about the cellulases distribution renaud berlemont, uci adam martiny lab
Post on 22-Dec-2015
219 views
TRANSCRIPT
![Page 1: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/1.jpg)
About the cellulases distribution
Renaud Berlemont, UCI
Adam Martiny Lab.
![Page 2: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/2.jpg)
About the GHx classification• CAZYdb Glycoside Hydrolases, …• Structure – Sequences Alignements : Families (>100) / Clans (14)• « Convergence – Divergence »
![Page 3: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/3.jpg)
Some statements
• Biochemically confirmed « cellulases » = CMCases
![Page 4: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/4.jpg)
Some statements
• Biochemically confirmed « cellulases » = CMCases
• Many cellulases are active on other substrates (e.g. xylan)
• Many « cellulases » are non-cellulolytic !?• CMCases ≠ Cellulases
![Page 5: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/5.jpg)
• 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)
![Page 6: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/6.jpg)
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
![Page 7: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/7.jpg)
Hypothesis 2a
Question 2
How 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.
![Page 8: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/8.jpg)
pSEED - FigFams• Sequenced genomes (patricbrc db - 4089)
In order to analyze as much as possible sequenced genomes
![Page 9: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/9.jpg)
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,
![Page 10: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/10.jpg)
Methodology
FigFam IDs
CAZYdb
E.C. 3.2.1.4
GHx
Pfam (pro. + euk.) InterPRo (pro.)
PfGHx.FASTA IprGHx.FASTA
pSEED
PEG IDs
Home-made Script :SEQ PEG ID
GH families
Figfam IDs
Several Figfam IDs correspondTo one GHx families because Signal
Peptides and accessory domainsAre not conserved …
![Page 11: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/11.jpg)
Methodology
FigFam IDs
GHx pSEED
Genomes Annotations
GHx Occurrence In
Sequenced genomes
AlignmentStatistic
Bacterialgroups
CBM2 …
Bacterialgroups
Bacterialgroups
…
Occurrence / List
Occurrence / List
Figfam IDs
Genomes annotations (pSEED)
GHx distribution
![Page 12: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/12.jpg)
A huge data-set
A ActinobacteriaB AequfacieC Bactero./ChlorobiD Chlam./ Verruco.E ChloroflexiF ChrysiogenetesG CyanobacteriaH DeferibacterI Deinoco./ThermusJ DictyoglomiK ElusomicrobiaL Fibrob./ Acidobact.M FirmicutesN FusobacteriaO NitrospiraeP GemmatimonadetesQ PlanctomycesR ProteobacteriaS SpirochaetesT SynergistetesU TenericutesV Thermodesulfobact.W Thermotogae
Huge bias : A + C + M + R = 88% of the sequenced genomes…
![Page 13: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/13.jpg)
Average Gene Content (AGC)
A ActinobacteriaB AequfacieC Bactero./ChlorobiD Chlam./ Verruco.E ChloroflexiF ChrysiogenetesG CyanobacteriaH DeferibacterI Deinoco./ThermusJ DictyoglomiK ElusomicrobiaL Fibrob./ Acidobact.M FirmicutesN FusobacteriaO NitrospiraeP GemmatimonadetesQ PlanctomycesR ProteobacteriaS SpirochaetesT SynergistetesU TenericutesV Thermodesulfobact.W Thermotogae
Life style (Auto Vs. Hetero)Host association
…
“HKG”Multi-function
…
![Page 14: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/14.jpg)
GHx distribution in Genomes
Life Style
Autotrophic : Aequifacie
CyanobacteriaChrysiogenetes
Nitrospirae
Host associated: Chlam./ Verruco.
ElusomicrobiaFibrob./ Acidobact.*
FusobacteriaSpirochaetesTenericutes
![Page 15: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/15.jpg)
GHx distribution in GenomesGHx functions
« house keeping »
GH6endoglucanase ; cellobiohydrolase
GH18… endo-β-N-acetylglucosaminidase …
Q: Planctomycetes
U: Tenericutes - Mycoplasma
![Page 16: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/16.jpg)
GHx distribution in GenomesGHx functions
GHx families « specialization »
GH6endoglucanase ; cellobiohydrolase
GH5chitosanase ; β-mannosidase ; cellulase ; glucan β-1,3-glucosidase ; licheninase
; glucan endo-1,6-β-glucosidase mannan endo-β-1,4-mannosidase ; endo-β-1,4-xylanase
; cellulose β-1,4-cellobiosidase ; β-1,3-mannanase ; xyloglucan-specific endo-β-1,4-glucanase
; mannan transglycosylase ; endo-β-1,6-galactanase ; endoglycoceramidase
How is it possible to know if an Enzyme from the GH5
is a cellulase?
