udp-glucuronic acid decarboxylases of bacteroides fragilis ... · one aliquot of each culture was...

JOURNAL OF BACTERIOLOGY, Oct. 2011, p. 5252–5259 Vol. 193, No. 190021-9193/11/$12.00 doi:10.1128/JB.05337-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and TheirPrevalence in Bacteria�†

Michael J. Coyne, C. Mark Fletcher, Barbara Reinap, and Laurie E. Comstock*Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received 18 May 2011/Accepted 20 July 2011

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated formnecessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronicacid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation ofUDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA andRsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxy-lases of Bacteroides fragilis, designated BfUxs1 and BfUxs2. Bfuxs1 is located in a conserved region of the B.fragilis genome, whereas Bfuxs2 is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus.Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of bothgenes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the B. fragilisNCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly asNAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Usingavailable structural and mutational data, we devised a sequence pattern to detect bacterial genes encodingUDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diversebacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These datasuggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterialglycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in Bacteroidesspecies, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containingglycan.

The pentose sugar xylose is a common component of the cellwalls of plants (44), fungal glycans (33), and glycosaminogly-cans of higher organisms (26). Xylose is rarely described as acomponent of bacterial glycans. Bacterial molecules shown tocontain xylose include the O antigen of Pseudomonas aerugi-nosa serotype O7 (10, 25), the core oligosaccharide of thelipopolysaccharide (LPS) of Sinorhizobium meliloti (20), theLPS core oligosaccharide of Ralstonia solanacearum (19),the LPS of Leptospira species (23, 35), and an S-layer glyco-protein of Parabacteroides distasonis (15).

The incorporation of xylose into glycans requires its additionfrom the nucleotide-activated precursor UDP-xylose. UDP-xylose was first shown to be synthesized from UDP-glucuronicacid (UDP-GlcA) by a UDP-GlcA decarboxylase in the fungusCryptococcus laurentii (5), and this enzymatic activity has sincebeen identified in plants and animals. The UDP-xylose syn-thase (Uxs) family enzymes found in animals produce UDP-xylose by decarboxylation of UDP-GlcA using NAD (NAD�).Similarly, the enzymes of the UDP-apiose/UDP-xylose syn-thase (Uaxs) family found in plants convert UDP-GlcA toUDP-apiose and UDP-xylose (21).

Only three bacterial enzymes catalyzing oxidative decarbox-ylation of UDP-GlcA to UDP-xylose have been described.SmUxs1 from Sinorhizobium meliloti (20) leads to the synthesis

of UDP-xylose as an end product. ArnA of Escherichia coli is abifunctional enzyme that has a C-terminal portion with UDP-GlcA decarboxylase activity, catalyzing the C-4 oxidation and C-6decarboxylation of UDP-GlcA. This enzyme’s N-terminal portionalso has formyl transferase activity, and both activities participatein a pathway leading ultimately to the synthesis of UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N). RsU4kpxs, from Ralstoniasolanacearum (19), is similar to the C-terminal portion ofArnA, has UDP-GlcA decarboxylase activity, and producesUDP-xylose as an end product. However, much of the UDP-xylose of this organism is further converted to UDP-L-Ara4Nby a formyl transferase, similar to the N-terminal portion ofArnA, encoded by an adjacent gene.

UDP-GlcA decarboxylases are members of the short-chaindehydrogenase/reductase (SDR) superfamily that also includeepimerases, dehydratases, and reductases that use nucleotide-activated monosaccharides as substrates. Enzymes in this largefamily of proteins share a Rossmann-fold NAD(P)H/NAD(P)(�) binding domain. Genes encoding SDR enzymesare often annotated solely as NAD-dependent epimerases/de-hydratases, and therefore, many UDP-GlcA decarboxylasegenes are likely incorrectly annotated.

Intestinal Bacteroidales species are the most abundantGram-negative bacteria of the human colonic microbiota,reaching densities of more than 1010 bacteria per gram offeces (45). Intestinal Bacteroidales species encode an exten-sive number of proteins dedicated to the synthesis of glyco-sylated molecules, including many capsular polysaccharides(27), glycoproteins (17), extracellular polysaccharides (11),and glycosylated S-layer proteins (15). Many of these mol-

* Corresponding author. Mailing address: Channing Laboratory,181 Longwood Avenue, Boston, MA 02115. Phone: (617) 525-7822.Fax: (617) 264-5193. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 29 July 2011.

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ecules are necessary for the organism to colonize the mam-malian intestine (12, 13, 17), and one has been shown toprovide beneficial properties to the host in the intestine (30)and abscess-promoting properties in extraintestinal sites(40).

