Specificity of Polysaccharide Use in
Intestinal Bacteroides Species Determines
Diet-Induced Microbiota Alterations
Erica D. Sonnenburg,1,3Hongjun Zheng,2,3Payal Joglekar,1Steven K. Higginbottom,1Susan J. Firbank,2
David N. Bolam,2,* and Justin L. Sonnenburg1,*
1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
2Institute for Cell and Molecular Biosciences, Newcastle University, Medical School, Newcastle upon Tyne, NE2 4HH, UK
3These authors contributed equally to this work
*Correspondence: firstname.lastname@example.org (D.N.B.), email@example.com (J.L.S.)
The intestinal microbiota impacts many facets of
Diet impacts microbiota composition, yet mecha-
nisms that link dietary changes to microbiota alter-
ations remain ill-defined. Here we elucidate the basis
of Bacteroides proliferation in response to fructans,
a class of fructose-based dietary polysaccharides.
Structural and genetic analysis disclosed a fructose-
binding, hybrid two-component signaling sensor that
controls the fructan utilization locus in Bacteroides
thetaiotaomicron. Gene content of this locus differs
among Bacteroides species and dictates the speci-
ficity and breadth of utilizable fructans. BT1760,
an extracellular b2-6 endo-fructanase, distinguishes
B. thetaiotaomicron genetically and functionally, and
enables the use of the b2-6-linked fructan levan. The
genetic and functional differences between Bacter-
in the presence of dietary fructans. Gene sequences
that distinguish species’ metabolic capacity serve as
potential biomarkers in microbiomic datasets to
The trillions of microbial cells that reside within the intestine
shape aspects of host metabolism and immune function and
extend the physiological definition of humans (Backhed et al.,
with greater than 90% of the cells belonging to the Firmicutes or
sition is highly personalized (Turnbaugh et al., 2009).
Community membership and function of the microbiota can
change due to numerous variables including antibiotic treat-
ment, inflammation, or changes in diet (Dethlefsen et al., 2008;
Frank et al., 2007; Jernberg et al., 2007; Ley et al., 2006). Pro-
tracted loss of the typical composition has been associated
with several disorders including inflammatory bowel diseases
(Frank et al., 2007). In addition, changes in composition have
been associated with obesity and weight loss; however, factors
that cause these changes are not well defined (Duncan
et al., 2008; Ley et al., 2006). The alterations in community
membership, whether chronic or short-term, are accompanied
by changes in the microbiota’s collective genome, or micro-
biome, and the patterns and metabolic capabilities it specifies
(Turnbaugh et al., 2009). Therefore, the mechanisms that link
relevant variables, such as changes in diet, to changes in the mi-
crobiome, are integral to understanding how environmental
factors and behavior influence human biology.
Many complex plant polysaccharides in the human diet are
resistant to host-mediated degradation due to either insolubility
or lack of human-encoded hydrolytic enzymes (Flint et al., 2008;
Louis et al., 2007; Sonnenburg et al., 2005). These carbohy-
drates are not absorbed in the upper gastrointestinal tract and
serve as a major source of carbon and energy for the distal gut
microbial community. Polysaccharide degradation is one of the
core functions encoded in the microbiome (Lozupone et al.,
2008; Turnbaugh et al., 2007). Broad expansion of the genes
and operons dedicated to degrading and consuming polysac-
charides has occurred within the genomes of microbiota-
resident species (Xu et al., 2003, 2007), a logical outcome of
the intense competition for these resources. It is, therefore,
expected that alterations in the type and quantity of polysaccha-
rides consumed can result in changes in the microbiota commu-
nity composition and function.
Inulin- and levan-type fructans (homopolymers of b2-1 or b2-6
fructose units, respectively) are common dietary plant polysac-
charides that feed the intestinal microbiota (Roberfroid et al.,
1993). Multiple bacterial taxa in the gut utilize fructans, including
members of Firmicutes, Bacteroides, and Bifidobacterium,
(Duncan et al., 2003; Rossi et al., 2005; Van der Meulen et al.,
2006), and dietary fructan can result in expansion of Actinobac-
2000; Ramirez-Farias et al., 2008). Lack of predictability in how
the microbiota responds to such dietary interventions reflects
our limited understanding of nutrient sensing and utilization by
members of the intestinal microbiota.
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1241
Bacteroides, a major genera in the human microbiota, have
a widely expanded capacity to use diverse types of dietary poly-
saccharides (Xu et al., 2007). Much of the glycan degrading and
import machinery within Bacteroides genomes are encoded
within clusters of coregulated genes known as polysaccharide
utilization loci (PULs). B. thetaiotaomicron (Bt), a prototypic
member of the Bacteroides, possesses 88 PULs, which differ
in polysaccharide specificity (Martens et al., 2008). The defining
characteristic of a PUL is the presence of a pair of genes homol-
ogous to Bt susD and susC, which encode outer membrane
proteins that bind and import starch oligosaccharides, respec-
tively (Figure 1A) (Martens et al., 2009; Shipman et al., 2000).
The pair of susC and susD homologs is usually associated with
genes that encode the machinery necessary to convert extracel-
lular polysaccharides into intracellular monosaccharides, such
as glycoside hydrolases (susA, susB, and susG in Figure 1A).
In addition to machinery for polysaccharide acquisition, most
PULs contain, or are closely linked to, a gene or genes encod-
ing an inner membrane-associated sensor-regulator system,
including the novel hybrid two-component systems (HTCS)
(Sonnenburg et al., 2006). Bt’s genome encodes 32 of these
Bt1754 Bt1762Bt1763 Bt1760 Bt1759 Bt1757 Bt1758
BT1754BT1757 BT1763 BT1765 BT3082
Absorbance (OD 600)
3.54.5 5.56.5 8.8
SusC SusD SusB SusA SusRSusESusF SusG
Figure 1. Bt’s Use of Fructose-Containing
Carbohydrates Corresponds to Induction
of the Polysaccharide Utilization Locus
BT1757-1763 and BT1765
(A) Genomic organization of Bt’s Sus locus (top)
and putative fructan utilization locus (bottom).
Genes of similarfunction are coded by color; inter-
vening unrelated genes are white; genes without
corresponding homologs are gray.
(B) Gene expression patterns of differentially regu-
lated susC and susD homologs from Bt grown in
rich medium (TYG) at five time points from early
log (3.5 hr) to stationary phase (8.8 hr) in duplicate.
Colors indicate standard deviations above (red)
(C) Growth curves of Bt in minimal medium con-
taining indicated carbon source at 0.5% w/v.
