About the cellulases distribution

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About the cellulases distribution

• Renaud Berlemont, UCI
• Adam Martiny Lab.

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About the GHx classification

• CAZYdb ? Glycoside Hydrolases,
• Structure Sequences Alignements Families
  (gt100) / Clans (14)
•  Convergence Divergence 

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Some statements

• Biochemically confirmed  cellulases  CMCases

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Some statements

• Biochemically confirmed  cellulases  CMCases
• Many cellulases are active on other substrates
  (e.g. xylan)
• Many  cellulases  are non-cellulolytic !?
• CMCases ? Cellulases

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• Cellulose production
• GH8 (Romling, 2002) Biofilm / Interaction (w.
  plant)
• GH5 (Berlemont, 2009) - Biofilm
• GH6 (Delbrassine, in prep) Cell differenciation
• GH6 (Tunicate, animal)
• GH9 (KORrigan, plant)

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Some statements

• Biochemically confirmed  cellulases  CMCases
• Many cellulases are active on other substrates
  (e.g. xylan)
• Many  cellulases  are non-cellulolytic
• CMCases ? Cellulases
• Best studied cellulose degraders all belong to
  the Firmicutes group (e.g. Clostridium)
• 20 genomes of cellulose degraders have been
  completely sequenced

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Hypothesis 2a
Question 2How are extracellular enzyme genes
distributed among microbial taxa ?
Some extracellular enzymes are broadly
distributed across taxa while others are
constrained to a small number of taxa.
Hypothesis 2b
The occurrence of different extracellular enzyme
genes among taxa will be correlated. Some genes
will show patterns of over-dispersion while
others will show co-occurrence.
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pSEED - FigFams

• Sequenced genomes (patricbrc db - 4089)

In order to analyze as much as possible sequenced
genomes
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pSEED - FigFams

•  FIGfams are sets of protein sequences that are
  similar along the full length of the proteins.
  Proteins are thought of as implementing one or
  more abstract functional roles, and all of the
  members of a single FIGfam are believed to
  implement precisely the same set of functional
  roles .

 Unambiguous coherent annotation
system   3.2.1.4 1,4-beta-D-endoglucanase,
1,4-beta-D-glucan-4-glucanohydrolase,
beta-1,4-endoglucan hydrolase,
beta-1,4-endoglucanase, endoglucanase,
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Methodology
GH families
Figfam IDs
Several Figfam IDs correspond To one GHx families
because Signal Peptides and accessory
domains Are not conserved
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Methodology
Figfam IDs
Genomes annotations (pSEED)
GHx distribution
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A huge data-set
A ActinobacteriaB AequfacieC
Bactero./ChlorobiD Chlam./ Verruco.E
ChloroflexiF ChrysiogenetesG
CyanobacteriaH DeferibacterI
Deinoco./ThermusJ DictyoglomiK
ElusomicrobiaL Fibrob./ Acidobact.M Firmicutes
N FusobacteriaO NitrospiraeP GemmatimonadetesQ
PlanctomycesR ProteobacteriaS SpirochaetesT Syn
ergistetesU TenericutesV Thermodesulfobact.W Th
ermotogae
Huge bias A C M R 88 of the sequenced
genomes
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Average Gene Content (AGC)
A ActinobacteriaB AequfacieC
Bactero./ChlorobiD Chlam./ Verruco.E
ChloroflexiF ChrysiogenetesG
CyanobacteriaH DeferibacterI
Deinoco./ThermusJ DictyoglomiK
ElusomicrobiaL Fibrob./ Acidobact.M Firmicutes
N FusobacteriaO NitrospiraeP GemmatimonadetesQ
PlanctomycesR ProteobacteriaS SpirochaetesT Syn
ergistetesU TenericutesV Thermodesulfobact.W Th
ermotogae
Life style (Auto Vs. Hetero) Host association
HKG Multi-function
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GHx distribution in Genomes
Life Style
Autotrophic Aequifacie Cyanobacteria Chrysiogen
etes Nitrospirae
Host associated Chlam./ Verruco.ElusomicrobiaF
ibrob./ Acidobact.FusobacteriaSpirochaetesTene
ricutes
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GHx distribution in Genomes
GHx functions  house keeping 
GH6 endoglucanase cellobiohydrolase
GH18 endo-ß-N-acetylglucosaminidase
Q Planctomycetes
U Tenericutes - Mycoplasma
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GHx distribution in Genomes
GHx functions GHx families  specialization 
GH6 endoglucanase cellobiohydrolase
GH5 chitosanase ß-mannosidase cellulase
glucan ß-1,3-glucosidase licheninase glucan
endo-1,6-ß-glucosidase mannan
endo-ß-1,4-mannosidase endo-ß-1,4-xylanase
cellulose ß-1,4-cellobiosidase ß-1,3-mannanase
xyloglucan-specific endo-ß-1,4-glucanase
mannan transglycosylase endo-ß-1,6-galactanase
endoglycoceramidase
How is it possible to know if an Enzyme from the
GH5 is a cellulase?
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Complex architectures
GH5 chitosanase (EC 3.2.1.132) ß-mannosidase (EC
3.2.1.25) cellulase (EC 3.2.1.4) glucan
ß-1,3-glucosidase (EC 3.2.1.58) licheninase (EC
3.2.1.73) glucan endo-1,6-ß-glucosidase (EC
3.2.1.75) mannan endo-ß-1,4-mannosidase (EC
3.2.1.78) endo-ß-1,4-xylanase (EC 3.2.1.8)
cellulose ß-1,4-cellobiosidase (EC 3.2.1.91)
ß-1,3-mannanase (EC 3.2.1.-) xyloglucan-specific
endo-ß-1,4-glucanase (EC 3.2.1.151) mannan
transglycosylase (EC 2.4.1.-) endo-ß-1,6-galacta
nase (EC 3.2.1.164) endoglycoceramidase (EC
3.2.1.123)
GH6 endoglucanase (EC 3.2.1.4) cellobiohydrolase
(EC 3.2.1.91)
GH6  cellulase 
GH5 Multifunction
Free cellulases from the GH6 are Associated to
the cellulose production In actynomycetes !
?
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Is there an efficient combination of enzymes ?
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Is there an efficient combination of enzymes ?
Some genes are abundant (GH5, 10, 16, 18,
19) Are these genes really involved in PCW
breakdown ?
Multi-domain
Why are Fibrobacteria so Efficient ?
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Is there an efficient combination of enzymes ?
The keys of the succes in Fibrobacteria
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Things to remember

