environmental and gut bacteroidetes - centre national de la

Environmental and gut Bacteroidetes:

the food connection

Gurvan MICHEL

UMR 7139: Marine plants and Biomolecules

Macroalgae evolved complex multicellularity independently from land plants, fungi and animals

Adapted from Baldauf, 2008

Me

-3OSO

OOSO3-

O

O

OH

Me OOSO3- Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the coastal primary production

Support the coastal food webs:from marine herbivorous animals

to human activities

(sea vegetables, hydrocolloids)

Me

-3OSO

OOSO3-

O

O

OH

Me OOSO3- Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the coastal primary production

Marine heterotrophic bacteria:key players in the recycling of algal biomass

Me

-3OSO

OOSO3-

O

O

OH

Me OOSO3- Polysaccharides

= ~50% algal biomass

Macroalgae: crucial role in the coastal primary production

• Recent availability of numerous marine bacterial genomes

• Annotation based on the knowledge of land plant degraders

How marine bacteria recycle algal biomass?

• Recent availability of numerous marine bacterial genomes

• Annotation based on the knowledge of land plant degraders

• Polysaccharides from seaweeds are different from land plant polysaccharides

• The catabolic pathways of algal polysaccharides are largely unknown

But

How marine bacteria recycle algal biomass?

• Recent availability of numerous marine bacterial genomes

• Annotation based on the knowledge of land plant degraders

• Polysaccharides from seaweeds are different from land plant polysaccharides

• The catabolic pathways of algal polysaccharides are largely unknown

But

How marine bacteria recycle algal biomass?

Critical lack of experimental knowledge on marine-specific

processes to correctly interpret marine (meta)genomic data

Zobellia galactanivorans: a model marine bacterium foralgae-bacteria interactions

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Genome sequencing (4738 genes, 5.5 MB)

• Large system for substrate detection and import

119 TonB-dependent receptors (TBDR) and 65 one/two-component systems

• Confirmation of the huge potential for polysaccharide degradation:

136 Glycoside hydrolases (GH) and 71 sulfatases !

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Zobellia galactanivorans: a model marine bacterium foralgae-bacteria interactions

Genome sequencing (4738 genes, 5.5 MB)

• Large system for substrate detection and import

119 TonB-dependent receptors (TBDR) and 65 one/two-component systems

• Confirmation of the huge potential for polysaccharide degradation:

136 Glycoside hydrolases (GH) and 71 sulfatases !

• Numerous putative operons specific for polysaccharide utilization

• Bacteroidetes isolated in Roscoff on a red alga

• Degrade most algal polysaccharides

Zg2624Zg2615Zg2613 Zg2614 Zg2616 Zg2617 Zg2618 Zg2619 Zg2620 Zg2621 Zg2622 Zg2623

Sugar kinase

transcriptionalfactor UNK

SusD-like

TBDR

Sugardehydrogenase

KgdF-like

PL17PL7Sugar

permease

SugardehydrogenaseSugar kinase

Zg2612

NRAMPtransporter

Poster 29

Zobellia galactanivorans: a model marine bacterium foralgae-bacteria interactions

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

Phylogenetic tree of the GH16 family from Zobellia

Allouch et al, (2003) JBC & ( 2004) Structure

Jam et al (2005) Biochem J

AgaB

AgaA

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

GH16 X70 UNK

GH16

GH16 CBM6

GH16

GH16 UNK

GH16 UNK

GH16

GH16

GH16

GH16 CBM16

GH16PKD

GH16

GH16 UNK CBM42

GH16 CBM6

GH16 CBM6 UNK

GH16

-agarases

-carrageenases

Laminarinases

A new sub-family of GH16 ?

~25% identity with AgaA and CgkA

Phylogenetic tree of the GH16 family from Zobellia

Zg3628 AgaF

Zg3640 AgaG

Zg1017 AgaH

Zg3376 AgaI

Zg2600 AgaE

Zg4267 AgaC

Zg4203 AgaA

Zg3573 AgaB

Zg4243 AgaD

Zg236 CgkA

Zg2431 LamA

Zg367 LamB

Zg1010 LamC

Zg1014 LamD

Zg1021

Z3347

LicA Bl

74

100

96

63

59

95

67

48

46

33

74

37

38

41

GH16 X70 UNK

GH16

GH16 CBM6

GH16

GH16 UNK

GH16 UNK

GH16

GH16

GH16

GH16 CBM16

GH16PKD

GH16

GH16 UNK CBM42

GH16 CBM6

GH16 CBM6 UNK

GH16

-agarases

-carrageenases

Laminarinases

Phylogenetic tree of the GH16 family from Zobellia

Overexpression of Zg1017 & Zg2600

Zg1017

Zg2600

Activity screening on cell wall extracts from seaweeds

• Inactive on agarose and carrageenans

Zg1017

Zg2600

Activity screening on cell wall extracts from seaweeds

L6S = L-galactose-6-sulfateLA = 3,6-anhydro-L-galactose

Zg2600 (PorA) et Zg1017 (PorB) are the first beta-porphyranases

• Main end product: Porphyran disaccharide (L6S-G)• Hydrolysis of beta-1,4 glycosidic linkage

