Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on -mannan possess distinct manno- oligosaccharide-binding profiles

Ejby, M.; Guskov, A.; Pichler, Michael Jakob; Zanten, G. C.; Schoof, Erwin; Saburi, W.; Slotboom, D. J.; Hachem, M. Abou

Published in: Molecular Microbiology

DOI: 10.1111/mmi.14257

Publication date: 2019

Document version Peer reviewed version

Citation for published version (APA): Ejby, M., Guskov, A., Pichler, M. J., Zanten, G. C., Schoof, E., Saburi, W., Slotboom, D. J., & Hachem, M. A. (2019). Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on -mannan possess distinct manno-oligosaccharide-binding profiles. Molecular Microbiology, 112(1), 114-130. https://doi.org/10.1111/mmi.14257

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Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on -mannan possess distinct manno- oligosaccharide-binding profiles

Ejby, M.; Guskov, A.; Pichler, Michael Jakob; Zanten, G. C.; Schoof, Erwin; Saburi, W.; Slotboom, D. J.; Abou Hachem, Maher

Published in: Molecular Microbiology

Link to article, DOI: 10.1111/mmi.14257

Publication date: 2019

Document Version Peer reviewed version

Link back to DTU Orbit

Citation (APA): Ejby, M., Guskov, A., Pichler, M. J., Zanten, G. C., Schoof, E., Saburi, W., Slotboom, D. J., & Abou Hachem, M. (2019). Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on -mannan possess distinct manno-oligosaccharide-binding profiles. Molecular Microbiology, 112(1), 114-130. https://doi.org/10.1111/mmi.14257

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This article isThis article by protected copyright. All rightsreserved. 10.1111/mmi.14257doi: to lead copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot interests: of Conflict words: Key * 4 3 974 2 224, 1 Hachem Abou Ejby M. title: Running lactis Two Article :Research Article type 0000 ID : (Orcid ABOU HACHEM MAHER DR. D Correspondance University, Hokkaido C, Denmark 26, Frederiksberg Food Science, Rolighedsvej Dept. ofCopenhagen, University 4, Nijenborgh (GBB), Biotechnology & Sciences Biomolecular Inst. Gron. Groningen, of University Dep Denmark, of University Technical Accepted Article 0000 ID (Orcid : GUSKOV R. ALBERT 7 AGG

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human gut microbiota (HGM) contributes profoundly to maintaining local and systemic health as as health systemic and local maintaining to profoundly contributes (HGM) microbiota gut human ,

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- ABC uptake system. system. uptake ABC Structural analys Structural bt t but , Bl idn cassette binding

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(Clemente and polysaccharides polysaccharides and Bl he highlights highlights K MnBP1 . bifidobacterial lactis

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This article isThis article by protected copyright. All rightsreserved. characterized. biochemically and structurally are analyses proteomic and growth through verified is locus the by of metabolism and uptake the mediates that locus utilization mannan the identifies study This utilization mannan described been have bifidobacteria from (Okubo 2019) intestinalis phylum ovatus Bacteroides gut the with consistent is mannans to humans of exposure The (GG) gum guar and fib dietary occurring group hydroxyl C6 the at ca or (glucomannan) moieties glucosyl contain may which β comprising backbone common a share mannans Structurally, 2010) carob, e.g. endosperms, seed in reservoirs β date. clear to (Turroni more oligosaccharide of modularity the of aspect Another ATP co typically to are importer coupled is translocation domai binding the nucleotide cytoplasmic and (TMD) domains transmembrane two by is formed which transporter, the permease of the into are released ligands oligosaccharide SBPs, by lipid (SBP) proteins extracellular the by largely defined is importers ABC of specificity Garrido 2013, systems be (CAZymes) to predicted are genomes enzymes bifidobacterial active carbohydrate the of few a only - Accepted ArticleMannans Bifidobacterium animalis Bifidobacterium (Tomlin

than one SBP per transport system as observed in human gut commensals commensals gut human in observed as system transport per SBP one than . or ,

M

et al. et (Turroni et al. et (Bågenholm na ws lo eotd o stimulate to reported also was annan

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t al. et , 1994) , , 2010, Milani 2010, , structural structural

in Gram positive bacteria positive Gram in t al. et

t al. et 1986) , strategy of of strategy wih s model a is which ,

are are e

21, Parche 2011, ,

and mice and rs, mannan hydrocolloids, e.g. hydrocolloids, mannan rs, t al. et 2017) , widely used as food food as used widely parts in the the in parts - Rcnl, the Recently, . localised on the same locus, consistent with their functional association. association. functional their with consistent locus, same the on localised i adto t aeyain (Fig acetylation to addition n

2017) , et al. et

subsp. subsp. members of this genus genus this membersof , especially especially ,

(Berger , 2016) ,

. lactis t al. et eiells cl wall cell hemicellulose ey recen Very

ns. The genes encoding the different modules of an ABC ABC an of modules different the encoding genes The ns. lcn utilis glycan

et al. et . The functional significance of the multip the of significance functional The . (Ejby fermentation fermentation

competitive competitive ATCC27673 ATCC27673 2007) , (Kulcinskaja guar, legumes, coconuts and coffee and coconuts legumes, guar, ATP

extracellular. By contrast, numerous carbohydrate uptake uptake carbohydrate numerous contrast, By extracellular. additives additives , 2014, Tomlin 2014, ,

et al. et

- h poieain of proliferation the idn cass binding This

tly, tly, r ecdd y bifidob by encoded are , 2016, Ejby 2016, , ing has not been been has not k -

(Gallagher primary degrader of mannan mannan of degrader primary onjac glucomannan (KGM), glucomannan onjac specific ABC importers is the occurrence of of occurrence the is importers ABC specific the the study promotes our understanding of mannan mannan of understanding our promotes study of galactomannan has been demonstrated by by demonstrated been has galactomannan of

(Loquasto t al. et generalist from the the from generalist .

n be galactosyl be n 1 fermentation of of fermentation butyrate producing producing butyrate . T . A

ette (ABC) (ABC) ette

et al. et 21, Morrill 2013, , ). Besides their relevance as naturally naturally as relevance their Besides ). he composite composite

(Cantarel et al. et

two explored et al. et -

(1 , 1986) , et al. et Bifidobacterium →

, 2013) , ABC importer associated associated importer ABC , 2004, Prajapati 2004, , 4) , 2013) , - im

. decorated linked mannosyl residues, residues, mannosyl linked

t al. et to to date. (Lundqvist Functional β Functional - these fib these a porters porters anchored solute binding binding solute anchored . After the initial capture capture initial the After . tra Te fiiy and affinity The cteria. dominant dominant

t al. et . (Scheller & Ulvskov, Ulvskov, & (Scheller from 2009) , Fimicute The functionality of functionality The licity of SBP is not is SBP of licity

(Andersen Carob e 2015) , (galactomannan) L Rosa (La Bifidobacterium rs spp. in humans humans in spp. -

yrlss by hydrolysis t al. et

Bacte in the human human the in

et al. et - noe by encoded mannanases

Roseburia Roseburia bean gum gum bean , but the the but , 2002) , r , 2013) , iodetes iodetes

β t al. et t al. et SBPs SBPs MOS . , , .

This article isThis article by protected copyright. All rightsreserved. supernatant glucose of ABC efficiently g of lack degrade mann putative a possesses B 5 family Previously protein solute binding two with cassette system binding endo secreted a on relies animalis B. of system utilisation mannan The ovatus co (Fig culture the of 85% approximately for accounting hours 12 after strain this grew animalis substrates(Fig. both on twostrains seemed the similar of relative proportions co the of rate growth the by al. of presence the in growth the To gum. from mannan Guar substituted galactosyl highly 1 (Fig. acidification by judged as (IVN) mannan nut ivory gluco Konjac on B mannans Bifid Results supplementation dietary health on based microbiota the manipulate to interventions oligosaccharide and bifidobacteria by catabolism . .

- , 2017) ,

animalis βMOS

Acceptedanimalis carob the rate as growth asimilar showed glucose on culture Article the evaluate the competitiveness of thecompetitiveness evaluate obacterium transport system system transport .

a conserved strictly

nd

βMOS

subfamily 8 subfamily o eiy h lclzto o ti enzyme this of localization the verify to subsp.

.

ot o mna. y contrast, By mannan. on rowth in this organism this in , The co The n oul mannans soluble on B s

the subsp.

subsp. subsp. . (

0.27±0.02 U 0.27±0.02 lactis , but but , lactis

extra - animalis - mannan (KGM), while only limited growth was growth limited only while (KGM), mannan growth was performed on the intact polymer and on a hydrolysate thereof prepared prepared thereof hydrolysate a on and polymer intact the on performed was growth

lactis rapidly reached a 50:50 level, which persisted until the end end of theculture the until which level, persisted reached a50:50 rapidly lactis lacks the ABC the lacks

- (GH5

ellr endo cellular efficiently competed with with competed efficiently in the the in

-

β cultures appeared higher on the intact polymer intact the on higher appeared cultures ATCC27673

an utilisation locus locus utilisation an Bl subsp. - mL _8 . mannanase from mannanase W - MUL ) (Carbohydrate Active Enzymes database at database Enzymes Active ) (Carbohydrate 04 - model 1 e analysed manna analysed e , )

with only aminor only with (Morrill possess l actis (Fig this strain this

- uptake system uptake β mannan utilising mannan - anns ( mannanase

. displayed displayed

ATCC27673 2 es

A) t al. et

n dnia GH5 identical an , B . animalis B. in the utilisation of of utilisation the in suggesting (MUL) . 2015) , animalis B na grow uptake a

Bl ovatus . identified in the present study, study, present the in identified fraction present in present fraction se activity on cells grow cells on activity se

Man5_8) Man5_8) . grows efficiently efficiently grows that encodes intracellular enzymes necessary to to necessary enzymes intracellular encodes that

th s

commensal commensal

a bohmcly characterized biochemically was The The

, which ,

that this transporter is the the is transporter this that subsp. subsp.

on soluble galacto soluble on subsp. subsp.

throughout the exponential phase and out and phase exponential the throughout B ciiy was activity _8

C , - eogn t te glycos the to belonging

hydrolysate, but the ratio between the ratio but hydrolysate, lactis is valuable for the design of efficient efficient of design the for valuable is endo Carob galactomannan, galactomannan, Carob ). No growth was observed on the the on observed was growth No ). lactis Bacteroides Bacteroides observed on crystalline insoluble insoluble crystalline on observed

-

cell pellets mannanase (Morrill and competitively competitively and

than than ATCC27673 - β http://www.cazy.org/) fr http://www.cazy.org/) - found found - mannanase and an ATP an and mannanase mannan from Carob and and Carob from mannan ing 1 the hydrolysate, but the the but hydrolysate, the D,

et al. et ovatus

on ( E 0.055±0.001 U 0.055±0.001 to promote human human promote to n ecds a encodes and consistent with the the with consistent sole largely largely ). ). . carob , 2015) , 1 Interestingly, Interestingly,

, E

(Bågenholm uptake uptake we performed performed we ide ). The control control The ). which .

