Supporting Information - PNAS · 25/6/2010 · Best PSI-BLAST result (identity %, E-value)...

Supporting InformationGloux et al. 10.1073/pnas.1000066107

setoeahcranerCairetcaboretnEairetcaboetorP

-setucimriF

puorgiborolhC/setedioretcaBaiborcimocurreV

airetcabosuFsamsalpocyM

GB-11G11H

i l o c a i h c i r e h c s E ) y t i v i t c a G B ( 2 1 K 2 8 7 9 3 4 4 0 _ P Z

A m u l i h p o m r e h t m u l l e c o r e a n 0 2 3 1 - Z / 5 2 7 6 M S D ) n o i t a t o n n a G B ( 6 3 9 3 7 5 2 0 0 _ P Y

s u v a n g s u c c o c o n i m u R ) y t i v i t c a G B ( 1 E 8 8 7 1 8 5 4 3 : I G

s u s o n m a h r s u l l i c a b o t c a L 1 - 2 S M L ) n o i t a t o n n a G B ( 2 8 7 9 3 4 4 0 _ P Z

s n e g n i r f r e p . m u i d i r t solC ) y t i v i t c a G B ( 9 3 2 8 C T C N 2 7 8 2 4 6 2 0 _ P Z

setucimriFpuorgGB-11G11H

Fig. S1. Distance tree view of H11G11-BG Blastp results and localization of the Firmicutes H11G11-BG group and of several BGs of the UidA group.

H11G11

C7D2

C. BartlettiZP_02210507

S. variabileZP_03776158

B. formatexigensZP_03687412

R. gnavusZP_02040206


Paenibacillus sp. ZP_02847334


B.

S. ZP_03776204


R.ZP_03753494


S. satellesZP_04455048

S. variabile_ZP_03774451

F. prausnitziiZP_02090684

E. coli P05804

L. gasseri gi12802352

R. gnavus gi34581788

C. perfringens B1RQV9

H11

G11

-like

BG

(Firm

icut

es)

Kno

wn

BG

s

variabile

inulinivorans

capillosusZP_02038110

PD81

9476

PD00

2797

PD33

9503

PD00

2797

PDA

2N0N

2

PD00

2163

PDA

253F

2

PD88

4831

PDA

1N8L

2

Fig. S2. Comparative domain organization of unique Firmicutes BGs and known BGs. Domain arrangements of proteins in families were performed using theProDom database on September 2009. Conserved characterized domains are represented by colored boxes and documented in ProDom database; un-characterized conserved domains are numbered.

Gloux et al. www.pnas.org/cgi/content/short/1000066107 1 of 9

www.pnas.org/cgi/content/short/1000066107

-New PS00719 pattern: [NT]-x-[LIVMFYWD]-R-[STACNL](2)-H-Y-[PQ]-x(4)-[LIVMFYWS](2)-x(3)-[DN]-x(2) -G- [LIVMFYWA](4)

>H11G11 CAT TAT CAG>ZP_02210507 CAC TAT CAA C. bartlettii>ZP_03776158 CAC TAC CAG S. variabile>ZP_036887412 CAC TAT CAG B. formatexigens >C7D2 CAT TAT CAG >ZP_02040206 CAT TAT CAG R. gnavus >ZP_02041835 CAT TAT CAG R. gnavus >ZP_02847334 CAT TAC CAG Paenibacillus sp.>ZP_02042987 CAT TAT CAG R. gnavus>ZP_02038110 CAC TAT CAG B. capillosus>ZP_03776204 CAC TAC CAG S. variabile >ZP_03686296 CAC TAC CAG B. formatexigens>ZP_03753494 CAC TAT CAG R. inulinivorans>ZP_03685645 CAT TAT CAG B. formatexigens>ZP_04455048 CAC TAT CAG S. satelles>ZP_3774451 CAC TAC CAG S. variabile>EDP22313 CAC TAC CAG F. prausnitzii>ZP_02065512 CAT TAT CCG B. ovatus>ZP_03478350 CAT TAC CCG P. johnsonii>ZP_02031489 CAT TAT CCG P. merdae>P05804 CAT TAC CCT E. coli >C2JTS9 CAT TAT CCA L. rhamnosus>C1CAWO CAT TAT CCA S. pneumoniae >Q3DSU4 CAT TAT CCT S. agalactiae>C2CG53 CAC TAC CCT A. tetradius>B1RQV9 CAT TAT CCA C. perfringens>B9MLH3 CAT TAT CCT A. thermophilum>Q6W7J7 CAT TAT CCT R. gnavus