(D) RNA abundance for genes relevant to fructan
tive to growth in minimal medium plus glucose.
Standard errors of expression levels from three
biological replicate cultures are shown.
HTCS, which may mediate the rapid and
tine. Here, we dissect a Bt PUL required
for utilization of fructans to better under-
stand how Bacteroides species acquire
and process this common class of dietary
carbohydrates. In addition, we provide
evidence that the associated HTCS con-
trols the expression of the fructan PUL
and that monomeric fructose is the acti-
vating signal that binds directly to the
periplasmic sensor domain of the regula-
tory protein. These data provide an
example of a well-defined ligand for a member of this class of
oides species, corresponding to a range of fructan utilization
capability across the genus. Using model intestinal microbiotas
can have disparate effects on community composition, depend-
biota. These studies suggest that within personal microbiomic
functions. Inference of function from these biomarkers should
provide predictive power in determining how an individual’s mi-
crobiota will respond to changes in diet and other interventions.
BT1757-BT1763 and BT1765 Form a Putative
Polysaccharide Utilization Locus that Is Transcribed
Early in Bt’s Growth in Rich Media
BT1757-BT1763 and BT1765 encodes eight open reading
frames on the negative strand of the Bt genome, including one
1242 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.
susC/susD homolog pair (BT1763 and BT1762), a putative outer
membrane lipoprotein (BT1761), a putative inner membrane
monosaccharide importer (BT1758), a putative fructokinase
(BT1757), and three putative glycoside hydrolases (BT1759,
BT1760, BT1765) (Figure 1A). These glycoside hydrolases are
members of Glycoside Hydrolase Family 32 (GH32), a family of
enzymes specific for fructans (Cantarel et al., 2009). One of
these, BT1760, possesses a N-terminal lipidation motif and is
predicted to reside on the cell surface; the other two, BT1759
and BT1765, are predicted to be periplasmic and intracellular,
respectively (www.cbs.dtu.dk/services/LipoP/ and www.cbs.
dtu.dk/services/SignalP/). Directly adjacent to the locus is a
putative inner membrane-associated sensor regulator of the
HTCS family, BT1754. These data suggest that this PUL
encodes the proteins required for Bt’s use of fructans.
Expression profiling of Bt in rich medium has revealed the
upregulation of several PULs, each of which is confined to
a discrete phase of growth (Sonnenburg et al., 2006). Analysis
of Bt transcriptional profiles at five time points that spanned
from early log to stationary phase in vitro in rich medium,
compared to basal expression in minimal medium (MM) contain-
ing glucose as the sole carbohydrate, revealed that 14 pairs of
susC/susD homologs were induced greater than 20-fold at one
or more time points during the growth (Figure 1B) (Gene Expres-
sion Omnibus database, www.ncbi.nlm.nih.gov/geo/; accession
numbers,GSM40897–40926). Theputative fructanPUL showed
upregulation early in Bt’s growth suggesting it is responsive to
(Figure 1B). Genes within this PUL are coexpressed both in vitro
in rich medium and in vivo in Bt mono-associated gnotobiotic
mice fed a polysaccharide-rich diet (Figure S1Aavailable online),
consistent with the functional relatedness of adjacent genes and
operon predictions in Bt (Westover et al., 2005). Bt increases
expression of this PUL in vivo while downregulating the vast
majority of other PULs when bi-associated in the gnotobiotic
mouse intestine with the methanogenic archeon, Methanobrevi-
bacter smithii (Samuel and Gordon, 2006). The upregulation of
the putative fructan PUL is concomitant with increased densities
of Bt in vivo, suggesting that expression of this locus is associ-
ated with growth potentiation of Bt.
Bt Upregulates Its Putative Fructan PUL When Grown
on Fructose-Containing Carbohydrates
We inoculated minimal medium containing specific fructose-
based carbohydrates as the only carbon and energy source
with Bt to test if the bacterium is competent to grow on fructans.
Bt grew on a broad range of fructose-based glycans, including
free fructose, sucrose, levan (high MW fructose polymer with
(FOS; short-chain b2-1 polymers of 2–10 fructose units)
(Figure 1C; see Figure S2 for carbohydrate structures). However,
Bt grew poorly on inulin (b2-1 fructose polymer with an average
degree of polymerization of ?25), with growth only apparent
three days after inoculation. Doubling times on simple mono-
saccharides and disaccharide were similar to one another
(Table S1). In contrast, growth rates of Bt between the different
fructans showed large linkage-dependent differences: b2-6
levan resulted in the fastest doubling time (2.7 hr), while b2-1
FOS and inulin were significantly slower (doubling times of
5.6 hr and 96.4 hr, respectively) (Table S1).
To determine whether these fructose-based substrates
induced expression of genes associated with the putative fruc-
tan PUL, Bt was grown in either glucose or one of five fruc-
tose-containing substrates (fructose, sucrose, levan, FOS, or
inulin) as the sole carbohydrate. Cells were harvested at mid-
log phase for quantitative RT-PCR (qPCR) analysis, and RNA
levels of the 30and the 50ends of the operon, BT1757 (encoding
the fructokinase) and BT1763 (encoding the SusC-like protein),
respectively, were used as an indicator of PUL expression
lated in all media containing fructose, whether as a free mono-
saccharide or in glycosidic linkage. Across all conditions,
expression of BT1757, BT1763, and BT1765 showed coordi-
nated increases consistent with the predicted operon structure.
However, BT1754 (the PUL-associated putative HTCS) showed
no significant induction under all conditions tested. Therefore,
the operon that encodes the structural genes of Bt’s putative
fructan PUL is transcriptionally responsive to fructose-contain-
ing carbohydrates. Published surveys of Bt gene expression in
numerous carbohydrates support that upregulation of the fruc-
tan PUL is specific to fructose-containing substrates (Martens
et al., 2009; Sonnenburg et al., 2005).
