Table Of ContentBottacinietal.MicrobialCellFactories2014,13(Suppl1):S4
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PROCEEDINGS Open Access
Diversity, ecology and intestinal function of
bifidobacteria
Francesca Bottacini1,2, Marco Ventura2, Douwe van Sinderen1, Mary O’Connell Motherway1*
From 11th International Symposium on Lactic Acid Bacteria
Egmond aan Zee, the Netherlands. 31 August - 4 September 2014
Abstract
The human gastrointestinal tract represents an environment which is a densely populated home for a microbiota
that has evolved to positively contribute to host health. At birth the essentially sterile gastrointestinal tract (GIT) is
rapidly colonized by microorganisms that originate from the mother and the surrounding environment. Within a
short timeframe a microbiota establishes within the (breastfed) infant’s GIT where bifidobacteria are among the
dominant members, although their numerical dominance disappears following weaning. The numerous health
benefits associated with bifidobacteria, and the consequent commercial relevance resulting from their
incorporation into functional foods, has led to intensified research aimed at the molecular understanding of
claimed probiotic attributes of this genus. In this review we provide the current status on the diversity and ecology
of bifidobacteria. In addition, we will discuss the molecular mechanisms that allow this intriguing group of bacteria
to colonize and persist in the GIT, so as to facilitate interaction with its host.
Introduction believed to be the most dominant bacterial group pre-
Bifidobacteria are typical gut inhabitants, and represent sent in the gut microbiota of vaginally delivered, breast-
non-motile, non-sporulating, non-gas producing, sac- fed infants [4]. They also are known to stably colonize
charolytic Gram-positive bacteria that belong to the the GIT of various other eukaryotic hosts, including
familyBifidobacteriaceaeandthephylumActinobacteria, mammals, birds and insects [5,6]. Their discovery has
the latter being one of the largest and most populated been attributed to Henri Tissier who in 1899 isolated a
taxonomicunitsinthedomainBacteria,andbeingcom- so-named Bacillus bifidus from breast-fed infant faeces
posed of six classes and 16 orders [1]. Actinobacteria [7]; bifidobacteria were incorrectly assigned to the genus
exhibit a wide spectrum of morphologies and metabolic Lactobacillus for much of the 20th century, and only
properties,andpossessgenomesofa highG+Ccontent, relatively recently obtained appropriate classification as
rangingfrom46% toover70% (forexample membersof a separate genus, Bifidobacterium [8].
the Corynebacterium genus) [1]. Furthermore, certain
representativesofthisphylumproducearangeofimpor- Taxonomic diversity and ecology
tant secondary metabolites, including those that are The Bifidobacterium genus
exploited as antibiotics by the pharmaceutical industry The Bifidobacterium genus currently comprises 48
(producedbyStreptomycesspp.)[2,3]. recognized species (Table 1) [9-13]. According to taxo-
During the last two to three decades bifidobacteria nomic classification based on comparative analyses of
have become the subject of intensifying scientific scru- 16S rRNA-encoding DNA and concatenated multilocus
tiny because they represent an abundant bacterial com- sequences, representing a number of conserved house-
ponent of the human GIT microbiota, while they are keeping genes (clpC, dnaJ, xfp, dnaB, rpoC and purF),
bifidobacterial species have been clustered into six main
phylogenetic clusters, consisting of the Bifidobacterium
*Correspondence:[email protected]
1AlimentaryPharmabioticCentreandSchoolofMicrobiology,National boum, Bifidobacterium asteroides, Bifidobacterium ado-
UniversityofIreland,Cork,WesternRoad,Cork,Ireland lescentis, Bifidobacterium pullorum, Bifidobacterium
Fulllistofauthorinformationisavailableattheendofthearticle
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reproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublicDomainDedicationwaiver
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Table 1Listofcurrently recognized bifidobacterial species withtheir isolation sources.
