Phylogenetic distribution of potential cellulases in bacteria

R. Berlemont & A. Martiny

The cellulose degradation

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Project Goals

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 genedistributions with microbial taxa responses to environmental change?4) Are microbial communities and their functions resilient to environmental change?

4 main questions :

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 CAZy

GH

fam

ily

Str

uctu

re

Mec

hani

sm

Hit

s

Enz

ymes

ch

arac

teri

zed

*

End

ocel

lula

se

(3.2

.1.4

)

Exo

cell

ulas

e (3

.2.1

.74/

91/1

76)

-gl

ucos

idas

e (3

.2.1

.21)

Oth

er

char

acte

rize

d ac

tivi

ty (

hits

)

Cel

lulo

se

synt

hesi

s

GH1 (-)8 R 2890 87 1 48 5 (43) GH3 (-)8 R 3212 91 5 55 8 (50)

GH5 (-)8 R 1952 206

152 2 6 (63) (Berlemont et al, 2009)

GH6 Atypical

/ barrel

I 231 16 13 4 (de Jong et al, 2009)

GH8 (/)6 I 538 55 26 4 (33) (Romling,

2002) GH9 (/)6 I 468 75 66 6 1 1 (1)

GH12 β-jelly

roll R 144 22 21 1 (1)

GH44 (/)6 I 55 10 8 1 (4) GH45 (/)6 I 13 13 3 GH48 (/)6 I 128 13 4 11

-

End

o/ex

o -

Accessing the genomes• SEED environment

• PATRIC database

• GH : 1, 3, 5, 6, 8, 9, 12, 44, 45, 48

CAZy Vs. us CAZy

GH

fam

ily

Str

uctu

re

Mec

hani

sm

Hit

s

Enz

ymes

ch

arac

teri

zed

*

End

ocel

lula

se

(3.2

.1.4

)

Exo

cell

ulas

e (3

.2.1

.74/

91/1

76)

-gl

ucos

idas

e (3

.2.1

.21)

Oth

er

char

acte

rize

d ac

tivi

ty (

hits

)

Cel

lulo

se

synt

hesi

s

Iden

tifi

ed

orth

olog

s

(SE

ED

)

GH1 (-)8 R 2890 87 1 48 5 (43) 9071 GH3 (-)8 R 3212 91 5 55 8 (50) 7105

GH5 (-)8 R 1952 206

152 2 6 (63) (Berlemont et al, 2009)

1727

GH6 Atypical

/ barrel

I 231 16 13 4 (de Jong et al, 2009)

699

GH8 (/)6 I 538 55 26 4 (33) (Romling,

2002) 702 GH8 & 380 BcsZ

GH9 (/)6 I 468 75 66 6 1 1 (1) 160

GH12 β-jelly

roll R 144 22 21 1 (1)

2095

GH44 (/)6 I 55 10 8 1 (4) 1 GH45 (/)6 I 13 13 3 580 GH48 (/)6 I 128 13 4 11 3

-

End

o/ex

o -

CAZY : all the NCBI genomes (including genome fragments) – 9,631 genes

SEED : Only the sequenced genomes (3711) are considered - 22,523 genes

Variations across phylaGH rich:

Acidobacteria,

Bacteroidetes,

Digtyoglomi,

Fibrobacteres,

Thermotogae,

Verrucomicrobia

Lentisphaerae

(Actinobacteria,

Chloroflexi,

Firmicutes)

Variations across subphyla

• Actinobacteria

• 402 genomes

• Niches (soil : Streptomycetales)

• ‘GH-rich’

Str

ain

spec

ific

GH

di

strib

utio

n

0 GH1 GH>1 GH

16S rRNA phylogeny

GH associations

End

o/ex

o -

BcsZ

Spearman Correlation

Glycoside Hydrolase abundance

-

Synergistic model

Endocellulases

Exocellulases

-glucosidases

Com

plementarities

Redundancy

Group I

Group II

Group IIIa

Group IIIb

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

put. cell. degr.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.

Poor strains … and their life styles

RuBisCO

“Poor phyla, subphyla and strains” ?

e.g. : Cyanobacteria

Life-style vs. GH content

Autotrophs

Intracellular

Actinobacteria

Our first “delivrable”

• 16s rRNA Phylogeny (Neighbor Joining)• Glycoside hydrolases based-clustering (Bray Curtis)

• distance between the matrices, CADM (Mantel)

Phylogeny vs. GH content

Phylogeny vs. GH content16

s rR

NA

phy

loge

nyG

H – based clustering

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 !