Metabolism of Sialic Acid by Bifidobacterium breve UCC2003

Muireann Egan,a Mary O’Connell Motherway,a Marco Ventura,b Douwe van Sinderena

School of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Irelanda; Laboratory of Probiogenomics, Department of Life Sciences,University of Parma, Parma, Italyb

Bifidobacteria constitute a specific group of commensal bacteria that inhabit the gastrointestinal tracts of humans and othermammals. Bifidobacterium breve UCC2003 has previously been shown to utilize several plant-derived carbohydrates that in-clude cellodextrins, starch, and galactan. In the present study, we investigated the ability of this strain to utilize the mucin- andhuman milk oligosaccharide (HMO)-derived carbohydrate sialic acid. Using a combination of transcriptomic and functionalgenomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demon-strate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3=-sialyllactose, an abundant HMO, byanother infant gut bifidobacterial strain, Bifidobacterium bifidum PRL2010.

Bifidobacteria are Gram-positive, anaerobic, typically Y-shapedbacteria that are members of the Bifidobacteriaceae family and

the Actinobacteria phylum. Discovered in 1900 (1), bifidobacteriaare naturally found in the digestive tracts of mammals and insectsbut are also isolated from the human oral cavity and sewage (2).The large intestine is the natural habitat for a large and diversebacterial community, which is known to contribute significantlyto the well-being of humans. This positive contribution includesmetabolic activities to provide energy and nutrients to the host (3,4), development of the immune system (5, 6), and protectionagainst pathogenic bacteria (7).

Bifidobacteria are saccharolytic organisms, and their survivaland growth in the large intestine require a variety of extracellularand cytoplasmic glycosyl hydrolases, which they employ to me-tabolize carbohydrates prevalent in this environment. The char-acteristic central metabolic pathway in bifidobacteria is the fruc-tose-6-phosphate phosphoketolase pathway, also known as thebifid shunt (8). Because of the diversity of carbohydrate sourcespresent in the gastrointestinal tract and the dependence of bifido-bacteria on such carbon and energy sources, it is not surprisingthat an estimated 8% of the typical bifidobacterial genome is de-voted to carbohydrate metabolism (2, 9).

Bifidobacterium breve UCC2003 has already been shown to uti-lize quite a variety of plant-derived poly-, oligo-, and monosac-charides, including cellodextrins, galactan, starch, raffinose, andmelezitose (10–15), which reflects the apparent flexibility of thecarbohydrate utilization profile of this strain. Interestingly, B.breve strains are numerically prevalent in the gut microbiota ofhealthy, breast-fed infants (16), although the ability of membersof this bifidobacterial species to utilize human milk and host-derived carbohydrates is relatively unexplored (see below).

Sialic acids comprise a large family of nine-carbon monosac-charides called neuraminic acids, the most common of which isN-acetylneuraminic acid (Neu5Ac), for which the name of sialicacid is also used. Approximately 16% of human milk oligosaccha-rides (HMOs) are sialylated (17), with 3=-sialyllactose and 6=-sia-lyllactose being abundant sialylated HMO components (18–20).The total sialic acid concentrations are highest in colostrum,reaching levels of 5.04 mmol liter�1, and they subsequently de-crease by almost 80% after 3 months (21). Sialic acid also featuresprominently at the surface-exposed end of human colonic mucin(22), and in healthy adults, approximately 300 �g of sialic acid/mg

of colonic mucin is present (23). The concentration of sialic acidin gastric juice has been found to decrease with age, with levels of100 �g ml�1 in young adults �25 years old compared to less than20 �g ml�1 in elderly people (24).

Representative Clostridium perfringens strains were the firstbacteria shown to be capable of utilizing sialic acid as the solecarbon source (25, 26). Nevertheless, sialic acid metabolism wasfirst explored in 1983 in Escherichia coli, with the identification ofa transporter, designated nanT, and an N-acetylneuraminatelyase, designated nanA (27, 28), followed by the description of thecomplete, five-step metabolic pathway in 1999 (Fig. 1) (29). Othergut inhabitants, including the pathogen Vibrio cholerae (30) andthe commensals Bacteroides fragilis and Lactobacillus sakei (31,32), have since been shown to utilize sialic acid as a carbon source.

The ability of Bifidobacterium species to metabolize sialic acidhas not been widely studied. Genes predicted to be involved in thebreakdown of sialic acid, which correspond to those outlined inFig. 1, were identified in the genome of Bifidobacterium longumsubsp. infantis ATCC 15697, and in accordance, this strain wasshown to be capable of utilizing sialic acid (33) or the HMO sia-lylated lacto-N-tetraose as a sole carbon source (34). A recentstudy of the consumption of HMOs by strains of B. breve revealedthat all of those tested can utilize sialylated lacto-N-tetraose (35).Another study examined the utilization of the monosaccharideconstituents of HMO by five bifidobacterial strains representingfour bifidobacterial species (B. longum, Bifidobacterium bifidum,B. breve, and Bifidobacterium adolescentis) and found that of thestrains examined, only B. longum subsp. infantis ATCC 15697 andB. breve ATCC 15700 are capable of utilizing sialic acid (36). Thissuggests that the ability to utilize sialic acid and/or sialylatedHMOs is a species- or strain-specific attribute.

Received 3 April 2014 Accepted 4 May 2014

Published ahead of print 9 May 2014

Editor: G. T. Macfarlane

Address correspondence to Douwe van Sinderen, d.vansinderen@ucc.ie.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01114-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01114-14

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The aim of this study was to investigate how B. breve UCC2003utilizes sialic acid, a characteristic that is likely to support theability of particular bifidobacterial species to colonize the infantgut. Furthermore, the manner in which B. breve UCC2003 cancross feed on sialic acid derived from the metabolism of 3=-sia-lyllactose by B. bifidum PRL2010 was characterized.