These bacteria have a tremendous capacity to degrade thenumerous plant and host polysaccharides that are abundant inthe colonic ecosystem. Bacteroides genomes in general encodea large number of xylosidases, which allow the bacteria todegrade xylose-containing polymers as an energy source. Wehave found that these bacteria often include monosaccharidesthat are abundant in the intestinal ecosystem in their ownglycosylated molecules (13). Therefore, we sought to explorewhether xylose was likely present in the glycans of intestinalBacteroidales species.

Elucidating the structures of glycosylated molecules of Bac-teroides is a difficult process, as the glycosylated molecule firstmust be purified from other glycans that often are of similarsize and have similar charge properties. Despite the impor-tance of these molecules to the biology of the bacteria and totheir interactions with the host in both symbiotic and patho-genic relationships, the structures of very few glycans of theintestinal Bacteroidales are known, nor is there much informa-tion regarding their compositions. Therefore, we searched forgenes encoding UDP-GlcA decarboxylases as an indicationthat xylose might be a constituent of the glycans of thesebacteria.

In this report, we characterize two UDP-glucuronic acid(UDP-GlcA) decarboxylases of Bacteroides fragilis. Using aresidue pattern to distinguish UDP-GlcA decarboxylases fromother enzymes of the short-chain dehydrogenase/reductase(SDR) superfamily, we found similar proteins encoded by spe-cies throughout the Bacteroides genus and by numerous otherbacterial species. Therefore, xylose is likely present in theglycans of most Bacteroides species and is more common thanpreviously appreciated in bacterial glycans in general.

MATERIALS AND METHODS

All primers used in this study are listed in Table S1 in the supplementalmaterial.

Cloning BF2189 and BF1555. BF2189 was PCR amplified as a 998-bp productusing high-fidelity Taq polymerase (Invitrogen). The product was digested withBamHI (Invitrogen) and cloned into the BamHI site of vector pET16b to createan N-terminal His-tagged protein. BF1555 was amplified and cloned in a similarmanner. BF1555 is annotated in the B. fragilis NCTC 9343 genome as having analternate start codon, followed immediately by an ATG codon, which we con-sidered to be the start codon for this study as each of the four major bacterialgene prediction programs (Prodigal 2.50, Glimmer 3, GeneMarkHMM 2.6r, andGeneMark 2.5m) predict that this gene starts with the ATG codon. The sequencefidelity and correct orientation of both clones were confirmed by sequencing, andeach was transformed into E. coli BL21(DE3) (37) for protein purification. Ascontrols, the pET16b vector alone and pDC22.6 (a pET28a-based clone contain-ing the Cryptococcus neoformans UXS1 gene [6]) were also transformed into E.coli BL21(DE3).

Enzyme purifications. E. coli BL21(DE3) cells containing recombinant vectorswere grown at 37°C in 3 ml of Luria broth supplemented with either ampicillin(100 �g/ml) or kanamycin (30 �g/ml, for pDC22.6) for 1.5 h and then used toinoculate 100 ml of the same medium. These cultures were grown until an opticaldensity at 600 nm of �0.4 was reached, and each was split into 50-ml aliquots.One aliquot of each culture was induced by adding 0.4 mM isopropyl �-D-1-thiogalactopyranoside (IPTG; Invitrogen), and incubation was continued for2.5 h. The cells were recovered by centrifugation at 4°C, and each pellet wasresuspended in 2.5 ml of 50 mM cold Tris-HCl (pH 7.8) and stored at �20°C.

Thoroughly resuspended Talon Dynabeads (50 �l, 2 mg; Invitrogen), which

are precharged with Co2�, were equilibrated for use according to the manufac-turer’s directions, except that the binding/wash buffer contained 50 mM sodiumphosphate buffer (pH 7.0), 500 mM NaCl, and 0.01% (vol/vol) Tween 20. Cellsfrom 25 ml of IPTG-induced culture (see above) were resuspended in 1.5 ml ofice-cold binding/wash buffer and lysed by sonication. DNase (Worthington Bio-chemical Corporation, Lakewood, NJ) was added to each sample at a finalconcentration of 1 �g/ml, and the suspensions were held on ice. The lysates werebriefly (15 s) centrifuged in a bench-top centrifuge to pellet the bulk of the celldebris, and 100 �l of each lysate was added to 50 �l of the washed and equili-brated paramagnetic beads. The tubes were allowed to stand on ice for 15 minwith occasional resuspension and then incubated end over end for 30 min at 4°C.The beads were collected magnetically and washed four times in 700 �l of thebinding and wash buffer. The enzyme-coated magnetic beads retain the enzymesin their native form and facilitate their removal postreaction and were thus useddirectly in the enzymatic activity assays.