Two genes within Bt’s genome that are not physically associ-
ated with the putative fructan PUL, a second putative periplas-
mic GH32 (BT3082) and a second putative fructokinase
(BT3305), were likely candidates to be involved in fructan utiliza-
tion. Analysis of BT3082 and BT3305 expression by qPCR
revealed that BT3082 was induced in all fructose-containing
media and showed a pattern of induction consistent with those
seen for BT1757, BT1763, and BT1765 (Figure 1D); however,
BT3305 showed no change in expression or a slightly reduced
expression in all conditions (data not shown). These data sug-
gest that the fructosidase, BT3082, but not the putative fructoki-
nase, BT3305, is part of the regulon of the putative fructan PUL.
for Efficient Fructan Utilization by Bt
We assessed the ability of an isogenic mutant of Bt lacking
the BT1754 gene to grow in a panel of fructose-based minimal
media to test if upregulation of the PUL was dependent upon
the HTCS signaling sensor. An in-frame, unmarked deletion of
BT1754 was constructed using a standard counter-selectable
colony morphology on solid medium and grew with a similar
doubling time to wild-type in MM-glucose (2.6 hr); however,
and levan) and showed retarded growth in fructose and sucrose
(Figure 2A and Table S1). Additionally, Bt-DBT1754 does not
exhibit prioritized upregulation ofthe putative fructan PUL during
growth in rich media (Figure S1B). Complementation of this
mutant was achieved by introducing the genomic fragment con-
taining BT1754 and its 50intergenic upstream promoter region in
trans. Growth of the DBT1754::BT1754 complemented mutant
to wild-type (Figure 2A and Table S1). These data demonstrate
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1243
The Periplasmic Domain of the Hybrid Two-Component
System BT1754 Binds to Monomeric Fructose
One of the key unanswered questions concerning the HTCS
family, and many other extracellular sensory systems, is the
identity of the molecular triggers for signaling events. The pre-
dicted inner-membrane localization of Bt’s HTCS family
members, including BT1754, suggests that the periplasmic
region likely serves as the sensor/receptor, similar to classic
two-component systems. Analysis of the sequence of BT1754
revealed a typical HTCS architecture with an N-terminal pre-
dicted periplasmic sensor domain flanked by two transmem-
brane regions and a C-terminal cytoplasmic histidine kinase
domain, a phosphoacceptor domain and a response regulator
(including a receiver and an HTH_AraC-type DNA binding
domain) (Figure 2B and Figure S3). Uniquely within Bt’s HTCS,
the sensor domain displays homology to Type I bacterial peri-
plasmic binding proteins (PBPs) (Dwyer and Hellinga, 2004). As
PBPs are known to bind small molecules such as sugars, we ex-
pressed the periplasmic domain of BT1754 (BT1754-PD; resi-
dues 29–343) in a recombinant form and tested for binding to
a range of monosaccharides and fructan-derived oligosaccha-
rides to see if direct interaction with a specific carbohydrate is
the means of signal perception in BT1754. The isothermal calo-
FructoseSucrose Levan FOS
0 24 48
0 24 48
0 24 48
0 24 48
Absorbance (OD 600)
Ka = 4.6 ±1.1 105 M-1
G = -7.7 ±0.2 kcal.mol-1
H = -15.4 ±0.8 kcal.mol-1
T S = -7.7 ±1.0 kcal.mol-1
n = 0.9 ±0.3
0 2 4 6 8 10 12 14
0 102030 40 50 6070
kcal/mole of injectant
0 20 40 60 80 100 120
0.0 0.5 1.0 1.5 2.0
kcal/mole of injectant
0 10 20 30 40 50 60 70
kcal/mole of injectant
Figure 2. BT1754 HTCS Binds Fructose and Is
(A) Growth curves of Bt-DBT1754 compared to wild-type
Bt (WT) and the complemented mutant (DBT1754::
BT1754) on fructose-based carbon sources.
(B) Domain organization of BT1754.
(C) Interaction of the N-terminal periplasmic domain of
by isothermal calorimetry, showing the raw injection heats
site binding model (fructose only).
Values are averages and SDs of three independent
rimetry data reveal that BT1754-PD binds
specifically to fructose, with a Kd of ?2 mM
and a stoichiometry of 1:1, but does not interact
with either b2-1- or b2-6-linked fructooligosac-
including glucose and ribose (Figure 2C).
Structure of BT1754 Periplasmic Sensor
To understand the mechanism of signal percep-
tion in more detail, we determined the structure
of BT1754-PD in complex with fructose to
2.66A˚. The closest homolog with known struc-
ture, a ribose-binding PBP from Thermoanaero-
bacter tengcongensis (TtRBP), PDB 2IOY, was
used as a molecular replacement search model.
Successful molecular replacement resulted in
a dimer in the asymmetric unit. A least-squares
alignment of the final model with TtRBP gave
a root mean square deviation of 1.2 A˚for 269
alpha carbons despite the relatively low sequence identity,
indicative of the high structural conservation of this family.
The BT1754-PD structure comprises a typical two-subdomain
PBP-fold, with each subdomain consisting of a core of six
b strands flanked by two or three a helices (Figure 3A). The poly-
peptide chain forms a hinge by crossing between the two
subdomains three times along one side, the last of these exiting
the PBP-fold and then forming a long a-helix, which extends
back along the length of the protein to the N-terminal region
The C-terminal helix of BT1754-PD provides the predominant
interface for homo-dimerization and is the main structural differ-
ence between classical soluble PBPs such as the TtRBP and
BT1754-PD (Figure 3B). Though there are several hydrogen
bonds to retain the turn between the PBP-fold and the helix,
once the polypeptide has progressed beyond the first residue
of the helix (Asn306), the remainder of the contacts, both inter-
and intramolecular, are nonpolar. The dimer, generating a buried
surface area of 2640 A˚2, appears to be biologically relevant as
both the N- and C-termini of each molecule are oriented such
that they face in the same direction and, therefore, both mole-
cules are positioned correctly for insertion into the membrane
1244 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.
Crystals were grown in the presence of fructose, and electron
density indicative of a fructose molecule in the b-furanose form
was observed in the cleft between the two subdomains, the
typical binding site of PBP family proteins (Dwyer and Hellinga,
2004)(Figure 3A). The sugar ring is sandwiched between two
tryptophan residues, one from each subdomain (Trp45 and
Trp196), with Tyr271 and Pro168 also forming hydrophobic
contacts along the C4-C6 edge of the fructose ring (Figure 3C).
All remaining interactions with the sugar are polar, with multiple
H-bonds formed between side chains, mainly Arg and Asp, and
the hyroxyls and ring oxygen of the fructose (Figure 3D). In
common with other PBPs, solvent is excluded from the binding
site itself. The structural and biochemical data for BT1754-PD
binding to fructose are consistent with the increased expression
of the fructan PUL observed in minimal medium containing only
fructose (Figure 1D). Furthermore, the structural data suggest
that signal transduction in BT1754 is driven by a conformational
change of the periplasmic domain on fructose binding that is
transmitted across the membrane via a ‘‘piston-like’’ movement
of the TM helices, similar to that postulated for other sensor
kinases (Falke and Erbse, 2009).