This strain strain This mannan on soluble soluble on

in culture culture in . Notably, Notably, . hydrolase hydrolase .

system system grow

mL full full B o

B or m a et et s - - . . -

This article isThis article by protected copyright. All rightsreserved. by recognized data SPR with agreement good in were constants a unfavorable by driven (IT binding calorimetry 1:1 titration a isothermal revealed using determined were mannotriose and mannobiose both for stoichiometry binding and parameters thermodynamic The Bl glucosyl and (MG) gluco on range same the in Bl 3 (DP) polymerisation and measured was affinity highest The ( mannooligomers galac xylose or glucose characterized βMOS The manno proteins binding two The internal in sidechain α putative a to contrast uptake from strain are system ABC associated the and SBPs two these encoding genes the that revealed which analysis, genomic a with galactomannan carob locus same designated 2A β intracellular glucose. on repressed ma 1 )

MnBP MnBP (Fig. n Accepted Article putative two encodes system ABC this Interestingly, ). n Bl t an ooligosaccharides . - MnBP

oligosaccharide (βMOS) s atvt fo te lcs clue ugse ta te ciiy s nue o mannan on induced is activity the that suggested culture glucose the from activity ase Interestingly, the GH1 the Interestingly, 2 - 2 2 mannobioses, . A B compared to mannotriose to compared Bl were were , niaig h secret the indicating ),

the the

binding entropy (Table (Table entropy binding

. 2 MnBP1 and MnBP1 similar trend was observed with galactosyl decorated mannobiose mannobiose decorated galactosyl with was observed similar trend

- , respectively. Both proteins displayed displayed proteins Both respectively. , idn proteins binding ~5 kcal mol~5 Both Both ansds (H) n a β a and (GH2) mannosidase Bl in the top five most upregulated proteins proteins upregulated most five top the in the the Fig. S1) S1) Fig. - for 04 - ized galacto mannose (GM), mannose ) proteins

the longer the − only transport proteins proteins transport only as compared to the glucose proteome (Table 1, Table Table 1, (Table proteome glucose the to compared as counterpart or 6

where The . - r raffinose or By contrast, contrast, By Bl galactosidase of GH36, which may confer the hydrolysis of galactosyl galactosyl of hydrolysis the confer may which GH36, of galactosidase Bl - 1 bind to able were

non MnBP2. MnBP2. additional binding energy binding additional MnBP1 and MnBP1 had had . animalis B. Bl

- β -

mannan MnBP possess complementary and partially overlapping specificities overlapping partially and complementary possess βMOS - βMOS glucosidase in the locus the in glucosidase

Bl o esrbe fiiy to affinity measurable no (Elbourne f o o te enzyme the of ion o

MnBP1 and and MnBP1 (Table 2 (Table 3 respectively, while no while respectively, r B

, ) mannotriose with with mannotriose

mannobiose mannobiose oth these SBPs together with the GH2 the with together SBPs these oth . 1 Fig. S Fig.

. This This .

favorable (Table 1, 1, (Table

showed showed T Bl oligosaccharides oligosaccharides

hirteen hirteen MnBP2 MnBP2 subsp. subsp. -

to at least one of the binding proteins binding the of one least at to ) lcsds (GH1 glucosidase 2 et al. et , differential affinity for disaccharides was also observed observed also was disaccharides for affinity differential itnus the distinguish

) while while . The affinity trend and the magnitude of the binding binding the of magnitude the and trend affinity The . K

Table Table

nhly hne wih a off was which change, enthalpy (Table (Table d , 2017) , was bound with with was bound iad rsmln βO ad ie gluco mixed and βMOS resembling ligands Bl

lactis of the ABC transporter that confers uptake of β of uptake confers that transporter ABC the of values of 4 µM and 33 µM 33 and µM 4 of values the affinity of affinity the MnBP2 by by similar

S1 K to the the to

Bl 2, d

in the proteomic the in ). was not upregulated on galactomannan, in in galactomannan, on upregulated not was ATCC27673 ATCC27673

in support of their crucial role for for role crucial their of support in values of of values

b MnBP βMOS Table Table ( inding

maltotriose, cellotetraose, xylotriose, xylotriose, cellotetraose, maltotriose,

monosaccharides monosaccharides B. affinit ) ee rdcd eobnnl and recombinantly produced were utr supernatant. culture

and animalis 1 - 3) at least 257 at least Bl Bl binding proteins, which we have have we which proteins, binding

of of compared to compared to

ies , 81 n B tasot system transport ABC an MnBP MnBP confirming that that confirming these ligands these

n for for MUL M and M

subsp. analysis of cells grown on grown cells of analysis 1 and and 1 1 βMOS β

to the disaccharide disaccharide the to S1 - 0 (Table (Table mannosidase for mannosyl for - encod ( fold 71 mannose, galactose, galactose, mannose, . hs s consisten is This ). Bl lactis Bl (Fig.

MnBP2 n

with a degree of of degree a with MnBP

was observed to to observed was - for M lower The lack of of lack The es set by a large large a by set 2 mannobiose mannobiose )

2 . ATCC27673 ATCC27673 C

a predicted predicted a 2 , . Bl affinity affinity

T

from the the from - towards towards MnBP glucose able C) and and C)

βMOS βMOS

(Fig. (Fig.

was and

2 β to to is ). 1 - - - t

This article isThis article by protected copyright. All rightsreserved. 60 about being mannotriose for 5 Table 2, to affinity S ( resonance plasmon surface ( N63G mannobiose: of recognition Two for this isalsovalid suggest that M5 and M4 for affinities of accommodation the mannosyl ( charge the of loss the to substitution ( Bl difference between interactions polar D338 W303/30 (Fig. OH) by recognized the between sets data Bl two the spanning sheet domain the at interface located site binding ligand the with region hinge tripartite a by joined size different classification structural to according Å the as of structures Crystal unit. end mannosyl penultimate reducing A Bl 3C, Fig. 2 , respectively , MnBP2. loop hinge This respectively. MBP2, variant )

AcceptedMnBP Article . The . recognition recognition mannobiose , K287/285 K287/285 , , ige m single 3 Bl whereas B

MnBP2 complex with mannotriose with complex MnBP2 . - in 1

loss of the aspar the of loss 2_G6 (Fig. (Fig.

D). ) lcrn d Electron unit residue residue D) mannobiose between between .

This substitution abolishes an abolishes substitution This is likely to to likely is By contrast, the contrast, By .

The The two s Bl utants were constructed to to constructed were utants Another difference is is difference Another dense dense 1

3 of (

the the at position at N (N A MnBP1/ Table Table s oprd o h wl tp protein type wild the to compared as

) . The ). these residues these between . The binding of these mutants to mannobiose and mannotriose was explored with with explored was mannotriose and mannobiose to mutants these of binding The . ζ ansl t oiin 2 position at mannosyl domains

mannosyl at position 3 is stabil is 3 position at mannosyl Bl – two Bl niis o te bound the for ensities polar contacts contacts polar

C2 MnBP 4 MnBP abolish or abolish and mannotriose respectively respectively mannotriose and ).

- Bl diinl ansl units mannosyl additional distance ig rgo cmrssto hr β short two comprises region hinge

OH) a oan ad t and domains Both Both s the the gine side gine MnBP2 .

Bl

- 4 1 and and 1 SPR Bl fold 1 in complex with with complex in 1 T

MnBP , S99 S99 , C2 and he MnBP1 transport proteins proteins transport

. in )

- increase from 2.4 Å to 3.4 Å). 3.4 Åto 2.4 from increase weaken the the weaken

OH and N and OH

T terminal reducing reducing terminal

and and to some degree some to

Bl n plr neatos ewe C6 between interactions polar and

(O Bl by by he 1 chain in chain MnBP

the the MnBP and 1

(Berntsson - in – isothermal titration titration isothermal kinetics of binding were grea were binding of kinetics the side chains of of chains side the appeared like variant ( variant like

C6 he loop loop he extra hydrogen bond bond hydrogen extra substitution of of substitution

Bl evaluate the role of of role the evaluate Bl

2 - βMOS ε were solved to solved were is stabilised by by stabilised is MnBP 1 OH) 2

MnBP2 Bl

H . is the substitution of N63 in in N63 of substitution the is N223/221, N223/221,

Thus, t Thus, MnBP1_N63G - mannobiose bond bond flexibl

adopt a canonical SBP fold ( fold SBP canonical a adopt and Q307/305 and

t al. et D380 1 positions

dniid h ligand the identified ized by stacking interactions onto interactions stacking by ized Bl (

as compared to to compared as i. S3) Fig. contributes to contributes to the C6 the to ( he binding site binding he ansl unit mannosyl MnBP Bl with , e as judged by the the by judged as e 2010) , – MnBP1_N63G 38 n D380 and Y388 C3 Bl D3 W calorimetry ( calorimetry resolution MnBP2. , mannotriose , 283/281 - 5 21 OH 2_G6 aromatic

- .

the strands arranged in in arranged strands was suggestive of lower stringency lower of suggestive was - caused a 63 a caused between

), which comprises two domains of of domains two comprises which ), The space available and the similar similar the and available space The OH of the mannosyl at position 2 position at mannosyl the of OH N

Notably, t Notably, Bl (N

and and

63 of of 63 1 corresponding gain in affinity for for affinity in gain corresponding MnBP ε N 2

substrate affinity by binding the the binding by affinity substrate -

(N tly affected by these mutations, these by affected tly s Bl C3 O ) is

of the ligand ligand the of - of 1. of stacking onto W216/214 and and W216/214 onto stacking gained 1 MnBP H n ete O either and OH ε1 )

spacious enough to to enough spacious N - Bl - ITC G337/335 idn site binding OH) 1

- he poor electron density electron poor he 63 and manno and

poor electron density electron poor

and and MnBP – - O5) to N336 N336 to 6 Bl fold Y )