Enzymatic reactions. Each bead preparation was resuspended in 100 �l ofenzyme reaction mixture consisting of 65 mM morpholinepropanesulfonic acid(MOPS) (pH 7.4), 1 mM NAD (NAD�; Sigma), and 1 mM UDP-glucuronic acid(UDP-GlcA; Sigma) or 1 mM UDP-galacturonic acid (UDP-GalA; CarboSourceServices, Atlanta, GA). The reaction mixtures were incubated at 30°C whilerotating end over end for 1 h. The enzymes were removed magnetically after thereaction.

High-performance anion-exchange chromatography. Samples (25 �l) of thereactions and standards were analyzed by high-performance anion-exchangechromatography using a Dionex (Sunnyvale, CA) pulsed amperometric detectorand a CarboPac PA1 carbohydrate column. The detector sensitivity was set at300 nA with a 0.05-V applied pulse potential. Compounds were eluted with 825mM sodium acetate in 20 mM sodium hydroxide, with a flow rate of 1 ml perminute. Standards included UDP-xylose, UDP-arabinose, UDP-glucuronic acid,and UDP-galacturonic acid (CarboSource Services).

Creation of single and double deletion mutants. Deletions of Bfuxs1 (BF2189)and Bfuxs2 (BF1555) were constructed such that 834 bp of the 936-bp Bfuxs1 and826 bp of the 942-bp Bfuxs2 were removed by allelic replacement. DNA segmentsupstream and downstream of each region to be deleted were PCR amplified andcloned by three-way ligation into the Bacteroides conjugal suicide vector pJST55(39). The resulting plasmids were conjugally transferred into wild-type B. fragilisNCTC 9343, and cointegrates were selected on the basis of erythromycin resis-tance encoded by the vector. The cointegrate strains were passaged, plated onnonselective medium, and replica plated on medium containing erythromycin.Erythromycin-sensitive colonies were screened by PCR to detect colonies thathad acquired the mutant genotype. The �Bfuxs1 �Bfuxs2 double mutant wasconstructed by first deleting Bfuxs1 and then Bfuxs2.

SDS-PAGE and Western blot analysis. Bacterial lysates from 18-h culturesgrown in basal medium (32) were separated using NuPAGE 4 to 12% gradientSDS-polyacrylamide gels with morpholineethanesulfonic acid (MES) buffer (In-vitrogen). The contents of the gels were transferred to polyvinylidene difluoridemembranes (Invitrogen) and probed with rabbit antiserum specific to individualcapsular polysaccharides of B. fragilis NCTC 9343 (27), to the glycan of the B.fragilis glycoproteins, or to the protein component of glycoprotein BF2494 (17).The �Bfuxs1 �Bfuxs2 adsorbed antiserum was prepared as previously described(27), except that the �Bfuxs1 �Bfuxs2 mutant was used to adsorb antibodies tothe wild-type strain. Following this adsorption, only antibodies to immunogenicmolecules produced by the wild-type strain but not by the mutant remained. Bycomparing the reactivities of this antiserum to wild-type and mutant extracts,molecules that are synthesized by the wild type but lost in the mutant can beidentified. All blots were then probed with alkaline phosphatase-conjugated goatanti-rabbit secondary antibodies and developed colorimetrically using 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (Kirkegaard & Perry).

Bioinformatic analyses. All sequences were retrieved from GenBank in Jan-uary 2011. Similar sequences were retrieved with BLAST (3, 4). Taxonomyassignment is based on NCBI’s taxonomy database. Alignments were routinelyperformed with ClustalW2 (28). Custom Perl scripts were used throughout theseanalyses. Secondary structure prediction was performed using the SABLE pro-gram (1).

To identify proteins encoded by bacterial genomes with predicted UDP-GlcA decar-boxylase activity, a BLAST search was performed using the amino acid sequence ofBfUxs1 to query NCBI’s nonredundant database. Similar proteins (E value, �10�3)encoded by bacterial genomes with completed or draft genomic sequences were re-tained. These proteins were then analyzed for those matching a Perl regular expression,G..G..G.{17,27}(D�E).{76,84}TSE.{26,28}Y.{3}K.{106,127}?R, devised to detectUDP-GlcA decarboxylases, as described above. Each sequence on this list of candidateUDP-GlcA decarboxylases was then classified as closest to one of the three types ofbacterial enzymes described to have UDP-GlcA decarboxylase activity: Uxs1, ArnA, and

