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About the cellulases distribution
Renaud Berlemont, UCI
Adam Martiny Lab.
About the GHx classification• CAZYdb Glycoside Hydrolases, …• Structure – Sequences Alignements : Families (>100) / Clans (14)• « Convergence – Divergence »
Some statements
• Biochemically confirmed « cellulases » = CMCases
Some statements
• Biochemically confirmed « cellulases » = CMCases
• Many cellulases are active on other substrates (e.g. xylan)
• Many « cellulases » are non-cellulolytic !?• CMCases ≠ Cellulases
• 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)
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
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.
pSEED - FigFams• Sequenced genomes (patricbrc db - 4089)
In order to analyze as much as possible sequenced genomes
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,
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 …
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
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…
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
…
GHx distribution in Genomes
Life Style
Autotrophic : Aequifacie
CyanobacteriaChrysiogenetes
Nitrospirae
Host associated: Chlam./ Verruco.
ElusomicrobiaFibrob./ Acidobact.*
FusobacteriaSpirochaetesTenericutes
GHx distribution in GenomesGHx functions
« house keeping »
GH6endoglucanase ; cellobiohydrolase
GH18… endo-β-N-acetylglucosaminidase …
Q: Planctomycetes
U: Tenericutes - Mycoplasma
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?
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 !
?
Is there an efficient combination of enzymes ?
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 ?
Is there an efficient combination of enzymes ?
The keys of the succes in Fibrobacteria
• 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…
What’s next
Looking at the GHx-distribution in subgroups (e.g Proteobacteria, Firmicutes, …)
Detailed table of the GHx distribution amongst (sub)-taxa
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
Do the cellulose degradation potential vary in environment ?
Some cases studies …
GHx distribution in metagenomes% of
CBM linked GHx
Warnecke 2007
Hess 2011
Bacteroidetes, Fibrobacteria,Clostridia, …
Spirochaetes, Fibrobacter, Bacteroidetes, …
…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
…Vs. Our study
Reno 2012 (probably)
Actinobacteria, Alphaproteobacteria,Bacteroidetes, …
Warnecke 2007
Hess 2011
Bacteroidetes, Fibrobacteria,Clostridia, …
Spirochaetes, Fibrobacter, Bacteroidetes, …
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
?
• 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…
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
Nitrogen fertilization
Nemergut, 2008, The effects of chronic nitrogen fertilization on alpinetundra soil microbial communities: implications forcarbon and nitrogen cycling.
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
24 samples
• TruSeq TM DNA (Illumina)
• 24 samples
• 22 samples ready to be sequenced
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
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