice that were while consuming a PR or GS diet (n ϭ 5 mice per treatment switched from a PR diet to a simple sugar diet (35% glucose͞ group per strain). Hierarchical clustering of all genes revealed 35% sucrose; GS) devoid of polysaccharides for 14 days before that the BT3172::pGERM data set obtained from mice on the being colonized with B. thetaiotaomicron for 10 days (n ϭ 3–5 PR diet clustered more closely with the WT B. thetaiotaomicron mice per treatment group; each cecal sample was analyzed PR diet data set than with the BT3172::pGERM GS data set independently with a GeneChip). The cecum was selected (Fig. 5A, which is published as supporting information on the because this anatomically distinct region, located between the PNAS web site). These findings underscore the impact of diet on

small intestine and colon, contains a large bacterial population the B. thetaiotaomicron transcriptome. MICROBIOLOGY that is readily harvested. We have shown that the density of Comparison of WT and mutant B. thetaiotaomicron harvested colonization of the ceca of mice in groups ii and iii is not from the ceca of PR diet-treated animals revealed statistically significantly different (1011–1012 colony forming units͞ml lumi- significant differences in the expression of 686 genes (14% of the nal contents), and that removing polysaccharides from the diet genome): 440 genes were up-regulated in BT3172::pGERM causes B. thetaiotaomicron to redirect its glycan foraging to the relative to WT, whereas 246 genes were down-regulated [P Ͻ mucus overlying the cecal epithelium, with attendant induction 0.01; false discovery rate, Յ0.4%; see Fig. 5 B and C and Table of genes that degrade host-derived glycans (e.g., sialidases, 2, which are published as supporting information on the PNAS fucosidases, ␤-hexosaminidases, and a mucin-desulfating sulfa- web site, for a Clusters of Orthologous Genes (COG)-based tase; see ref. 3) categorization of regulated genes]. The absence of a BT3172

Sonnenburg et al. PNAS ͉ June 6, 2006 ͉ vol. 103 ͉ no. 23 ͉ 8835 Downloaded by guest on September 24, 2021 (mainly as ␤-linked mannose residues) and in fungal and mam- malian glycans (primarily as ␣-linked mannose). Our genome contains no mannanases and 16 mannosidases (15 ␣-mannosi- dases and 1 ␤-mannosidase). The carbohydrate-active enzymes (CAZy) database (www.cazy.org; see ref. 2) divides B. thetaiotaomi- cron’s 241 glycoside hydrolases and polysaccharide lyases into 45 families. B. thetaiotaomicron is well equipped to liberate both ␣- and ␤-anomers of mannose: it possesses 25 ␣-mannosidases, 10 ␣-man- nanases, and 5 ␤-mannosidases. Glycoside hydrolase family 92 was most affected by loss of BT3172 (see Fig. 6A, which is published as supporting informa- tion on the PNAS web site). This family contains 23 members, all of which are predicted to be ␣-mannosidases capable of cleaving disaccharides or more complex glycans with terminal mannose residues. Loss of BT3172 significantly reduced expres- sion of five family 92 ␣-mannosidases in vivo: two of the genes encoding these enzymes are in a single operon (BT3962 and BT3963; see Fig. 6 B and C). Loss of BT3172 did not affect expression of B. thetaiotaomicron’s 10 mannanases.