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RsU4kpxs. Each UDP-GlcA decarboxylase was further analyzed using a custom BLASTdatabase to determine whether it was more similar (based on the lowest E value) to theC-terminal UDP-GlcA decarboxylase domain of the ArnA protein (NCBI accession no.2BLL_A, GI:66361562) or to UDP-glucuronic acid decarboxylase 1 (Uxs1) from Homosapiens (NCBI accession no. NP_079352.2, GI:42516563). As ArnA proteins have aformyl transferase domain at the N terminus, the UDP-GlcA decarboxylases were alsoanalyzed with the Pfam database (version 25.0 of March 2011). If the protein returneda significant match to PF00551.13 (Formyl_trans_N) and/or PF02911.12 (Formyl-_trans_C)—two Pfam motifs found in the N-terminal formyl transferase domain ofArnA—it was assigned as an ArnA type of UDP-GlcA decarboxylase. As RsU4kpxs-type UDP-GlcA decarboxylase proteins are more similar to the C terminus of ArnAthan to that of Uxs1 but have the formyltransferase activity of ArnA encoded by anadjacent gene, the RsU4kpxs-type proteins were identified as being more similar toArnA than to Uxs1, lacking a formyltransferase domain, and as having an adjacent geneencoding a protein with a significant match to PF00551.13 (Formyl_trans_N) and/orPF02911.12 (Formyl_trans_C).

RESULTS

Identification of putative UDP-GlcA decarboxylases of B.fragilis. To determine whether B. fragilis may synthesize glycanscontaining xylose, we searched the B. fragilis NCTC 9343 ge-nome for genes encoding orthologs of the human UDP-glucu-ronic acid decarboxylase protein UXS1 (NCBI accession no.AY147934.1). Two B. fragilis orthologs encoded by BF2189and BF1555 (both 76% similar to the human ortholog and 87%similar to each other) were identified (Fig. 1). Each of theseproducts is annotated as an NAD-dependent epimerase/dehy-dratase, which is a general category applied to diverse productsinvolved in the synthesis and modification of nucleotide-linkedsugars. When the amino acid sequences of BF2189 and BF1555are used to query the RCSB Protein Data Bank (PDB) (34),

the closest structural match is human UDP-glucuronate decar-boxylase (PDB entry 2B69; alignment E value, 5.8 � 10�105).BF2189, which we have designated Bfuxs1, is conserved in theB. fragilis genome and is the first gene of a putative two-geneoperon. BF1555, designated Bfuxs2, is contained within thepolysaccharide F (PSF) capsular polysaccharide biosynthesislocus of strain NCTC 9343, which is a heterogeneous region ofthe B. fragilis genome.

The predicted secondary structures of BfUxs1, BfUxs2,and the human USX1 ortholog are remarkably similar (Fig.1). As SDR superfamily members, UDP-GlcA decarboxyl-ase enzymes conform to the structural characteristics of thissuperfamily. SABLE (1) predicts that each protein has analternating series of alpha helices and beta strands (Fig. 1)comprising the structural motif known as a Rossmann fold.The sequence motif GX(X)GXXG, involved in NAD� bind-ing, is also present (G8G11G14 in both BfUxs1 and BfUxs2),with the third glycine residue appropriately positioned inthe first helix (29). Eighteen residues after the glycine-richsequence pattern, after the second beta strand, there is aconserved aspartic acid residue (D32), which indicates thatthe enzyme utilizes NAD(H) as a nicotinamide cofactor(24). The conserved catalytic tetrad of N-[S/T]-Y-K (14, 18)is present as well (represented by N98T115Y144K146). Theseresidues catalyze the oxidation of the substrate to a 4-ketointermediate via hydride transfer between the substrate andthe nicotinamide cofactor, the hallmark reaction of all SDRenzymes (14).

Within the large SDR superfamily, there are features of

FIG. 1. Similarities of BfUxs1 (NCBI accession no. CAH07883) and BfUxs2 (accession no. CAH07260) with the human UDP-GlcA decar-boxylase UXS1 (accession no. AAN39844). BOXSHADE alignment of the three proteins with the relevant SDR superfamily residues arehighlighted with blue asterisks. Below the primary sequence are the secondary structures predicted by SABLE (1). Helical areas are indicated bya red cylindrical line, while beta sheet areas are indicated by a gold arrow.