Firmicutes H11G11-BG group

Bacteroidetes H11G11-BG group

known BGs (uidA homologs)

Fig. S3. A specific “HYQ” motif in the H11G11-BG group from Firmicutes. Codon usage within the HYP conserved domain of pattern 1 (Prosite PS00719) wascompared between H11G11-like BGs and known BGs (UidA homologs).

IepyT

IIepyT

Ty IIIep

VIepyT

,1122GOC BleM /+aN, esoibilem retropmys dna detalersretropsnart [ etardyhobraC dnatropsnart ]msilobatem

,29700RGIT hpg , ragus ( edisocylG - edisotneP -edinoruxeH retropsnart)

,1122GOC BleM /+aN, esoibilem retropmys dna detalersretropsnart [ etardyhobraC dnatropsnart msilobatem ]







,52920KRP,52920KRP etanoruculg esaremosi


,1122GOC BleM /+aN, esoibilem retropmys dnasretropsnart [ etardyhobraC dnatropsnart msilobatem ]



,4350GOC MroN +aN, - nevird gurditlum xulffe pmup]smsinahcemesnefeD[ e9 13

e2 62

e3 62

e4 52

e6 33

e1 251

e3 23

e3 04

e7 34

e4 86

e8 15

e3 37

e2 76

e1 96

e5 76

e9 - 13

e2 - 62

e3 - 62

e4 - 52

e6 - 33

e1 - 251

e3 - 23

e3 - 04

e7 - 34

e4 - 86

e8 - 15

e3 - 37

e2 - 76

e1 - 96

e5 - 76

GB niamoddevresnoctsebretropmySretropmyS puorgretropmyStih)seigolomohpuorglanretni()eulaV-E(

mulunargilodbuS elibairav 67151MSD 40267730PZ dyhylg 70267730PZ

alletnayrB snegixetamrof 96441MSD

succoconimuR suvang 4192CCTA

airubesoR snaroviniluni 14861MSD

sedioretcaB susollipac 99792CCTA

54658630PZ 64658630PZalletnayrB snegixetamrof 96441MSD

69268630PZ 79268630PZ

78924020PZ 88924020PZ

49435730PZ 39435730PZ

01183020PZ 21183020PZ

15447730PZ 25447730PZmulunargilodbuS elibairav 67151MSD

iiztinsuarp 31322PDE 41322PDE

aihtrowelttuhS selletas 00641MSD 84055440PZ 94055440PZ

53374820PZsullicabineaP RDJ.ps -2

11G11H 02eneg 12eneg

85167730PZ 75167730PZmulunargilodbuS elibairav 67151MSD


43374820PZ 43374820PZ-


2D7C 6eneg 7eneg

53814020PZ 73814020PZ

60204020PZ 70204020PZ

muiretcabilaceaF 2/12M

Fig. S4. Genetic environments of the unique Firmicutes BGs. The conserved genetic environments close to the homolog Firmicutes BGs were restricted tosymporters whose homologies were grouped into four types determined after ClustalW multiple alignment and homology search. Red arrows represent thedifferent BG genes and green arrows the associated symporters. The best conserved domains and E values were those found after Blastp against nonredundantprotein sequences (NCBI).