![Page 17: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/17.jpg)
Complex architecturesGH5
chitosanase (EC 3.2.1.132); β-mannosidase (EC 3.2.1.25); cellulase (EC 3.2.1.4); glucan β-1,3-glucosidase (EC 3.2.1.58); licheninase
(EC 3.2.1.73); glucan endo-1,6-β-glucosidase (EC 3.2.1.75) mannan endo-β-1,4-mannosidase (EC 3.2.1.78); endo-β-1,4-xylanase
(EC 3.2.1.8); cellulose β-1,4-cellobiosidase (EC 3.2.1.91); β-1,3-mannanase (EC 3.2.1.-); xyloglucan-specific endo-β-1,4-glucanase
(EC 3.2.1.151); mannan transglycosylase (EC 2.4.1.-); endo-β-1,6-galactanase (EC 3.2.1.164);
endoglycoceramidase (EC 3.2.1.123)
GH6endoglucanase (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 !
?
![Page 18: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/18.jpg)
Is there an efficient combination of enzymes ?
![Page 19: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/19.jpg)
Some genes are abundant
(GH5, 10, 16, 18, 19)Are these genes really involved in
PCW breakdown ?
Why are Fibrobacteria so Efficient ?
Multi-domain
Is there an efficient combination of enzymes ?
![Page 20: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/20.jpg)
Is there an efficient combination of enzymes ?
The keys of the succes in Fibrobacteria
![Page 21: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/21.jpg)
• 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 »
Things to remember…
![Page 22: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/22.jpg)
What’s next
Looking at the GHx-distribution in subgroups (e.g Proteobacteria, Firmicutes, …)
Detailed table of the GHx distribution amongst (sub)-taxa
![Page 23: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/23.jpg)
Potential publication ?
• What is the phylogenetic distribution of GHx’s and CBM-GHx’s
• 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/absence of
GHx’s
![Page 24: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/24.jpg)
Do the cellulose degradation potential vary in environment ?
![Page 25: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/25.jpg)
Some cases studies …
GHx distribution in metagenomes% of
CBM linked GHx
Warnecke 2007
Hess 2011
Bacteroidetes, Fibrobacteria,Clostridia, …
Spirochaetes, Fibrobacter, Bacteroidetes, …
![Page 26: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/26.jpg)
…Vs. Our studyUsing the SSU…
L1 L2 L3 L4 L5 L6 PL
Per
cent
of
hits
to
bact
eria
l SS
U-r
RN
Ase
quen
ces
0
20
40
60
80
100
120Fibrobacter/AcidobacterBacteroidetesCyanobacteriaFirmicutesGammaProteobacteriaBetaProteobacteriaAlphaProteobacteriaActinoBacteriaOthers
![Page 27: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/27.jpg)
…Vs. Our study
Reno 2012 (probably)
Actinobacteria, Alphaproteobacteria,Bacteroidetes, …
Warnecke 2007
Hess 2011
Bacteroidetes, Fibrobacteria,Clostridia, …
Spirochaetes, Fibrobacter, Bacteroidetes, …
![Page 28: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/28.jpg)
Metagenomes Clustrering
Environment selects for different populations (with different GHx)
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
16S rRNA
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
GHx
?
![Page 29: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/29.jpg)
• 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, GH9]LL > [GH6, GH8, GH9]CR
– [CMB-GHx]LL > [CBM-GHx]CR
Things to remember…
![Page 30: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/30.jpg)
What’s 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
![Page 31: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/31.jpg)
![Page 32: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/32.jpg)
Nitrogen fertilization
Nemergut, 2008, The effects of chronic nitrogen fertilization on alpinetundra soil microbial communities: implications forcarbon and nitrogen cycling.
![Page 33: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/33.jpg)
16S
rR
NA
GH
x
GH
y
GH
z
16S
rR
NA
GH
x
GH
y
GH
z
16S
rR
NA
GH
z
cont
rol
cont
rol
16S
rR
NA
GH
z
cont
rol
cont
rol
16S
rR
NA
GH
z
cont
rol
cont
rol
![Page 34: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/34.jpg)
24 samples
• TruSeq TM DNA (Illumina)
• 24 samples
• 22 samples ready to be sequenced
![Page 35: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/35.jpg)
Complex architectures
Cel5CBM2
Xyl8 Cel5CBM2
Cel5
Amount of FigFam IDs corresponding to a 2-domain protein
Plant Cell Wall
Amount of FigFam IDs ≠ Amount of genes
![Page 36: About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab](https://reader035.vdocuments.site/reader035/viewer/2022062421/56649d795503460f94a5be6e/html5/thumbnails/36.jpg)
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Metagenomes Clustrering
Environment selects for different GHx potential
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
Leaf Litter
Leaf Litter (tr. 1)
Leaf Litter (tr. 2)
Cow Rmuen
Termites
Wood feeding insects
Human metagenome
GOS
16S rRNA GHx