MATERIALS AND METHODSBacterial strains, plasmids, media, and culture conditions. The bacterialstrains and plasmids used in this study are listed in Table 1. B. breveUCC2003 was routinely cultured in reinforced clostridial medium (OxoidLtd., Basingstoke, Hampshire, United Kingdom). B. bifidum PRL2010 wascultured in modified de Man, Rogosa, and Sharpe (mMRS) mediummade from first principles (37) but excluding a carbohydrate source andsupplemented with 0.05% (wt/vol) L-cysteine HCl and 1% (wt/vol) lac-tose (unless otherwise specified). Carbohydrate utilization by bifidobac-teria was examined in mMRS medium supplemented with 0.05% (wt/vol)L-cysteine HCl and a particular carbohydrate source (0.5%, wt/vol). It waspreviously shown that without the addition of a carbohydrate, mMRSmedium does not support the growth of B. breve (38). The carbohydratesused were lactose (Sigma-Aldrich, Steinheim, Germany), sialic acid, and3=- and 6=-sialyllactose (Carbosynth, Compton, Berkshire, United King-dom). A 0.5% (wt/vol) concentration of carbohydrate was consideredsufficient to analyze the growth capabilities of a strain on a particularcarbon source. Addition of sialic acid at this concentration to mMRSmedium resulted in a decrease in the pH to 6.0; hence, the pH was read-justed to 6.8 following the addition of the sugar and the medium was

subsequently filter sterilized. Bifidobacterial cultures were incubated un-der anaerobic conditions in a modular, atmosphere-controlled system(Davidson and Hardy, Belfast, Ireland) at 37°C. E. coli was cultured inLuria-Bertani (LB) broth at 37°C with agitation (39). Lactococcus lactisstrains were grown in M17 medium supplemented with 0.5% (wt/vol)glucose at 30°C (40). Where appropriate, the growth medium used con-tained tetracycline (Tet; 10 �g ml�1), chloramphenicol (Cm; 5 �g ml�1

for L. lactis and E. coli, 2.5 �g ml�1 for B. breve), erythromycin (Em; 100�g ml�1), or kanamycin (Kan; 50 �g ml�1). Recombinant E. coli cellscontaining pORI19 were selected on LB agar containing Em and Kan andsupplemented with X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopy-ranoside; 40 �g ml�1) and 1 mM IPTG (isopropyl-�-D-thiogalactopyra-noside).

In order to determine bacterial growth profiles and final optical den-sities (ODs), 10 ml of freshly prepared mMRS medium including a par-ticular carbohydrate source (see above) was inoculated with 100 �l (1%)of a stationary-phase culture of a particular strain. Uninoculated mMRSmedium was used as a negative control. Cultures were incubated anaero-bically for 24 to 36 h. Measurements of OD at 600 nm (OD600) wererecorded unless otherwise stated.

Nucleotide sequence analysis. Sequence data were obtained from theArtemis-mediated genome annotations of B. breve UCC2003 (41, 42).Database searches were performed by using the nonredundant sequencedatabase accessible at the National Center for Biotechnology Informationwebsite (http://www.ncbi.nlm.nih.gov) with BLAST (43). Sequence anal-ysis was performed by using the Seqbuilder and Seqman programs of theDNASTAR software package (DNASTAR, Madison, WI).

DNA manipulations. Chromosomal DNA was isolated from B. breveUCC2003 as previously described (44). Plasmid DNA was isolated from E.coli, L. lactis, and B. breve with the Roche High Pure plasmid isolation kit(Roche Diagnostics, Basel, Switzerland). An initial lysis step was per-formed with 30 mg ml�1 of lysozyme for 30 min at 37°C prior to plasmidisolation from L. lactis or B. breve. The single-stranded oligonucleotideprimers used in this study were synthesized by Eurofins (Ebersberg, Ger-many) (see Table S1 in the supplemental material). Standard PCRs wereperformed with Taq master mix (Qiagen GmbH, Hilden, Germany). B.breve colony PCRs were carried out as described previously (45). PCRfragments were purified with the Roche High Pure PCR purification kit(Roche Diagnostics). Electroporation of plasmid DNA into E. coli, L. lac-tis, or B. breve was performed as previously described (39, 46, 47).

Analysis of global gene expression with B. breve DNA microarrays.Global gene expression was determined during log-phase growth of B.breve UCC2003 in mMRS medium supplemented with 0.5% sialic acid.The transcriptome obtained was compared to that obtained from log-phase B. breve UCC2003 cells grown in mMRS medium supplementedwith 0.5% ribose. DNA microarrays containing oligonucleotide primersrepresenting each of the 1,864 open reading frames identified in the ge-nome of B. breve UCC2003 were designed and obtained from AgilentTechnologies (Palo Alto, CA). Cell disruption, RNA isolation, RNA qual-ity control, and cDNA synthesis and labeling were performed as describedpreviously (48). Labeled cDNA was hybridized with the Agilent GeneExpression hybridization kit (part no. 5188-5242) as described in the Agi-lent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual(publication no. G4140-90050). Following hybridization, microarrayswere washed in accordance with Agilent’s standard procedures andscanned with an Agilent DNA microarray scanner (model G2565A). Thescans generated were converted to data files with Agilent’s Feature Extrac-tion software (version 9.5). DNA microarray data were processed as pre-viously described (49–51). Differential-expression tests were performedwith the Cyber-T implementation of a variant of the t test (52).

Construction of B. breve UCC2003 insertion mutants. An internalfragment of Bbr_0161 (designated nanK here; 370 bp, representingcodons 148 through to 271 of the 336 codons of this gene), Bbr_0164(designated nanB; 504 bp, representing codons 226 through to 394 of the521 codons of this gene), Bbr_0165 (designated nanC; 355 bp, represent-

FIG 1 Metabolism of sialic acid by E. coli as previously described (29). Sialicacid enters the cell through a transporter encoded by the nanT gene. Intracel-lular sialic acid is cleaved by the nanA-encoded N-acetylneuraminate lyase,producing pyruvate and N-acetylmannosamine. N-Acetylmannosamine isphosphorylated by the nanK-encoded N-acetylmannosamine kinase, formingN-acetylmannosamine-6-phosphate, which is in turn converted to N-acetylg-lucosamine-6-phosphate by the nanE-encoded N-acetylmannosamine-6-phosphate epimerase. N-Acetylglucosamine-6-phosphate then enters theamino sugar degradation pathway, where this compound is first deacetylatedby nagA-encoded N-acetylglucosamine-6-phosphate deacetylase and subse-quently converted to fructose-6-phosphate with the concomitant release ofammonia by the nagB-encoded glucosamine-6-phosphate deaminase.

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TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmid Relevant feature(s) Reference or sourcea

E. coli strainsXL1-blue supE44 hsdR17 recA1 gyrA96 thi relA1 lac F= [proAB� laclq lacZ�M15 Tn10(Tetr)] StratageneXL1-blue/pBC1.2-nanA XL1-blue containing pBC1.2-nanA This studyEC101 Cloning host; repA� kmr 82EC101/pNZ-M.BbrII�M.BbrIII EC101 harboring pNZ8048 derivative containing bbrIIM and bbrIIIM 45

L. lactis strainsNZ9000 MG1363, pepN::nisRK, nisin-inducible overexpression host 56NZ9000/pNZ-nanA NZ9000 containing pNZ-nanA This studyNZ9000/pNZ-nanK NZ9000 containing pNZ-nanK This studyNZ9000/pNZ-nanE NZ9000 containing pNZ-nanE This studyNZ9000/pNZ-nagA2 NZ9000 containing pNZ-nagA2 This studyNZ9000/pNZ-nagB1 NZ9000 containing pNZ-nagB1 This studyNZ9000/pNZ44-nanA NZ9000 containing pNZ44-nanA This study