Species Typestrain Origin Sequencingstatus
Bifidobacteriumactinocoloniiforme DSM22766 Bumblebeedigestivetract -
Bifidobacteriumadolescentis ATCC15705 Adultfaeces COMPLETE
Bifidobacteriumangulatum ATCC27535 Adultfaeces DRAFT
Bifidobacteriumanimalissubsp.animalis ATCC25527 Sewage COMPLETE
Bifidobacteriumanimalissubsp.lactis DSM10140 Fermentedmilk COMPLETE
Bifidobacteriumasteroides ATCC25910 Beeintestine COMPLETE
Bifidobacteriumbiavatii DSM23969 Tamarindfaeces -
Bifidobacteriumbifidum ATCC29521 Infantfaeces COMPLETE
Bifidobacteriumbohemicum DSM22767 Bumblebeedigestivetract -
Bifidobacteriumbombi DSM19703 Bumblebeedigestivetract -
Bifidobacteriumboum ATCC27917 Bovinerumen -
Bifidobacteriumbreve ATCC15700 Infantfaeces COMPLETE
Bifidobacteriumcallitrichos DSM23973 Marmosetfaeces -
Bifidobacteriumcatenulatum ATCC27539 Adultfaeces DRAFT
Bifidobacteriumchoerinum ATCC27686 Pigletfaeces -
Bifidobacteriumcoryneforme ATCC25911 Beeintestine -
Bifidobacteriumcrudilactis LMG23609 Rawmilkcheese -
Bifidobacteriumcuniculi ATCC27916 Rabbitfaeces -
Bifidobacteriumdentium ATCC27534 Oralcavity COMPLETE
Bifidobacteriumgallicum ATCC49850 Humanfaeces DRAFT
Bifidobacteriumgallinarum ATCC33777 Chickencaecum -
Bifidobacteriumindicum ATCC25912 Beeintestine -
Bifidobacteriumkashiwanohense DSM21854 Infantfaeces -
Bifidobacteriumlongumsubsp.infantis ATCC15697 Infantfaeces COMPLETE
Bifidobacteriumlongumsubsp.longum ATCC15707 Adultfaeces COMPLETE
Bifidobacteriumlongumsubsp.suis ATCC27533 Pigletfaeces -
Bifidobacteriummagnum ATCC27540 Rabbitfaeces -
Bifidobacteriummerycicum ATCC49391 Bovinerumen -
Bifidobacteriumminimum ATCC27538 Sewage -
Bifidobacteriummongoliense DSM21395 Fermentedmilk -
Bifidobacteriummoukalabense JCM18751 Gorillafaeces -
Bifidobacteriumpseudocatenulatum ATCC27919 Infantfaeces DRAFT
Bifidobacteriumpseudolongumsubsp.globosum ATCC25865 Bovinerumen -
Bifidobacteriumpseudolongumsubsp.pseudolongum ATCC25526 Pigfaeces -
Bifidobacteriumpsychraerophilum LMG21775 Porcinecaecum -
Bifidobacteriumpullorum ATCC27685 Chickenfaeces -
Bifidobacteriumreuteri DSM23975 Marmosetfaeces -
Bifidobacteriumruminantium ATCC49390 Bovinerumen -
Bifidobacteriumsaeculare ATCC49392 Rabbitfaeces -
Bifidobacteriumsanguini DSM23967 Tamarindfaeces -
Bifidobacteriumscardovii DSM13734 Humansources -
Bifidobacteriumstellenboschense DSM23968 Tamarindfaeces -
Bifidobacteriumstercoris JCM15918 Adultfaeces -
Bifidobacteriumsubtile ATCC27537 Sewage -
Bifidobacteriumthermacidophilumsubsp.porcinum DSM17755 Pigletfaeces -
Bifidobacteriumthermacidophilumsubsp.thermoacidophilum DSM15837 Anaerobicdigester -
Bifidobacteriumthermophilum ATCC25525 Pigletfaeces COMPLETE
Bifidobacteriumtsurumiense JCM13495 Hamsterdentalplaque -
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longum, and Bifidobacterium pseudolongum phylogenetic bifidobacteria,andthecorrespondingchargedtRNAsare
groups, where Bifidobacterium asteroides, isolated from believedtobeproducedwiththeinvolvementofspecific
the insect gut, appears to be the closest relative of the Asn- and Gln-tRNA amidotransferases, performing
ancient progenitor of the genus Bifidobacterium [9]. transamidation of misacetylated Asp-tRNA(Asn) and
Several of the currently recognized species have only Glu-tRNA(Gln) [20,21]. The organization of the bifido-
very recently been isolated, such as Bifidobacterium acti- bacterial chromosomealsoappearstobeconsistentwith
nocoloniiforme, Bifidobacterium bohemicum, Bifidobac- that ofa typical bacterial genome, showing a set ofcon-
terium bombi, Bifidobacterium biavatii, Bifidobacterium served genes around the predicted origin of replication
reuteri, Bifidobacterium callitrichos, Bifidobacterium (including rpmH, dnaA, dnaN and recF) and a region
sanguini, Bifidobacterium stellenboschense, Bifidobacter- rich in AT and multiple DnaA-binding boxes in proxi-
ium stercoris, Bifidobacterium kashiwanohense and Bifi- mity of the gene specifying the presumed chromosomal
dobacterium moukalabense [10-13]. Interestingly, as initiator of replication (dnaA) [22]. Finally, a switch in
shown in the 16S rDNA-sequence-based Neighbour- thedirectionofthe GCskew[the(G-C)/(G+C)value] is
joining tree in Figure 1, three additional phylogenetic observed in bifidobacteria at the origin and terminus of
clusters seem to be present in Bifidobacterium, repre- replication,sothatthe leadingstrandtendstobe higher
senting the B. crudilactis, B. bohemicum and B. scardovii inGuanine content ascomparedtoitsCytosine content
groups. Moreover, microbiota analysis by 16S rRNA- [1,23]. The copy number of rRNA-specifying loci repre-
encoding DNA sequences has indicated that more bifi- sents another variability in bifidobacterial genomes, and
dobacterial species are yet to be discovered [4,14], and rangesbetweentwoandfive(Table2).