Fig. S5. Amino acid motifs conserved in symporters associated with the Firmicutes BGs. The different types of putative symporters and their identities/sim-ilarities within types were determined after homology search and ClustalW Multiple alignment. The patterns proposed were obtained by using the discoverpatterns tool PRATT (http://www.expasy.ch/tools/pratt/).


http://www.expasy.ch/tools/pratt/www.pnas.org/cgi/content/short/1000066107

00+E00,0

80-E00,5

70-E00,1

70-E05,1

70-E00,2

70-E05,2

BnI EnI U1FMnI

d.n d.n d.n

stnafnI

Freq

uenc

y in

met

agen

omes

(h

its/b

p)

00+E00,0

80-E00,5

70-E00,1

70-E05,1

70-E00,2

70-E05,2

8bus7busY2FX2FW2FV2FT1FS1FRnIDnIAnI

11G11H)%14(muidirtsolC

70501220_PZiitteltrab.C

85167730_PZelibairav.S

31322PDEiiztinsuarp.F

4943573_PZsnaroviniluni.R

43374820_PZ.pssullicabineaP

43374820_PZ.pssullicabineaP

21556020_PZsutavo.B

05387430_PZiinosnhoj.P

98413020_PZeadrem.P

98413020_PZeadrem.P

5E1Q1CiitoverpsuccocoreanA

07TF8BesneinfahmuiretcabotifluseD

7J7W6Qsuvang.R

3HLM9BmulihpomrehtmullecoreanA

9VQR1BsnegnirfrepmuidirtsolC

35GC2CsuidartetsuccocoreanA

35GC2CsuidartetsuccocoreanA

4USD3QeaitcalagasuccocotpertS

0WAC1CeainomuenpsuccocotpertS

9STJ2CsusonmahrsullicabotcaL

9STJ2CsusonmahrsullicabotcaL

40850P21Kiloc.E

nerdlihcdnastludA

Freq

uenc

y in

met

agen

omes

(h

its/b

p)

sGBnwonKsgolomohAdiU

sGB-11G11HsgolomohsetucimriF

sGB-11G11HsgolomohsetedioretcaB

Fig. S6. Information details about frequencies and distribution of the unique and known BGs among individuals. This figure presents the detailed results ofFig. 7 with the frequency per each BG gene tested.



Table

S1.

Gen

esiden

tified

aspotential

BGs

Clone

No.of

aminoacids

BestPS

I-BLA

STresult

(iden

tity

%,E-va

lue)

Simila

rity

with

glyco

sylhyd

rolase

family

2Prosite

patterns

Activity,

arch

itecture,an

dgen

etic

context

Putative

function

COG

functional

category

H3D

458

5gi29

3462

82-aspartyl-tRNA

synthetase

(Bacteroides

thetaiotaomicronVPI-548

2)(91%

,0.0).

1:<50

%,2:<50

%*

PNP-G

deg

lucu

ronidation

Aminoacid

activa

tion

COG01

73Aspartyl-

tRNA

synthetase

C11

H2

165

gi118

7487

50-2C-m

ethyl- D-erythritol2,4-

cyclodiphosphatesynthase

(Marinomonas

sp.MW

YL1

)(59%

,1e

-51

).Pa

ttern2C

-methyl- D-erythritol2,4-

cyclodiphosphatesynthasesignature

(100

%).

1:<50

%,2:<50

%PN

P-G

deg

lucu

ronidation

Enzy

mes

ofthemethyl

erythritolphosphate

pathway

(terpen

oid

biosynthesis).Interaction

withthehost.

COG02

45Lipid

tran

sport

andmetab

olism

H11

G11

755

gi293

4616

7β-galactosidase(Bacteroides

thetaiotaomicronVPI-548

2)(40%

,3e

-118

)

1:82

%,2:93

%PN

P-G

deg

lucu

ronidation.

Glucu

ronidepermea

seupstream

.Noβ-galactosidaseactivity.