• Huge dataset
• Distribution of GHx amongst taxa
• Not all the GHx are equivalent
• Multifunction, house keeping and specialized GHx
  families
• Not all the taxa are equivelent
• Life style, metabolism
• Future  Multi-domain 

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Whats next

• Looking at the GHx-distribution in subgroups (e.g
  Proteobacteria, Firmicutes, )
• ? Detailed table of the GHx distribution amongst
  (sub)-taxa

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Potential publication ?

• What is the phylogenetic distribution of GHxs
  and CBM-GHxs
• Catabolism regulation analysis in Actynobacteria
  CebR (GHx vs CBM-GHx)
• Presence/absence of regulating sequences upstream
  the GHx-coding sequences
• Environmental factors life style,
  metabolism,
• Gene Gain/loss 16S rRNA Vs. presence/abse
  nce of GHxs

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Do the cellulose degradation potential vary in
environment ?
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Some cases studies
GHx distribution in metagenomes
of CBM linked GHx
Warnecke 2007
Spirochaetes, Fibrobacter, Bacteroidetes,
Hess 2011
Bacteroidetes, Fibrobacteria, Clostridia,
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Vs. Our study
Using the SSU
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Vs. Our study
Reno 2012 (probably)
Actinobacteria, Alphaproteobacteria, Bacteroidetes
,
Warnecke 2007
Spirochaetes, Fibrobacter, Bacteroidetes,
Hess 2011
Bacteroidetes, Fibrobacteria, Clostridia,
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Metagenomes Clustrering
16S rRNA
GHx
?
Environment selects for different populations
(with different GHx)
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Things to remember

• Different recipes for efficient PCW breakdown
• Depending on the ecosystem
• Leaf litter ? Cow Rumen
• Bacterial content
• GH content
• Regarding the ecosystems, bacteria display
  different strategies to access plant polymers
• GH6, GH8, GH9LL gt GH6, GH8, GH9CR
• CMB-GHxLL gt CBM-GHxCR

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Whats next

• Leaf Litter Metagenome
• 22 samples ready to be sequenced
• (TruSeq TM DNA -Illumina) (first year)
• samples to be prepared (second year)
• Compare
• GHx/16s rRNA in sequenced genomes
• vs.
• GHx/16s rRNA in Leaf Litter
• Compare different treatments, metagenomes

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Nitrogen fertilization
Nemergut, 2008, The effects of chronic nitrogen
fertilization on alpine tundra soil microbial
communities implications for carbon and nitrogen
cycling.
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24 samples

• TruSeq TM DNA (Illumina)
• 24 samples
• 22 samples ready to be sequenced

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Complex architectures
CBM2
CBM2
Cel5
Xyl8
Cel5
Cel5
Amount of FigFam IDs corresponding to a 2-domain
protein
Plant Cell Wall
Amount of FigFam IDs ? Amount of genes
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Metagenomes Clustrering
16S rRNA
GHx
Environment selects for different GHx potential