for this reason we may expect further expansion or In silico gene predictions show that representatives of
refinement of this classification. the Bifidobacterium genus contain an average of 2012
Bifidobacteria have been isolated from a variety of openreadingframes(ORFs)pergenome,wheregenomes
ecological niches, such as sewage, fermented milk and ofB.animalissubsp.lactisandB.longumsubsp.infantis
anaerobic digestion facilities, yet are most frequently possessthesmallestandlargestnumberofORFs,respec-
associated with the GIT of humans and animals (in gen- tively, being consistent with their genome size. It is also
eral where the offspring of the bifidobacterial host is possible that the lower number of genes observed in
raised with parental care which may ensure direct trans- B. animalis subsp. lactis genomes have been caused by
mission from mother to child/progeny) [15-17]. theirwide-spreadutilizationincommercialpreparations,
Theirecologicaladaptationmayalsodifferamongspe- leading tophenomena ofgene lossandgenomedecayas
cies, some of them for instance can be present in differ- aresultoftheadaptationtoanutrient-richenvironment,
ent niches, such as in the case of Bifidobacterium being in agreement with what was also observed in cer-
adolescentis, Bifidobacterium animalis, Bifidobacterium tainlacticacidbacteria[24-26].
dentiumandBifidobacteriumcatenulatum(referredtoas
cosmopolitanbifidobacterialtaxa),whileothersappearto Comparative genomics of bifidobacteria
be much more niche-specific, for example Bifidobacter- ComparativegenomicanalysisofthegenusBifidobacter-
ium cuniculi,Bifidobacterium angulatumandBifidobac- ium involving genome sequences of nine bifidobacterial
terium gallinarum (so-called specialized bifidobacterial species,B.longumsubsp.longum,B.longumsubsp.infan-
taxa)[18]. tis, B. adolescentis, B. dentium, B. bifidum, B. animalis
subsp. lactis, B. angulatum, B.catenulatum and B. galli-
General genome features cumandfullnucleotidesequencealignmentrevealedthat
Bifidobacterialchromosomeshaveagenomethatranges thesegenomesarenotcolinear,showingafrequentinter-
insizebetween1.9and2.8Mbp,withrepresentativesof ruption of chromosomal synteny, thereby inferring the
the B. animalis species possessing the smallest genome, existenceofsignificantgenomediversitywithinmembers
and a representative of B. longum subsp. infantis har- of the genus Bifidobacterium caused by chromosomal
bouringthelargestreportedgenome(Table2).Bifidobac- rearrangment events. The Bifidobacterium core genome
terial genomes are predicted to encode a substantial was determined to consist of 506 orthologues that are
numberoftRNAmolecules,whichaveragesat52tRNA- sharedbyallninebifidobacterialspecies.Functionalanno-
encoding genes per genome, with an outlier in the case tation established thatthesecoregenesprimarily encode
of the B. longum subsp. infantis ATCC 15697 genome, housekeeping functions, including those dedicated to
whichcontains areported79tRNA-encodinggenes[19] replication,transcriptionandtranslation,aswellasgenes
(Table 2). Interestingly, despite the fact that bifidobac- associatedwithadaptationtoa particularniche environ-
teria possess a tRNA-encoding gene for all common ment,e.g.genesassociatedwithcarbohydratemetabolism,
amino acids, genes encoding the amino acyl-tRNA signaltransductionandcellenvelopebiogenesis[27].The
synthetases for glutamine and asparagine are absent in number of truly unique genes (present in a single
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Figure 1 Rooted neighbour-joining tree based on the alignment (1265 nucleotides) of the 16S rRNA gene (96% of homology)
sequencefromthe48sofarrecognizedbifidobacterialtaxa(Dec.,2013).Thephylogeneticgroups,ashighlightedingreyshaded
quadrangles,aretheresultofapreviouslyperformedmultilocusanalysis[9],integratedwiththeinformationresultingfromthealignmentofthe
16Ssequenceofnewbifidobacterialspecies.