β-Glucu

ronidase,

glucu

ronide

metab

olism.

COG32

50Carbohyd

rate

tran

sport

andmetab

olism

233

and

≥60

5

ABC-typ

ean

timicrobialpep

tidetran

sport

system

:gi158

9681

9-ABCtran

sporter,

ATP

-bindingprotein

(Carnobacterium

sp.

AT7

)(70%

,5e

-93).ABCtran

sporter,

permea

seprotein

(Carnobacterium

sp.

AT7

)(28%

,1e

-54).

1:<50

%,2:<50

%(forboth

proteins)

PNP-G

deg

lucu

ronidation

permea

se.Pa

tternglyco

syl

hyd

rolase

family

16active

site

(65%

).Xyloglucanen

do-

tran

sglyco

sylase

Cterm

inal

domain.

SalX-A

BC-typ

ean

timicrobial

pep

tidetran

sport

system

.COG11

36.2

defen

semechan

isms

331

gi122

5826

56sporulationprotein

and

relatedproteins(Teth39

DRAFT

_034

0)(Thermoan

aerobacterethan

olicusATC

C33

223)

(35%

,1e

-44).

1:<50

%,2:55

%PN

P-G

deg

lucu

ronidation,cell

wallhyd

rolase,MraY

family

signature

1(53%

)

Sporulationstag

eII,

protein

DFirm

icutes(IPR

0142

25).

COG23

85Sp

orulation

protein

andrelated

proteins

H11

B1

916

gi149

8117

21serine/threonineprotein

kinase(Roseobactersp.Azw

K-3b)

(39%

,3e

-44).Le

ngth:44

4am

inoacids.

1:<50

%,2:51

%PN

P-G

deg

lucu

ronidation.

UDP-glyco

syltransferase/glyco

gen

phosphorylase

superfamily

.Domainglyco

syltran

sferase

group1.

TPRrepea

tSL

1domain.

Patternhex

apep

tide-repea

t–co

ntainingtran

sferasesignature

(68%

)

Bifunctional

protein,signal

tran

sductionmechan

isms,

polysaccharideproduction/

tran

sport

interactionwith

thehost.

COG

gen

eral

function

prediction.

Gen

esiden

tified

onfosm

idinsertsco

nferringdeg

lucu

ronidationactivity

werean

alyz

edusingPS

I-BLA

STsearch

onNCBIa

gainst

nonredundan

tprotein

sequen

cesofGen

Ban

k(relea

se16

3.0,

http://www.ncb

i.nlm

.nih.gov/BLA

ST/)

and

EXPA

SY(http://www.exp

asy.ch

/tools/blast/)

datab

ases.Domain

arch

itectures,

potential

functions,

and

associated

patternswere

analyz

edusing

InterProScan

(http://www.ebi.a

c.uk/

InterProScan

/),MyH

itsfrom

theSw

issInstitute

ofBioinform

atics(http://www.isb-sib.ch/),PR

OSITE

(http://www.exp

asy.org/prosite/),SM

ART(http://sm

art.em

bl-heidelberg.de/),PF

AM

(http://www.san

ger.ac.uk/

Software/Pfam

/),MotifScan

(http://myh

its.isb-sib.ch/cgi-bin/m

otif_scan

),an

dSU

PERFA

MILY

(http://supfam.org/SUPE

RFA

MILY/).Proteinsim

plicated

intran

sport

system

swerean

alyzed

withtheTran

sport

Clas-

sificationDatab

ase(http://www.tcd

b.org/).