B. breve strainsUCC2003 Isolate from nursling stool sample 47UCC2003-nanA pORI19-tet-nanA insertion mutant of UCC2003 This studyUCC2003-nanK pORI19-tet-nanK insertion mutant of UCC2003 This studyUCC2003-nanB pORI19-tet-nanB insertion mutant of UCC2003 This studyUCC2003-nanC pORI19-tet-nanC insertion mutant of UCC2003 This studyUCC2003-nagA2 pORI19-tet-nagA2 insertion mutant of UCC2003 This studyUCC2003-nanA/pBC1.2-nanA pORI19-tet-nanA insertion mutant of UCC2003 harboring complementation

construct pBC1.2-nanAThis study

JCM7017 Isolate from human feces JCMJCM7019 Isolate from adult feces JCMNCFB2257 Isolate from infant intestine NCFBNCFB2258 Isolate from infant intestine NCFBNCIMB8815 Isolate from human feces NCIMBNIZO658 Isolate from human feces NIZOLMG13208 Isolate from infant intestine LMGUCC2007 Isolate from nursling stool sample UCCUCC2005 Isolate from infant intestine UCCNCTC11815 Isolate from infant intestine NCTC461B Isolate from infant and adult feces PRL689B Isolate from infant feces 6212L Mother’s milk 62

B. bifidum PRL2010 Isolate from infant feces 64

PlasmidspAM5 pBC1-puC19-Tcr 54pNZ44 pNZ8048 containing constitutive p44 promoter from lactococcal chromosome 53pNZ44-nanA pNZ44 harboring nanA downstream of p44 promoter This studypBC1.2 pBC1-pSC101-Cmr 54pBC1.2-nanA pBC1-pSC101-Cmr harboring nanA downstream of p44 promoter This studypORI19 Emr, RepA�, ori�, cloning vector 82pORI19-tet-nanA Internal 301-bp fragment of nanA and tetW cloned in pORI19 This studypORI19-tet-nank Internal 370-bp fragment of nanK and tetW cloned in pORI19 This studypORI19-tet-nanB Internal 504-bp fragment of nanB and tetW cloned in pORI19 This studypORI19-tet-nanC Internal 355-bp fragment of nanC and tetW cloned in pORI19 This studypORI19-tet-nagA2 Internal 402-bp fragment of nagA2 and tetW cloned in pORI19 This studypNZ8150 Cmr, nisin-inducible translational fusion vector 55pNZ-nanA Cmr, pNZ8150 derivative containing translational fusion of nanA-containing

DNA fragment to nisin-inducible promoterThis study

pNZ-nanK Cmr, pNZ8150 derivative containing translational fusion of nanK-containingDNA fragment to nisin-inducible promoter

This study

pNZ-nanE Cmr, pNZ8150 derivative containing translational fusion of nanE-containingDNA fragment to nisin-inducible promoter

This study

pNZ-nagA2 Cmr, pNZ8150 derivative containing translational fusion of nagA2-containingDNA fragment to nisin-inducible promoter

This study

pNZ-nagB1 Cmr, pNZ8150 derivative containing translational fusion of nagB1-containingDNA fragment to nisin-inducible promoter

This study

a JCM, Japanese Collection of Microorganisms; NCFB, National Collection of Food Bacteria; NCIMB, National Collection of Industrial and Marine Bacteria; NIZO, NetherlandsInstitute for Dairy Research; LMG, Belgian Coordinated Collection of Microorganisms; UCC, University College Cork; NCTC, National Collection of Type Cultures; PRL, CultureCollection of Probiogenomics, University of Parma.

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ing codons 27 through to 145 of the 319 codons of this gene), Bbr_0168(designated nanA; 301 bp, representing codons 116 through to 216 of the321 codons of this gene), and Bbr_1247 (designated nagA2; 402 bp, rep-resenting codons 107 through to 241 of the 426 codons of this gene) wereamplified by PCR with B. breve UCC2003 chromosomal DNA as the tem-plate and primer pairs NanKF and NanKR, NanBF and NanBR, NanCFand NanCR, NanAF and NanAR, and NagA2F and NagA2R, respectively.The insertion mutants were constructed as described previously (45).Site-specific recombination of potential Tetr mutant isolates was con-firmed by colony PCR with primers TetWF and TetWR to verify tetW geneintegration and primers NanKconfirm, NanBconfirm, NanCconfirm,NanAconfirm, and NagA2confirm (positioned upstream of the selectedinternal fragments of nanK, nanB, nanC, nanA, and nagA2, respectively)in combination with primer TetWF to confirm integration at the correctchromosomal location.

Complementation of B. breve UCC2003-nanA. A DNA fragment en-compassing nanA was generated by PCR amplification from B. breveUCC2003 chromosomal DNA with Pfu II DNA polymerase (Agilent,Cork, Ireland) and primers NanApNZ44F and NanApNZ44R. The result-ing fragment was digested with PstI and XbaI and ligated to similarlydigested pNZ44 (53). The ligation mixture was introduced into L. lactisNZ9000 by electrotransformation, and transformants were selected onthe basis of Cm resistance. The plasmid contents of a number of Cmr

transformants were screened by restriction analysis. The integrity of thecloned insert of one of the recombinant plasmids, designated pNZ44-nanA, was confirmed by sequencing. The nanA coding sequence, togetherwith the constitutive p44 lactococcal promoter, specified by pNZ44, wasamplified by PCR from a representative pNZ44-nanA plasmid with Pfu IIDNA polymerase and primers P44FORWARD and NanApNZ44R. Theresulting DNA fragment was digested with BamHI and XbaI and ligated tosimilarly digested pBC1.2 (54). The ligation mixture was introduced intoE. coli XL1-Blue by electrotransformation, and transformants were se-lected on the basis of Tet and Cm resistance. Transformants were checkedfor plasmid content by colony PCR and restriction analysis of plasmidDNA. Positively identified clones were verified by sequencing, and one ofthe plasmids thus identified was designated pBC1.2-nanA. PlasmidpBC1.2-nanA was introduced into B. breve UCC2003-nanA by electro-transformation, and transformants were selected on the basis of Tet andCm resistance.