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pili
Sortase-pendent 1 0 1 0 3 3 7 0 2 2
e
d
enceofEPS Yes No Yes mnant) No Yes No No Yes No
Pres (Re
ofe
encephag Yes No Yes No Yes Yes Yes Yes Yes Yes
so
Prepr
s
m
M
R/ste 2 1 2 3 7 3 2 8 6 0
y
e. s
v
tati of
epresen PresenceCRISPR Yes Yes Yes Yes No Yes Yes No No No
r
d
e
nc of
seque esenceplasmid No No No Yes No No No No Yes No
y Pr
el
t
e
compl rRNAcontent 5 3 4 2 3 2 4 4 4 4
e
aston tRNAcontent 54 52 52 44 52 54 55 79 57 47
e
atl +C% 9.1 0.4 0.5 0.1 2.7 8.7 8.5 9.8 0.3 0.1
G 5 6 6 6 6 5 5 5 6 6
h
t
ecieswi ORFcontent 1631 1538 1565 1658 1706 1985 2127 2416 1799 1845
p
s
terium Length(Mbp) 2.08 1.94 1.93 2.16 2.21 2.42 2.63 2.83 2.26 2.29
c
a
Bifidobralfeaturesof Organism adolescentisATCC animalissubsp.animalis animalissubsp.lactis asteroidsPRL2011 bifidumPRL2010 breveUCC2003 dentiumBd1 longumsubsp.infantis longumsubsp.longum thermophilumRBL67
e m m m m m m m m m m
Table2Gen Bifidobacteriu15703 BifidobacteriuATCC25527 BifidobacteriuDSM10140 Bifidobacteriu Bifidobacteriu Bifidobacteriu Bifidobacteriu BifidobacteriuATCC15697 BifidobacteriuNCC2705 Bifidobacteriu
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reference genome but absent in all other analysed gen- 49-56 nm while their tails ranged in length from
omes),variesbetween21and 230genes inthe nine gen- 76-268nm.Infact,thepresenceofoneormoreprophage-
omes analysed. The majority of such unique genes have likeelementshasbeenobservedinseveralbifidobacterial
no functional annotation, thereby suggesting that these genomes(Table2)[32].Thenotionthatprophage-likeele-
genesencodefornovelbiosyntheticorbifidobacterial-host mentsmay actasa transmission vehicle of genesthatdo
interactionmolecules. not appear to be essential for phage functions (e.g.,
Our recent analysis of the pan-genome of the B. breve hypothetical proteins andglycosyl hydrolase enzymes) is
taxon, adopting complete genome sequences of eight evidence for their potential involvement in lateral gene
B.brevestrains,namelyB.breveUCC2003,B.breveNCFB transfer,andthe acquisitionofsuchelementsmay result
2258,B. breve ATCC20213,B. breve JCM7017,B.breve in an enhancement of ecological fitness in the receiving
JCM 7019, B. breve ACS-071-V-Sch8b, B. breve 12L, memberspopulatingthesameecologicaniche[33].Inter-
B. breve S27 established that these genomes are highly estingly,bifidobacterial prophageinsertion hasinseveral
syntenic,withtheexceptionofB.breveACS-071-V-Sch8b casesbeenshowntooccurata35-bpsequencelocatedat
and B. breve JCM 7017 that harbour inversions in their theendofthegenespecifyingtRNAMet[30].Moreover,an
genomes of1.1 Mband 126Kb, respectively. Analysis of evolutionaryanalysisconductedonsuspectedbifidobacter-
the core and dispensable genome highlighted that the ial prophage sequences revealed in a number of cases a
B. breve coregenome is comprised of 1,323 core gene highlevelofsequenceidentitywithprophagespresentin
familiesandasexpectedtheseencodefunctionsdedicated high and low G+C content Gram-positive bacteria, sup-
tocellularhousekeeping.FromtheeightB.brevegenomes portingthehypothesisofphage-mediatedDNAexchange
924familiesofvariablegeneswereidentifiedandofthese betweenActinobacteriaandFirmicutes,perhapsfacilitated
426couldbeclassifiedastrulyuniquegenes.Thevariable bythefactthatthesebacteriaincertaincasesshareacom-
genesencodedproteinsinvolvedincapsularpolysacchar- monniche[32].
idebiosynthesis,phageresistance(restriction-modification The majority of bifidobacterial strains do not harbour
systems and CRISPR loci), production of sortase depen- any plasmid and, if they do, a given isolate rarely con-
dent pili, and carbohydrate transport and utilisation. tains more than one such extrachromosomal genetic
An extension of the pangenome analysis to include gen- element, which then range in size from 1.5-15 Kb. So
omes of 6 publically available B. longum subsp. longum far, B. longum subsp. longum and B. breve represent the
sequencesestablishedthat564genefamilieswereuniquely species with the highest number of strains in which
presentinB.breveandabsentinB.longumsubsp.longum, plasmids have been identified, with 18 reported plasmid-
andwhile50%oftheseencodedhypotheticalfunctionsthe harbouring representatives of B. longum subsp. longum
other50%werefoundtoencodeB.brevespecificglycosyl and three of B. breve [29]. Analysis of their replication
hydrolases, ABCtransporters, CRISPR genesand mobile (Rep) proteins has indicated that the majority of identi-
elements[28]. fied bifidobacterial plasmids replicate by means of the
so-called rolling circle mechanism (RCR), while other
Mobilome functions, if encoded, still remain to a large degree
The pool of mobile elements so far found in bifidobac- unknown [29].