*1an

d2forPS

0071

9an

dPS

0060

8Prosite

patterns,respective

ly.Conserved

patternswerefoundonPR

OSITE

(http://www.exp

asy.org/prosite/),a

ndsimila

rities

weredetermined

onPB

IL(http://npsa-pbil.ibcp

.fr/)

usingPA

TTINPR

OT.


http://www.ncbi.nlm.nih.gov/BLAST/http://www.ncbi.nlm.nih.gov/BLAST/http://www.expasy.ch/tools/blast/http://www.ebi.ac.uk/InterProScan/http://www.ebi.ac.uk/InterProScan/http://www.isb-sib.ch/http://www.expasy.org/prosite/http://smart.embl-heidelberg.de/http://www.sanger.ac.uk/Software/Pfam/http://www.sanger.ac.uk/Software/Pfam/http://myhits.isb-sib.ch/cgi-bin/motif_scanhttp://supfam.org/SUPERFAMILY/http://www.tcdb.org/http://www.expasy.org/prosite/http://npsa-pbil.ibcp.fr/www.pnas.org/cgi/content/short/1000066107

Table S2. Glycosyl hydrolase family 2 signatures conservation in the unique BG and homologs

Metagenomic clone or genomeNCBI reference andno. of amino acids

Prosite pattern PS00719(position and similarity %)

Prosite pattern PS00608(position and similarity %)

Metagenomic clone H11G11 309–334, 82% 362–376, 93%Metagenomic clone C7D2 314–339, 86% 369–383, 90%R. gnavus ATCC 29149 ZP_02041835 (747 aa) 304–329, 76% 359–373, 90%

ZP_02040205 (757 aa) 316–341, 86% 371–385, 90%ZP_02042987 (640 aa) 298–323, 86% 351–365, 89%

S. variabile DSM 15176 ZP_03776158 (735 aa) 312–337, 86% 365–379, 81%ZP_03774451 (727 aa) 319–344, 86% 372–386, 93%ZP_03776204 (639 aa) 329–354, 86% 382–396, 89%

B.a formatexigens DSM 14469 ZP_03687412 (756 aa) 322–347, 86% 377–391, 90%ZP_03685645 (756 aa) 331–356, 86% 384–398, 71%ZP_03686296 (642 aa) 305–330, 76% 358–372, 77%

F. prausnitzii M21/2 EDP22313 (735 aa) 302–327, 86% 355–369, 93%R. inulinivorans DSM 16841 ZP_03753494 (640 aa) 298–323, 86% 351–365, 93%C. bartlettii DSM 16795 ZP_02210507 (746 aa) 311–336, 76% 364–378, 93%B. capillosus ATCC 29799 ZP_02038110 (641 aa) 299–324, 86% 352–366, 89%S. satelles DSM 14600 ZP_04455048 (766 aa) 321–346, 86% 374–388, 93%Paenibacillus sp. JDR-2 ZP_02847334 (767 aa) 326–351, 86% 381–395, 90%

Homologies of signatures (Prosite patterns) of glycosyl hydrolase family 2 (galactosidases/glucuronidases) and unique BG homologs were studied using thePATTINPROT tool (http://npsa-pbil.ibcp.fr/).


http://npsa-pbil.ibcp.fr/www.pnas.org/cgi/content/short/1000066107

Table

S3.

UidA

homologsresearch

ingen

omes

possessingtheuniqueBG

sequen

ce

Knownan

dco

nfirm

edBG-positive

strains

Protein

sources

for

homologyresearch

BesttBlastn,localiz

ation,

andhomology(%

iden

tity,

%simila

rity)

Aminoacid

sequen

cehomologywithH11

G11

(localiz

ation,%

iden

tity,%

simila

rity)

Assigned

asH11

G11

BG-like

group(positiononco

ntig,

%H11

G11

iden

tity,%

H11

G11

simila

rity)

S.va

riab

ileDSM

1517

6P0

5804

E.co

liS_va

riab

ile-1.0.1_C

ont0.25

(198

49–21

483)

26%

,42

%(198

73–22

053)

48%

,61

%ZP

0377

4451

(198

73–22

056)

45%

,57

%gi128

0235

2L.

gasseri

S_va

riab

ile-1.0.1_C

ont0.19

(592

10–60

709)