The Effect of BT3172 Deficiency on Capsular Polysaccharide Locus Gene Expression in Vivo Indicates a Defect in Sensing Polysaccharides. The B. thetaiotaomicron genome contains eight capsular poly- saccharide synthesis (CPS) loci, each composed of genes in- volved in the decoration of the microbe’s surface with polysac- charides. Elegant work by Comstock and coworkers (13) has shown that by regulating CPS locus expression Bacteroides fragilis can match its surface glycan features with the glycan landscape of its habitat. This molecular mimicry may help Bacteroides species evade a host immune response. Transcription profiling of WT B. thetaiotaomicron revealed that CPS3 (BT0595–BT0614) is normally expressed at higher levels than the other CPS loci when polysaccharides are scarce (i.e., during growth in MM-G) and is down-regulated when cells are (i) exposed to tryptone͞yeast extract͞glucose (TYG) me- dium, which is rich in ␣-linked mannans as well as other Saccharomyces cerevisiae-derived polysaccharides, or (ii) reside in the ceca of mice consuming a PR diet. In contrast, CPS4 (BT1336–BT1358) is selectively up-regulated under conditions in which polysaccharides are abundant (i.e., in the ceca of mice fed a PR diet and in TYG medium) and is down-regulated in Fig. 1. Effects of nutrient environment on expression of B. thetaiotaomicron MM-G (ref. 3 and data not shown). genes encoding hybrid two-component proteins in vitro and in the ceca of B. thetaiotaomicron containing the BT3172::pGERM allele gnotobiotic mice. (A) GeneChip profiling of HTCS gene expression during has the opposite pattern of expression of CPS3: it up-regulates growth of WT B. thetaiotaomicron in a batch culture fermenter containing this locus relative to WT during growth in TYG (a 3.1-fold MM-G or collected from the cecal contents of monoassociated mice fed a GS increase in the aggregate signal produced by all probesets that diet or a PR diet (n ϭ 3 and 5 animals, respectively; each analyzed separately). recognize the products of CPS3; P Ͻ 0.001) and in the ceca of Cells were harvested at five time points during growth from log to stationary mice fed a PR diet (a 4.6-fold increase in the aggregate signal; phase in MM-G (n ϭ 2 samples per time point). Four patterns of HTCS Ͻ expression are observed; those that are either most highly expressed in MM-G, P 0.001; see Fig. 7, which is published as supporting infor- in vivo on a GS diet, in vivo on a PR diet, or in vivo on both diets compared with mation on the PNAS web site). This aberrant pattern of CPS3 MM-G. Colors indicate standard deviation above (red) and below (green) the expression suggests that removal of BT3172 causes B. thetaio- mean level of expression (black) of a gene across all conditions. (B) Cocoloni- taomicron to ‘‘misinterpret’’ its nutrient landscape as being zation of GF mice gavaged once with equivalent numbers of isogenic WT and polysaccharide-deficient when it is replete with glycans. BT3172::pGERM strains and killed 10 days later. Loss of BT3172 reduces levels of cecal colonization in animals fed a PR but not a GS diet. Mean values Ϯ SEM Loss of BT3172 Produces a Change in Prioritized Utilization of Glycans. are plotted. **, P Ͻ 0.01. GeneChip profiling of WT B. thetaiotaomicron during growth from log to stationary phase in TYG medium disclosed a prioritized pattern of glycan utilization; it first expresses glyco- transcript did not produce statistically significant difference in side hydrolases that liberate fructose from fructans, followed by the expression of any other HTCS family member. COG M (cell ͞ enzymes that degrade mannans, then starch, and finally hex- wall membrane biogenesis, includes genes involved in capsular osamine-containing glycans (Fig. 2). These findings indicate that polysaccharide biosynthesis) and COG G (carbohydrate trans- WT B. thetaiotaomicron prioritizes expression of genes involved port and metabolism, includes glycoside hydrolases) represented in the acquisition of carbohydrates that are most easily shunted the dominant functional categories of responses to BT3172 into the glycolytic pathway (i.e., fructose and mannose). Con- deficiency. sistent with this notion, the WT strain up-regulates genes Mannans (polymannose containing glycans) and mannosides involved in glycolysis in the early log phase and down-regulates (D-mannose connected to other sugars by various glycosidic these genes as it approaches stationary phase (Fig. 3A). linkages) are abundantly represented in dietary plant glycans BT3172::pGERM shows a similar pattern of nutrient utiliza-