O

38 Å MnBP 1 ( in in about 100 about assigned into into assigned T δ1 ,

, G based on SPR on based 6 able 2 able

and 157 fold 157 and E284/282 2.0 Å 2.0 Bl and and 1 , 6

. 1N MnBP1/ n anti an

in in the differential differential the in T 1 pentaose OH of C2 or C2 of OH Bl he most striking striking most he

at the interface interface the at (

, to Bl , Table Table , oiin 1 position Y339/337 and Y339/337 MnBP2. MnBP2. n Bl in 2. - MnBP1 a glycine a δ1 fold affinity fold 2 -

aall β parallel (O Bl Å r O or allow the the allow cluster B cluster - MnBP2 MnBP2 MnBP ε2

and 2.0 and drop in in drop as well well as 5 (

– Table Table , in all in

δ2

T Fig. Fig. ) C1 and and due

for for h C

of of in in in is is 2 6 - -

This article isThis article by protected copyright. All rightsreserved. co (1 excluding of 1 and 2 positions at stringent more was mannosyl for preference oligosaccharid drop affinity unit mannosyl of recognition preferential The is set numbering) 1 position through established mannopentaose βMnOS of structures crystal The manno for specificity of Basis animalis the knowledge. our to unexplored remains systems such of functionality longum Bifidobacterium single a are they and O potential therapeutic the of import Restoring barrier F Discussion mutants (Fig. e.g. iber ligo → unterparts, which demonstrates demonstrates which unterparts, within hydrogen bonding distance from from distance bonding hydrogen within s affinity for affinity Accepted Article

manno the increase of affinity of of affinity of increase the preference preference 6) - 4 saccharide specific specific saccharide poor diet is associated associated is diet poor

ance to combat to ance - ) healthy healthy (Desai galactosyl at the reducing end was end reducing the at galactosyl , although , . .

subsp W related

irboa y osig pcfc rus osdrd o e health be to considered groups specific boosting by microbiota - oligosaccharide

oligosaccharide specific ABC systems that confers confers that systems ABC specific oligosaccharide

by two hydrogen bonds bonds hydrogen two by e at position 2 position at

common in in common a of of

et al. et es (Table (Table es k the GGM trisaccharide (Table (Table trisaccharide GGM the HGM

lactis for the for er beyond beyond 2−3 orders of magnitude for magnitude of orders 2−3

the magnitude of the kinetic rate constants could not be determined reliably determined be not could constants rate kinetic the of magnitude the non lcrn de electron , 2016, Porter & Martens, 2016) Martens, & Porter 2016, ,

h rcgiin f h ail C2 axial the of recognition the to ATCC 27673 -

disease states disease su axial C2 axial position catabolise soitd with associated human gut gut human b

ABC importer genes importer ABC 2 restricts space for equatorial for space restricts Bl strates - subsp. subsp. . ). specific MnBP1 and and MnBP1 - Bl with oligosaccharides T n -

OH in mannosyl. mannosyl. in OH MnBP e oeac fr glu for tolerance he s , e.g. e.g. , 3 t ws observed was ity tolerance to to tolerance

infantis

compared to positions 1−3 1−3 positions to compared . non a shift shift a

Clostridia

related to dysbiosis. dysbiosis. to related ABC transporter is observed in genomes of HGM, HGM, of genomes in observed is transporter ABC cello - 2_G6 digestible glycans digestible ( Fig

Bl

only only the indole indole the n irba cmoiin n a and composition microbial in - (Garrido specific . MnBP2 oligosacchar 1

N

. s 3). 3). 2

galacotsyl decorations decorations galacotsyl manno

are ubiquit are T )

is in is was associated with a marked a with associated was . about 10 about he occurrence of more than one SBP one than more of occurrence he

This recognition recognition This Stacking Similarly, the C2 the Similarly,

that protect that

accord provide a structural structural a provide - t al. et n bacterial in the complex structure of of structure complex the in oy ws highest was cosyl gluco -

itrogen of W303/301 and the the and W303/301 of itrogen OH C2 i - Increased f Increased

d fold lower as compared to the unsubstituted unsubstituted the to compared as lower fold ous in bifidobacterial in ous provides a rational basis rational a provides - es.

OH group OH 21, Garrido 2011, , - ru of group with the binding data, which which data, binding the with no W216/W214 onto oligosaccharides as compared to manno to compared as oligosaccharides

( A Fig. S Fig. s ffinity to to ffinity

, as judged by the the by judged as , groups groups epithelial cells from microbial insult microbial from cells epithelial efficient efficient pattern - OH of the the of OH unctional i unctional Here we Here 3

present in galactomannan (Table (Table galactomannan in present s h reduc the ) . in The specificity for βMnOS for specificity The

i deay interventions. dietary via basis basis

t oiin 3 position at βMnOS the glucosyl epimer, which which epimer, glucosyl the is likely to be important in important be to likely is rwh n mannan on growth

weakening of the mucin mucin the of weakening describe describe t al. et - increase increase nsight into nsight

mannosyl at position 3 position at mannosyl promoting for the the for glycan utilization loci loci utilization glycan ing end mannosyl at at mannosyl end ing ( Bl substituted with α with substituted severe drop or loss or drop severe MnBP1/ for for 2015) , N in association to association in Bl ε two SBP two decrease decrease 2 harnessing the harnessing wh , pcfct for specificity MnBP of of

disclosed an disclosed the is Q223/221. Q223/221. , Bl ereas the the ereas

of great great of for both for abilities abilities but MnBP2 1 s

by by in in

from from with with e.g. e.g. the the k

B. off is - - .

This article isThis article by protected copyright. All rightsreserved. GlnPQ the of domains transmembrane the mutant in increase the abolishing of provides whereas histidine either animalis Bl that showed analysis Phylogenetic animal of Homologues proteins solute binding multiple preferences uptake by of Diversification de xylan cross confers which arabino the for capture their after ligands of Cp position at contacts aromatic and polar additional the ( orthogonal of binding manno end reducing the accommodates 1 Position 2) occ (Chekan counterp of Comparison the of products 3 DP of oligomers to proteins transport the of preference 2015) animalis of binding plausible a provides of which accommodation cavity, exposed solvent a into explana oriented is 1 position at mannosyl 2 ,

MnBP1 Fig AcceptedMnBP Article luded, as compared to the more shallow and open binding site binding open and shallow more the to compared as luded,

. and the lack of growth on this substrate this on growth of lack the and

Bl and and S1 (Fig. (Fig. tion tion

graders.

in bonding hydrogen of capable are which , r fo te oeaey hrohlc bacterium thermophilic moderately the from art Bl MnBP1 or or MnBP1 support for the striking impact of a single substitution on substitution single a of impact striking the for support et al. et subsp.

subsp. - is )

. βMnOS with adjacent galact adjacent with βMnOS MnBP2 k or human human h non the

likely to likely Fig. of Although the Although f 4 or f Bl

). , 2014) , for the the for - mannanase extracellular

lactis Bl xylooligosacchar

MnBP2 lactis F

S the the

Bl unctional differences unctional 4 MnBP2 N63 polar bond to the mannosyl at position 2 is evident from the considerable considerable the from evident is 2 position at mannosyl the to bond polar N63 - f - the the like like ). a MnBP1/ reducing end in in end reducing - ecal feeding of bifidob of feeding lower lower fiiy for affinity

Bl Bl From the functional the From

β reveals encoded two different copies, whereas most strains encoded a single copy of copy single a encoded strains most whereas copies, different two encoded same substitution at position position at substitution same - MnBP2. MnBP2. MnBP1 N63G variant N63G MnBP1 possess - mannanase on on mannanase

like isolates isolates

binding site of the the of site binding or or the the . Indeed, the measured the Indeed, . Bl . Bl Depending on the taxonomic group, only a few strains, mainly from mainly strains, few a only group, taxonomic the on Depending pronounced pronounced MnBP2 dissociation dissociation

MnBP1 MnBP1 a glycine a i e.g h former The d galactosyl bifidobacterial bifidobacterial e transport protein from the same organism organism same the from protein transport e . Cp from

dens a osyl substitu osyl were to the only structurally and biochemically characterized characterized biochemically and structurally only the to (Morrill MnBP are encoded by the genomes of a variety of bifidobacteria of variety a of genomes the by encoded are cteria on on cteria

or more rarely rarely more or point of view, the occlusion of the the of occlusion the view, of point differences: 1) differences: B. rate constant ( constant rate e -

ly galactosylated mannan from Guar gum Guar from mannan galactosylated ly ABC ABC reported substituted

animalis

βMOS , and 3) the orientation of the the of orientation the 3) and , and and are distinguished by by distinguished are as opposed to opposed as

et al. βMOS xylan from the the from k of of t off ions

3

s , 2015) is relatively relatively is 3

between a tandem repeat of two SBPs fused to to fused SBPs two of repeat tandem a between

yl in the bifidobacter the in yl atccu lactis Lactococcus , whereas sufficient space seems to to seems space sufficient whereas , values are in the same range as those me those as range same the in are values subsp. subsp. similar a

in the bifidobacterial proteins as compared to to compared as proteins bifidobacterial the in (Rogowski , - binding proteins could be classified as either as classified be could proteins binding

βMnOS

an k 4 consistent with the lack of activity of the the of activity of lack the with consistent The off also r . alanine ) of of ) eciprocal eciprocal

the less su less the lactis

binding site of site binding adneois polysaccharolyticus Caldanaerobius correlates to the dominant hydrolysis hydrolysis dominant the to correlates the ligands the manner to the corresponding corresponding the to manner . occluded of of affinity A similar cavity may allow the the allow may cavity similar A

and and et al. et ( Fig. Fig. Cp an effects effects

B. bstituted galactomannan bstituted MnBP asparagine

, 2015) ,

5 ht confer that for adolescentis ) , ial proteins as opposed to to opposed as proteins ial , which may which ,

. the C6 the O for the the for M2 and M3 and M2 ligand sites binding Bl

ur mutational analysis analysis mutational ur (Chekan MnBP1/

(Ejby and co and - glutamine , OH group of the the of group OH - Bl binding s

(Morrill MnBP2 G61NMnBP2 the uptake uptake the clade

t al. et - limit et al. et Bl growth with with growth .