25%

,44

%(590

90–61

003)

55%

,69

%ZP

0377

6204

(590

90–61

009)

43%

,54

%gi345

8178

8R.gnav

us

S_va

riab

ile-1.0.1_C

ont0.19

(591

92–60

709)

25%,41

%B.ova

tusATC

C84

83P0

5804

E.co

liB_o

vatus-MSIQ_C

ont517

(133

275–

1316

08)

26%

,44%

(133

134–

1316

08)25

%,44%

<50

simila

rity

gi128

0235

2L.

gasseri

B_o

vatus-MSIQ_C

ont460

(264

053–

2656

39)27

%,

42%

(263

942–

2657

77)40

%,54

%ZP

0206

5512

(263

912–

2659

09)34

%,46

%

gi345

8178

8R.gnav

us

B_o

vatus-MSIQ_C

ont517

(133

293–

1315

90)26

%,

41%

(133

134–

1316

08)25

%–44

%<50

simila

rity

P.johnsoniiDSM

1831

5P0

5804

E.co

liP_

johnsonii-1.0_

Cont16.36

(888

373

12)28

%,42

%(901

5–70

90)40

%,55

%ZP

0347

8350

(703

9 –90

75)

34%

,46

%gi128

0235

2L.

gasseri

P_johnsonii-1.0_

Cont16.36

(894

6–73

12)27

%,43

%(901

570

90)40

%,55

%

gi345

8178

8R.gnav

us

P_johnsonii-1.0_

Cont16.36

(894

0–73

18)25

%,41

%(901

5–70

90)40

%,55

%

F.prausnitziiM21

/2P0

5804

E.co

liF_prausnitzii_M21

2-2.0.1_

Cont750

(314

70–

3326

0)35

%,53

%

(314

97–33

233)

25%

,41

%<50

simila

rity

gi128

0235

2L.

gasseri

F_prausnitzii_M21

2-2.0.1_

Cont750

(314

73–

3324

2)42

%,57

%gi345

8178

8R.gnav

us

F_prausnitzii_M21

2-2.0.1_

Cont750

(314

73–

3323

3)38

%,53

%R.gnav

usATC

C29

149

P058

04E.

coli

R_g

nav

us-1.0.1_

Cont39.1

(267

4–41

13)27

%,41

%(249

1–44

07)53

%,68

%ZP

0204

2987

(249

1–44

10)

45%

,57

%gi128

0235

2L.

gasseri

R_g

nav

us-1.0.1_C

ont39.1

(261

1–41

13)26

%,45

%gi345

8178

8R.gnav

us

R_g

nav

us-1.0.1_

Cont244

(468

95–48

577)

25%

,41

%(468

92–49

153)

46%

,60

%ZP

0204

0206

(468

86–49

156)

44%

,57

%B.capillosusATC

C29

799

P058

04E.

coli

B_cap

illosus-2.0.1_

Cont317

(459

01–47

340)

27%,39

%(457

21–47

634)

52%

,67

%ZP

0203

8110

(457

15–47

637)

44%

,56

%gi128

0235

2L.

gasseri

B_cap

illosus -2.0.1_

Cont317

(457

30–47

340)

26%,44

%gi345

8178

8R.gnav

us

B_cap

illosus-2.0.1_

Cont317

(457

24–47

352)

24%,41

%



Table

S3.Cont.