Plasmid constructions. For the construction of plasmids pNZ-nanK,pNZ-nanE, pNZ-nanA, pNZ-nagB1, and pNZ-nagA2, DNA fragmentsencompassing the predicted N-acetylmannosamine kinase-encodinggene nanK (Bbr_0161), N-acetylmannosamine-6-phosphate epimerase-encoding gene nanE (Bbr_0162), N-acetylneuraminate lyase-encodinggene nanA (Bbr_0168), glucosamine-6-phosphate deaminase-encodinggene nagB1 (Bbr_0169), and N-acetylglucosamine-6-phosphate deacety-lase-encoding gene nagA2 (Bbr_1247) were generated by PCR amplifica-tion of chromosomal DNA of B. breve UCC2003 with Pfu II DNA poly-merase and primers nanKFOR and nanKREV, nanEFOR and nanEREV,nanAFOR and nanAREV, nagB1FOR and nagB1REV, and nagA2FORand nagA2REV, respectively. An in-frame His10-encoding sequence wasincorporated into forward primers nanKFOR, nanEFOR, and nagA2FORand reverse primers nanAREV and nagB1REV to facilitate downstreamprotein purification. The amplicons generated were digested with EcoRVand XbaI and ligated into ScaI- and XbaI-digested, nisin-inducible trans-lational fusion plasmid pNZ8150 (55). The ligation mixtures were intro-duced into L. lactis NZ9000 by electrotransformation, and transformantswere then selected on the basis of Cm resistance. The plasmid contents ofa number of Cmr transformants were screened by restriction analysis, andthe integrity of positively identified clones was verified by sequencing.

Protein overproduction and purification. Nisin-inducible gene ex-pression and protein overproduction were performed as described previ-ously (10, 12, 13). In brief, 400 ml of M17 broth supplemented with 0.5%(wt/vol) glucose was inoculated with a 2% inoculum of a particular L.lactis strain, followed by incubation at 30°C until an OD600 of 0.5 was

reached, at which point protein expression was induced by the addition ofcell-free supernatant of a nisin-producing strain (56), followed by contin-ued incubation for a further 2 h. Cells were harvested by centrifugation,and protein purification was achieved with the PrepEase histidine-taggedprotein purification maxikit (USB, Germany) according to the manufac-turer’s instructions. Elution fractions were analyzed by SDS-polyacryl-amide gel electrophoresis. After electrophoresis, the gels were fixed andstained with Coomassie brilliant blue to identify fractions containing thepurified protein. The molecular weights of the proteins were estimated bycomparison with rainbow-prestained, low-molecular-weight proteinmarkers (New England BioLabs, Herefordshire, United Kingdom). Pro-tein concentrations were determined by the Bradford method (57).

HPTLC. High-performance thin-layer chromatography (HPTLC)analysis was used for qualitative determination of the activity of each ofthe purified enzymes. Purified NanAHis, NanKHis, NanEHis, NagA2His, orNagB1His (final concentration, 20 �g ml�1) or a combination thereofwas incubated with sialic acid, N-acetylmannosamine, N-acetylman-nosamine-6-phosphate, N-acetylglucosamine-6-phosphate, or glucosa-mine-6-phosphate, respectively. Enzymatic activity assays were per-formed at 37°C in a total volume of 1 ml with a reaction buffer containing20 mM Tris-HCl (pH 8.0), 10 mM KCl, and 5 mM MgSO4 · 7H2O alongwith a 5-mg ml�1 concentration of a particular carbohydrate substrate.This concentration of carbohydrate was considered sufficient for qualita-tive analysis. In reaction mixtures including NanKHis, 100 �l of 100 mMATP was added. The reactions were terminated after 24 h by incubation at65°C for 10 min. HPTLC analysis was performed as previously described(10). An aliquot (1 �l) of the reaction mixture was spotted onto a SilicaGel 60 plate (10 by 10 cm; Merck) with a Nanomat 4 (Camag, Switzer-land). The chromatogram was developed with a butanol-isopropanol-water (3:12:4, vol/vol/vol) solvent system in a horizontal developingchamber. Ascending development was repeated twice at room tempera-ture. The plate was allowed to dry in a fume hood and then sprayed evenlywith 20% (vol/vol) sulfuric acid in ethanol. The plate was dried and heatedto 120°C for 10 min to visualize sugar-representing spots. Reaction prod-ucts were identified by comparison with relevant carbohydrate standards.

Growth of B. bifidum PRL2010 and B. breve UCC2003 or UCC2003-nanA on 3=-sialyllactose. An overnight culture of B. bifidum PRL2010(1%) was used to inoculate mMRS broth supplemented with 0.05% (wt/vol) L-cysteine HCl and 0.5% (wt/vol) 3=-sialyllactose and cultivated for24 h at 37°C under anaerobic conditions. The cells were removed bycentrifugation at 9,000 � g for 5 min. Growth of B. bifidum PRL2010resulted in a pH decrease to 5.1; therefore, the pH of the cell-free super-natant (CFS) was readjusted to 6.8 and the CFS was filter sterilized. Thismedium was supplemented with 0.01% (wt/vol) lactose and 0.05% (wt/vol) L-cysteine HCl prior to inoculation with an overnight culture (0.01%)of B. breve UCC2003 or UCC2003-nanA. Growth was monitored for 72 h,with samples taken at 6- or 12-h intervals. All of the samples collected wereserially diluted in sterile Ringer solution and plated on reinforced clos-tridial agar. Viable counts were determined by counting colonies on agarplates with dilutions that yielded between 30 and 300 CFU.

HPAEC-PAD. Carbohydrate analysis of CFS samples taken at 6- or12-h intervals during the 72-h growth experiment was performed by high-performance anion-exchange chromatography with pulsed amperomet-ric detection (HPAEC-PAD) with a Dionex ICS-3000 system (Dionex,Sunnyvale, CA). Oligosaccharide fractions (25-�l aliquots) were sepa-rated on a CarboPac PA1 analytical-exchange column with dimensions of250 by 4 mm (Dionex) with a CarboPac PA1 guard column with dimen-sions of 50 by 4 mm (Dionex) and a pulsed electrochemical detector in thePAD mode. Elution was performed at a constant flow rate of 1.0 ml min�1

at 30°C with the following eluents for the analysis: 200 mM NaOH (A),100 mM NaOH–550 mM sodium acetate (NaAC) (B), and Milli-Q water(C). The following linear gradient of NaAC with 100 mM NaOH was used:0 to 50 min, 0 mM; 50 to 51 min, 16 mM; 51 to 56 min, 100 mM; 56.1 to61 min, 0 mM. Detection was achieved with a Dionex ED40 detector in thePAD mode. The Chromeleon software version 6.70 (Dionex Corpora-

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tion) was used for the integration and evaluation of the chromato-grams obtained. The chromatographic profiles corresponding to 3=-sialyllactose, sialic acid, and lactose were qualitatively examined toevaluate 3=-sialyllactose hydrolysis and subsequent metabolism of lac-tose and/or sialic acid.

Microarray data accession number. The microarray data obtained inthis study have been deposited in the NCBI Gene Expression Omnibusdatabase under GEO series accession no. GSE56291.