MnBP2 the hinder The effect effect The is

2013) , ( site , 2014) , the l the Fig almost almost asured asured

et al. et . N63 or a a or . The The and

os 5

B. B. of of of is is ). s , , , ,

This article isThis article by protected copyright. All rightsreserved. motifs proteome in supernatant competitors. to leakage minimizing (CAZymes) cell al. in only not strategy Firmicutes common most the be to seems surfaces system uptake oligosaccharide attached membrane outer gum Guar substituted highly including genus loc utilization commensal (polysaccharide dominant the of members al. to contrast In microbiota of system utilization mannan The from Bacteroidetes. co the from ligands distinct of oligosaccharides to underscores of polymerization intracellular β the and transporter ABC the of components the of rest the with together study glycans host target that seven to up encode that loci h Bl animalis niche ecological specific pro to likely is importer. ABC specific oligosaccharide same the to associated SBPs separate of difference functional of report in asparagine and glutamine ost glycans from human milk or mucin by by mucin or milk human from glycans ost MnBP1 Accepted2005) , 2013) , Article - attached attached were similarly highly highly similarly were

(Ejby in the the in

species is unclear, but this maybe an evolutionary transition towards the more common common more the towards transition evolutionary an maybe this but unclear, is species The expansion of expansion The . Indeed, the modular mannanase of GH26 from GH26 of mannanase modular the Indeed, . - , the metabolism of mannan has not been addressed in detail. Recent work shows that that shows work Recent detail. in addressed been not has mannan of metabolism the , consistent like in higher animal guts. animal higher in like (Cockburn members -

growth competition assays with with assays competition growth the the may

abundant dietary glycans such as xylan asxylan such glycans dietary abundant et al. et (Kulcinskaja ie n advantage an vide . animalis B. key key enzyme enzyme

contribute to maintaining the proximity of the substrate close to the cells, thereby thereby cells, the to close substrate the of proximity the maintaining to contribute , 2016, Leth 2016, ,

contribut with

e t al. t (Garrido .

rmr structure primary enzymes, its

The rationale of maintaining the the maintaining of rationale The , 2015, Leth 2015, , high affinity capture capture affinity high

upregulated and were in the the in were and upregulated t al. et subsp. subsp.

Lactococcus lactis Lactococcus ion of of ion adjacent putative oligosaccharide specific SBP in addition to enzymes enzymes to addition in SBP specific oligosaccharide putative adjacent secretion

size and composition and and composition and size

i) al. et yial ue by used typically

et al. et βMOS ht noe h poen apparatus protein the encode that 2013) , by by transporters in addition to periplasmic enzymes and and enzymes periplasmic to addition in lactis , 2018) ,

. animalis B. The The A similar strategy might have been evolved for targeting related targeting for evolved been have might strategy similar A , 2011, Garrido 2011, , adapting adapting

.

(Bågenholm etal. hs is This

Tbe 1). (Table β (Fig . - B. bifidum B. . anns atvt was activity mannanase , Ba ovatu Ba , 2018, Møller 2018, , The competitiveness of of competitiveness The oua c Modular .. t e.g. .

to defining the preference of gut microbiota members members microbiota gut of preference the defining to to (Fulyani n line in of ligands of 2

) Bacteroides subsp. subsp. the competitive competitive the Bacteroides

n te enzyme the and et al. et e ots mecha sortase he (Leth

s

et al. et The and and , which is a model glycan utili glycan model a is which ,

with with

top five proteins i proteins five top ell et al. et support , 2017) ,

lactis

B. longum B. et al. et

by the evolution of of evolution the by , 2015) , - ih xrsin of expression high et al. et B. adolescentis B. attached attached the lack of identifiable cell attachment attachment cell identifiable of lack the

Bl

, 2016) , members

, 2018, Rogowski , 2018, nP besides MnBP2 , 2012) , is atypical compared to to compared atypical is . This . ing possess mannan utilization PULs PULs utilization mannan possess . Bacteroides Notably, both SBPs both Notably, idw f agtd glycans targeted of window

B. animalis B. competitive growth on preferred preferred on growth competitive

. su mainly mainly

was not not was carbohydrate active enzymes enzymes active carbohydrate

To our knowledge, this is first first is this knowledge, our To strategy

bsp. bsp. Displaying . nism widely employed by by employed widely nism for for and in bifidobacteria bifidobacteria inand n the differential proteome differential the n -

mannosidase required for for required mannosidase has been proposed to be be to proposed been has i radw o mannans of breakdown TonB dependent SusCD SusCD dependent TonB nfantis localised to the culture culture the to localised , but also in different different in also but , Bl

identified in the cell cell the in identified relies

subs subs MnBP1 like proteins proteins like MnBP1 Bl this this

et al. MnBP1 in the the in MnBP1 ,

s which possesses possesses which enzymes at cell cell at enzymes lactis lactis on ation generalist generalist ation uptake system system uptake , 2015, , 2015, in the present present the in

one or more more or one other gut gut other is evident evident is (Hinz Ejby

to a a to

B. et et et

This article isThis article by protected copyright. All rightsreserved. respectively. mg, 15 of amounts desalted B using chromatography column h 24 (Sigma), cellobiose acceptor M mg 0.56 pho mannnooligosaccharide 4 al. (1, Denmark) (Brabrand, Inc. Nutrition and Health DuPont from Louis, maltotriose raffinose, xylose, galactose, mannose, glucomannan g m Chemicals procedures Experimental taxa. prefe differential these of advantage taking allows groups taxonomic different by metabolism its of understanding better a justifies mannan of content GH2 upregulated the by depolymerisation confer enzyme this that A loci includes that glycans complex more (Bågenholm from energy harvesting allows that with growth The bifidobacteria amongst proposed been has sharing resource and behaviour social our bifidoba Man) Man) alactosyl annobiose putative α putative 2013) , Accepted4 Article

) knowledge of ,

- high affinity of the SBP of the the of SBP the of affinity high Glc and and Glc terminated

bifidobacte O USA) MO,

cteria. Th cteria. mL ih melt M4 Ra ad as Piaepi, A, n freeze and PA), Philadelphia, Haas, and (Roam MB4 Amberlite with were prepared by by prepared were - . mannotriose –

Man

Gluco -

- 1

Ba et al. et All chemicals were of analytical grade. The following carbohydrate ligands were used: used: were ligands carbohydrate following The grade. analytical of were chemicals All Ioy u mannan nut Ivory , galactosidase of GH36 encoded by a different locus was highly upregulated, suggesting suggesting upregulated, highly was locus different a by encoded GH36 of galactosidase (M2) through to to through (M2)

intracellular intracellular Man Ra

. by other h other by

β MP2, 0.1 M 0.1 MP2, ovatus by heating by - e - ria, which outlines a plausible a outlines which ria, , 2017) , 4Manβ . β

mannotrisaccharides

secretion of a CAZyme a of secretion in - β 4Glc, were prepared as described elsewhere elsewhere described as prepared were 4Glc,

- natural foods, foods, natural Galacto l te trisacchar the All ,

(Fig l wt purities with all the removal of galactosyl sidechains in galactomannan, prior to complete complete to prior galactomannan, in sidechains galactosyl of removal the

- 4Glc, as compared to the simpler utilisation system of of system utilisation simpler the to compared as u

β man gut microbiota members microbiota gut man sphorylase sphorylase - . ( mannosidases

100˚C for 5 min 5 for 100˚C 1 α - oligosaccharides oligosaccharides E - Man mannnose 1 mannnose Man ) mannohexaose io . , Carob galactomannan galactomannan Carob , On the other hand, other the On - of phosphorolysis reverse e P (Bio P2 Gel - this glycan is a common food additive and hydrocolloid, which which hydrocolloid, and additive food common a is glycan this β β β - - nS pae system uptake MnOS 4Glcβ

(1→ Ra (Man ides ides > MP2 MP2

5 wr from were 95%

and and is highly atypical and and atypical highly is 4

- phosphate phosphate ) - β - β 4Glc, and and 4Glc, ), migrated migrated - Glc, or Glc or Glc, - ansds (i. 6 (Fig. mannosidase

(Kawahara 4Manβ

β and t and ( - model for the utilisation of simple mannans (Fig. (Fig. mannans simple of utilisation the for model (M6) Rad; 1.6 1.6 Rad; [β - glucosidases - , HGM members. members. HGM

D and and he trisaccharides were purified by gel by purified were trisaccharides he - , Gal Ba - dicyclohexylamine salt dicyclohexylamine 4Glc, a Man 6 s . . 1 - p G

-

β The rationale for secretion for rationale The m × cm ovatus - α ige spot single al. et a gum uar - (1,4)] Megazyme Megazyme

- (1→ β ee al were D seem

. -

Man conserved , - 4Glcβ Gluco , galactosyl 0 cm 100 , 2012) , 4

e.g n=1 has not been previously reported to reported previously been not has (Kawahara possesses a more elaborate system system elaborate more a possesses ) s, β - –

Man - Table ,

dney rnhd ur gum Guar branched densely . This insight on the preference o preference the on insight This 5 uioocs albus Ruminococcus - 4 however, to enable competitive competitive enable to however, so - - ee from were 4Man were 42 mg, 16 mg, and and mg, 16 mg, 42 were 4Man mannno Glc D s

). -

Wclw Ireland) (Wicklow, ) Glc rm Megazyme from R n h TC nlss n 2 in analysis TLC the in

- β w h poe fatos were fractions pooled The

mannobiose ea - in the mannan utilisation utilisation mannan the in

4 ere B. animalis B. p

ction mixture ction 1). Glc, and and Glc, rences to rences ) et al. et -

disaccharides, disaccharides,

(Liu were kindly supplied supplied kindly were incubated at 37˚C for for 37˚C at incubated (Milani

Besides the dietary dietary the Besides Sigma , 2012, Hamura 2012, ,

- et al. et dried. Obtained Obtained dried. ,

is unclear, but but unclear, is

target specific specific target Man

- et al. et and subsp. lrc (St. Aldrich , 2015) , . Glucose, Glucose, . s

-

. β (2.5 mL (2.5 filtration

- , 2015) , β K 6 4 - β 1 Glc Man onjac onjac lactis - - , 0.2 0.2 , α 1,4 - 6

D β et et ). - - - f - - , .