Genetic and Biochemical Basis of b2-6 Fructan
Specificity of Bt
The lack of linkage recognition by the HTCS sensor suggested
that the b2-6-linkage specificity of Bt’s fructan use was encoded
within the structural genes of the fructan PUL. We firstfocused on
catalyze the depolymerization of fructans (Cantarel et al., 2009).
withinthe fructanPUL;the otherGH32familymember, BT3082, is
not encoded within the PUL, but is coregulated (Figure 1D).
Complex with Fructose
(A) Representation of the homodimer of BT1754-
PD present in the asymmetric unit, with each
monomer separated by a dotted line; molecule of
fructose (pink); the flexible hinge between the
two subdomains (circle).
(B) Overlay of BT1754-PD (green) with TtRBP
(blue); the extended C-terminal helix in BT1754-
PD (bracket) is unique to BT1754.
(C) Side view of the binding site illustrating hydro-
phobic interactions of BT1754-PD and fructose.
Fo-Fc electron density prior to modeling the single
molecule of fructose in the b-furanose form is
shown (blue mesh contoured at 3s).
(D) Top view of the binding site of BT1754-PD illus-
trating the numerous H-bonds (dotted black lines)
3. Structure ofBT1754-PD in
To test whether the only putative cell
surface GH32 in Bt, BT1760, is required
for levan utilization, an in-frame, un-
marked deletion of BT1760 was con-
structed. Bt-DBT1760 exhibited normal
colony morphology on solid medium and
grew with a normal doubling time in
MM-glucose. Bt-DBT1760 did not grow on levan, but showed
normal growth on all other media tested including b2-1-linked
FOS (Figure 4A), with doubling times comparable to wild-type
in fructose, sucrose, and FOS (Table S1). Complementation in
trans of this mutant was achieved by fusing the upstream inter-
genic promoter region of BT1765 to the 50end of the genomic
fragment containing BT1760. Levan growth was restored, albeit
at a reduced rate, in the complemented Bt-DBT1760::BT1760
strain (Figure 4A), confirming the requirement of this gycoside
hydrolase for utilization of the b2-6 linked fructan.
We next assessed whether BT1760 is a b2-6-specific fructa-
nase. Activity of a recombinant form of BT1760 was tested
against a range of b2-6 and b2-1 fructan oligo- and polysaccha-
rides. The data show that BT1760 is indeed a b2-6-fructan
specific enzymewithnodetectable activityagainstb2-1fructans
or fructooligosaccharides (Table S2). TLC analysis of levan
digestion by BT1760 revealed that a mixture of different sized
oligosaccharides was produced. Mono-, di-, tri-, and tetra-
levanoligosaccharides accumulated as the main products as
the reaction proceeded (Figure 4B). These data demonstrate
that BT1760 is a b2-6-specific endo-acting fructanase.
To determine whether the b2-6 fructoside hydrolase activity
of BT1760 could be detected on the cell surface, we measured
the activity of washed whole Bt cells against levan and inulin.
Fructose-grown wild-type cells could degrade the b2-6 polymer
but had no detectable activity against inulin, mirroring the spec-
ificity of recombinant BT1760 (Figure 4C and data not shown).
This levanase activity was completely lost in the Bt-DBT1760
Bt-DBT1760::BT1760 strain (Figure 4C). Moreover, cells grown
on glucose displayed ?100-fold lower levan activity, confirming
that the levan-specific hydrolysis is inducible by fructose (data
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1245
not shown). Cytoplasmic and periplasmic marker enzyme
assays demonstrated that no cell lysis or leakage occurred in
the assay conditions used; therefore, the hydrolase activity
detected could only be extracellular (data not shown). These
data indicate BT1760 is indeed localized on the surface of
the bacterium. The localization and activity are consistent with
the hydrolase serving as a key step for converting long-chain
levan into oligosaccharides for SusC/SusD-homolog-mediated
Structural insight into the nature of SusD and a SusD homolog
binding to oligosaccharides suggests that linkage is an impor-
tant determinant in cell surface structural recognition of oligo-
saccharides (Koropatkin et al., 2008, 2009). We tested whether
BT1760 was the sole specificity determinant in Bt’s efficient
use of levan, or whether the SusD homolog within the fructan
PUL, BT1762, also exhibited specificity for the b2-6 linkage.
We constructed a Bt mutant in which BT1762 was deleted,
and we tested the ability of this mutant to grow in minimal media
in which levan is the sole carbon source. Bt-DBT1762 showed
significantly retarded growth on levan compared with wild-type
and growth of this mutant in levan was largely restored upon
BT1762 complementation. (Figure 5A and Table S1). Absence
of BT1762, however, did not affect extracellular levan degrada-
tion, supporting that BT1760 is responsible for cell surface
levan degradation (Figure 4C). To determine the specificity of
BT1762 directly, the protein was expressed in a recombinant
form lacking its signal peptide and lipidation motif, and its
interaction with levan and inulin was assessed by isothermal
calorimetry (Figure 5B). The data show that BT1762 binds to
the b2-6 fructose polymer but displays no affinity for the b2-1
equivalent. BT1762 displays a Kd of ?40 mM for levan, similar
to the affinity of the prototypic SusD for cyclodextrins (Koropat-
kin et al., 2008).
0 1m 10m 1h 5h
0 1m 10m 1h 5h
0 1m 10m 1h 5h
0 1m 10m 1h 5h
Absorbance (OD 600)
Frc equivalents (µg/ml)
0 1 2 3 4 5
Figure 4. BT1760 Encodes an Extracellular
Endo-Levanase Required for Bt Growth in
(A) Growth curves of Bt-DBT1760 compared to the
complemented mutant (DBT1760::BT1760) in le-
van (top) or FOS (bottom panel).
(B) TLC analysis of the products of levan digestion
by the Bt GH32 enzymes, BT1760, BT1759,
BT1765, and BT3082. Frc, fructose; L2, levan-
biose; L3, levantriose; L4, levantetraose.
(C) Degradation of levan by Bt cells grown in
minimal medium plus fructose.