teriaisrepresentedbyinsertionsequences,prophage-like
elements and plasmids [29,30]. Regarding insertion CRISPR loci and restriction modification systems
sequences,representativesofthemainfamiliesthathave As mentioned above, bifidobacteria were until relatively
been reported to be present in bifidobacterial genomes recentlynotthoughttobepronetophageinfectionssince
are IS3, IS21, IS30, IS110, IS150, IS256, IS607/200 and theirwasnoevidenceofinfection,however,genomeana-
ISL3, where in general representatives of IS30 appear to lysisofseveralbifidobacterialstrainsrevealedthepresence
be the most abundant and active in bifidobacteria. In of predicted phage resistance systems, in particular
fact,ithasbeensuggestedthatIS30isactiveinB.longum CRISPRandrestrictionmodification(R-M)systems.The
subsp.longumandinvolvedincausinggenomedeletions former anti-phage system was not only found in B. ani-
and rearrangements,supportingthe suggested role ofIS malissubsp.lactis,butalsoinoneormorerepresentatives
elementsinenvironmentaladaptationofbifidobacteriain ofB.animalissubsp. animalis, B.longum subsp.longum,
general[29]. B.breve,B.bifidum,B.dentium,B.adolescentis, B.aster-
In contrast to what was initially thought, bifidobacteria oides,B.angulatumandB.catenulatumspecies[21,29].
appear to be subject to phage infections [30]. Sgorbati Restriction/modification (R-M) systems are ubiquitous
et al [31] assessed fourteen strains of B. longum by UV among prokaryotes and generally comprise of a restric-
and mitomycin C induction for the release of prophage. tion endonuclease (REase) and cognate methyltransfer-
BacteriophagewereidentifiedfromfouroftheB.longum ase (MTase) [34]. R-M systems serve primarily as
cultures, with the phage head diameters ranging from defensive instruments that protect prokaryotic cells
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against invading DNA such as promiscuous plasmids or Intestinal function
infecting bacteriophage as the unmodified incoming Adaptation to the gastrointestinal environment
DNA is targeted by the REase component of the R-M. Inordertosurvivegastrointestinaltransitandtransiently
The host DNA is resistant to cleavage as the recognition colonise theGIT,bifidobacteria mustbeable tocounter
sites of the endonuclease are modified by the cognate the adverse conditions of the oral cavity, stomach and
methyltransferase at adenosyl or cytosyl residues. R-M smallintestine.Inparticular,exposuretooxygenorother
systems are classified into four groups (designated type oxygen-derived free radicals, organic acids, and bile, as
I, II, III and IV) on the basis of their subunit composi- wellasosmoticstresscanhaveanegativeimpactonbifi-
tion, co-factor requirement, recognition sequence struc- dobacterialcellviabilityandconsequentlyprobioticfunc-
ture and the cleavage site relative to the recognition tionality. Bifidobacteria cope with these stressful
sequence[35].REaseactivityinBifidobacteriumwasfirst conditionsbyadoptingarepertoireofmolecularchaper-
described by Khosaka et al. [36,37], who identified the ones, bile efflux transporters, bile salt hydrolases, two-
restriction endonucleases BbeI from B. breve YIT4006, component systems andATPases[49-55]. Tight control
and BinSI and BinSII fromB. longum subsp. infantis ofthesestress-inducedcopingmechanismsallowsbifido-
S76e. Subsequently, REase activity was demonstrated in bacteria to rapidly reactto various andsometimes com-
strainsofB. adolescentis, B.bifidum,B. lactentis(subse- plex environmental challenges. This regulation is
quently reclassified as B. longum subsp. infantis) and governed by an interactive nextwork of regulators that
B. longum subsp. longum [38-40]. In silico analysis of include ClgR, HspR, HrcA, and LexA. The regulons are
sequencedbifidobacterialgenomesshowsthatthe num- highly conserved among sequenced bifidobacterial gen-
berofR-Msystemsvariesnotonlybetweenbifidobacter- omessuggestingthatauniversalsystemforadaptationto
ial species, but also between strains of a particular osmotic,oxidativeandacidstressexistsamongallmem-
species(Table2).The genome of B.thermophilumlacks bers of the Bifidobacterium genus [55]. Bile tolerance is
R-M systems, while strains of B. bifidum harbour the amongthemostcrucialpropertiesforaprobioticbacter-
genetic determinants to encode between four and seven ium as it determines a strain’s ability to survive transit
R-M systems and genes encoding up to eight R-M sys- through the small intestine. The active extrusion of bile
tems have been identified on the sequenced genomes of acidsandsaltsthataccumulateinthecytoplasmthrough
strains of B. longum subsp. infantis. The ability to cir- effluxpumpsisacommommechanismemployedbybac-
cumvent the R-M complement of strains of B. longum teria to counter bile toxicity. Multidrug transporters
andB. adolescentishas allowed with the introduction of belongingto theATP-bindingcassette ormajorfacilita-
E.coli-bifidobacterialshuttlevectorsintothesestrainsby tor family have been described as mediating bile toler-
electroporation [41,42], while for B. breve strains it has ance in strains of B. longum subsp. longum and B. breve
allowedthecreationofinsertionmutantsviasitespecific UCC2003 [49,56,57]. In B. breve UCC2003 Bbr_0838
homologous recombination or transposon mutagenesis encodingamultidrugtransporterofthemajorfacilitator
[43],thereby advancing our understandingof bifidobac- familyisstronglyinducedduringexposuretobile.Inacti-
terialgenomics,physiologyandmetabolism[28,44,45]. vationofBbr_0838throughinsertionalmutagenesisren-
deredthemutantstrain,UCC2003-838,moresensitiveto
Evidence of horizontal gene transfer cholicacidcomparedtotheparentstraindemonstrating
Bacterial genome evolution occurs through various that cholate is the main bile component detoxified by
mechanisms,which includegeneduplication,chromoso- Bbr_0838[49]. Survival in the highly complexandcom-
mal rearrangements, vertical DNA exchange and intra- petitiveenvironmentoftheGITrequiresthatcommensal
species horizontal gene transfer (HGT), events that may bacteria including bifidobacteria can protect themselves
facilitaterapidenvironmentaladaptation[46].Forthisrea- against host proteases. Some bifidobacterial species
son, defining the precise evolutionary distance between achieve protection against human proteases, such as a-
bacterialtaxaisacomplexanddifficulttask,andthepre- antitrypsin and human neutrophil elastase, by a serine
senceofacommonancestormaynotbesufficientinmea- protease inhibitor (Serpin), the production of which is
suringthetruedistancebetweentwophylogeneticgroups, regulated by an environmental sensing two component
when also taking the presence of common functions for regulatorysystem[51,53].