This article isThis article by protected copyright. All rightsreserved. Origin of ovatus cultures pure from harvested DNA of ng 10 and 1 0.1, 0.01, Bacterial 15 for 60°C s, 15 for 95°C at done 2 for 40 by 50°Cfollowed of cycle one of consisted and amplifications both for same Mix, Master al. al. Bacteriodes Real Fast 7500 a and USA) Biosystems, (Applied Bacteriodes Relative Denmark). Aps, Wernerm (Saveen Scientific Thermo spectrophotometer 1000 NanoDrop (Bio matrizx InstaGene™ a by above) (see (qPCR) PCR quantitative and extraction DNA at stored NaCl and (8000 centrifugation by collected were c were samples The h. 24.6 and 12 6.8, 3.6, 0, at analysis qPCR for anaerobically co a inoculate 2015) 10% recombinant a with treatment by produced latter the of hydrolysate grow animalis B. UK) Ltd., Scientific media u carbohydrate SEM LAB in °C 37 at grown Anaerobic USA) D Fisher ESI by propanol/1 2 , 2010) , 2012) , Accepted ArticleSigma (99.9%, O ing (w/v) (w/v) . . Equal .

respectively , Scientific). . -

S n NR ESI NMR. and MS

the strains as above above as strains the

count . and

- growth and competition assay competition and growth Samples were analyzed analyzed were Samples butanol/water (12/3/4, v/v/v). The chemical structures of the trisaccharides were verified verified were trisaccharides the of structures chemical The v/v/v). (12/3/4, butanol/water

carob was quantified was amplified using primers targeting the 16S rRNA gene gene rRNA 16S the targeting primers using amplified was quantified was

10μM of each p each of 10μM subsp. subsp. and and - amount ( amount and growth was followed by measuring by followed was growth and Bifidobacteri culture to to culture nder anaerobic conditions using a Whitley DG250 anaerobic workstation (Don Whitley Whitley (Don workstation anaerobic DG250 Whitley a using conditions anaerobic nder s in the samples the in s

cycles of 95°C for 15 for 95°C of cycles -

galactomannan, 37 37 galactomannan, B The products gave a signal at 527.16 527.16 at signal a gave products The - , St. Louis, MO, USA MO, Louis, St. , 20 °C until further analysis. further °Cuntil 20 ifidobacterium . Growth was monitored by measuring measuring by monitored was Growth . .

lactis h saitcl infcne a aaye uig a using analysed was significance statistical The OD OD

600 a were quantified by targeting the elongation elongation the targeting by quantified were a ATCC27673 ATCC27673 600 - nm rimer (Table (Table rimer MS was performed performed was MS in the presence of of presence the in nm

units a t 0, 3.6, 6.8, 12 and 24.6 h were h 24.6 and 12 6.8, 3.6, 0, t ≈

s and increasing to 95°C at 2% ramp rate ramp 2% at 95°C to increasing and s 0.1 in in 0.1 in triplicates in ee eemnd y PR sn SB gen methodology green SYBR using qPCR by determined were )

s and 60°C and s of exponentially growing exponentially of °C

x

,

g and and LAB SEM LAB (Barrangou ) using Bruker AMX500 (500 M (500 AMX500 Bruker using ) – S overnight , 10 min, 4 °C). The pellets The °C). 4 min, 10 , -

Rad, California) and quantification was carried out with a a with out carried was quantification and California) Rad, 2

) and 5 and ) B atrie ovatus Bacteroides

.

– animalis animalis

in a total volume of 20 μL usi μL 20 of volume total a in

DNA was extracted from pellets from 2 mL culture culture mL 2 from pellets from extracted was DNA for 1 min. Finally a dissociation curve analysis was analysis curve dissociation a Finally min. 1 for 0.5 % (w/v) glucose, glucose, (w/v) % 0.5 using an Executive Mass Spectrometer (Thermo (Thermo Spectrometer Mass Executive an using -

from ) Time PCR System (Applied Biosystems, USA). USA). Biosystems, (Applied System PCR Time

ng template DNA. The PCR conditions were the the were conditions PCR The DNA. template ng

medium. The co The medium. OD al. et m subsp. subsp. 600 / z OD

2003) , . animalis B.

[M+Na] for 2 for

600

lactis cells form both both form cells nm

5 h. Samples h. 5 estimated based on sta on based estimated ooled

. animalis B. ATCC 8483, 8483, ATCC

and pH and + supplemented with 0.5% (w/v) (w/v) 0.5% with supplemented β .

- - NMR spectra were recorded in recorded were spectra NMR ATCC 27673 27673 ATCC culture was incubated at 37 °C 37 at incubated was culture mannanase ( mannanase were washed with 0.9% (w/v) (w/v) 0.9% with washed were

factor Tu gene ( gene Tu factor C subsp. subsp. immediately on ice and cells cells and ice on immediately

arob galactomannan and and galactomannan arob min and 95°C for 10 min min 10 for 95°C and min Hz, Bruker, Billerica, MA, MA, Billerica, Bruker, Hz, .

The competition assay of assay competition The (Bergström one

(2 mL) (2 ng 10 μL Fast SYBR SYBR Fast μL 10 ng subsp. subsp. lactis strains were strains - tailed tailed was performed by by performed was 2 µM 2

and Bl and

(Morri were collected collected were lactis lactis (Bergström

tuf ndard curves curves ndard et al. et t - Bl et using test ) ) - Man5_8 ll

(Sheu 04 were 04 and and , 2012) , used to to used

t al. et Ba.

et et et a . , ,

This article isThis article by protected copyright. All rightsreserved. 3×10 were spectra MS Full method. top10 MS2 acid, gradient minute 260 a over eluted were Peptides (0.1% A 50 a to line P acquisition MS data (MS) analysis. Mass Spectrometry for Acetonitrile re and Speedvac, Eppendorf an in concentrated were peptides 500 clean into 1700 1 of 80% µ 200 (Rappsilber (v/v) 2% adding by quenched was activity Promega) grade, (MS of s ratio Wako):protein grade, (MS LysC with incubated and 8.5 pH standard. using determined centrifugat 4 at II, (TissueLyser Tris µ according was preparation Sample C x g,5min at4°C) (5000 centrifugation Cells glucose or galactomannan Carob (w/v) 0.5% with supplemented medium SEM LAB The s analysis Proteomic amples were diluted to 1:10 with 10% 10% with 1:10 to diluted were amples eptides eptides l lysis buffer (consisting of of (consisting buffer lysis l Accepted Article before NaCl (w/v) 0.9% cold ice with twice washed were pellets ell

% o differential proteome of of proteome differential Pir o as pcrmty te etds ee eatd n in on desalted were peptides the spectrometry, mass to Prior . C pH

6 (v/v) 45°C, 250 nl nl 250 45°C, g l tip, and the and tip, l

were grown in four biological four in grown were

or maximum injection time of 20 of time injection maximum or ) (w/v) (w/v) . After .

in a Bioruptor sonication water bath (Diagenode). (Diagenode). bath water sonication Bioruptor a in 8.5). 8.5). samples samples Digestion was carried out out carried was Digestion (w/v) (w/v) Acetonitrile, 0.1% Acetonitrile, ion (14.000 x g, 10 min at 4°C) and protein concentrations in the supernatants was was supernatants the in concentrations protein and 4°C) at min 10 g, x (14.000 ion

cm C18 reverse C18 cm et al. et TFA, 3% TFA,

washing the tips twice with 100 with twice tips the washing Sample

µ omc cd n ae) t 750 at water) in acid Formic l Eppendorf tubes using 40% 40% using tubes Eppendorf l Qiagen) , 2007) ,

were loaded onto a 2 a onto loaded were

C18 material activated with 40 µ 40 with activated material C18 min Bafr assay Bradford a

and ample Preparation ample s : protein ratio protein

- (v/v) (v/v) 1 were ) , Label mat C18 of discs 2 sample, each For . and the Q the and boiled -

Acetonitrile, after which the samples were loaded using centrifugation at at centrifugation using loaded were samples the which after Acetonitrile, phase analytical column (Thermo EasySpray ES803) using 100% Buffer Buffer 100% using ES803) EasySpray (Thermo column analytical phase (w/v) (w/v) B. an B. 6 M Guanidinium Hydrochloride, 10 mM TCEP, 40 mM CAA, 100 mM 100 CAA, mM 40 TCEP, mM 10 Hydrochloride, Guanidinium M 6 lysed mechanically using bead beating ( beating bead using mechanically lysed - free Quantitative Proteomics Analysis Proteomics free Quantitative

( 95 imalis

formic acid. The tips were subsequently equilibrated 2x with 40 with 2x equilibrated subsequently were tips The acid. formic to Kulak et al. et Kulak to - of of with Exactive instrument (Thermo Fisher Scientific) was run in a DD a in run was Scientific) Fisher (Thermo instrument Exactive o

C, replicates and and replicates (w/v) (w/v) (Thermo Fisher Scientific) against a bovine serum albumin albumin serum bovine a against Scientific) Fisher (Thermo 1:100 and samples samples and 1:100 .

5 minutes 5 ms and a scan range of 300 of range scan a and ms

cm C18 trap column (ThermoFisher 164705), connected in connected 164705), (ThermoFisher column trap C18 cm 20 subsp. subsp. Acetonitrile, 25 m 25 Acetonitrile, collected at a resolution of 70,000, with an AGC target of of target AGC an with 70,000, of resolution a at collected

µ trifluoroacetic acid (TFA) to a final concentration of 1% of concentration final a to (TFA) acid trifluoroacetic g

µ (v/v) protein bar, using the the using bar, lactis l of 0.1% of l )

(Kulak (6−60%,

after which which after l of Methanol (HPLC grade, Sigma), then 40 then Sigma), grade, (HPLC Methanol of l harvested Acetonitrile, 0.1 Acetonitrile,

ATCC 27673 cells 27673 ATCC

diluted 1:3 with 10% 10% with 1:3 diluted

et al. et were (w/v) (w/v) M Tris pH 8.5 and digested with digested and 8.5 pH Tris M 80% - noul cell Insoluble

constituted in 1% in constituted in the in hro Easy Thermo , 2014) ,

they were sonicated on high (3x 10 s, 10 (3x high on sonicated were they incubated erial (3M Empore) were packed in packed were Empore) (3M erial (v/v) (v/v) formic acid, the peptides were eluted eluted were peptides the acid, formic –

1750 %

log phase log . Briefly, cells Briefly, . acetonitrile, 0.1% 0.1% acetonitrile, 3mm beads, 30 beads, 3mm - (w/v) (w/v) house packed C18 C18 packed house 1:50 resuspension grown anaerobically in 4 ml 4 in anaerobically grown

m/z. The MS2 spectra were were spectra MS2 The m/z. overnight at 37 at overnight debris LC 1000 HPLC system. system. HPLC 1000 LC Acetonitrile, 25 mM Tris Tris mM 25 Acetonitrile,

formic acid. The eluted eluted The acid. formic t 37 at ( (w/v) (w/v) OD

o were fr 4 for C 600 were lysed in in lysed were

TFA, 2% TFA, in lysis buffer lysis in

Hz for 1 min 1 for Hz was analysed. analysed. was = 0.6 =

(w/v) removed removed o C. Enzyme Enzyme C.

Stagetips − h 0.8) 0.8) . trypsin trypsin formic formic

Next, Next, (v7v) µ l of of l

by by 60 by µ a - - ) l .