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BfUxs1 and BfUxs2 that strongly suggest that they are UDP-GlcA decarboxylases. Analysis of the crystal structure of thehuman USX1 protein (PDB identification [ID] no. 2B69) isunpublished; however, two research groups have indepen-dently published analyses of the structure (PDB ID no. 1U9Jand 2BLL) of the C-terminal portion of E. coli ArnA (18, 42).The C-terminal portion of this bifunctional enzyme catalyzesthe NAD�-dependent oxidative decarboxylation of UDP-GlcA as part of a pathway leading to production of UDP-4-amino-4-deoxy-L-arabinose (7, 18, 42). Analysis of the crystalstructure of the C-terminal decarboxylase region of ArnA re-vealed that R619 and S433 are important for decarboxylaseactivity (7, 18, 42). In addition, site-directed mutations ofS433A, E434A, and E434Q of ArnA affected UDP-GlcA de-carboxylase activity. These three residues are represented byS116, E117, and R276 in BfUxs1 and BfUxs2 and are also con-served in the human USX1 decarboxylase, represented by S203,E204, and R361 (Fig. 2). Alignment of BfUxs1 and BfUxs2 withother functionally characterized UDP-GlcA decarboxylasesfrom diverse species shows that they all retain these Ser, Glu,and Arg residues, whereas other bacterial SDR enzymes withnondecarboxylase activity do not (Fig. 2).

Enzymatic characterization of BfUxs1 and BfUxs2. To de-termine whether BfUxs1 and BfUxs2 have UDP-GlcA decar-boxylase activity, we purified the proteins and performed en-zymatic analyses. The characterized His-tagged UDP-GlcAdecarboxylase of Cryptococcus neoformans (6) was included asa positive control, and the purifications of each of these en-zymes are shown in Fig. 3A. The conversion of UDP-GlcA toUDP-xylose was monitored by high-performance anion-ex-change chromatography. The products of both the BfUxs1 andBfUxs2 reactions eluted identically to the UDP-xylose controlat approximately 6.24 min (Fig. 3), easily distinguishable fromthe UDP-GlcA substrate, which elutes at approximately 14.5min. These peaks were also indistinguishable from that pro-

duced by the Cryptococcus neoformans USX1p UDP-GlcA de-carboxylase (6). To provide further evidence that BfUxs1 andBfUxs2 were catalyzing the conversion of UDP-GlcA to UDP-xylose, the reaction products were spiked with UDP-xyloseprior to analysis. For this analysis, there remains a single sharpUDP-xylose peak with no shouldering (Fig. 3H), providingfurther evidence that UDP-xylose is the reaction product.Arabinose is a five-carbon sugar that is an epimer of xylose andhas been detected in plant and bacterial glycans (8, 20). Theelution of UDP-arabinose at 5.43 min is easily distinguishedfrom that of UDP-xylose, demonstrating that this detectionmethod can differentiate these two closely related nucleotide-activated pentoses (Fig. 3I). Reactions performed under thesame conditions using protein purified in the same mannerfrom E. coli BL21(DE3) containing vector alone failed to pro-duce a UDP-xylose peak (Fig. 3D). No product was formedwhen UDP-galacturonic acid was substituted for UDP-GlcA asthe substrate (data not shown), demonstrating that these en-zymes are specific for UDP-GlcA.

Distribution of UDP-GlcA decarboxylase proteins. With theconfirmation that the BfUxs proteins have UDP-GlcA decar-boxylase activity, we sought to determine the prevalence ofUDP-GlcA decarboxylases within the domain Bacteria. First, aBLAST search was performed using the amino acid sequenceof BfUxs1 to query NCBI’s nonredundant database for similarproteins encoded by bacterial genomes with completed or draftgenomic sequences. This search returned 9,915 bacterial pro-teins with an E value of �10�3, virtually all of which containthe residues indicative of SDR superfamily enzymes. We thendevised a Perl regular expression to detect the pattern ofconserved residues of SDR superfamily members combinedwith those residues that have been shown by structural ormutagenesis analysis to be essential for UDP-GlcA decar-boxylase activity. The Perl regular expression used to querythe 9,915 proteins returned from the BLAST search is