Error bars show the SDs from three independent
Recent studies have indicated that
gene found downstream of the susD
homolog also encodes a polysaccha-
ride-binding lipoprotein (Martens et al.,
2009). Although the products of these
obvious sequence homology to one
another they appear to be functionally
conserved. To explore the role of the
susE-positioned gene from the Bt fructan PUL, BT1761, we as-
sessed the ability of a recombinant form of the protein to interact
with inulin and levan. The data reveal that BT1761 bound specif-
ically to levan (Figure S4). Reducing sugar and TLC assays with
BT1761 and BT1762 against inulin and levan revealed that
neither protein had any detectable degradative capacity (data
not shown). Together, these genetic and biochemical data
show that the cell surface components of Bt’s fructan PUL
exhibit b2-6 linkage specificity.
Bt Has Three GH32 Enzymes that Are Not Linkage
To understand the pattern of fructan degradation in Bt in more
detail we biochemically characterized the three other GH32s
expressed during growth on fructose-containing media, the
predicted periplasmic BT1759 and BT3082 and the predicted
intracellular BT1765. The data revealed that all three of these
enzymes are exo-acting fructosidases that release fructose
from both b2-1 and b2-6 fructans, although some differences
in their kinetic characteristics were observed (Figure 4B and
Table S2). BT1759 and BT3082 act equally well on inulin and
levan, as well as oligosaccharides of these polymers, although
BT3082 appears to be overall a more efficient enzyme with
?2- to 4-fold higher kcat/KM values than BT1759 for most
substrates, driven mainly by its higher turnover number. Consid-
ering the b2-6 fructan preference of Bt, it is interesting that both
enzymes display lower KMvalues (?2- to 8-fold) for b2-1 oligo-
saccharides compared to their b2-6 equivalents (Table S2).
BT1759 and BT3082 also cleave sucrose, a trait shared with
other bacterial fructosidases, although both have a higher KM
for the disaccharide than for larger b2-1 kesto-oligosaccharides.
By contrast, BT1765 much prefers sucrose over any of the other
oligo- or polysaccharides tested, although the enzyme is also
1246 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.
able to efficiently hydrolyse levanbiose (Table S2). The predicted
cytoplasmic location of BT1765 and substrate specificity
suggest that some of the disaccharide products of levan (and
possibly FOS) digestion are transported across the inner
membrane before they are degraded by the periplasmic fructo-
sidases (see Figure S5).
The Fructan PUL Is Variably Conserved in Sequenced
Bacteroides, which Have Differing Capacity to Utilize
We performed a comparative genomic analysis focused on Bt’s
fructan utilization locus between five sequenced species of
Bacteroides to gain further insight into the mechanism of fructan
use for this major group of gut resident microbes. Using the
N-terminal fructose-binding domain of the HTCS BT1754 to
query a BLAST database consisting of the Bacteroides species
B. caccae, B. vulgatus, B. uniformis, B. fragilis, and B. ovatus,
we have identified a single orthologous HTCS in each species,
with the exception of B. fragilis, which harbors two BT1754-like
genes. Sequence identity between the periplasmic sensor
domains of the BT1754 orthologs was high for all but one,
ranging from 93% for the B. ovatus protein to 58% for the B. vul-
gatus domain. Furthermore, the residues involved in fructose
binding in BT1754 are almost completely conserved among
orthologs, consistent with conservation of the ligand sensed by
each HTCS (Figure S3). The periplasmic domain of one of the
Absorbance (OD 600)
Levan ( 2-6 fructan)
FOS ( 2-1 fructooligosaccharides)
Inulin ( 2-1 fructan)
Levan ( 2-6 fructan)
Ka = 2.3 ±0.1 104 M-1
G = -5.9 ±0.0 kcal.mol-1
H = -7.8 ±0.3 kcal.mol-1
T S = -1.9 ±0.3 kcal.mol-1
n = 1.0 ±0.0
n = 1.0 ±0.0
Ka = 2.3 ±0.1 104 M-1
G = -5.9 ±0.0 kcal.mol-1
H = -7.8 ±0.3 kcal.mol-1
T S = -1.9 ±0.3 kcal.mol-1
0 12 24
0 20 40 60 80 100 120 140
0 20 40 60 80 100 120 140 160
kcal/mole of injectant
kcal/mole of injectant
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0 10 20 30 40
Figure 5. The SusD-Homolog Encoded by BT1762
Is Required for Efficient Bt Utilizaton of Levan
and Binds b2-6 but Not b2-1 Fructan
(A) Growth curves of wild-type Bt, Bt-DBT1762, and
Bt-DBT1762::BT1762 in levan (left) or FOS (right).
(B) Interaction of BT1762 with fructans as assessed by
isothermal calorimetry. Levan binding data integrated
and fit to a single site binding model (bottom left).
Values are averages and SDs of three independent
two B. fragilis orthologs (BF4326) displayed
only 36% identity with BT1754-PD, and this
domain was unique in its lack of fully conserved
fructose binding residues (Figure S3). Regions
adjacent to the HTCS in each genome were
analyzed and found to display local synteny
with the Bt locus (Figure 6, left panel), including
the presence of open reading frames that
are predicted to play a role in utilization of
fructose-containing carbohydrates. In all six
Bacteroides species, the HTCS is adjacent
to a predicted fructokinase, a putative inner
GH32-family glycoside hydrolases. In each
genome, except that of B. vulgatus, the syntenic
regions also contain a susC/susD homologous
The presence of an apparent fructan PUL in
multiple Bacteroides species suggested that
fructan utilization is shared between members
of this genus. Testing for growth on fructose-based glycans
revealed that all six species are competent for growth on fruc-
tose (Figure 6, right panel), sucrose and FOS (Table S1). All Bac-
teroides species tested, except B. vulgatus, were able to grow
efficiently using one of the long-chain fructans, inulin or levan.
The inability of B. vulgatus to grow on long-chain fructans is
consistent with the absence of a susC/susD-like pair within its
locus. B. caccae, B.ovatus, B. fragilis and B. uniformis can utilize
with Bt inulin use, which is only observed after three days
Bt is the only species tested able to use levan, which was
particularly striking when considering the overall similarity in
PUL structure between Bt, B. caccae, and B. ovatus. However,
examination of PUL gene content of the two inulin-utilizing
species revealed genes encoding PL19 enzymes, a family that
is known to include members capable of degrading the b2-1
fructan. Additionally, Bt’s extracellular b2-6-specific GH32,
BT1760, does not possess an orthologous gene in the other
species (Figure S6). Notably, two other sequenced Bt strains
utilize levan more efficiently than inulin in vitro (data not shown),
similar to the type strain. Both of these strains possess orthologs
to the type strain’s BT1760 (Figure S6). Together these data
species correspond to differences in the gene content of their
respective fructan PULs.