This article isThis article by protected copyright. All rightsreserved. primers directed site lightning Quickchange 11_Blac_ 2005) al., et (Dummler Germany) Heidelberg, Biochemistry, for Center EMBL, Stier, Gunter from gift kind respectively number t amplify (DSMZ, lactis E deviations standard. a as thereafter were Reactions corresponding the form i resuspended 0.2% phosphate sodium mM 30 a in out carried were Activity ≤0.01 p of and identified were peptides 2 least at if present Feature Minora built the using done quantitation protein and FDR, 1% a to filtered were results All modification. N protein on Acetyl and (N,Q) Deamidation accession:CP003941.1). database (NCBI were spectra Label 2.2. Discoverer th with repository ( Consortium ProteomeXchange monitore con quality lysate cell complex running by consistency for 60 to set 60 1×10 of value target AGC an with 17,500, of resolution a at obtained xp

Accepted Article 1.7e of threshold intensity an and of 25 energy collision ms,a normalised ression and purification of the manno the of purification and ression

ATCC27673 using primers using wv C (w/v) s

DNS reducing sugar assay sugar reducing DNS ,

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thiogalactopyranoside) to 100 µM and growth was continued for 16 h. Cells were harvested by by harvested were Cells h. 16 for continued was growth and µM 100 to thiogalactopyranoside) w mature polypeptides lacking the native signal peptide (amino acid (amino peptide signal native the lacking polypeptides mature r cevd f uig E poes (oac Ec Vrs nuclear Virus Etch (Tobacco protease TEV using off cleaved ere b a high pressure homogenizer and centrifuged (25 min, 30000 30000 min, (25 centrifuged and homogenizer pressure high a

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This article isThis article by protected copyright. All rightsreserved. preparation. proteomics sample the with assistance technical for acknowledged Switzerland) Facility Radiation equipment calorimetry 00297B Denmark Fund Research Independent by the wassupported work This Acknowledgements > S queries as proteins the of Homologues Bioi Version System, Graphics 2010) qual overall The manually. or Coot with buil were parts protein the after included were molecules Ligand suite. software Phenix the in performed were refinements and building model initial phasing, Experimental experimental the with method (SAD) K of structure PXIII (ESRF, beamline and PXI beamlines the at collected were Zn M 0.05 5.5, pH MES M 0.1 of solution reservoir a with grown were Complexes solution. reservoir s either in diffusion NaCl) mM 150 and 6.5 UK). Labtech, (TTP robot handling liquid 1 screen crystallization Structure cacodylate determination structure and Crystallization 95% were retrieved, aligned and and aligned were retrieved, 95% 2 PtCl Accepted Article- acetate and 18% PEG 8000 PEG 18% and acetate nformatics

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T fr PD coasi t MP t MJP, to scholarship PhD a for DTU

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Grenoble, Grenoble, , Geol, rne ad ws Lgt Source Light Swiss and France) (Grenoble, , 2010) , itting or hanging drops and grew for 48 h at h 48 for grew and drops hanging or itting 0.2M

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1 (Emsley Julia Tanas Tanasi is is Tanasi Tanas Julia Kbc, 2010) (Kabsch,

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en. mM) using vapor vapor using mM) and and 01 M (0.1

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This article isThis article by protected copyright. All rightsreserved. AcceptedL.W., Murray, G.J., R.M., Kapral, Immormino, Keedy, D.A., J.J., III, Headd, W.B., Arendall, Chen, V.B., S.K. Nair, I.,and R.I., Cann, Mackie, V., D.,Revindran, V.,Dodd, Agarwal, I.H., Kwon, Chekan, J.R., The B.(2009) Henrissat, V., and T., Lombard, C.,Bernard, Rancurel, P.M., Coutinho, B.L., Cantarel, V., R Bågenholm, large to improving approach Acombined (2007) B.G. Fox, and P.G., Blommel, D. L., Slotboom, Schmitt, S.H.J., Smits, R.P.A., Berntsson, L.K., Vigsnaes, H.A., Gronlund, L.R., Schmidt, J.B., Andersen, Wilcks,A., Licht,T.R., A., Bergström, E.N.,Nyman, Karlsson, H., Grey, C.,Stålbrand, U., Axling, U., C.,Nilsson, P.,Linninge, Berger, K.,Falck, and Functional (2003) T.R. Klaenhammer, R., and R.,Cano, E., Hutkins, Altermann, R., Barrangou, Article B.,and Svensson, Goh, Y.J., S.J., M.,Lahtinen, Hachem, Abou R., J.M., Barrangou, Andersen, L. Hung, J.J., N., Headd, I.W., Echols, Davis, Chen,V.B., G., Bunkóczi, P.V., Afonine, P.D., Adams, References and manuscript, the the with WS by performed were which culture mixed the of qPCR MH contributions Author and MAH have conc have MAH and macromolecular crystallography. crystallography. macromolecular all MolProbity: (2010) D.C. Richardson, and J.S., Richardson, polysacch caldanaerobius by utilization formannan basis andbiochemical Structural (2014) Res. Nucleic Acids Carbohydrate 243. beta ofa structure crystal and Enzyme ovatus: synergy Bacte of locus utilization by apolysaccharide conferred catabolism Galactomannan H.(2017) Stalbrand, N.M., and Koropatkin, E.C., Martens, D.T., Logan, Y., T., Xiao, protease. virus etch tobacco substrate of classification Letters Microbiology forqPCR validated approach M.I. Bahl, K.F., and Michaelsen, High Fed a and Adlercreut C., M., Holm, acidophilus Lactobacillus involv ofanoperon analyses genomic comparative lactis Bifidobacterium (2 T.R. Klaenhammer, Biol Crystallogr. Crystallogr D Python comprehensive and T.C., J.S.,Terwilliger, Richardson, D.C., Richardson, G.J.,Grosse Kapral, X -

ray crystallography ray

eddy, S.K., Bouraoui, H., Morrill, J., Kulcinskaja, E., Bahr, C.M., Aurelius, O., Rogers, Rogers, O., Aurelius, C.M., Bahr, E., J., H.,Morrill,Kulcinskaja, Bouraoui, S.K., eddy, arolyticus strain ATCC BAA ATCC strain arolyticus - Fat Diet. Fat all authors read and contributed to the final tothe contributed readand all authors -

Active EnZymes database (CAZy): an expert an (CAZy): database EnZymes Active

eived the idea of the study. ME study. the of idea the eived 37 , -

Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Read, R., Oeffner, N.W., Moriarty, A.J., McCoy, R.W., Kunstleve, the preparation of the manno the of preparation the J. Agric. Food Chem. J. Food Agric.

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: 34965

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resource for Glycogenomics. Glycogenomics. for resource : 8957 - - Density Array (GULDA) (GULDA) Density Array atom structure validation f validation structure atom the - - 2617. -

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Nature Communications Nature - 3 sample processing applied to copy to applied processing sample : 570 : : 319 : Environ. Microbiol. Environ. - 39. - 428. Acta Crystallographica Section D Section Crystallographica Acta - - -

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affinity mannan affinity tifs. tifs. Applied and Environmental Microbiology Environmental and Applied Nat. Rev. Microbiol. Rev. Nat. BMC Biochem. BMC - rich gut environment. environment. gut rich 5 1,4 Appl. Environ. Microbiol. Environ. Appl. - 5510.

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mannanase from from mannanase 10 . -

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binding module and highlights the diversity diversity of the highlights and module binding

growth on dietary xylan in the human gut. gut. xylaninthe human dietary on growth 16 E.C. (2012) How glycan metabolism shapes the shapes metabolism glycan How (2012) E.C. nch of the tree of life. life. of the tree nch of .

10 - epimerase: Discovery of a new carbohydrate anewcarbohydrate of Discovery epimerase: - n, O., Tjerneld, F., and Stålbrand, H. (2002) (2002) H. Stålbrand, F.,and n, O.,Tjerneld, , Ruminococcus albus, involving two involvingtwo albus, Ruminococcus , number estimation estimation number : 323 : Appl. Environ. Microbiol Environ. Appl.

66

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Bifidobacterium animalis animalis Bifidobacterium mannanase carrying mannan carrying mannanase 79 . (2018) Differential bacterial capture capture bacterial Differential . (2018)

Carbohydr. Res. Carbohydr. : 133 -

132. - J. Biol. J. Chem. Biol. 140.

ut. ut. ISME J ISME

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in eukaryotic cells. eukaryotic in Scientific Reports Scientific : 6903 11

401 .

82 : 2834 -

6910. - 287 - : 1 : 980 : phosphates as phosphates specific specific - subsp. subsp. 4. : 42389 : - 2847.

-

Carbohydr. Carbohydr. 991. - Nat. Nat. Anal. Anal. binding binding

5 - a, A., a, lactis lactis Nat. Nat. . s.

-

Appl. Appl. This article isThis article by protected copyright. All rightsreserved. Accepted Micro (2016) Kroemer, G. B.,and Routy, M., L., Zitvogel, Ayyoub, Glycan M. (2017) Ventura, and Sinderen,D., J., van Mahony, S., C.,Duranti, F.,Milani, Turroni, A.,Ferrarini, Bidossi, Sanchez,B., A., Zomer, Kim, J.H., E.,Mulder,I., F., Foroni, F.,Bottacini, Turroni, The (1986) B.I. C.A.,andDuerden, Edwards, N.W., J., Read, Tomlin, gene tuf Use of (2010) H.Y. Tsen, H.C., and Chen, W.H., Lin, Y.C., W.Z.,Chiang, Hwang, S.J., Sheu, He P.,(2010) Ulvskov, and Scheller, H.V., Article C., A.,Morland, Basle, E.C., Lowe, N., Terrapon, T., Tryfona, J.C., Mortimer, Briggs,J.A., A., Rogowski, micro for Y. (2007) Protocol Ishihama, M.,and Mann, J., Rappsilber, gum: A bean Locust B.J.(2013) Nagar, N.P., and Randeria, N.G., Moradiya, G.K., V.D.,Jani, Prajapati, gut inthe and cooperativity Sharing thyneighbor: Love (2016) E.C. Martens, N.T.,and Porter, Eddy, M.P., I., H.,Schendel, M.,Barutcu, Beleut, E., I., Jankovic, Rezzonico, J., Parche, S., Amon, (1994) Mitsuoka, T. T.,and Yamamoto, M.J., T.,Kim, H.,Fujisawa, N., Takahashi, T., Ishihara, Okubo, O'C J.C.N., Poulsen, Nakai,H., A., Majumder, F., Fredslund, M.S., Møller, allaghan, A., and van Sinderen, D. (2016) Bifidobacteria and their role as members of the human the human of roleasmembers their and Bifidobacteria (2016) D. Sinderen, A., andvan allaghan, Immunosurveillance. Immunosurveillance. cross and utilization foraging. of analysis Genome M. (2010) Ventura, and Sinderen,D., A.,Kelly, D.,van Margolles, Mills, D., P., B.,Coutinho, M.,Henrissat, A., Giubellini,V.,Delledonne, system. fecal incubation Food Sci. quantitative real cultural and milk by in fermented Bifidobacteria of for based primers 263 Reviews,pp. Alto:Annual Palo (eds). Ort & Briggs D. W.R. Merchant, intestine. inthelarge allocation microbial resource dictates Glycancomplexity (2015) D.N. and Bolam, H.J., Gilbert, Dupree,P., E.C., Martens, Thomp T.E., Rogers, H.J., A.M.,Day, Zheng, 1906. StageTips. using proteomics peptidesfor of and storage fractionation versatile biopolymer. microbiota. longum Bifidobacterium A., Arigoni, F., Burkovski, metabolism. ofpartia Effects gutmicrobiota. NCFM. acidophilus Lactobacillus the Probiotic in Utilization Isomaltooligosaccharide That Confers Glucosidase 1,6 Glucan GH13_31 ofthe andStructure Enzymology (2012) M. Abou Hacheme, Bifidobacterium bifidum Bifidobacterium

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the PCR detection of probiotic Bifidobacterium species and enumeration enumeration species and probiotic Bifidobacterium of detection the PCR - J. Bacteriol. M527. - feeding activities by bifidobacteria. bifidobacteria. by feeding activities Cell Carbohydr. Polym. Carbohydr.