RESULTSGrowth of B. breve strains on sialic acid. In order to determine ifB. breve strains are capable of sialic acid metabolism, the growth of14 B. breve strains in mMRS medium supplemented with 0.5%(wt/vol) lactose or sialic acid was assessed by measuring the OD600

following 24 h of anaerobic growth at 37°C. All of the B. brevestrains were shown to grow well on lactose (OD600 of 1.2) (re-sults not shown). Eleven of the 14 B. breve strains examined, in-cluding prototypical strain UCC2003, were capable of moderategrowth on sialic acid, reaching OD600s of 0.6 to 0.9, while the threeremaining strains were shown to be unable to metabolize sialicacid (Fig. 2).

Genome response of B. breve UCC2003 to growth on sialicacid. In order to investigate which genes are involved in sialic acidmetabolism by B. breve UCC2003, global gene expression was de-termined by microarray analysis during growth of the strain inmMRS medium supplemented with sialic acid and compared withgene expression when it was grown in mMRS medium supple-mented with ribose. The pentose sugar ribose was considered anappropriate carbohydrate for comparative transcriptome analy-sis, as the genes involved in ribose metabolism are known, while ithas previously been used in several transcriptome studies of B.breve UCC2003 (10, 11, 13). Analysis of the DNA microarray datarevealed that the expression of a gene cluster comprising 12 openreading frames corresponding to locus tags Bbr_0160 to Bbr_0172was significantly upregulated when B. breve UCC2003 was grownon sialic acid relative to when the strain was grown on ribose(3.0-fold change, P � 0.001). In addition, two adjacent genes,corresponding to Bbr_1247 (nagA2) and Bbr_1248 (nagB3), werealso upregulated (see below; Table 2).

Genetic organization of the predicted nan-nag gene clusters.Our presumption, based on the microarray results, was that theproducts encoded by the Bbr_0160-to-Bbr_0172 gene cluster andthose of Bbr_1247 and Bbr_1248, schematically outlined in Fig. 3,are involved in the metabolism of sialic acid in B. breve UCC2003.The first gene of this upregulated cluster, Bbr_0160, encodes ahypothetical protein of unknown function. Bbr_0161, designatednanK here, encodes a putative N-acetylmannosamine kinase thatis predicted to be a member of the RhoA-binding kinase (ROK)protein family. The members of this family include transcriptionalregulators and sugar kinases (58). Predicted ROK kinases possessa conserved N-terminal ATP-binding motif, DxGxT, that can beidentified in the nanK gene of B. breve UCC2003 and is absentfrom ROK transcriptional regulators (59). The predicted functionof NanK is to phosphorylate N-acetylmannosamine, producingN-acetylmannosamine-6-phosphate. Bbr_0162, designated nanEhere, is predicted to encode an N-acetylmannosamine-6-phos-phate epimerase, the predicted function of which is the conversionof N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. Bbr_0163 encodes a putative hydrolase protein, thefunction of which, if any, in relation to sialic acid metabolism isunknown. The deduced protein products of Bbr_0164 toBbr_0167, designated nanB, nanC, nanD, and nanF, respectively,are predicted to represent an ABC transport system including aputative solute-binding protein, a permease, and two ATP-bind-ing proteins. Bbr_0168, designated nanA, encodes a putative N-acetylneuraminate lyase. The predicted function of NanA is thecleavage of sialic acid, producing N-acetylmannosamine and py-ruvate. Bbr_0169, designated nagB1, encodes a putative glucosa-mine-6-phosphate deaminase, the predicted function of which isthe conversion of glucosamine-6-phosphate to fructose-6-phos-phate, with the concomitant release of ammonia. Bbr_0170 is apseudogene that is predicted to encode a truncated, nonfunctionalNagA protein. Bbr_0171, designated nanH, encodes a predictedsialidase that, because of the lack of a signal sequence at the Nterminus, is presumed to be intracellular. Sialidases are responsi-ble for the removal of sialic acid residues from glycans. The nanHgene is followed by Bbr_0172, which encodes a putative ATPase inthe AAA� superfamily of ATPases, a large and diverse family ofenzymes that are found in all of the kingdoms of living organisms,

FIG 2 Final OD600 values obtained following 24 h of growth of 14 different B.breve strains in mMRS containing 0.5% (wt/vol) sialic acid as the sole carbonsource. The results are mean values obtained from two different experiments.Error bars represent standard deviations.

TABLE 2 Effect of sialic acid on the transcriptome of B. breve UCC2003

Locus tag (gene) Predicted functionLevel ofupregulationa

Bbr_0160 Conserved hypothetical protein 37.32Bbr_0161 (nanK) Conserved hypothetical protein in ROK

family59.01

Bbr_0162 (nanE) N-Acetylmannosamine-6-phosphate2-epimerase

49.91

Bbr_0163 Hydrolase 51.55Bbr_0164 (nanB) Substrate-binding protein 367.68Bbr_0165 (nanC) ABC transport system permease protein 451.08Bbr_0166 (nanD) ABC transport system ATP-binding protein 453.7Bbr_0167 (nanF) ABC transport system ATP-binding protein 435.43Bbr_0168 (nanA) N-Acetylneuraminate lyase 435.04Bbr_0169 (nagB1) Glucosamine-6-phosphate isomerase 275.38Bbr_0171 (nanH) Sialidase A 204.69Bbr_0172 ATPase 6.7Bbr_1247 (nagA2) N-Acetylglucosamine-6-phosphate

deacetylase3.07

Bbr_1248 (nagB3) Glucosamine-6-phosphate isomerase 4.11

a The cutoff point is 3-fold with a P value of �0.001.

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where they are essential in various cellular processes such as pro-teolysis and DNA replication (60). Its function, if any, in sialic acidmetabolism by B. breve UCC2003 is obscure. The Bbr_0160-to-Bbr_0172 gene cluster was renamed the nan-nag cluster, as it in-cludes a complete nan system, as defined in 2004 as “one thatminimally includes orthologues of genes encoding NanA, NanE,and NanK” (61), as well as a predicted transport system and thenagB1-encoded glucosamine-6-phosphate deaminase. In addi-tion to the nan-nag cluster, the Bbr_1247-Bbr_1248 locus is alsopredicted to be involved in sialic acid metabolism. Bbr_1247 (des-ignated nagA2) encodes a putative N-acetylglucosamine-6-phos-phate deacetylase, the predicted function of which is the removalof acetate from N-acetylglucosamine-6-phosphate, producingglucosamine-6-phosphate. Bbr_1248 (designated nagB3) encodesan additional glucosamine-6-phosphate deaminase (see above).Therefore, within these two loci, whose encompassing genes aretranscriptionally induced when B. breve UCC2003 is grown onsialic acid, we can identify elements of a complete sialic acid cata-bolic pathway corresponding to that previously outlined in E. coli(Fig. 1), with the sole difference being a nanBCDF-encoded ABCtransport system instead of the nanT-encoded transporter in E.