8836 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603249103 Sonnenburg et al. Downloaded by guest on September 24, 2021 Fig. 3. The absence of BT3172 impairs glycolysis. (A) GeneChip analysis of the expression of genes in the glycolytic pathway during growth of isogenic WT and BT3172::pGERM strains in batch culture fermenters containing TYG me- dium. (B) Concentration of metabolites in WT and mutant B. thetaiotaomicron strains harvested from the cecal contents of gnotobiotic mice fed a PR diet or during mid-log phase growth in a fermenter containing TYG medium. , P Ͻ Fig. 2. The prioritized pattern of glycan utilization by B. thetaiotaomicron * 0.05; , P Ͻ 0.01 is disrupted by BT3172::pGERM. GeneChip analysis of glycoside hydrolase- ** containing operons that display differential expression in WT B. thetaiotaomi- cron during growth from log to stationary phase in rich (TYG) medium is shown. The orderly pattern of induction and suppression of expression pro- they were below the limit of detection in cecal contents harvested vides insights about the prioritization of glycan consumption: fructans are from GF controls fed the same diet; data not shown). consumed initially, followed by mannans, starch, and finally hexosamine- Mannose enters the glycolytic pathway via the action of two containing glycans. Note that an operon involved in the degradation of enzymes, a hexokinase (BT2430), which phosphorylates man- mannose-containing glycans and import of liberated mannose (BT3962– nose to mannose 6-phosphate, and mannose 6-phosphate BT3965) is induced prematurely during early log phase in the BT3172::pGERM isomerase (BT0373), which converts mannose 6-phosphate to strain. fructose 6-phosphate. GeneChip profiling of BT3172::pGERM and WT B. thetaiotaomicron disclosed no statistically significant tion in TYG medium, with the exception that an operon differences in the expression of genes encoding either enzyme, both (BT3962–BT3965), containing three CAZy family 92 ␣-manno- in vitro and in vivo. However, mannose 6-phosphate levels were sidases, is up-regulated during the early log phase rather than the significantly elevated in the mutant strain, in vitro (mid-log-phase late log͞stationary phase (Fig. 2). The lack of a discernible cells) and in vivo (Fig. 3B). This elevation is consistent with the growth defect of the mutant in vitro and in monoassociation observed increase in fructose 6-phosphate and͞or reduction in experiments contrasts with its reduced fitness in the face of com- glycolysis. petition with WT in vivo (Fig. 1A). These data are consistent with the notion that there is functional redundancy in B. thetaiotaomi- BT3172 Expression Is Activated by Mannosides but Not by Monomeric cron’s nutrient sensing and acquisition machinery that effectively D-Mannose. These findings indicated that loss of BT3172 disrupts compensates for loss of BT3172 except in highly competitive B. thetaiotaomicron’s pattern of mannoside utilization and raised situations. the question of whether expression of this HTCS is regulated by The mutant exhibits a constant level of expression of glycolytic mannosides. Therefore, we used quantitative RT-PCR (see MICROBIOLOGY genes so that compared with WT, their expression is lower in the Table 3, which is published as supporting information on the early log phase and higher in the late log and stationary phase PNAS web site, for a list of all primers used for quantitative (Fig. 3A). Biochemical assays disclosed that glucose 6-phosphate RT-PCR) to measure BT3172 mRNA levels during mid-log- and fructose 6-phosphate levels are significantly higher in the phase growth in MM containing yeast extract (rich in ␣-linked mutant compared with the WT strain during mid-log-phase mannans) or 0.5% mannan. BT3172 expression is induced to growth in TYG medium and in the ceca of mice fed a PR diet similar degrees in MM–yeast extract and MM–yeast mannan (Fig. 3B), consistent with reduced glycolytic pathway activity in compared with MM-G (4.5 Ϯ 0.5- and 5.8 Ϯ 1.1-fold). Although, the absence of BT3172. (The presence of these metabolites in MM containing 0.5% D-mannose was not able to induce BT3172, cecal contents could be attributed to B. thetaiotaomicron because MM plus 0.5% ␣1,3-mannobiose up-regulated BT3172 4.0 Ϯ