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55 745

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-

746. 94

- 58 : 19514 4259. son, P., Hawkins, A.R., Yadav, M.P., Henrissat, B., Henrissat, M.P.,Yadav, A.R., P.,Hawkins, son, : 814 - : 1364 486.

-

821. - - 19519. 1369. Nat. Nat. Commun.

Trends Microbiol. Trends biome and Anticancer Anticancer and biome

degradation of guar gum by a gum by ofguar degradation -

purification, enrichm purification, Lo Leggio, L., Svensson, B.,and Svensson, Leggio, L., Lo Oggioni, M., Fitzgerald, G.F., M.,Fitzgerald, Oggioni,

12

6 : 9 - .

time PCR methods. methods. PCR time - Nat. Protoc. Nat. 19. - -

289. derived glycan glycan derived

- ent, pre ent,

α 2 : 1896 - . - J. J.

S. S. - - This article isThis article by protected copyright. All rightsreserved. S against enZ (Glc) glucose to using performed relative (Carob) galactomannan Carob a BLAC_ BLAC_ BLAC_06995 BLAC_05415 BLAC_07905 BLAC_07950 BLAC_01590 BLAC_01595 BLAC_00825 BLAC_01585 BLAC_00785 BLAC_00800 BLAC_00780 BLAC_00790 BLAC_03635 tag Locus glucose. and galactomannan Carob on grown 1 Table Tables

L

Acceptedog Article yme 2

fold changes of top top of changes fold

05995 07675 .

wiss (CAZ

Differential expressed proteins of of proteins expressed Differential -

Prot. y)

SignalP v.5.0 SignalP aiy affiliation family 1.57 1.76 2.08 2.16 2.48 3.21 3.56 3.80 3.90 4.38 4.62 5.16 5.21 6.52 6.64 Carob/Glc change Log

2

- fold

(log a

2 (

http://www.cbs.dtu.dk/services/SignalP/ fold change>2) change>2) fold . No No No No No No No No No No Yes No Yes No No SP d

Putative function of the proteins based on BLAST searches searches BLAST on based proteins the of function Putative b

GH 13 GH 36 GH 2 CAZy

iioatru animalis Bifidobacterium differential

c

Cystathionine beta Cystathionine α α P Acyl N A solute ABC transporter, β solute ABC transporter, permease ABC transporter, nucleotide ABC transporter, function Putative Transcriptional regulator Transcriptional binding ATP ABC transporter, A

- - - ropionyl - cetyl cyl M G G acylglucosamine 2 lucosidase alactosidase annosidase - - carrier CoA synthetase CoA ly - . CoA/propionyl

expressed identified in cells grown on grown cells in identified expressed

b - inl etds S) rdcin is prediction (SP) peptides Signal CoA carboxylase ß CoA carboxylase - protein

d

- ).

lyase

subsp. subsp. S - c

epimerase The Carbohydrate Active Active Carbohydrate The - - malonyltransferase CoA carboxylase - - protein binding protein binding

lactis

binding binding - subunit subunit protein

ATCC27673 ATCC27673 domain

This article isThis article by protected copyright. All rightsreserved. quantification. forreliable range within devi standard the with gluco in respectively units, glucosyl a MG GM GGM MMG MGM MGG mannotriose Galactosyl mannobiose Galactosyl Mannohexaose Mannopentaose Mannotetraose Mannotriose Mannobiose Ligand Table

S

Accepted of representation chematic Article

2 a .

Surface plasmon resonance binding analysis analysis of binding resonance Surface plasmon - -

( ( ( ( ( ( ( (1.76 (1.08 (1.27 (8.17 (1.87 K Bl 4.4 3 1 1 1 1.13 ± 0.0 1.96 ± 0.09 d . . . . MnBP

25 ± 5 8 36 ± (M) 0 2 1

± ± ± 0.03 ± 0.05 ± 0.05 ± 0.03 ± 0. ations ± 1.6 b 0. 0. 0. 0.0

1 25 47 1 12

1 5 8

)×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 .

The binding kinetics are reported for those data sets where the rate constants are are constants rate the where sets data those for reported are kinetics binding The ligand - 6 ------

5 5 4 4 6 6 6 7 7 8 7

k 3.09 5.12 6.06 1.29 1.50 structures is shown in in shown is structures - on manno

(M

× × × × × 10 10 10 10 10 - 1 s 4 5 5 6 6

- - 1 ) oligomers.

5.39 5.53 7.68 1.05 2.81 k

off

(s ×10 ×10 ×10 ×10 ×10 - 1 ) Bl

- - - - - b 2 2 2 1 1 iscain osat ( constants Dissociation

MnBP Fig. (1.27 ( ( ( (9.26 ( ( (1.67 ( (7.12 ( K Bl

8.13 ± 1 3 4 2.03 ± 0.0 1 4 d . . .08 .28 . MnBP

3 0 81 ± (M) 1 and 4 7 S1 ± ± ± ± 0.04 ± 1 ± 0.0 ± 0.0 ± 0.0 a 0 0. 0. 0. 1 , the “M” and “G” denote mannosyl and and mannosyl denote “G” and “M” the ,

.50 . . 2 35 32 14 03 30 Bl

4 1 1 )×10 1 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 )×10 MnBP2 )×10 - 3 ------6 4 4 7 5 6 7 8 4 -

7

K 1.62 2.13 3.95 5.13 8.51 k

d ae en o trip of means are ) on

(M × × × × × 10 10 10 10 10 - 1 s 5 3 5 5 5

- 1 )

1.50 5.98 6.57 6.59 6.06 0.08 k

off

(s × × × × × - 10 10 10 10 10 1

)

licates licates - - - - - 1 2 2 2 2

This article isThis article by protected copyright. All rightsreserved. b a Bl Bl Bl Bl SBP Table E The binding constant is less reliable due to increased affinity ( affinity increased to is lessreliabledue binding constant The MnBP1_N63G MnBP2 MnBP1 MnBP2_G61N Accepted Articlenthalpy

3 .

Manno

and stoichiometry and

- oligosaccharide b oligosaccharide Mannotriose Mannobiose Mannotriose Mannobiose Mannotriose Mannobiose Mannotriose Mannobiose Ligand

are not reliable not are

a

b

inding energetics analysed by isothermal titration calorimetry isothermal titration by analysed indingenergetics K (2.64 ± 2 (1.07 ± 0.1)×10 (7.81 ± 0.9)×10 (5.71 ± 0.5)×10 (5.18 ± 0.4)×10 (3.7 (2.62 ± 0.2)×10 (8.20 ± 0.8)×10 (M) d

5

± due to weak binding weak to due 6 .0 .7)×10 ) ×10

------5 6 6 7 3 7 7 - 9

------(kcal mol ∆ 11.71 6.77 6.97 7.15 8.58 3.31 8.98 8.30 G °

and inherent lack of information in the binding sigmoid. binding the in information of lack inherent and

see Fig. S2) see Fig. - 1 )

------(kcal mol ∆ 37.83 ±0.178 22.96 ±0.73 16.75 ±0.22 23.73 ±0.12 33.18 ±0.53 53.40 17.39 ±0.13 13.94 ±0.24 H °

± . 26.

- 1

)

6

------T 26.12 16.19 9.78 16.58 24.60 13.67 8.41 5.64 (kcal mol ∆ S °

- 1 )

(ITC).

0.80 ± 0.016 1.14 ± 0.017 0.77 ± 0.007 0.77 ± 0.002 0.83 ± 0.01 0.32 0.98 ± 0.01 0.83 ± 0.01 n

± 13.5

b a AverageB Clashscore (%) Ramachandran outliers favored(%) Ramachandran RMS angles( RMS bonds(Å) Proteinresidues Water Ligands Macromolecules Number of atoms R R Wilson B MeanI/σ(I) CC½ Completeness(%) Multiplicity Unique Unit cell Space group Resolution range (Å) Wavelength (Å) Beamline codePDB Table 4. This article isThis article by protected copyright. All rightsreserved.

