Phylogenetic distribution of
potential cellulases in bacteria
R. Berlemont & A. Martiny
The cellulose degradation
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Project Goals
4 main questions :
1) How do microbial taxa respond
to environmental changes?
2) How are extracellular enzyme
genes distributed among
microbial taxa?
3) Can we predict enzyme
function and litter decomposition
rates by combining enzyme gene
distributions with microbial taxa
responses to environmental
change?
4) Are microbial communities and
their functions resilient to
environmental change?
Analyze the Gene Content in
Available sequenced bacterial
genomes
CAZy classification
•
•
•
•
•
Endo/Exo-cellulases, -glucosidases
Glycoside Hydrolases, GH
>100 GH families (14 folds)
>100 activities
multifunctional enzymes contain catalytic
domains that belong to different GH
families
Mining the CAZy
Atypical
/
barrel
I
231
GH8
(/)6
I
GH9
(/)6
β-jelly
roll
(/)6
(/)6
(/)6
GH12
GH44
GH45
GH48
2
6 (63)
16
13
4
538
55
26
I
468
75
66
R
144
22
21
1 (1)
I
I
I
55
13
128
10
13
13
8
3
4
1 (4)
4 (33)
6
11
1
1 (1)
(Berlemont
et al, 2009)
(de Jong et
al, 2009)
(Romling,
2002)
(SEED)
GH6
152
orthologs
1952
5 (43)
8 (50)
Identified
R
48
55
synthesis
(-)8
1
5
Cellulose
GH5
87
91
206
Other
characterized
activity (hits)
2890
3212
-glucosidase
(3.2.1.21)
R
R
Exocellulase
(3.2.1.74/91/1
76)
Hits
(-)8
(-)8
Endocellulase
(3.2.1.4)
Mechanism
GH1
GH3
Enzymes
characterized
*
Structure
Endo/exo -
-
GH family
CAZy
9071
7105
1727
699
702 GH8 &
380 BcsZ
160
2095
1
580
3
Accessing the genomes
• SEED environment
• PATRIC database
• GH : 1, 3, 5, 6, 8, 9, 12, 44, 45, 48
CAZy Vs. us
Atypical
/
barrel
I
231
GH8
(/)6
I
GH9
(/)6
β-jelly
roll
(/)6
(/)6
(/)6
GH12
GH44
GH45
GH48
2
6 (63)
16
13
4
538
55
26
I
468
75
66
R
144
22
21
1 (1)
I
I
I
55
13
128
10
13
13
8
3
4
1 (4)
4 (33)
6
1
1 (1)
9071
7105
1727
(Berlemont
et al, 2009)
(de Jong et
al, 2009)
(Romling,
2002)
(SEED)
GH6
152
orthologs
1952
5 (43)
8 (50)
Identified
R
48
55
synthesis
(-)8
1
5
Cellulose
GH5
87
91
206
Other
characterized
activity (hits)
2890
3212
-glucosidase
(3.2.1.21)
R
R
Exocellulase
(3.2.1.74/91/1
76)
Hits
(-)8
(-)8
Endocellulase
(3.2.1.4)
Mechanism
GH1
GH3
Enzymes
characterized
*
Structure
Endo/exo -
-
GH family
CAZy
699
702 GH8 &
380 BcsZ
160
2095
11
CAZY : all the NCBI genomes (including genome fragments) – 9,631 genes
SEED : Only the sequenced genomes (3711) are considered - 22,523 genes
1
580
3
Variations across phyla
GH rich:
Acidobacteria,
Bacteroidetes,
Digtyoglomi,
Fibrobacteres,
Thermotogae,
Verrucomicrobia
Lentisphaerae
(Actinobacteria,
Chloroflexi,
Firmicutes)
Variations across subphyla
•
•
•
•
Actinobacteria
402 genomes
Niches (soil : Streptomycetales)
‘GH-rich’
Strain specific GH
distribution
16S rRNA phylogeny
0 GH
1 GH
>1 GH
GH associations
Redundancy
Complementarities
Endocellulases
Spearman Correlation
Glycoside Hydrolase abundance
Endo/exo -
Synergistic model
Group IIIa
Group IIIb
Exocellulases
-glucosidases
Group I
BcsZ
Group II
GH associations
• Nothing ! (can’t degrade cellulose) (21%)
• No Group II – III : opportunists (Opp.) (44%)
• 100% Gr.I and 0% Gr.II:Gr.III
• putative Cellulose Degraders (pCD) (35%)
• 100% GII/III and GI (94%)
•pCD vs. Opp. ?
GH associations
Cellulose degraders and opportunists
• Actinobacteria
+ Bacteroidetes
+ Proteobacteria
+ Firmicutes = 86 %
Sequenced strains
• putative Cell. Degr.,
Opportunists, in all phyla
•  35 % pCD [16-56%]
• G1pCD = (1.7-4.4) G1Opp.
Opportunists
put. cell. degr.
Poor strains … and their life styles
“Poor phyla, subphyla and strains” ?
e.g. : Cyanobacteria
RuBisCO
Life-style vs. GH content
Autotrophs
Intracellular
Actinobacteria
Phylogeny vs. GH content
Our first “delivrable”
• 16s rRNA Phylogeny (Neighbor Joining)
• Glycoside hydrolases based-clustering (Bray Curtis)
• distance between the matrices, CADM (Mantel)
Phylogeny vs. GH content
?
GH – based clustering
16s rRNA phylogeny
402 Actinobacteria
Conclusions
• No information concerning the Activity of these enzymes
• 3711 sequenced genomes ≠ natural population (pathogens)
• Genomic perspectives
– New “pipeline” for genes distribution/association
– Tell me what is your GH content, I ‘ll tell you who you are!
• Ecological perspectives
– Functional redundancy in sequenced genomes (substrate, pH, …),
– 44% opportunists, 35% putative cellulose degraders
• Implications for ecosystems processes
– -glucosidases reveal nothing about the cellulose degradation !
– More than 35% of the strains have endo/exo-cellulases !
– Cellulose degraders have multiple copies of these genes !