Knownan

dco

nfirm

edBG-positive

strains

Protein

sources

for

homologyresearch

BesttBlastn,localiz

ation,

andhomology(%

iden

tity,

%simila

rity)

Aminoacid

sequen

cehomologywithH11

G11

(localiz

ation,%

iden

tity,%

simila

rity)

Assigned

asH11

G11

BG-like

group(positiononco

ntig,

%H11

G11

iden

tity,%

H11

G11

simila

rity)

B.form

atex

igen

sDSM

1446

9P0

5804

E.co

liB_form

atex

igen

s-1.0.1_

Cont23.1(74

444–

7625

2)43

%,57

%

(746

96–76

225)

26%

,41

%<50

simila

rity

gi128

0235

2L.

gasseri

B_form

atex

igen

s-1.0.1_

Cont2.1

(232

634–

2344

24)37

%,55

%

(232

901–

2344

15)28

%,44

%

gi345

8178

8R.gnav

us

B_form

atex

igen

s-1.0.1_

Cont2.1(232

643–

2344

21)40

%,54

%P.

merdae

ATC

C43

184

P058

04E.

coli

P_merdae

-MSIQ_C

ont63

(214

079–

2125

08)28

%,

42%

(214

211–

2122

86)41

%,55

%ZP

0203

1489

(214

271–

2122

38)34

%,46%

gi128

0235

2L.

gasseri

P_merdae

-MSIQ_C

ont63

(214

211–

2125

08)26

%,

43%

gi345

8178

8R.gnav

us

P_merdae

-MSIQ_C

ont63

(214

091–

2125

14)26

%,

41%

C.bartlettiiDSM

1679

5P0

5804

E.co

liC_b

artlettii-2.0.1_

Cont15.1

(406

77–42

332)

24%

,39

%(406

74–42

899)

58%

,70

%ZP

0221

0507

(406

74–42

911)

54%

,68

%gi128

0235

2L.

gasseri

C_b

artlettii-2.0.1_

Cont15.1

(406

56–42

332)

22%

,39

%gi345

8178

8R.gnav

us

C_b

artlettii-2.0.1_

Cont15.1

(407

22–42

33223

%,39

%)

R.inulin

ivoransDSM

1684

1P0

5804

E.co

liR_inulin

ivorans-

1.0.1_

Cont419

.1(752

33–

7373

4)26

%,42

%

(753

56–73

458)

53%

,67

%ZP

0375

3494

(753

56–73

437)

44%

,57

%

gi128

0235

2L.

gasseri

R_inulin

ivorans-

1.0.1_

Cont419

.1(752

36–

7373

4)27

%,45

%gi345

8178

8R.gnav

us

R_inulin

ivorans-

1.0.1_

Cont419

.1(752

33–

7373

1)25

%,40

%

Todeterminetherelationship

betwee

nthesestrain

activities

andtheirsequen

cepotentialitiesforboth

UidA(E.coliK12

P058

04)an

dH11

G11

-BGhomologs,UidAhomologsweresearch

edusingtblastnin

the

gen

omes

concerned

andfurther

aligned

toH11

G11

-BGhomologs.Comparativean

alysisofiden

tity

percentages

betwee

nthetw

oBGclassesallowed

iden

tificationofstrainsforwhichaH11

G11

-BGhomologwas

theonly

knownBG

gen

eab

leto

gen

eratean

activity.Th

esameap

proachwas

perform

edwiththethreeBacteroidetes

strainsstudied(uniqueBG

group)ex

ceptthat

theinco

mplete

16SrRNA

sequen

ceof

Parabacteroides

merdae

ATC

C43

184T

was

replacedbytheclosest

sequen

cefrom

uncu

lturedbacterium

cloneTS

4_a0

2f06

.



Table S4. Oligonucleotides and PCR conditions used in this study

Gene (metagenomic clone) Primer Sequences

Putative aspartyl-tRNA synthetase H3D4 F-H3D4-Synth-Gt 5′-GGA TCC AAT GTA TAG ATC ACA CAC CTG CGG AGA ATT G-3′R-H3D4-Synth-Gt 5′-CTG CAG TCA CTT AAA AGT GAC CTT TTT ATT CAT CAG GAA-3′