FIG. 2. Conserved UDP-GlcA decarboxylase residues. All of these SDR enzymes were aligned together for the BOXSHADE analysis.Segments of the proteins corresponding to the conserved SDR and UDP-GlcA decarboxylase residues are highlighted with green (�) and pink (�),respectively. Black and gray shading indicates consensus residues conserved in more than half the sequences and residues considered similar tothe consensus residues, respectively. Dots represent gaps in the sequences displayed. The UDP-GlcA decarboxylase sequences shown areSinorhizobium meliloti 1021 SmUxs1.1 (NCBI accession no. NP_436980) (20), Arabidopsis thaliana AtUxs1 (accession no. NP_850694) (22),Cryptococcus neoformans Uxs1p (accession no. AF385328_1) (6), Homo sapiens UXS1 (accession no. AAN39844) (31), Ralstonia solanacearumGMI1000 RsU4kpxs (accession no. NP_519440.1) (19), and the C-terminal portion of Escherichia coli ArnA (accession no. NP_416758) (42).Nondecarboxylase SDR family enzymes are the UDP-GlcNAc 4,6-dehydratases UngD1 (accession no. YP_211344) and UngD2 (accession no.YP_212462) from B. fragilis NCTC 9343 (12, 13), WbpM from Pseudomonas aeruginosa PAO1 (accession no. AAC45867) (9), RmlB dTDP-glucose4,6-dehydratase from Streptococcus suis (accession no. CAD49092) (2), GalE UDP-galactose 4-epimerase from Escherichia coli (accession no.BAA35421) (38), and GDP-mannose-4,6-dehydratase (Gmd) (accession no. YP_211522) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (Fcl) (accession no. YP_211521) from B. fragilis NCTC 9343 (12, 13). The activities of all listed enzymes were determined experimentally.


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G..G..G.{17,27}(D�E).{76,84}TSE.{26,28}Y.{3}K.{106,127}?R, where ? signifies that only the first R in the range shouldbe considered. This analysis identified 826 potential bacterialUDP-GlcA decarboxylases (see Table S2 in the supplementalmaterial). Proteins of proteobacterial origin predominated, com-prising 70.3% of the list (581 proteins), likely due to their relativeoverrepresentation in the database. Of the 1,116 proteobacterialstrains with sequence information available, 467 encode one ormore proteins matching the decarboxylase motif.

These 826 predicted bacterial UDP-GlcA decarboxylaseswere then further categorized into the three types of bacterialproteins with this described activity, Uxs, ArnA, andRsU4kpxs. Of the 826 UDP-GlcA decarboxylase proteins, 518are the Uxs type, 227 are the ArnA type, and 81 are theRsU4kpxs type. With only two exceptions, the ArnA types arecontained exclusively in species within the Proteobacteria phy-lum. The ArnA types are encoded mostly by gammaproteo-bacteria, and 94 of the 108 queried E. coli genomes encode aprotein with the characteristics of the larger bifunctional ArnAtype, suggesting that these bacteria synthesize UDP-4-amino-4-deoxy-L-arabinose. No E. coli genomes analyzed encodedUxs or RsU4kpxs types. The RsU4kpxs types are encodedlargely by betaproteobacteria and are highly represented inBurkholderia species.

Although the Proteobacteria contained nearly all of theArnA- and RsU4kpxs-type UDP-GlcA decarboxylases, thereare also 276 proteins of the Uxs type encoded by proteobac-terial genomes. In addition to containing sequences of theRsU4kpxs type, 66 of the 69 Burkholderia species analyzed alsoencode a Uxs-type enzyme, suggesting that xylose is likelyconsistently present in a glycan(s) of this genus.

Other bacteria with a high prevalence of Uxs types ofUDP-GlcA decarboxylase enzymes include Chloroflexi (9/12genomes), the Chlamydiae/Verrucomicrobia group (11/14 ge-nomes), Cyanobacteria (50/60 genomes) Fibrobacteres/Acidobacteria (7/8 genomes), Planctomycetes (7/7 genomes),and Leptospira spp. (6/6 genomes). Table S2 in the supple-mental material lists the distribution of proteomes with pre-dicted UDP-GlcA decarboxylases within the different phylafor species with draft or completed genomes. Of note is therarity of these proteins in the phyla Firmicutes (7/799 ge-nomes) and Fusobacteria (0/25 genomes).

Of the 44 sequenced strains classified by NCBI as belongingto the genus Bacteroides (excluding B. pectinophilus, which 16SrRNA analysis suggests is a member of the Firmicutes phylum),41 synthesize at least one Uxs-type UDP-GlcA decarboxylase(Fig. 4). The closely related species B. plebeius, B. coprocola,

FIG. 3. Analysis of reaction substrates and products by high-per-formance anion-exchange chromatography. (A) Coomassie-stained gelof the purified enzymes used in the reactions or the vector control.Molecular masses of standards are shown to the left in kDa. BfUxs1and BfUxs2 are approximately 38.7 kDa, whereas Uxs1p is approxi-mately 48.7 kDa. (B) UDP-glucuronic acid standard. (C) UDP-xylosestandard. (D to J) Enzymatic reaction mixtures containing the follow-ing His-tagged enzymes or controls: pET16b (vector only) (D), BfUxs1(E), BfUxs2 (F), C. neoformans Uxs1p (G), BfUxs1 spiked with UDP-xylose (H), UDP-arabinose standard (I), and UDP-arabinose andUDP-glucuronic acid (J).