Valuesin the parenthesis are for before refinement. Valuesin the parenthesis are the for highest resolution shell. - - free factor Water Ligands AcceptedMacromolecules Article

b

reflections

b Data collection and refinement statistics of thecomplexstructures of

-

factor

-

factor

a a

° )

a

a

Bl 33.1 25.8 22.8 23.7 5.1 0 97.4 0.98 0.011 428 499 37 3376 3912 0.22 (0.33 0.19(0.29 19.73 8.57 (1.12) 0.997 (0.456) 99.83 (99.98) 11.8 (10.8) 56799 (5602) 55.6 74.5 102.4 P 48.8 0.999 PXISLS 6I5R .28 MnBP1 2

1

- 2

1.6 (1.7 1

2 1

) - )

M2

-

1.6)

34.89 11.66 (1.33) 0.999 (0.656) 98.82 5.1 (5.2) 29709 (2838) 56.3 75.9 102.3 P 42.5 0.999 ESRF ID30A 6I5V Bl 43.3 42.0 44.9 44.7 3.65 0.24 95.2 1.37 0.013 421 336 43 3317 3696 0.26 (0.36 0.19 (0.30

2 MnBP1

1

-

2

2.0 (2.1

1 (97.02)

2

1

- ) ) M3

- -

2.0) 3

45.46 10.09 (0.94) 0.998 (0.445) 99.50 (97.57) 13.3 (12.8) 21351 (2049) 53.3 73.3 103.5 P 47.4 0.999 ESRF ID30A 6I5W Bl 55.6 71.2 56.2 57.3 9.2 0. 96 1. 0.016 427 81 99 33 3 0.26 (0.37 0.18 (0.33 547

71 02 2 MnBP1 67

1

2

- Bl

2.2 (2.3 1

MnBP1and 2 1

- ) ) M5

- -

3 2.2

)

Bl MnBP2 21.25 4.45 (0.64) 0.2751 90.38 (53.66) 3.0 (2.1) 28205 (1657) 52.2 64.6 133.3 P 40.6 0.999 PXIIISLS 6FUV Bl 29.7 27.8 21.2 22.45 7.47 0.28 97.4 1.43 0.015 427 479 122 3362 3 0.27 (0.34 0.18 (0.26 963

2 MnBP2 1

-

2

2.0 (2.1

. 1

(1.384) 2

1

- ) )

M3

-

2.0)

This article isThis article by protected copyright. All rightsreserved. proteins both respectively units, glucosyl and mannosyl of affinities The gluco mixed and (galman3) to through glucose of fractions cell and mannan on growth during upregulated highly regulator on located transcriptional type LacI a encoding a gene protein glucosidase 2 Figure from different (P<0.005) samples two last the during of concentration the (P=0.02), mannan Carob the on especially triplicates by determined (R Carob of hydrolysate (Green), cult nut ivory insoluble to shown is mannan addition in B panel in substrates of growth during medium of Acidification gum. Guar from galactomannan substituted highly on and glucomannan konjac galactomannan, ace ( units galactosyl and Figure a Mannotetraose Mannotriose Mannobiose Ligand Table

K d ed) and and ed)

Accepted Article yae ( tylated of theof mutant relative tothe wildtype protein.

ures n glucomannan in 5

s 1 . Binding of of . Binding

. . (Glc) and two adjacent transmembrane domain genes domain transmembrane adjacent two and of of

A) A) .

a different locus on locus different a

a B lhuh h eprmn sat wt s with starts experiment the Although

Ac

of of Schematic representation Schematic Mannan utilisation utilisation Mannan ) .

glucose glucose

mannohexaose affinit Relative C) . and Bacteroides animalis ). q GH Bl (1.12 (1.28 (1.18 K Bl C a 5 ie point time 5 at PCR . All cultures were cultures All . Yellow B)

d shown in a shown MnBP MnBP1 N63A and and N63A MnBP1

(M) 1 Bl

an ,

cultures lcsl (B glucosyl Growth of of Growth ± 0.05 ± 0.03 ± 0. MnBP control control a

subsp. 1 . diinly h C and C2 the Additionally ). 12 _N63G ATP

on glucose on )×10 )×10 )×10

- 1 and and 1 manno log f h sm sri a A, as strain same the of

( (man6 -

Grey binding cassette cassette binding the genome the - - - galactomannan galactomannan lactis loc 5 5 5

10

lue) iioatru animalis Bifidobacterium ies

Bl scale to account for the large difference in affinity largedifference in the for scale account to i

- 6 K biological 8 1.6 ). in in MnBP oligosaccharides ),

. . .

d 3 8

Bl based on a one tailed aone tailed on based of the the of of

ATCC27673 and and ATCC27673 . The data are means with standard standard with means are data The . E h mnoy rsde cn be can residues mannosyl The (relative) ×10 ×10 ×10 B MnBP2 G61N are also indicated. indicated. also are G61N MnBP2 ) wees h ratio the whereas , galactosyl . mannan

animalis subsp. lactis subsp. animalis The 1 2 1 and the and

2

and a GH36 α GH36 a and

variants variants Bl .

tr eaie rprin o te co the of proportions relative B) β B) a MnBP1 and and MnBP1 with with iplicate

.

transport system consisting of two solute binding binding solute two of consisting system transport The backbone consists of β of consists backbone The

- data is normalized to mannotriose to normalized is data mannobiose - mannan /or ignificantly lower fraction of of fraction lower ignificantly h mannanase the

measured by by measured s eemnd y ufc pamn resonance. plasmon surface by determined as s

The . , rw o crb galactomannan carob on grown

Bacteriodes ovatus Bacteriodes 3 oiin o te ansl nt cn be can units mannosyl the of positions C3 with standard deviations standard with followed by a β a by followed - t galactosidase enco galactosidase

- a test Not Not (1.20 ± 0.01)×10 (4.50 ± 1. K Bl Bl of

subsp. se

extracellular d

MnBP

nP twrs mannobiose towards MnBP2

(M) ATCC27673 activity activity Bifidobacterium bifidobacteria is significantly higher significantly is bifidobacteria conducted in in conducted determined (galman2), surface plasmon resonance. surface plasmon

Bl 2 30)×10 lactis _G61N substituted substituted GH5 of culture of - The mannosidase mannosidase

mannan

_8 - - on 9 6 deviation - . The loc The . ATCC27673 ATCC27673

1→4 1→4 - “M” and “G” designate designate “G” and “M” ded by third locus was was locus third by ded galactosyl Origin culture culture (Morrill carob

. D) . is not significantly significantly not is y → lne α linked 1→6 by supernatant mannosyl (Green) mannosyl 1.69 9.36 K a . . se d

B

-

( us

s (relative) galactomannan galactomannan

best ifid n ae D panel in

Growth of c of Growth ×10 ×10 of of

Bl

of biological biological of - t al. et encodes encodes mannotriose (Carob) o Man5_8 is is Man5_8 GH2 and a and GH2

- - b on 2 3 ligand for for ligand

a

cterium 2015) ,

(man2) (man2) s a Carob Carob

(SN)

a β a and and C) as as o - - -

This article isThis article by protected copyright. All rightsreserved. biochemical galactosidase de and up taken be to like are and proteins transport the to affinity relevant locus. same the by encoded o Internalised and disaccharides to binding proteins: binding associated two by capture following system oligom to mannan of degradation confers to medium mannan a to joint module catalytic (GH5_8) ATCC27673 Figure red. in are colored SBP one stra and identifiers as indicated are strain source and accessions genbank Bl of capable or asparagine glutamine, a possessing members of conservation Figure to mannotriose to m sensograms Bl 4 Figure mFobs axis Bl of site binding mannobiose Close B) 1−3. number with indicated are (Domain The Figure annob AcceptedMnBP ArticleMnBP mannotriose. with complex in MnBP2 Bl are shown. shown. are SBPs MnBP

3 – 6 5. iose to to iose .

2 2_ . .

. The The . DFcalc and σA DFcalc and

2, brown 2, A) - A schematic model showing the mannan utilization strategy of of strategy utilization mannan the showing model schematic A Phylogenetics of bifidobacterial mannan solute binding proteins. proteins. binding solute mannan bifidobacterial of Phylogenetics like proteins proteins like Ligand binding to to binding Ligand G hydrogen bonding to the mannosyl at position 2 similarly to similarly 2 position at mannosyl the to bonding hydrogen oss o a N an of consist

2. . Ribbon representation of the overall structure of structure overall the of representation Ribbon data 61 n t in and the fits of one binding site models (solid line) depicting the binding of of binding the depicting line) (solid models site binding one of fits the and

ioacaie are ligosaccharides A modular β modular A of GH36. The mannan utilization model is based on proteomic analysis, and the the and analysis, proteomic on based is model utilization mannan The GH36. of Bl Bl Bl N hlgntc tree Phylogenetic E) o rettos ifrn b a 18 a by differing orientations wo

the the ifrne lcrn est maps density electron Difference D) mannobiose mannotriose with complex in MnBP1 MnBP1. B) MnBP1. MnBP1_N63G MnBP1_N63G except for the for except

as analysed by surface plasmon resonance. Reference and baseline corrected corrected baseline and Reference resonance. plasmon surface by analysed as ). The two domains domains two The ). mannobiose position position - possess mainly glycine at this position (alanine in a single sequence). The The sequence). single a in (alanine position this at glycine mainly possess weighted weighted -

endomannanase comprising a glycoside hydrolase family 5 subfamily 8 8 subfamily 5 family hydrolase glycoside a comprising endomannanase -

terminal domain (Domain (Domain domain terminal ma N63 in in N63 Bl

α Bl G nno H MnBP - to to based on on based H1 (1 MnBP2, which preference ligands larger than a disaccharide. disaccharide. a than larger ligands preference which MnBP2, and and likely )

b mannotriose to mannotriose →

Bl iose to to iose β 6 are shown as a light meshblue light areas a shown

are linked by linked are - Bl MnBP1_N63G MnBP1_N63G 1, 1, ) glucosidase, which was not observed on observed not was which glucosidase, oe omny hsiie t hs oiin al potentially all position, this at histidine a commonly more

- MnBP1 and surrounding residues across across residues surrounding and MnBP1 o e erdd y β a by degraded be to aatsl usiue manno substituted galactosyl Two orient Two Bl h alignment the - Bl MnBP - up of the binding site of of site binding the of up binding binding MnBP º oain ln te x the along rotation º 0

2 Bl o te iad were ligands the for

ations differing by a 180 º rotation along the x the along rotation º 180 a by differing ations hinge 2. s el as well as MnBP CBM r ta ae nenlzd by internalized are that ers

C) F) mannobiose to to mannobiose F) , green) 1,

m in in regions shown regions

2_G61N

of family 10 is secreted into the growth growth the into secreted is 10 family of annotr Bl

Bl A MnBP1 in complex with mannotriose. with complex in MnBP1 ) shows that that shows ) MnBP1, which displays high affinity affinity high displays which MnBP1,

- the the ansds ad β a and mannosidase

iose to to iose n a agr C larger a and . .

mutants

Close - oligosacch Bl

Bl

calculated with coefficient coefficient with calculated ins that possess more than than more possess that ins Bl - in teal. Mannosyl subsites subsites Mannosyl teal. in - nP i c in MnBP1 br axis B MnBP1 - MnBP1. D) MnBP1.

up of the binding site of site binding the of up . a Bl A)

nched Bl animalis Bl

MnBP1

MnBP this MnBP1_N63G h sqec l sequence The C) a - . By comparison, comparison, By . a

terminal domain domain terminal rides

n Bifidobacterium Close by by galactomannan, galactomannan,

- 2_G61N omplex with with omplex ABC uptake uptake ABC like proteins proteins like a subsp. - manno

glucosidase glucosidase

putative - bind with with bind up of the the of up tr lactis lactis G ,

iose iose ogo and and

A α - ) - )

This article isThis article by protected copyright. All rightsreserved. Accepted Article non manno the of hydrolysis the catalyse to inferred is which but - oligosaccharides .

-

euig lcsl oeis n galacto in moieties glucosyl reducing -

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article