-1,3 -1,4• Inactive on agarose and carrageenans

PorBPorA

Crystal structure of the -porphyranases PorA and PorB

Hehemann et al (2010) Nature

Discovery of a new GH family in Zobellia

• 5 paralogous proteins distantly related to GH43 (~15% sequence identity)

• Always localized in gene clusters with CAZymes and sulfatases

• Zg3597 and Zg4663: soluble expressed in the MARINE-EXPRESS project

(Groisillier et al, 2010, Molecular Cell Factories)

• Purification and crystallization of Zg3597 and Zg4663

• Reducing sugar activity screening on our collection of algal polysaccharides

and cell wall extracts

Hypothesis: new GH specific for algal sulfated polysaccharides?

Unsuccessful…

?

?

?

Surrounded by -agarases, -porphyranases and -galactosidases

Zg4663: a -1,3-galactosidase specific for agarocolloids?

Neoagarobiose (DP2)

-1,3

Test of the activity of Zg4663 on oligo-agars

released by the -agarase AgaB

Thin layer chromatography

Neoagarobiose (DP2)

-1,3

1121 (DP7)

3,6-anhydro-L-galactose

815 (DP5)

509 (DP3)

m/z (M+Na+)

Test of the activity of Zg4663 on oligo-agars

released by the -agarase AgaB

Lane 2 and 4: addition of Zg4663

Size of the products determined by mass spectrometry (MALDI-TOF)

Thin layer chromatography

• Zg4663 is a 1,3- 3,6-anhydro-L-galactosidase (last step of agar degradation)

Neoagarobiose (DP2)

-1,3

• Adopts a five-bladed -propeller fold and forms a dimer by domain swapping

• Displays a zinc-dependent catalytic machinery

1121 (DP7)

3,6-anhydro-L-galactose

815 (DP5)

509 (DP3)

m/z (M+Na+)

• Zg4663 is a 1,3- 3,6-anhydro-L-galactosidase (last step of agar degradation)

Rebuffet et al (2011) Environmental Microbiology

GH16 GH117

All -agarases, -porphyranases and GH117 originate from marine bacteria

With the exception of Bacteroides plebeius, a Japanese gut bacterium !

Bacteroides plebeius received porphyran-related genes from ancestral marine bacteria

Metagenome analysis shows that porphyranases, agarases and GH117:

• are frequent in the gut microbiota of Japanese people (38%)

Cohort: 13 individuals; total read length: 0.73 Gb

• are absent in the gut microbiota of American and Danish people

Cohorts: 18 American (TRL: 1.83 Gb); 85 Danish (TRL: 391.58 Gb)

• are present in the gut microbiota of a minority of Spanish people (10 %)Cohort: 39 individuals; total read length: 185.13 Gb

Gut-metagenome analysis shows that porphyranases, agarases and GH177:

• are frequent in the gut microbiota of Japanese people (38%)

Cohort: 13 individuals; total read length: 0.73 Gb

• are absent in the gut microbiota of American and Danish people

Cohorts: 18 American (TRL: 1.83 Gb); 85 Danish (TRL: 391.58 Gb)

• are present in the gut microbiota of a minority of Spanish people (10 %)Cohort: 39 individuals; total read length: 185.13 Gb

Hehemann et al, (2010) Nature; Rebuffet et al (2011) Env Micro; Thomas et al (2011) Front Micro

1. HGT of agarolytic genes from marine bacteria associated to seafood seaweeds for Japanese likely animal seafood for Spanish

2. Hypothesis: regular HGT of CAZYme genes from plant-associated bacteria during the evolution of animals

alyA3 SDR SusC‐like SusD‐like alyA2

GntR‐like

permease

SDR

kdgK1

kdgF

Thomas et al, 2012

Marine Proteobacteria

Marine FlavobacteriaComplete, frequent, conserved clusters

Gut Bacteroides

sameecological

niche

dietmediated

Poster 29

LionelCladière

TristanBarbeyron

MirjamCzjzek

AlexandraJeudy

Jan-HendrikHehemann

MurielleJam

FrancoisThomas Etienne

Rebuffet

Thank you! Marine Glycobiology Group

JustynaRzonca

MPI BremenRudi AmannFrank-Oliver Glöckner

Synchrotron staffs(ESRF, SOLEIL)