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and B. coprophilus are the three exceptions. In each of these 41strains, the orthologous protein is more similar to BfUxs1 thanto BfUxs2 and is present in the same genomic context asBfuxs1. With the exception of the Parabacteroides species,which—like the Bacteroides—are abundant colonic organisms,other species of the order Bacteroidales, many of which are oralorganisms, rarely encode this enzymatic function. Analysis ofthe prevalence of UDP-GlcA decarboxylases within the othertwo orders of Bacteroidetes demonstrates that these proteinsare encoded consistently by Sphingobacteriales (9/9 genomes)

but variably within the order Flavobacteroidales (14/26 ge-nomes).

Phenotypic analysis of Bfuxs mutants. As Uxs-type UDP-GlcA decarboxylases are predicted to be encoded by nearly allBacteroides species analyzed, xylose may be present in a con-served glycan of the genus. Bacteroides fragilis synthesizes eightcapsular polysaccharides and numerous glycoproteins. To de-termine whether synthesis of any of these molecules relies onthe production of UDP-xylose, we analyzed glycan productionby �Bfuxs1, �Bfuxs2, and �Bfuxs1 �Bfuxs2 mutants.

We previously showed that numerous proteins of differentmolecular sizes are glycosylated with the same glycans (16, 17),and these glycoproteins are present in all extracellular com-partments (17) of B. fragilis. As shown by the Western immu-noblot in Fig. 5A, an antiserum specific to the glycan portion ofthe B. fragilis glycoproteins binds numerous glycoproteins pro-duced by all three mutants. There are a few low-molecular-weight molecules that appear to migrate differently in the dou-ble mutant; however, the deletion of Bfuxs1 and Bfuxs2 doesnot result in a general protein glycosylation defect or an alter-ation in the glycan that is added to proteins. This is alsoillustrated in Fig. 5B, where the size of a single glycoprotein,BF2494, is shown to be the same from both the wild-type and

FIG. 4. 16S rRNA gene cladogram of Bacteroidales strains. Strainswith a protein that matches the UDP-GlcA decarboxylase motif arehighlighted in blue. Genus abbreviations are as follows: Pre., Pre-votella; Azo., Azobacteroides; Pal., Paludibacter; Ali., Alistipes; Por.,Porphyromonas; Par., Parabacteroides; and B., Bacteroides.

FIG. 5. Western immunoblot analysis of �Bfuxs1, �Bfuxs2, and�Bfuxs1 �Bfuxs2 phenotypes. Whole-cell lysates of the indicatedstrains were probed with an antiserum specific to the glycan portion ofthe B. fragilis glycoproteins (A), an antiserum specific to the proteinportion of glycoprotein BF2494 (B), an adsorbed antiserum specific toPSF (C), and an adsorbed antiserum specific to immunogenic mole-cules produced by the wild type (WT) but not by the �Bfuxs1 �Bfuxs2mutant (D). Arrows in panel A indicate molecules with apparentdifferences in the double mutant.


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mutant strains. We previously demonstrated that alteration ofthe glycan results in a size reduction of every glycoprotein (17),a shift not evident in these mutants.

Analysis of the eight capsular polysaccharides revealed thatall but one (PSF) were synthesized, and the defect in synthesisof this molecule was observed only in the Bfuxs double mutant(Fig. 5C and data not shown). These data demonstrate thatboth Bfuxs1 and Bfuxs2 are expressed under the in vitro con-ditions used and that they are functional paralogs, as expectedbased on the analysis of their enzymatic activity (Fig. 3). Todetermine whether there is an additional xylose-containingglycan synthesized by B. fragilis that we not did detect withthese antisera, we made an adsorbed serum. A polyclonal an-tiserum generated to B. fragilis NCTC 9343 whole cells wasadsorbed with the �Bfuxs1 �Bfuxs2 mutant to remove antibod-ies to those molecules common to the wild type and mutant.Therefore, this adsorbed serum will contain antibodies to thosemolecules synthesized by the wild type but not by the �Bfuxs1�Bfuxs2 mutant. The only apparent difference in the reactivityof this serum between the two strains is to that of a high-molecular-weight polysaccharide consistent with the migrationof PSF (Fig. 5D).

DISCUSSION

Very few studies have shown xylose to be a component ofbacterial glycans. As there are currently no lectins commer-cially available that recognized xylose, its detection relies onglycan purification and compositional analysis. Most bacterialglycans that have been compositionally or structurally analyzedare those of pathogens which—with a few exceptions—do notcontain xylose. Identification of species that may synthesizexylose-containing glycans is further hindered by the incorrectannotation of the non-ArnA class of UDP-GlcA decarboxy-lases. These enzymes are frequently annotated as NAD-depen-dent epimerases/dehydratases.