coli (29). Interestingly, the genome of B. breve UCC2003 containstwo adjacent genes, corresponding to locus tags Bbr_0846 andBbr_0847, designated nagA1 and nagB2 here, which are predictedto encode additional N-acetylglucosamine-6-phosphate deacety-lase and glucosamine-6-phosphate deaminase activities, respec-tively, although these genes were not shown to be significantlyupregulated when B. breve UCC2003 was grown on sialic acid. TheNagA1 protein is 74% identical to NagA2, while the NagB1 pro-tein is 84% identical to NagB2 and 89% identical to NagB3. On thebasis of the comparative genome analysis presented in Fig. 3, thenan-nag cluster is highly conserved among the B. breve strainswhose genomes were recently published (62). The same genes canalso be identified in B. longum subsp. infantis ATCC 15697, an-other strain that can utilize sialic acid, although the order of thegenes in this particular nan-nag cluster has been slightly rear-ranged relative to that of B. breve UCC2003 (33) (Fig. 3). There areno other homologs of this cluster within the completed bifidobac-terial genomes currently available.

Disruption of the nanK, nanB, nanC, nanA, and nagA2 genesin B. breve UCC2003. In order to investigate if the disruption ofindividual genes in the nan cluster would affect the ability of B.

FIG 3 Comparison of the sialic acid gene cluster of B. breve UCC2003 with corresponding putative sialic acid utilization loci of other bifidobacteria. Each solidarrow represents an open reading frame. The length of each arrow is proportional to the size of the open reading frame, and the gene locus name, which isindicative of its putative function, is shown at the top. Orthologs are the same color. The amino acid sequence identity of each predicted protein to its equivalentprotein encoded by B. breve UCC2003, expressed as a percentage, is shown above each arrow.

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breve UCC2003 to utilize sialic acid, nanK, nanB, nanC, nanA, andnagA2 gene insertion mutants were constructed, resulting in B.breve strains UCC2003-nanK, UCC2003-nanB, UCC2003-nanC,UCC2003-nanA, and UCC2003-nagA2, respectively. These strainswere compared with B. breve UCC2003 for the ability to grow inmMRS medium supplemented with sialic acid. As expected, andin contrast to the wild type, there was a complete lack of growth of

B. breve UCC2003-nanA in medium containing sialic acid(Fig. 4A). In order to demonstrate that the protein product ofnanA is uniquely required for sialic acid metabolism in B. breveUCC2003, a complementation experiment was performed. ThenanA gene was cloned into pBC1.2 under the control of the con-stitutive p44 promoter (63) and introduced into B. breveUCC2003-nanA. Expression of NanA in B. breve UCC2003-nanA/

FIG 4 (A) Final OD600 values after 24 h of growth of B. breve UCC2003 and the B. breve insertion mutants UCC2003-nanA, UCC2003-nanK, UCC2003-nanB,UCC2003-nanC, and UCC2003-nagA2 in mMRS containing 0.5% (wt/vol) lactose or 0.5% (wt/vol) sialic acid as the sole carbon source. The results are meanvalues obtained from three separate experiments. Error bars represent standard deviations. (B) Final OD600 values after 24 h of growth of B. breve UCC2003, theinsertion mutants B. breve UCC2003-nanA and UCC2003-nanA/pBC1.2, and the complemented insertion mutant B. breve UCC2003-nanA/pBC1.2-nanA inmMRS containing 0.5% (wt/vol) lactose or 0.5% (wt/vol) sialic acid as the sole carbon source. The results are mean values obtained from three separateexperiments. Error bars represent standard deviations.

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pBC1.2-nanA allowed this strain to regain the ability to grow onsialic acid to an OD comparable to that reached by the wild type(Fig. 4B). The B. breve insertion mutants UCC2003-nanK,UCC2003-nanB, and UCC2003-nanC also failed to grow in me-dium containing sialic acid. However, the B. breve insertion mu-tant UCC2003-nagA2 reached OD600 levels comparable to thoseof the wild-type strain during growth on sialic acid. The growth ofthe insertion mutants was not impaired on lactose, where all of thestrains reached final OD600 levels comparable to that reached bythe wild-type strain (Fig. 4A).

Purification and biochemical characterization of NanA,NanK, NanE, NagA2, and NagB1. In order to investigate the pre-dicted enzymatic activities of NanA, NanK, NanE, NagA2, andNagB1, the genes encoding the respective enzymes were clonedinto L. lactis NZ9000 and subsequently overproduced and puri-fied. The NanAHis, NanKHis, NanEHis, NagA2His, and NagB1His

proteins thus produced exhibited the expected molecular massesof 34, 33, 23, 45, and 29 kDa, respectively (results not shown).Biochemical characterization was performed by incubating an in-dividual enzyme or a combination of enzymes with a particularsubstrate and analyzing the resulting products by HPTLC. Theresults are consistent with the predicted sialic acid breakdownpathway, as outlined above (Fig. 1) (29). Under the HPTLC con-ditions used, it was not possible to visualize sialic acid; however, asexpected, NanAHis, when incubated with its assumed substrate,was shown to produce a product with mobility properties similarto those of N-acetylmannosamine. The expected by-product ofthe reaction, pyruvate, was not visible under these conditions.Only in the presence of ATP was NanKHis shown to use N-acetyl-mannosamine as a substrate and generate a product with(HPTLC) mobility properties consistent with its expected prod-uct, N-acetylmannosamine-6-phosphate. Furthermore, whenboth NanAHis and NanKHis were incubated with sialic acid andATP, this also allowed the production of a presumed N-acetyl-mannosamine-6-phosphate. Since it was not possible to visualizeN-acetylglucosamine-6-phosphate or glucosamine-6-phosphateunder the HPTLC conditions used, NanEHis, NagB1His, andNagA2His were incubated together with N-acetylmannosamine-6-phosphate and, as expected, the combined activities of these threeenzymes generated a product exhibiting HPTLC mobility proper-ties consistent with those of fructose-6-phosphate. If any one ortwo of these enzymes were not included, this product was notgenerated, thus demonstrating the contribution of each of thesethree enzymes to this biochemical conversion (results not shown).Finally, when the five enzymes NanAHis, NanKHis, NanEHis,NagA2His, and NagB1His were incubated with only sialic acid as thesubstrate (plus ATP), a product with HPTLC mobility propertiesconsistent with those of the expected end product fructose-6-phosphate was generated (Fig. 5).

