About the cellulases distribution Renaud Berlemont UCI Adam

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 : – – – GH 8 (Romling, 2002) – Biofilm / Interaction (w. plant) GH 5 (Berlemont, 2009) - Biofilm GH 6 (Delbrassine, in prep) – Cell differenciation GH 6 (Tunicate, animal) GH 9 (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

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

p. SEED - Fig. Fams • Sequenced genomes (patricbrc db - 4089) In order to analyze as much as possible sequenced genomes

p. SEED - Fig. Fams « 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,

Methodology CAZYdb GH families E. C. 3. 2. 1. 4 GHx Pfam (pro. + euk. ) Inter. PRo (pro. ) Pf. GHx. FASTA Ipr. GHx. FASTA Home-made Script : SEQ PEG ID Figfam IDs p. SEED PEG IDs Fig. Fam IDs Several Figfam IDs correspond To one GHx families because Signal Peptides and accessory domains Are not conserved …

Methodology GHx p. SEED Fig. Fam IDs Genomes Annotations GHx Occurrence In Sequenced genomes Bacterial CBM 2 groups Bacterial Occurrence / groups List Genomes annotations (p. SEED) … … Statistic Figfam IDs Alignment GHx distribution

A huge data-set A B C D E F G H I J K L M N O P Q R S T U V W Huge bias : A + C + M + R = 88% of the sequenced genomes… Actinobacteria Aequfacie Bactero. /Chlorobi Chlam. / Verruco. Chloroflexi Chrysiogenetes Cyanobacteria Deferibacter Deinoco. /Thermus Dictyoglomi Elusomicrobia Fibrob. / Acidobact. Firmicutes Fusobacteria Nitrospirae Gemmatimonadetes Planctomyces Proteobacteria Spirochaetes Synergistetes Tenericutes Thermodesulfobact. Thermotogae

Average Gene Content (AGC) Life style (Auto Vs. Hetero) Host association … “HKG” Multi-function … A B C D E F G H I J K L M N O P Q R S T U V W Actinobacteria Aequfacie Bactero. /Chlorobi Chlam. / Verruco. Chloroflexi Chrysiogenetes Cyanobacteria Deferibacter Deinoco. /Thermus Dictyoglomi Elusomicrobia Fibrob. / Acidobact. Firmicutes Fusobacteria Nitrospirae Gemmatimonadetes Planctomyces Proteobacteria Spirochaetes Synergistetes Tenericutes Thermodesulfobact. Thermotogae

GHx distribution in Genomes Life Style Autotrophic : Aequifacie Cyanobacteria Chrysiogenetes Nitrospirae Host associated: Chlam. / Verruco. Elusomicrobia Fibrob. / Acidobact. * Fusobacteria Spirochaetes Tenericutes

GHx distribution in Genomes GHx functions « house keeping » GH 6 endoglucanase ; cellobiohydrolase GH 18 … endo-β-N-acetylglucosaminidase … Q: Planctomycetes U: Tenericutes - Mycoplasma

GHx distribution in Genomes GHx functions GHx families « specialization » GH 6 endoglucanase ; cellobiohydrolase GH 5 chitosanase ; 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 GH 5 is a cellulase?

Complex architectures GH 5 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) n o i t nc ltifu u M = 5 GH Free cellulases from the GH 6 are Associated to the cellulose production In actynomycetes ! ? » GH 6 llulase ce « endoglucanase (EC 3. 2. 1. 4); cellobiohydrolase (EC 3. 2. 1. 91) = 6 GH

Is there an efficient combination of enzymes ?

Is there an efficient combination of enzymes ? Some genes are abundant (GH 5, 10, 16, 18, 19) Are these genes really involved in PCW breakdown ? Multi-domain Why are Fibrobacteria so Efficient ?

Is there an efficient combination of enzymes ? The keys of the succes in Fibrobacteria

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 »

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 Ceb. R (GHx vs CBM-GHx) : – Presence/absence of regulating sequences upstream the GHx-coding sequences • Environmental factors : “life style”, “metabolism”, … • Gene Gain/loss : 16 S r. RNA 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 Spirochaetes, Fibrobacter, Bacteroidetes, … Hess 2011 Bacteroidetes, Fibrobacteria, Clostridia, …

…Vs. Our study Using the SSU…

…Vs. Our study Reno 2012 (probably) Actinobacteria, Alphaproteobacteria, Bacteroidetes, … Warnecke 2007 Spirochaetes, Fibrobacter, Bacteroidetes, … Hess 2011 Bacteroidetes, Fibrobacteria, Clostridia, …

Metagenomes Clustrering 16 S r. RNA GHx ? Environment selects for different populations (with different GHx)

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 – [GH 6, GH 8, GH 9]LL > [GH 6, GH 8, GH 9]CR – [CMB-GHx]LL > [CBM-GHx]CR

What’s next • Leaf Litter Metagenome – 22 samples ~ready to be sequenced (Tru. Seq TM DNA -Illumina) (first year) – samples to be prepared (second year) – Compare : [GHx/16 s r. RNA in sequenced genomes] vs. [GHx/16 s r. RNA in Leaf Litter] – Compare different treatments, metagenomes

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

24 samples • Tru. Seq TM DNA (Illumina) • 24 samples • 22 samples ready to be sequenced

Complex architectures Cel 5 CBM 2 Xyl 8 Cel 5 CBM 2 Amount of Fig. Fam IDs corresponding to a 2 -domain protein Pla nt Ce ll W all Amount of Fig. Fam IDs ≠ Amount of genes

Metagenomes Clustrering 16 S r. RNA GHx Environment selects for different GHx potential