Sonnenburg et al. PNAS ͉ June 6, 2006 ͉ vol. 103 ͉ no. 23 ͉ 8837 Downloaded by guest on September 24, 2021 0.6-fold above MM-G controls. Together, these data reveal that ␣-linked mannosides, but not monomeric mannose, modulates BT3172 expression. Comparison of mid-log-phase WT and BT3172:pGERM strains grown in MM–␣1,3-mannobiose disclosed that loss of BT3172 reduces expression of the two CAZy-group 92 ␣-mannosidases contained in a single operon (BT3962 and BT3963) by 3.3 Ϯ 0.5- and 5.6 Ϯ 0.9-fold, respectively (P Ͻ 0.001). These two genes also show reduced expression in the ceca of mice fed a PR diet when BT3172 is missing. Thus, our results reveal a positive feedback loop in which mannose present in glycosidic linkages induces BT3172 expression. BT3172, in turn, induces expression of the ␣-mannosidases that degrade these mannosides. Glycosidic linkage-dependent regulation of gene expression is known to occur in B. thetaiotaomicron’s starch utilization system (Sus) operon, which is induced by maltose and higher order starches, but not by glucose alone (14). Linkage- specific activation is consistent with the idea that at least some B. thetaiotaomicron HTCS incorporate all of their input and output domains into one polypeptide so that they can sense very specific nutrients in their habitat and mobilize appropriate cellular transcriptional and metabolic responses without the need for signal amplification. One way to coordinate such responses would be to assemble enzymes involved in processing the products of HTCS-regulated glycoside hydrolases on the HTCS itself: i.e., the HTCS would serve as a scaffold for bringing enzymes and their substrates together to form a multicomponent Fig. 4. Model of BT3172-directed metabolic regulation. B. thetaiotaomicron metabolic machine. has 32 HTCS proteins that incorporate the elements of classical two- component signaling proteins [a sensor, phosphoacceptor (HisKA), histidine kinase (HATPase࿝c), response regulator receiver (Response࿝reg), and signal The Sensor Domain of BT3172 Interacts with Enzymes Involved in output domain] in one polypeptide. Structural diversity among HTCS proteins Glucose Metabolism. To search for protein partners of BT3172, we is greatest among their sensor domains. This domain in BT3172 (Met-1-Glu- performed a yeast two-hybrid screen using the N-terminal sensor 745) contains three predicted transmembrane spanning elements, producing domain (Met-1-Glu-745) of BT3172 as bait and the protein periplasmic- and cytoplasmic-facing elements. Expression of BT3172 is in- products of a B. thetaiotaomicron cDNA library as preys. This duced by mannosides; the HTCS, in turn, induces expression of ␣-mannosi- cDNA library, containing 330,000 clones, was prepared from RNA dases required for breakdown of mannosides. Asp-146-Ser-299 of the cyto- isolated from B. thetaiotaomicron harvested during growth in TYG plasmic portion of BT3172’s sensor domain serves as a site for binding two proteins: glucose 6-phosphate isomerase (EC 5.3.1.9; key enzyme in the gly- and MM-G at all of the time points shown in Figs. 1 and 2. The colytic pathway) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49; cata- cDNA library of preys was screened at 6-fold coverage by using the lyzes the first step in the pentose phosphate shunt). Loss of BT3172 diminishes strict selection criteria described in Supporting Text, which is pub- glycolytic pathway activity in vivo and in vitro. The organization of BT3172 and lished as supporting information on the PNAS web site. its interacting partners links sensing the availability of carbohydrates in the Our screen yielded two cytoplasmic proteins that interact with environment with regulation of bacterial carbohydrate metabolism. BT3172’s sensor domain: (i) glucose 6-phosphate isomerase (BT2124, Gly-228-Gly-352), which produces fructose 6- phosphate, the substrate for phosphofructokinase (rate-limiting statistically significant differences in the levels of 6-phosphoglu- enzyme in