geneticadaptationtoacommonnicheintoaccount.
HGT appears to play an important role in increasing Interaction with the host environment
the competitiveness of bacteria in their ecological niche, The mechanisms by which bifidobacteria interact within
and in bifidobacteria DNA regions acquired through the gut environment and adhere to the host surfaces are
HGT are frequently present in clusters that are ran- still under investigation and the recent availability of
domly dispersed across the genome and in many cases several bifidobacterial genome sequences underlined the
being flanked by mobile elements [21,47,48]. presence of a number of macromolecules associated
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with the cellular envelope and involved in host-microbe a promoter reorientation reverses this situation using a
interactions [48,58,59]. The capsular or surface exopoly- mechanism that is likely to be similar to that observed
saccharide (sEPS) is one such macromolecule which was in Bacteroides fragilis [67,69]. More specifically, this pro-
shown to contribute to host colonization and persis- moter inversion is presumed to be catalyzed by the pre-
tence by facilitating bifidobacterial long-term coloniza- sence of an inverted repeat sequence in the intergenic
tion of host cells [1,60,61]. Another extracellular region between eps2A and eps2B, and the activity of an
structure which is crucial in the process of colonization as yet unidentified site-specific DNA invertase/recombi-
of the intestinal mucosa is represented by appendices nase [67]. Recent comparitive analysis on eight fully
called fimbriae or pili, found in both enteric and oral sequenced B. breve genomes revealed the presence of an
bifidobacteria [21,62-64]. They can be involved in the intact eps2 locus in B. breve JCM 7017, B. breve JCM
establishment of host cell contact and adhesion to the 7019, B. breve 689b and B. breve S27, while the genomes
epithelial cells, in cellular aggregation or in biofilm for- of B. breve ACS-071-V-Sch8b, B. breve NCFB 2258 and
mation, but they may also stimulate a response by the B. breve 12L appear to contain only a remnant EPS clus-
host immune system [48,59]. Bifidobacteria have long ter, where the gene encoding the priming glycosyl trans-
been recognized for their ability to prevent pathogen ferase is present, yet lacking several genes commonly
infection, however, the precise mechanism has remained associated with EPS biosynthesis [28]. Aside from
elusive. Recent research has demonstrated that gut B. breve, putative EPS-specifying regions are also present
pathogen protection conferred by bifidobacteria is asso- in the genomes of most other bifidobacterial species, for
ciated with LuxS and AI-2 production via a mechanism example a complete locus has been identified in mem-
that may be correlated with iron acquisition (Christiaen bers of B. animalis subsp. lactis, B. longum subsp.
et al., submitted). longum and B. pseudocatenulatum [61,68,70] (Table 2).
Interestingly, the G+C content of these regions deviates
Exopolysaccharide production from that of the genome and suggests their acquisition
A bacterial sEPS usually consists of a complex extracel- through a HGT mechanism [48]. Further functional
lular structure composed of a repetition of mono/oligo- analyses are needed to determine the structural diversity
saccharides linked through glycosidic bonds, which of bifidobacterial EPS polymers and their associated bio-
determines the properties of the homo/heteropolymeric logical function.
structure. The bacterial sEPS was first studied and char-
acterized in pathogens, where such macromolecules play Pili in bifidobacteria
a crucial role as a virulence factor in the interaction of The mechanism of interaction between gut microbes
the bacterium with its host through modulation of the and the intestinal mucosa may involve hair-like appen-
immune system [65]. dices, named pili or fimbriae, which are exposed on and
Differently from pathogens, the sEPS in commensal attached to the cellular surface. Apart from the process
bacteria (such as bifidobacteria) has only recently of host colonization, pili may also be involved in other
received scientific attention and very little is known cellular functions, which include protein secretion and
about its precise biological role. The involvement of conjugation [71].