Growth of B. bifidum PRL2010 and B. breve UCC2003 orUCC2003-nanA on 3=-sialyllactose. Growth of B. bifidumPRL2010 and B. breve UCC2003 on mMRS medium supple-mented with 0.5% (wt/vol) 3=-sialyllactose, a trisaccharide con-sisting of sialic acid and lactose bound by an -(2¡3) linkage, wasfirst assessed by measuring the OD600 for 36 h under anaerobicconditions. 3=-Sialyllactose was found to support the growth of B.bifidum PRL2010, reaching an OD600 of approximately 0.8 (Fig.6A). Analysis of the CFS after 24 h of growth by HPAEC-PAD andcomparison with nonfermented standards of 3=-sialyllactose,sialic acid, and lactose showed that 3=-sialyllactose was consumed

from the medium but that the consumption of this trisaccharidecoincided with the accumulation in the medium of a carbohydratewith HPAEC-PAD properties consistent with those of sialic acid.It has previously been shown that B. bifidum PRL2010 encodestwo putative extracellular exosialidases on its genome, each pre-sumed to be specific for -(2¡3) or -(2¡6) linkages (64). Fromthis information, we assumed that the extracellular sialidase of B.bifidum PRL2010 cleaves 3=-sialyllactose to produce sialic acid,which it cannot utilize (i.e., B. bifidum PRL2010 does not exhibitgrowth in a medium containing sialic acid as a sole carbon source[results not shown]), and lactose, which it can utilize, resulting insialic acid accumulation in the medium. Consistent with this ob-servation, the genome of B. bifidum PRL2010 lacks a sialic acidutilization cluster (64). In contrast, it was found that 3=-sialyllac-tose and its -(2¡6)-linked counterpart 6=-sialyllactose were un-able to support the growth of B. breve UCC2003 (results notshown). In order to establish if B. breve UCC2003 could grow inthe 3=-sialyllactose-supplemented mMRS medium in which B. bi-fidum PRL2010 was previously grown for 24 h (spent medium), asmall amount of lactose, 0.01% (wt/vol), was added to the spentmedium prior to inoculation to initiate growth. A small inoculum(0.01%) of B. breve UCC2003 was used to allow the strain to un-dergo multiple growth generations. Growth on lactose was re-quired for the initial increase in cell numbers during the first 12 h;however, HPAEC-PAD analysis confirmed that lactose was fullyutilized by B. breve UCC2003 after 12 h of incubation, after whichgrowth and viability were dependent on the ability to utilize thesialic acid in the spent medium. Between 12 and 30 h, B. breveUCC2003 was able to reach a viable count of almost 109 CFU ml�1

from a starting point of 108 CFU ml�1 and was able to maintainthese viable cell numbers until 72 h (Fig. 6B). HPAEC analysis ofthe cell-free supernatant after 30 h of growth shows the absence ofthe sialic acid-associated peak, implying that the sialic acid re-leased as a result of the exosialidase activity of B. bifidum PRL2010was entirely utilized to support the growth of B. breve UCC2003(Fig. 6C). Consistent with this scenario, B. breve UCC2003-nanA(which is incapable of growth in sialic acid [see above]) was shownto be incapable of growth in this spent medium, with viable countsdropping from their initial inoculation number of 105 CFU ml�1

after 72 h (Fig. 6B). As expected, HPAEC analysis demonstrated

FIG 5 Hydrolysis of sialic acid to fructose-6-phosphate by the combined ac-tivities of purified NanAHis, NanKHis, NanEHis, NagA2His, and NagB1His.Lanes: 1, 5 mg ml�1 (wt/vol) standards (N-acetylmannosamine [A], N-acetyl-mannosamine-6-phosphate [B], and fructose-6-phosphate [C]) in kinase buf-fer; 2, NanAHis plus sialic acid; 3, NanKHis plus N-acetylmannosamine; 4,NanAHis and NanKHis plus sialic acid; 5, NanEHis, NagA2His, and NagB1His

plus N-acetylmannosamine-6-phosphate; 6, NanAHis, NanKHis, NanEHis,NagA2His, and NagB1His plus sialic acid.

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FIG 6 (A) Growth profiles of B. bifidum PRL2010 in mMRS containing 0.5% (wt/vol) lactose or 0.5% (wt/vol) 3=-sialyllactose. The results are mean valuesobtained from two separate experiments. Error bars represent standard deviations. (B) Growth profiles of B. breve UCC2003 and UCC2003-nanA in mMRScontaining 0.5% (wt/vol) 3=-sialyllactose previously fermented by B. bifidum PRL2010 for 24 h. The results are mean values obtained from two separateexperiments. Error bars represent standard deviations. (C) HPAEC profiles of mMRS plus 0.5% (wt/vol) 3=-sialyllactose (I), mMRS plus 0.5% (wt/vol) sialic acid(II), mMRS plus 0.5% (wt/vol) 3=-sialyllactose after 24 h of growth of B. bifidum PRL2010 (III), the medium from condition III after 30 h of growth of B. breveUCC2003 (IV), and the medium from condition III after 30 h of growth of B. breve UCC2003-nanA (V).

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that sialic acid is not metabolized by B. breve UCC2003-nanA(Fig. 6C).

DISCUSSION

B. breve strains represent a dominant commensal group in thebreast-fed infant gut microbiota, but it is only recently that thefactors that contribute to this dominance have become a subject ofscientific scrutiny. It has already been shown that B. breve strainscan liberate N-glycans from host glycoproteins and glycoproteinsfrom breast milk (65), and a more recent study has demonstratedthat B. breve strains can grow on the HMOs lacto-N-tetraose,lacto-N-neotetraose, and sialylated lacto-N-tetraose (35). In thisreport, we describe the functional characterization of a locus ded-icated to the uptake and utilization of sialic acid. To our knowl-edge, this is the first study to adopt a functional-genomic ap-proach to gain an understanding of sialic acid metabolism by aBifidobacterium species.

Of the 14 B. breve strains tested for growth on sialic acid as asole carbon source, 11 were shown to be capable of growth. DNAmicroarray analysis of B. breve UCC2003 grown in sialic acid re-vealed a locus of 12 genes predicted to be involved in sialic aciduptake and catabolism, as well as 2 genes specifying putativeN-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, designated nagA2 and nagB3, respec-tively, located at an unlinked position on the genome. The genomeof B. breve UCC2003 also contains additional nagA and nagBgenes at the Bbr_0846-Bbr_0847 locus, which were designatednagA1 and nagB2, respectively. Since the enzymatic activities en-coded by nagAB genes are also required for the metabolism ofamino sugars such as N-acetylglucosamine and N-acetylgalac-tosamine (66, 67), the presence of multiple nagAB copies on the B.breve UCC2003 genome may reflect the metabolic versatilitythat allows it to utilize such amino sugars when they are re-leased from host-derived carbohydrates such as mucin andHMO (20, 22). Previously performed comparative genomic hy-bridization (CGH) analysis revealed that all of the B. breve strainstested harbor the nan-nag genes in their genomes, thus indicatingthat the ability to utilize sialic acid is well conserved in the B. brevespecies (42). An apparent inconsistency is the presence of thesegenes in the genomes of B. breve NIZO 658, LMG13208, andUCC2007, which are unable to grow on sialic acid; however, itmust be considered that the nature of CGH analysis means thatonly the presence or absence of genes is revealed but that of anymutations in a gene or promoter that may result in loss of functionis not.