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1247
Genomic Content of Bacteroides Species Predicts
Changes in Microbiota Composition Induced by an
The differences in ability to utilize fructans between the Bacter-
oides species implies that the relative success of a species
within a gut ecosystem may be determined, in part, by the abun-
dance and type of fructan in the host diet. Furthermore, the
comparison of genomic sequences and differences in fructan
use between species suggests that personalized predictions of
microbiota response to specific dietary polysaccharides may
be made based on metagenomic microbiome sequence data.
We constructed defined two-member communities of Bacter-
oides species within the intestines of gnotobiotic mice to test
how model microbiotas respond in vivo to dietary inulin, which,
unlike levan, is available in pure form in quantities sufficient to
conduct such a study. Our in vivo experiment aimed to test
how differing functionalities embedded within the genomes of
two different two-species model microbiotas influence inulin-
induced changes in community composition.
Due to B. caccae’s superior ability to use inulin compared to
Bt, we tested whether B. caccae would become dominant over
Bt within the intestines of mice fed an inulin-supplemented
diet. Conversely, Bt’s poor growth on inulin is better than
B. vulgatus, which is unable to utilize inulin, suggesting that Bt
might benefit from inulin when colonized with B. vulgatus. Two
groups of 8–12-week-old, germ-free mice were colonized with
equivalent quantities (108colony forming units, CFU) of Bt and
B. caccae or Bt and B. vulgatus. Each mouse was maintained
on a standard polysaccharide-rich diet for the first 7 days of
colonization and then switched to a diet in which the sole
polysaccharide was inulin (10% w/w) for an additional 14 days
(Figure 7A). Mice were individually housed throughout the exper-
iment to ensure no cross inoculation could occur and bedding
was changed every two days. Total bacterial colonization
density was determined by assessing the CFUs in feces over
21 days. The change in each species’ relative abundance before
and after dietary inulin supplementation was assessed using
species-specific primers in a quantitative PCR assay.
Our results disclosed that total fecal bacterial densities over
the course of the experiment did not differ significantly upon
dietary shift (total densities ranged from 1010–1011bacteria/ml
of fecal material). Relative densities were determined on days
4 and 6 (standard diet) and on days 13 and 21 (6 and 14 days
after dietary switch). In the Bt/B. caccae, bi-associated mice,
before the diet switch (day 6 postcolonization in mice fed a
standard diet), Bt comprised 87 ± 3% of the community, indi-
cating that Bt is better adapted than B. caccae to these in vivo
conditions. Six days after a change to the inulin-based diet, Bt
levels dropped to 80 ± 4%, and B. caccae increased to 20 ±
4%. After two weeks consuming the inulin diet, the relative
proportion of the two species showed a more drastic shift in
favor of B. caccae: Bt representation decreased to approxi-
mately 49 ± 6% versus 51 ± 6% B. caccae (p = 8 3 10?5, day
21 versus day 6; n = 7 mice; Student’s t test) (Figure 7B). In
Figure 6. Comparative Genomic and Functional Analysis of Fructan Utilization among Bacteroides Species
Fructan-utilization loci from Bacteroides species (left). Common predicted functions are color coded, intervening unrelated genes are white. PL19, polysaccha-
ride lyase family 19; GH32, glycoside hydrolase family 32. Growth curves (right) of each Bacteroides species in fructose-based carbohydrates.
1248 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.
contrast, the Bt/B. vulgatus bi-associated mice did not exhibit
any significant trend in changed community composition after
6 days on an inulin-based diet, but Bt increased in abundance
from 74 ± 3% on day 6 to 84 ± 5% on day 21 (p = 0.1; n = 3
mice) on the inulin-enriched diet (Figure 7C). The delayed and
gatus bi-association is consistent with poor inulin use by Bt and
no inulin use by B. vulgatus (Figure 6). Together, these data are
consistent with dietary polysaccharide-induced changes in the
microbiota composition that are predictable based on the resi-
dent species’ ability to use that polysaccharide.
In the previous experiment, inulin was the sole polysaccharide
in the diet. We wondered whether we would observe the same
inulin-induced increase in B. caccae relative to Bt if other
polysaccharides were also present in the diet. To test this,
gnotobiotic mice were co-colonized with Bt and B. caccae and
maintained on the standard diet with inulin supplementation in
the water (1% w/v). Over the 14 days the mice ingested an
average of 117 ± 6 mg of inulin daily via the water (compared
to 355 ±7mg/day with the inulin diet). Fecal samples were tested
by qPCR over the course of the 21-day experiment for relative
levels of Bt or B. caccae. These data revealed no statistical
difference in the change in relative colonization between mice
fed inulin-supplemented water compared to controls that
received the same standard diet for 21 days, but received no
inulin (Figure 7D). These data suggest that when mice were fed
a diet rich in carbohydrates, the presence of inulin did not
provide enough of an advantage to B. caccae to allow it to out-
compete Bt; however, the amount of inulin supplied in the water
05 1015 2025
05 10 15 2025
Difference in % representation
of B. caccae
Figure 7. Effect of Dietary Fructans on Bac-
teorides Competition within the Intestine
(A) Experimental design for in vivo experiments.
(B) Average relative fecal proportion (% total
bacteria) of Bt and B. caccae at 4, 6, 14, and
21 days after colonization; n = 7 mice.
(C) Average relative fecal proportion (% total
bacteria) of Bt and B. vulgatus at 4, 6, 14, and
21 days after colonization; n = 3 mice.
(D) Increase in proportion (%) of B. caccae over Bt
from day 6 (1 day prior to diet change) to day 21
(14 days after diet change). All groups received
indicated; n = 3-7 individually housed mice.
(E) Average relative fecal proportion (% total
bacteria) of inulin-utilizing Bt(In+) and B. caccae
at 4, 6, 14, and 21 days after colonization; n = 7
individually housed mice.
Values are averages and standard errors.
(117mg/day average) was less than the
amount derived from the inulin diet
(355mg/day average) potentially con-
tributing to the lack of the B. caccae
We decided to feed mice a custom
diet deficient in all polysaccharides and
supplement inulin in the water to determine whether a lower
dose of inulin in the absence of other polysaccharides was
sufficient to provide B. caccae a competitive advantage over
Btinvivo. Underthisexperimental paradigm the miceconsumed
an average of 97mg of inulin per day. After 14 days on inulin-
water supplementation, the proportion of B. caccae increased
by 26 ± 8% (Figure 7D). While not as robust an increase as
observed in the inulin-only diet experiment (which showed a
36 ± 7% increase in B. caccae), these data demonstrate that
reduced inulin consumption in the absence of competing
polysaccharides, offers a significant competitive advantage to
inulin-utilizing B. caccae, consistent with the flexible nutrient
saccharides present in the standard diet allows Bt to compete
effectively with B. caccae even in the presence of inulin.