Based on enzymatic analyses demonstrating that B. fragilisNCTC 9343 synthesizes two UDP-GlcA decarboxylases, wedevised methods to detect each of the three types of UDP-GlcA decarboxylases encoded in the genomes of other bacte-ria. To do this, we first began by finding all proteins similar toBfUxs1 and then used a Perl regular expression to identifythose SDR enzymes with a residue pattern characteristic ofUDP-GlcA decarboxylases. Each of the three types of UDP-GlcA decarboxylases was then differentiated by comparing theBLAST E values of each to those of known ArnA and Uxsprototypes and by the presence or absence of domains indic-ative of formyl transferase activity. The list of bacteria encod-ing proteins similar to BfUxs1 and containing the UDP-GlcAdecarboxylases pattern described in this study includes diversespecies such as Helicobacter hepaticus, Burkholderia spp., vari-ous soil and aquatic bacteria, and cyanobacteria, to name afew. The likely success of identifying true UDP-GlcA decar-boxylases using this pattern search is evidenced by our findingsin Leptospira species. All six of the Leptospira strains withgenomic sequences have Uxs types of UDP-GlcA decarboxy-lases, and the LPS of Leptospira species has previously beenshown to contain xylose (23, 35).

Uxs-type UDP-GlcA decarboxylases are relatively well con-served in the genus Bacteroides, with nearly all species encod-

ing at least one UDP-GlcA decarboxylase. Each of these Bac-teroides proteins is very similar to BfUxs1 and less so toBfUxs2. The genetic regions containing the Bfuxs1 orthologshave similar architecture, with Bfuxs1 being the first gene in atwo-gene operon in all Bacteroides species analyzed. The down-stream gene encodes a predicted hybrid two-component histi-dine sensor kinase, which is 85 to 94% similar to those ofdifferent Bacteroides species. Only two other B. fragilis strains,2_1_16 and 3_2_5, have two proteins that match the UDP-GlcA decarboxylase pattern. Analysis of the two proteins fromboth of these strains shows that one of each set is 99% identicalto BfUxs1, and the other is 99% identical to BfUxs2. As in B.fragilis NCTC 9343, the Bfuxs2 orthologs of these strains are incapsular polysaccharide loci that are closely related at theDNA level to the B. fragilis NCTC 9343 PSF locus. Thus, it isquite likely that both Bacteroides sp. 2_1_16 and Bacteroides sp.3_2_5 produce a capsular polysaccharide related or identical toPSF. Bfuxs2 orthologs were likely acquired by horizontal DNAtransfer, whereas the Bfuxs1 orthologs are likely endogenous tothe genus.

The question remains as to what molecule(s) utilizes theUDP-xylose synthesized by Bacteroides BfUxs1 orthologs orwhether the UDP-xylose is further processed by an enzymeencoded by an unlinked gene. The only molecule clearly shownto be impacted by the loss of Bfuxs1 and Bfuxs2 is PSF. As thePSF region is heterogeneous, and most Bacteroides speciescontain only a Bfuxs1 ortholog, the UDP-xylose produced bythese bacteria likely serves another purpose. Analysis of pre-vious transcriptional profiling of in vitro-grown bacteria (12)reveals that Bfuxs1 is actively transcribed in vitro, further con-firmed by its ability to provide UDP-xylose for PSF synthesis inthe Bfuxs2 mutant (see above). Transcriptional profiling assaysof the related species Bacteroides thetaiotaomicron VPI 5482grown in culture or isolated from the cecum of mice (36)(GEO data set record GDS1849) demonstrate that the Bfuxs1ortholog BT_1059 (NCBI accession no. AAO76166.1 [43]) isexpressed at high levels under both conditions, making it un-likely that the predicted conserved xylose-containing moleculewas not detected due to lack of expression. Another possibilityis that this molecule is not immunogenic. The phenotypicanalyses performed in this study depended on this moleculeinducing an antibody response in rabbits for detection.Clearly, further studies are needed to assess the importanceof UDP-xylose synthesis within Bacteroides species. Based onthe expression profile of these genes in vivo and their wide-spread occurrence throughout the genus, xylose-containingglycans are likely an important component of the bacterialsurface and contribute to their success in the intestinal envi-ronment.

ACKNOWLEDGMENTS

We thank Tamara Doering for providing pDC22.6.This work was funded by NIH/NIAID grant AI067711.

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