The uptake of sialic acid is likely to be facilitated by an ABCtransport system encoded by the nanBCDF genes. Disruption ofnanB or nanC in B. breve UCC2003 was shown to result in animpairment of growth on sialic acid, thus proving that this pre-dicted transport system is solely responsible for sialic acid uptake.Previous studies have shown that transport of sialic acid into bac-terial cells can be achieved by ABC transport systems, as seen inHaemophilus ducreyi (68), transporters of the major facilitatorsuperfamily of proteins, as in E. coli (28, 69) and Bt. fragilis (31)and/or tripartite ATP-independent periplasmic transporters, asseen first in Haemophilus influenzae (70) and in V. cholerae (71).ABC transport systems have previously been shown to be involvedin carbohydrate uptake in B. breve UCC2003 (10, 12, 13, 72).

Similarly, mutations in nanA and nanK were shown to causeimpaired growth on sialic acid, implying that the enzymes en-

coded by these genes are essential for sialic acid metabolism. Incontrast, the insertion mutant B. breve UCC2003-nagA2 did notdisplay impaired growth on sialic acid, suggesting either that thisgene is not involved in sialic acid metabolism or that another geneis able to compensate for the mutation, a possible candidate forwhich would be the nagA1 gene mentioned above.

Analysis of the substrate specificities of the enzymes NanA,NanK, NanE, NagA2, and NagB1 indicates that sialic acid metab-olism by B. breve UCC2003 occurs by means of a five-step pathwaysimilar to that previously characterized in E. coli (Fig. 1) (29). Ananalogous pathway has been described in H. influenzae (73),Staphylococcus aureus (74), and L. sakei (32), yet it differs slightlyfrom that found in Bt. fragilis. It was shown that Bt. fragilis doesnot encode a nanK gene but instead encodes a novel N-acetylman-nosamine epimerase, encoded by nanE, which converts N-acetyl-mannosamine to N-acetylglucosamine, which is then phosphory-lated by a rokA-encoded kinase (31). While individual enzymes ofthis pathway have been purified and characterized previously,such as NanA from C. perfringens (75), H. influenzae (76), andLactobacillus plantarum (77) and the previously mentioned NanEenzyme from Bt. fragilis (31), to our knowledge, this is the firsttime that the full catabolic pathway of sialic acid has been recon-stituted in vitro.

In order for B. breve UCC2003 to utilize sialic acid, it must firstbe released from glycoconjugates such as mucin or HMO, inwhich it is most commonly found. Sialidases, which release sialicacid from glycans, have previously been described for infant-de-rived bifidobacteria, including two intracellular sialidases from B.longum subsp. infantis ATCC 15697 (34) and an extracellular siali-dase from B. bifidum JCM1254 (78). Two predicted extracellularexo--sialidases were also identified in the genome of B. bifidumPRL2010 (64). A putative intracellular sialidase is located in thenan-nag locus of B. breve UCC2003, but its substrate is unknown.The inability of B. breve UCC2003 to grow on 3=- or 6=-sialyllac-tose indicates that this carbohydrate is not the correct substrate forthis sialidase, or it may also be that B. breve UCC2003 does notencode an appropriate transport system for its uptake. Its cognatesubstrate may, in fact, be sialylated lacto-N-tetraose, which haspreviously been shown to support the growth of various B. brevestrains (35).

Interestingly, experiments testing B. breve UCC2003 growth inspent medium in which B. bifidum PRL2010 had previously grownon 3=-sialyllactose (as the sole carbon source) showed that B. breveUCC2003 can cross feed on the sialic acid released by the exosiali-dase activity of B. bifidum PRL2010. These results suggest that B.bifidum PRL2010 uses its sialidase activity to gain access to thelactose component of this HMO, leaving the sialic acid for B. breveUCC2003 (and others) to forage. These results show that althoughthe sialic acid in the gut is predominantly glycosidically bound(24), a lack of sialidase activity would not necessarily disadvantagethe proliferation of a cross-feeding bifidobacterial strain such as B.breve UCC2003 in the infant gut. Similarly, a recent study showedthat two pathogens, Salmonella enterica serovar Typhimuriumand Clostridium difficile, can scavenge sialic acid released by thesialidase activity of Bacteroides thetaiotaomicron in a gnotobioticmouse (79). Within the same study, it was shown that antibiotic-treated mice had higher levels of free sialic acid in their ceca thantheir untreated counterparts. The authors proposed that in theuntreated gut, sialic acid is utilized by members of the gut micro-biota. Antibiotic treatment causes an acute disturbance of this

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complex microbiota, yet enough sialidase-producing bacteria re-main, resulting in elevated levels of sialic acid, which can be ex-ploited by a sialic acid-utilizing pathogen such as C. difficile (79).

Our results, combined with previous knowledge of the utiliza-tion of sialic acid by pathogens, e.g., C. difficile, suggest that themetabolism of sialic acid by certain bifidobacterial strains such asB. breve UCC2003 may provide nutritional immunity and com-petition for opportunistic pathogens in a healthy gut environ-ment, thus inhibiting or moderating their proliferation. It alsosuggests a role for sialic acid-utilizing B. breve strains as probioticprophylaxis, particularly during antibiotic treatment, or in probi-otic treatment of C. difficile infection, as recently suggested (80).Recently, B. breve strains were identified as suitable probiotics forthe treatment of enteric disorders in infants (81). Our results sug-gest that a combination of (extracellular) sialidase-encoding B.bifidum strains and sialic acid-utilizing B. breve strains can im-prove B. breve strain colonization of and persistence in the infantgut, thus potentially conferring the aforementioned benefits onthe infant host.

Previous research has shown that B. breve UCC2003, as well asother B. breve strains, is capable of utilizing a number of constit-uents of an adult, plant-based diet (10–15), yet these data did notexplain the prevalence of this species in the infant gut. The presentstudy, which demonstrates how B. breve UCC2003 can utilizesialic acid, advances our understanding of the prevalence of B.breve in the infant gut, where cross feeding may play an importantrole in the establishment of a microbiota with a high abundanceand diversity of bifidobacterial species.

ACKNOWLEDGMENTS

The Alimentary Pharmabiotic Centre is a research center funded by Sci-ence Foundation Ireland (SFI) through the Irish Government’s NationalDevelopment Plan. We and our work were supported by SFI (grants 07/CE/B1368 and SFI/12/RC/2273) and an HRB postdoctoral fellowship(grant PDTM/20011/9) awarded to M.O.M.

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