We finally demonstrate the importance of inulin utilization for
conferring a competitive advantage in hosts fed an inulin-rich
fructan-utilization locus from the susC-like gene through the
GH32-encoding gene (BC02727-BC02731) was cloned and
expressed in a strain of Bt that is compromised in its ability to
utilize levan (Bt-DBT1763) under the control of the BT1763
promoter (data not shown). The resulting strain, Bt(In+), exhibits
efficient growth in minimal medium containing inulin, similar to
B. caccae (Figure S7). Repeating our original in vivo competition
experiment with Bt(In+) revealed that conferring inulin use ability
the presence of an inulin-based diet (Figure 7E). This result
confirms that the specificity of dietary polysaccharide use is
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1249
the key functionality that dictates the alterations in the model
microbiota that we observe. These results support our hypoth-
esis thatchanges in microbiota community membership brought
tional knowledge of resident microbial populations. They also
suggest that diet can be a dominant determinant in dictating
changes in microbiota composition.
Inulin (b2-1 fructan) and levan (b2-6 fructan) are polysaccharides
that are abundant in the human diet, but are resistant to host-
mediated digestion in the upper gastrointestinal tract. These
glycans instead serve as a carbon and energy source for
the bacteria that reside in the distal intestine. Bacteroides the-
taiotaomicron, a resident of the human GI tract, encodes a fruc-
tan utilization locus, BT1757-63 and BT1765, the gene products
of which enable efficient acquisition and use of levan-type
The fructan PUL is adjacent to a hybrid two-component
system sensor-regulator, BT1754, which binds only to mono-
meric fructose, a signal sufficient to induce transcription of
the locus. While the upregulation of polysaccharide utilization
machinery in response to a monosaccharide may seem unex-
pected, this signal is a likely consequence of the environment
in which Bt resides. Within the natural habitat of the large bowel,
free fructose and simple disaccharides, such as sucrose, do not
occur at appreciable levels as the host absorbs such sugars
within the small intestine. Therefore, the regulation of this locus
evolved in the absence of selective pressure to discriminate
free monosaccharide from polysaccharides. In addition, unlike
many other monosaccharides, fructose is found in only a single
class of polysaccharide, namely homopolymeric fructans. Bt
appears to use the liberated fructose as a proxy (i.e., indicator)
for fructan, which results in upregulation of the machinery to
utilize the polysaccharide. This is consistent with previous
data that demonstrate Bt’s constitutive, low-level expression
of PULs in conditions lacking the relevant substrates (Martens
et al., 2009; Sonnenburg et al., 2005), as well as the low-level
cell surface levanase activity we observe with whole cells
grown in glucose. The constitutive expression suggests that
Bt employs a strategy of being prepared to degrade multiple
polysaccharides immediately upon their arrival into the distal
gut environment. Specific liberated carbohydrates that result
from the degradation serve as signals that augment expression
of the appropriate PUL via a specific sensor-regulator such as
The binding of BT1754 to monomeric fructose also results in
a failure of the sensor to differentiate b2-1 and b2-6 linkages
despite Bt being much more efficient in use of the levan-type
fructans. Specificity of signal is instead derived from the cell
surface structural components of the PUL, which serve as the
‘‘gateway’’ for substrates crossing the outer membrane. The
cell surface SusD homolog, BT1762, the susE-positioned gene
product, BT1761, and the endo-levanase, BT1760, all contribute
to the specific import of b2-6 fructans into Bt’s periplasm.
ride degradation and binding machinery to provide fructose
derived from b2-6 fructan to the periplasm where the sensor is
Despite Bt’s inability to utilize inulin efficiently it is able to grow
well on FOS, a short chain b2-1 fructan. Notably, the fructan PUL
of Bt is upregulated during growth in vitro in minimal medium
containing FOS or inulin. Bt’s ability to grow in FOS at a rate
that is significantly faster than inulin is likely due to the difference
in degree of polymerization between the two substrates.
Whether small oligosaccharides from these substrates undergo
passive diffusion into the periplasm or are accessed via another
mechanism requires further investigation.
Among the Bacteroides species tested, Bt appears to be
unique in its ability to utilize levan, whereas other species are
adept at utilizing polymeric b2-1 fructans. Such phenotypic
differences, combined with dietary variation between individ-
uals, could provide the basis for the striking person-to-person
variability observed for Bacteroidetes in human microbiota
enumeration studies (Eckburg et al., 2005). Our in vivo studies
illustrate that species well-adapted to use inulin gain a competi-
tiveadvantage whenhosts arefed aninulin-based diet. Although
a genetic loss-of function experiment, in which inulin use is
compromised, could be used to test whether the observed
changes in species abundance are due to inulin use, we have
used a gain-of-function experiment, in which inulin use is
conferred upon Bt, to illustrate this point unequivocally. These
results suggest that some aspects of diet-induced changes in
microbiota composition may be predetermined based on the
intrinsic capacity of an individual species to use the substrates
riched in different polysaccharides, or polysaccharide-deficient
diets, could result in microbiotas of very different species
composition. Future studies that follow species and gene
tion of levan- or inulin-based diets will provide insight into the
rapidity with which members of a complex community adapt at
a functional, compositional, and genetic level. How such niche
specialization occurs over the course of evolution and the role
that diet plays in determining a species’ glycan utilization reper-
toire remain important yet difficult questions to address.
As the age of personal genomes approaches, some aspects of
diet and medical therapies will be customized based on geno-
type. Diet can also be personalized to optimize microbiota
analysis of an individual’s microbiota. A prerequisite for incorpo-
preventative medicine is to attain a mechanistic understanding
of the most dominant aspects of microbiota function. Here we
present a case study of how understanding the mechanisms
that link the microbome to microbiota function may enable indi-
vidualized predictions of microbiota response to perturbations.