sEPS in providing tolerance to bile salts and low pH has During colonization bifidobacteria are believed to
previously been eluded to [66], while a recent study con- establish an initial contact with the host surface, fol-
ducted in B. breve UCC2003 demonstrated that its sur- lowed by a more tight and specific adhesion [48]. Two
face-located EPS promotes in vivo persistence by different types of pili are held responsible for this pro-
mediating evasion of the B cell-mediated adaptive cess: the type IVb or so-called tight adherence pili (Tad
immune response in the murine gut and preventing the pili) and the sortase-dependent pili. Both of these extra-
production of proinflammatory cytokines such as IFN-g, cellular structures are composed of assembled pilin sub-
TNF-a and IL-12 [67]. units where in the case of Tad pili they are linked by
The genome of B. breve UCC2003 contains two pre- non-covalent interactions and attached to the membrane
dicted EPS-encoding gene clusters: eps1, elements of lipid bilayer, while in the case of sortase-dependent pili
which are similar to a cluster involved in the formation they are covalently anchored to the cell wall and their
of a cell wall-associated phospho-polysaccharide or pelli- subunit assembly involves the establishment of covalent
cle in L. lactis subsp. cremoris MG1363 [68] and eps2, bonds catalyzed by a dedicated transpeptidase enzyme
which is responsible for sEPS production. (so-called sortase) [72].
Interestingly, the eps2 locus is organized in two adja- Tad pili were first characterized in the Gram-negative
cent and oppositely oriented operons (called eps2A and coccobacillus Actinobacillus actinomycetemcomitans,
eps2B, Figure 2), one of which is constitutively tran- where they are shown to be required for adhesion to
scribed, while the other is transcriptionally silent unless host surfaces, colonization and pathogenesis [73,74].
Bottacinietal.MicrobialCellFactories2014,13(Suppl1):S4 Page9of15
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Figure2GenomeatlasofB.breveUCC2003.Surroundingthegenomeareschematicrepresentationsofselectedgenesorgeneclustersthat
havebeencharacterisedthroughcomparativeandfunctionalgenomicapproachesusingB.breveUCC2003asamodelBifidobacteriumstrain.
Theprovenfunctionofeachgene/geneclusterisindicatedtogetherwiththegenenameandgeneID,seetextforfurtherexplanationsand
relevantreferences.
The assembly apparatus is composed of an ATPase [64]. Oneormoresortase-dependentpilus-encodingloci
(TadA) associated with two membrane proteins (TadB arepresentinthegenomeofmost,butnotall,bifidobac-
and TadC), together constituting the secretion system, a terial species (Table 2), whereB. dentium represents the
septum site-determining protein (TadZ), which directs bifidobacterial species with the highest number of pilus-
the pilus secretion machinery to a cell-polar position, encodingloci(Table2)[21]. Atypical sortase-dependent
and finally a peptidase (TadV), which cleaves a leader pilus-specifyinglocusinabifidobacterialgenomeconsists
peptide from the prepilin precursors [75]. This locus is of a gene cluster composed of one or two pilus subunit-
present in both Bacteria and Archea, and is for this rea- encoding genes with a dedicated sortase-encoding gene
son termed a Widespread Colonization Island (WCI) placedinanadjacentposition[64].
because of its common presence among and apparent Various sortase-dependent pilus-specifying clusters
mobility between phylogenetically distant micro-organ- found in other bacteria are thought to have been
isms [76]. Highly homologous Tad pili-encoding genes acquired through lateral transfer due to their deviating
are present in all currently sequenced bifidobacterial (G+C) content, and a phylogenetic analysis conducted
genomes, which enforces their presumed role in the on bifidobacterial gene clusters predicted to be involved
establishment of a direct interaction with their host. A in the biosynthesis of sortase-dependent pilus-like struc-
recent description of the genome sequence of B. breve tures was shown to be consistent with this notion [71].
UCC2003, together with an in vivo transcriptomic and Notably, a recent study conducted on B. bifidum
mutational analysis, confirmed the involvement of Tad PRL2010 established an additional function of sortase-
pili in murine gut colonization by B. breve [48]. dependentpiliastheywerenotonlyshowntobeinvolved
In contrast, sortase-dependent pili have been observed inthespecificbindingtoextracellularmatrixcomponents
decoratingthebifidobacterialcellsurfaceandtheirexpres- (in this case fibronectin and collagen), but were also
sionseemstobestronglydependentongrowthconditions shown to be responsible for bacterial aggregation [59].