glycolysis) and (ii) glucose 6-phosphate dehydroge- conate, an intermediate in this pathway (data not shown). nase (BT1221, Thr-309-Gly-464), which catalyzes the first committed step of the pentose phosphate pathway. A third Interactions Between BT3172 and These Two Proteins Are Specific for interacting protein was identified (BT0924, Trp-77-Gly-152) that This HTCS and Limited to a Discrete Cytoplasmic Region of Its Sensor shares homology to ATP-binding cassette (ABC) transporter Domain. Follow-up, directed, yeast two-hybrid assays established substrate-binding proteins. BT0924 is part of an operon that also that the BT3172 sensor domain interacted with the intact encodes an ABC transporter ATP-binding protein and an ABC isomerase and dehydrogenase. BT2124 and BT1221 were sub- transporter permease. Loss of BT3172 does not affect the sequently screened against the sensor domains of 29 other HTCS expression of this operon in vitro or in vivo. Directed yeast proteins (Table 4, which is published as supporting information two-hybrid screens revealed that BT0924 also interacts with on the PNAS web site). The results demonstrated that the the sensor domains of two other HTCS proteins (BT026 interactions between the isomerase and dehydrogenase were and BT3302). Based on these findings, we focused on our specific for the BT3172 sensor domain. subsequent analyses on the significance of the interactions To identify the region of BT3172’s sensor domain responsible between BT3172 and the two cytoplasmic enzymes involved in for these protein–protein interactions, four truncation mutants glucose metabolism. were constructed: Met-1-Asn-601, Met-1-Pro-451, Met-1-Ser- Although loss of BT3172 affects glycolytic activity, it does not 299, and Met-1-Lys-145. The isomerase and dehydrogenase appear to affect the pentose phosphate pathway. Enzymes interacted with Met-1-Ser-299 but not Met-1-Lys-145, implying catalyzing the first three steps in this pathway (glucose 6- that Asp-146-Ser-299 is the critical BT3172 domain. This con- phosphate 3 glucono 1,5-lactone 6-phosphate 3 6-phospho- clusion was further validated by replacement of Asp-146-Ser-299 gluconate 3 ribulose 5-phosphate) are all encoded by genes in BT3172 with the analogous region of another HTCS (Lys- contained in a single operon (BT1220–BT1222). None of these 145-Ser-298 of BT4137): the substitution resulted in the loss of genes are differentially expressed in the WT compared with BT3172’s ability to interact with all three protein partners. BT3172::pGERM strains, in vitro or in vivo, nor are there Glucose 6-phosphate isomerase and glucose 6-phosphate de-

8838 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603249103 Sonnenburg et al. Downloaded by guest on September 24, 2021 hydrogenase are cytoplasmic proteins. The program TMPRED lection of glycoside hydrolases and polysaccharide lyases among (www.ch.embnet.org͞software͞TMPRED࿝form.html) predicts sequenced Bacteroides, suggesting that its evolved collection of three transmembrane segments in the sensor domain of BT3172 HTCS paralogs, with their structurally diversified sensor do- (Leu-5-Ala-25, Val-393-Ala-41, and Thr-755-Ile-771), resulting mains, provides a means for controlling its elaborate glycan in its subdivision into periplasmic and cytoplasmic components. processing apparatus in an integrated and responsive manner. The region of BT3172 sensor domain that interacts with the These features could explain B. thetaiotaomicron’s abundant isomerase and dehydrogenase (Arg-146-Ser-299) is predicted to be representation in our densely populated, competitive distal gut located on the cytoplasmic face of the inner membrane (Fig. 4). ecosystem. Identifying the ligands recognized by HTCS sensor Yeast two-hybrid screens using the predicted periplasmic face domains may provide a starting point for developing strategies of BT3172’s sensor domain (Gly-421-Glu-745) as bait did not for intentionally manipulating the biological activities of B. identify any interacting proteins. Screens using the entire intra- thetaiotaomicron and perhaps other Bacteroides in vivo and for cellular portion of BT3172 (Lys-827-Ile-1310) or just its histidine matching diet to B. thetaiotaomicron’s niche. kinase, phosphotransfer, response regulator, or