We have taken two-species model microbiotas that collectively
possess close to 10,000 genes and predicted how they will
respond to a specific dietary cue based on a functional under-
standing of the ?20 relevant genes. A similar distillation of full
microbiomic datasets that contain > 106genes, to a relevant
subset, will be required to make microbiota management
1250 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.
tractable. With an ever-increasing understanding of how the
biology of host and microbiota integrate, we may soon be able
to use genomic and microbiomic sequence data to intentionally
program or reprogram the emergent properties of the host-
Bacteria were cultured in TYG and MM as described previously (Martens etal.,
2008; Sonnenburg et al., 2005). The following bacteria were used:
Bt (VPI-5482), B. caccae (ATCC-43185), B. ovatus (ATCC-8483), B. fragilis
(NCTC-9343), B. uniformis (ATCC-8492), and B. vulgatus (ATCC-8482).
Growth curves in MM were obtained using a Powerwave (Biotek) reading
OD600 every 30 min from anaerobic cultures at 37?C.
Quantitative RT-PCR Analysis
Quantitative RT-PCR was performed using gene-specific primers as
described previously (Table S3) with SYBR Green (ABgene) in a MX3000P
thermocycler (Strategene) (Martens et al., 2008).
Gene Deletion and Complementation in Bt
In-frame (nonpolar) gene deletions for mutants were generated using counter-
selectable allele exchange (Martens et al., 2008). PCR amplified genes for
complementation were ligated into the pNBU2-tetQb vector and conjugated
into Bt via E. coli S17.1 l-pir (Martens et al., 2008). Resulting clones were
screened by PCR and sequenced to confirm isolates.
Genes for expression were amplified from Bt genomic DNA using the primers
stated in Table S3 and cloned into pRSETA (Invitrogen) or pET22b (Novagen).
Protein Expression and Purification
Recombinant proteins were expressed in E. coli C41 or BL21 cells and purified
in a single step using metal affinity chromatography as described previously
(Bolam et al., 2004).
Sources and Preparation of Carbohydrates
Monosaccharides, sucrose, and chicory inulin for enzymatic and binding
assays were obtained from Sigma. Growth of Bacteroides strains, qRT-PCR,
and mouse experiments used inulin, FOS (Beneo-Orafti group; OraftiHP, Oraf-
tiP95, respectively) and levan (Sigma; 66674). Kestooligosaccharides were
fromMegazyme.Levanoligosaccharideswereproducedby partial acidhydro-
lysis (1 M HCl at 25?C for 20 min - 1 hr) of levan (Montana Polysaccharides).
NaOH-neutralized samples were separated on BioGel P2 (BioRad) size exclu-
Isothermal Titration Calorimetry
Measurements were carried out essentially as described previously (Bolam
et al., 2004), except that a Microcal VP-ITC machine was used, and proteins
were dialyzed into 20 mM Tris-HCl (pH 8.0). The assumption that n = 1 for
BT1762 binding to levan was based on the structure of the starch binding
SusD (Koropatkin et al., 2008).
Samples were spotted onto foil backed silica plates and placed in a glass tank
equilibrated with butanol:acetic acid:H2O (2:2:1). Sugars were visualized using
orcinol-sulphuric acid (sulphuric acid:ethanol:H2O 3:70:20 v/v, orcinol 1%
w/v), 90?C for 5–10 min.
All assays were carried out at 37?C in 20 mM Tris-HCl (pH 8.0). Activity of
BT1760 wasdetermined by quantifying the amount of reducing sugar released
using the DNSA assay (Miller, 1959). Free fructose was determined using
amodified fructose detection kit(MegazymeInternational). Kinetic parameters
were determined by fitting initial rates versus substrate concentration
lis-Menten equation using nonlinear regression (Graphpad Prism, v5.0).
Enzyme Localization Studies
Cultures grown on 0.5% (w/v) fructose or glucose were harvested by centrifu-
gation (OD600?1.0). PBS-washed cells and 0.5% levan or inulin in 20 mM Tris-
HCl, pH8.0, were incubated at 37?C. Reducing sugar present was quantified
using DNSA reagent (Miller, 1959). Activities of the periplasmic marker alkaline
phosphatase and cytoplasmic marker glucose-6-phophate dehydrogenase
were compared to lysed cells to ensure no cell lysis/leakage occurred.
Bacterial Colonization and Density Determination of Germ-Free
Germ-free Swiss-Webster mice were maintained in gnotobiotic isolators and
fed an autoclaved standard diet (Purina LabDiet 5K67) or custom diet (Bio-
Serv, http://bio-serv.com/), in accordance with A-PLAC, the Stanford IACUC.
Mice were bi-associated using oral gavage (108CFU of each bacterial
species). Relative densities of bacteria were determined by qPCR using
strain-specific primers (Table S3) (Martens et al., 2008).
Crystallization, Structure Determination, and Refinement
Crystals formed in 0.7 M K/Na phosphate, 0.1 M HEPES (pH 8.0) (protein at
8 mg/ml with 5 mM fructose). Diffraction data, collected at Diamond Light
Source (Oxford, UK) on a tiled ADSC Q315 CCD detector were processed
withMOSFLM(Leslie,1992).Scaling ofdata,searchmodelgeneration, molec-
ular replacement and structure refinement were carried out using SCALA,
CHAINSAW, MOLREP and REFMAC (Collaborative Computational Project,
1994), respectively, with model rebuilding in COOT (Emsley and Cowtan,
Protein Data Bank coordinates have been deposited under the accession
Supplemental Information includes four tables, seven figures, and Supple-
mental References and can be found with this article online at doi:10.1016/
We thank Karla Kirkegaard and Stanley Falkow for valuable comments and
Sara Fisher for editing the manuscript. Inulin and FOS for mouse experiments
were a kind gift from Beneo-Orafti. Levan was a kind gift from Montana Poly-
saccharides. We thank Jeffrey Gordon and members of the Gordon Lab for
valuable advice; Carl Morland for excellent technical assistance; and Eric
Martens and Andrew Goodman for development of genetic tools used in this
paper. Some Bacteroides genomic data were produced by The Genome
Center at Washington University School of Medicine in St. Louis (genome.
wustl.edu). This work was funded in part by grants from National Institutes
of Health through the NIH Director’s New Innovator Award Program (DP2-
OD006515) the NIDDK (K01-DK077053), the Stanford Digestive Disease
Center (PO3-DK56339) and the BBSRC (BB/F014163/1).
Received: October 15, 2009
Revised: January 20, 2010
Accepted: April 27, 2010
Published: June 24, 2010
Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc. 1251
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Note Added in Proof
PL19 enzymes have recently been reclassified into glycoside hydrolase family
91 (see www.cazy.org/GH91.html).
1252 Cell 141, 1241–1252, June 25, 2010 ª2010 Elsevier Inc.