Bottacinietal.MicrobialCellFactories2014,13(Suppl1):S4 Page10of15
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Perhapsthisaggregationphenotypemayallowindividual bifidobacteria to establish as gut commensals, which
cells of this gut commensal to adhere to each other in also includes their beneficial effect pertaining to patho-
order to enhance the colonization of the host mucosa, gen protection/exclusion [87]. Furthermore, our results
using a mechanism similar to the one described for the indicate that LuxS activity is involved in iron acquisi-
probiotic strain Lactobacillus rhamnosus GG [77,78]. tion, a property that gives B. breve UCC2003 a competi-
Moreover, this study demonstrated that such pili play a tive advantage in iron-limited environments such as the
roleinmodulatingthehostimmuneresponse,ontheone gastrointestinal tract.
hand inducinga high level ofexpressionofTNF-acyto-
kines known to be produced at local level as a result of Production of bioactive metabolites
inflammatory disease, while on the other hand acting as Metabolic end products, such as SCFA, vitamins, polyun-
low-levelinducersofotherproinflammatorycytokines(e. saturated fatty acids such as conjugated linoleic acid also
g., IL-12), associated with systemic responses [59]. contribute to intestinal functionality and probiotic attri-
Furthermore, the induction of TNF-a exerted by pili of butes of bifidobacteria. SCFAs are the end products of
PRL2010 cells may be crucial for the initiation of cross- bifidobacterial fermentation of complex carbohydrates in
talkamongimmunecellswithoutcausinganyinflamma- the GIT, and have been found to be key for human
tion or detrimental effects [79]. In fact, the infant’s metabolism as they stimulate water and sodium absorp-
immune system is immature and the presence of pro- tion, lower luminal pH and the bioavailability of toxic
inflammatorystimulisuchasthoseprovokedbypiliofB. amines [88]. Recently, itwas shown that acetate produced
bifidumPRL2010maybeessentialtoachieveappropriate by bifidobacteria could enhance intestinal defence
developmentalimmunologicalprogramming[59]. mediated by epithelial cells and thereby protecting the
host against infection by E. coli O157:H7 [89]. In addi-
AI-2 production by bifidobacteria tion, while bifidobacteria do not produce butyrate as an
A wide range of Gram-negative and Gram-positive bac- end product of fermentation, de Vuyst and Leroy have
terial species produce the quorum sensing molecule demonstrated the importance of cross-feeding on acetate
AI-2 and for this reason it is often referred to as the by butyrate-producing bacteria in the gut [90]. Butyrate is
interspecies signaling molecule. The key enzyme for the primary energy source for colonocytes and has
AI-2 production is LuxS, which is an essential part of attracted much research interest due to the possibility of
the activated methyl cycle, involved in recycling S-ade- its use for the prevention of colon cancer [91]. Conju-
nosylhomocysteine. More specifically, LuxS catalyzes the gated linoleic acid (CLA) refers to a mixture of positional
cleavage of S-ribosyl-homocysteine to homocysteine and and geometric isomers of the essential fatty acid linoleic
4,5-dihydroxy-2,3-pentanedione (DPD), which subse- acid (C18:2, cis-9, cis-12 octadecadienoic acid). CLA has
quently leads to the production of AI-2 [80]. Although been reported to be produced by some human bacterial
AI-2 is commonly linked to virulence and pathogenicity isolates from different bacterial groups that include
[81,82], it has recently been shown that probiotic Lacto- Lactobacillus, Propionibacterium, Bifidobacterium, Pedio-
bacillus strains, including Lactobacillus acidophilus coccus, Enterococcus and Lactococcus. Among bifidobac-
NCFM, Lactobacillus rhamnosus GG and Lactobacillus teria, strains of B. breve have been show to produce high
reuteri, each harbour a functional luxS gene and pro- levels of CLA [92]. CLA has been shown to exert several
duce AI-2 [83-86]. We recently established that all Bifi- health benefitsand hasbeen demonstrated tohave potent
dobacterium strains sequenced to date harbor a luxS anti-inflammatory, immunomodulatory, anti-obese and
gene, and our investigations have demonstrated that all anti-carcinogenic activity, along with the ability to
tested bifidobacterial strains, representing 11 species of improve biomarkers of cardio-vascular health [93].
this genus, were capable of producing AI-2. Through
insertional inactivation and subsequent complementa- Carbohydrate transport and metabolism by bifidobacteria
tion experiments we demonstrated that a functional One way by which gut commensals exert their beneficial
luxS gene is necessary for bifidobacterial AI-2 produc- effect on their hosts is by degrading diet-derived carbo-
tion. In addition, we observed downregulation of genes hydrates that cannot be digested by host enzymes, such
associated with iron transport in a luxS insertion mutant as plant-derived glycans (e.g., glucans, fructans, xylans,
strain, UCC2003-luxS, during in vitro growth. Consis- resistant starch, pectins, cellulose, arabinoxylan, and
tent with this result UCC2003-luxS was shown to be their respective oligosaccharide degradation products),
more sensitive to various iron chelators, and unable to and host glycans (e.g., Human Milk Oligosaccharides or
colonize the murine gastrointestinal tract, while this HMO, and mucin-type O-and N-glycans).
mutant also conferred less protection against Salmonella Of suchcarbohydrates,bifidobacteria candegrade cer-
infection in a C. elegans nematode model. Collectively tainpolysaccharidesbyextracellularenzymesintomono-
these data demonstrate the importance of LuxS for and/oroligosaccharides,whicharetheninternalizedusing