Putative 2C-methyl-D-erythritol 2,4-cyclodiphosphatesynthase C11H2

F-C11H2-Ery 5′-GGA TCC AAT GAG TTC GGG AAT GAT GAA ATT CAG A-3′

R-C11H2-Ery 5′-CTG CAG CTA TTC CCT GTA AAT CAG GGC GAC CGC-3′Putative serine/threonine protein kinase H11B1 F-H11B1 5′-GAA TTC GAT GCC AGC AGA AGG CCA GCC AGA C-3′

R-H11B1 5′-CTG CAG TTA ATT GAT TTC AAT TCC GGA GTT TTT-3′Putative sporulation protein H11G11 F-H11G11-Spo 5′-GGA TCC AAT GAA AAC ATA CGG TAT TTT ATG CTT -3′

R-H11G11-Spo 5′-CTG CAG TCA AAT AAT TTC GGT GTT CGG ATA GTA-3′Putative ABC transporter, permease protein H11G11 F-H11G11-Perm 5′-GGA TCC AAT GAG AAA AGA TTT TCA GCG GGA AAT A-3′

R-H11G11-Perm 5′-CTG CAG TTA TAC CAA AGC GGC AAC AAG GAA GAA TAT-3′Putative β-galactosidase H11G11 F-H11G11-Bgal 5′-GGA TCC AAT GCG AGA AGT AAT AAA TAT AAA CAA-3′

R-H11G11-Bgal 5′-CTG CAG TTA TTT CTG CTT TTT CTT AAT CTT GTT-3′

Underlining indicates restriction enzymes sites (BamHI and PstI except for putative serine/threonine protein kinase H11B1: EcoRI and PstI). PCR was conductedunder the following conditions: putative aspartyl-tRNA synthetase H3D4—(F0.3 μM, R0.3 μM), PCR (15′ 95 °C, 5 cycles: 94 °C 1′, 62 °C 1′, 72 °C 2′, 5 cycles: 94 °C 1′,57 °C 1′, 72 °C 2′, 10 cycles: 94 °C 1′, 52 °C 1′, 72 °C 2′10 and10-min elongation step at 72 °C); putative serine/threonineprotein kinaseH11B1—(F0.2 μM,R0.2 μM), PCR(15′ 95 °C, 5 cycles: 94 °C 1′, 68 °C 1′, 72 °C 2′, 5 cycles: 94 °C 1′, 63 °C 1′ and 72 °C 2′, 20 cycles: 94 °C 1′, 58 °C 1′, 72 °C 3′ and 10-min elongation step at 72 °C); putativeABC transporter, permease proteinH11G11—(F0.2 μM,R0.2 μM), PCR (15′ 95 °C, 5 cycles: 94 °C 1′, 66 °C 1′, 72 °C 1′ 30 20 cycles: 94 °C 1′, 61 °C 1′, 72 °C 1′ 30 and10-minelongation step at 72 °C); putative 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase C11H2—(F0.3 μM, R0.3 μM, 3% DMSO); PCR (15 ′95 °C, 5 cycles: 94 °C 1′,68 °C 1′, 72 °C 30′′, 20 cycles: 94 °C 1′, 62 °C 1′, 72 °C 45′′ and 10-min elongation step at 72 °C); putative sporulation protein H11G11—(F0.3 μM, R0.3 μM); PCR (15′95 °C, 5 cycles: 94 °C 1′, 63 °C 1′, 72 °C 1′, 20 cycles: 94 °C 1′, 58 °C 1′, 72 °C 1′ and 10-min elongation step at 72 °C); putative β-galactosidase H11G11—(F0.6 μM,R0.6 μM,3% DMSO), (15′ 95 °C, 5 cycles: 94°C 1′, 52 °C 1′, 72 °C 2′, 20 cycles: 94 °C 1′, 47 °C 1′, 72 °C 2′ and 10-min elongation step at 72 °C).


Supporting Information - PNAS · 25/6/2010 · Best PSI-BLAST result (identity %, E-value)...

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Transcript of Supporting Information - PNAS · 25/6/2010 · Best PSI-BLAST result (identity %, E-value)...