Environmental and gut Bacteroidetes: the food connection Gurvan MICHEL UMR 7139: Marine plants and Biomolecules
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)