helix-turn-helix domains, also failed to identify any interacting proteins that Materials and Methods satisfied our stringent selection criteria. Generation and Growth an Isogenic BT3172::pGERM Mutant. The methods used for targeted disruption of BT3172 and growth of Prospectus. GeneChip studies of B. thetaiotaomicron grown in WT and mutant strains in a BioFlo-110 batch culture fermentor vitro in MM-G and TYG media or harvested from the ceca of under defined nutrient conditions are similar to those reported mice fed a PR-diet, have shown that B. thetaiotaomicron con- in earlier publications for other B. thetaiotaomicron genes and stitutively expresses, at least at low levels, members of all of its strains (3, 15). Further details can be found in Supporting Text. glycoside hydrolase families (secreted and cytoplasmic), as well as a majority of its SusC͞D paralogs (3). These products of the GF Mice. All experiments employing mice were performed using B. thetaiotaomicron glycobiome presumably allow the organism protocols approved by the Washington University Animal Studies to constantly survey and import samples of glycans present in its Committee. NMRI-KI mice were maintained in plastic gnotobiotic gut habitat into its periplasmic space where metabolic responses isolators (16) under a strict 12-h light cycle. Animals were fed a standard autoclaved chow diet (B&KUniversal, Hull, U.K.) or an are initiated. Our analysis of the operations of BT3172, sum- ͞ marized in Fig. 4, supports this view. The presence of mannosides irradiated diet composed of 35% (wt wt) glucose, 35% sucrose, and induces BT3172 expression, either through their direct interac- 20% protein (Bio-Serv, Frenchtown, NJ). For further details about tion with this HTCS or with other cellular signaling proteins. colonization and purification of RNA from and analyses of me- BT3172 induction by mannosides, in turn, induces expression of tabolites in cecal contents, see Supporting Text. secreted ␣-mannosidases that aid in the harvest of the perceived nutrient. The cytoplasmic portion of BT3172’s sensor domain GeneChip Analysis. cDNA targets were prepared from both in vivo serves as a scaffold for recruiting two key enzymes that regulate and in vitro RNA samples by using protocols described in the E. metabolism of monosaccharides generated from harvested car- coli Antisense Genome Array manual (Affymetrix). The cDNA product was isolated (QiaQuick Spin columns; Qiagen, Valencia, bohydrates. Interaction with these enzymes is a unique charac- CA), fragmented (DNase-I; Amersham Pharmacia Biosciences), teristic of this B. thetaiotaomicron HTCS and specific for a biotinylated (bioArray Terminal Labeling Kit; Enzo) and hy- common region of the cytoplasmic face of its sensor domain. bridized to custom B. thetaiotaomicron GeneChips (3). Regulation of these site-specific protein–protein interactions would provide a means for B. thetaiotaomicron to dynamically Yeast Two-Hybrid Screens. A B. thetaiotaomicron cDNA library couple carbohydrate availability with utilization. The role of the was produced by using the Matchmaker Library Construction kit phosphorelay system incorporated into BT3172 in modulating and the GAL4 activation domain fusion vector (BD Biosciences). these protein–protein interactions, or in communicating with RNA was prepared from samples obtained during growth of WT other signaling molecules remains to be defined, as does the role ࿝ B. thetaiotaomicron from log to stationary phase in batch culture of its C-terminal helix–turn–helix (HTH AraC) domain. Meta- fermenter vessels containing TYG or MM-G. For further details bolic intermediates may themselves be important mediators of about the screen, see Table 4 and Supporting Text. BT3172–protein interactions. B. thetaiotaomicron contains the largest collection of HTCS We thank David O’Donnell, Maria Karlsson, Janaki Guruge, and among sequenced human gut-associated Bacteroides, both in Sabrina Wagoner for invaluable assistance with experiments involving turns of absolute number and when corrected for genome size. gnotobiotic mice. This work was supported by National Institutes of The B. thetaiotaomicron genome also encodes the largest col- Health Grants DK30292 and DK52574.

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