1. About the cellulases distribution Renaud Berlemont, UCI Adam Martiny Lab.

2. About the GHx classification CAZYdb  Glycoside Hydrolases, … Structure – Sequences Alignements : Families (>100) / Clans (14) « Convergence – Divergence »

3. Some statements Biochemically confirmed « cellulases » = CMCases

4. Some statements Biochemically confirmed « cellulases » = CMCases Many cellulases are active on other substrates (e.g. xylan) Many « cellulases » are non-cellulolytic !? CMCases ≠ Cellulases

5. 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)

6. 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

7. 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.

8. pSEED - FigFams Sequenced genomes (patricbrc db - 4089) In order to analyze as much as possible sequenced genomes

9. 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,

10. Methodology FigFam IDs CAZYdb E.C. 3.2.1.4 GHx Pfam (pro. + euk.)InterPRo (pro.) PfGHx.FASTAIprGHx.FASTA pSEED PEG IDs Home-made Script : SEQ  PEG ID GH families Figfam IDs Several Figfam IDs correspond To one GHx families because Signal Peptides and accessory domains Are not conserved …

11. Methodology FigFam IDs GHx pSEED Genomes Annotations GHx Occurrence In Sequenced genomes AlignmentStatistic Bacterial groups CBM2… Bacterial groups Bacterial groups … Occurrence / List Occurrence / List Figfam IDs Genomes annotations (pSEED) GHx distribution

12. A huge data-set A Actinobacteria B Aequfacie C Bactero./Chlorobi D Chlam./ Verruco. E Chloroflexi F Chrysiogenetes G Cyanobacteria H Deferibacter I Deinoco./Thermus J Dictyoglomi K Elusomicrobia LFibrob./ Acidobact. MFirmicutes NFusobacteria ONitrospirae PGemmatimonadetes QPlanctomyces RProteobacteria SSpirochaetes TSynergistetes UTenericutes VThermodesulfobact. WThermotogae Huge bias : A + C + M + R = 88% of the sequenced genomes…

13. Average Gene Content (AGC) A Actinobacteria B Aequfacie C Bactero./Chlorobi D Chlam./ Verruco. E Chloroflexi F Chrysiogenetes G Cyanobacteria H Deferibacter I Deinoco./Thermus J Dictyoglomi K Elusomicrobia LFibrob./ Acidobact. MFirmicutes NFusobacteria ONitrospirae PGemmatimonadetes QPlanctomyces RProteobacteria SSpirochaetes TSynergistetes UTenericutes VThermodesulfobact. WThermotogae Life style (Auto Vs. Hetero) Host association … “HKG” Multi-function …

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

15. GHx distribution in Genomes GHx functions « house keeping » GH6 endoglucanase ; cellobiohydrolase GH18 … endo-β-N-acetylglucosaminidase … Q: Planctomycetes U: Tenericutes - Mycoplasma

16. GHx distribution in Genomes GHx functions GHx families « specialization » GH6 endoglucanase ; cellobiohydrolase GH5 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 GH5 is a cellulase?

17. Complex architectures GH5 chitosanase (EC 3.2.1.132); β-mannosidase (EC 3.2.1.25); cellulase3.2.1.1323.2.1.25 (EC 3.2.1.4); glucan β-1,3-glucosidase (EC 3.2.1.58); licheninase3.2.1.43.2.1.58 (EC 3.2.1.73); glucan endo-1,6-β-glucosidase (EC 3.2.1.75)3.2.1.733.2.1.75 mannan endo-β-1,4-mannosidase (EC 3.2.1.78); endo-β-1,4-xylanase3.2.1.78 (EC 3.2.1.8); cellulose β-1,4-cellobiosidase (EC 3.2.1.91);3.2.1.83.2.1.91 β-1,3-mannanase (EC 3.2.1.-); xyloglucan-specific endo-β-1,4-glucanase3.2.1.- (EC 3.2.1.151); mannan transglycosylase (EC 2.4.1.-);3.2.1.1512.4.1.- endo-β-1,6-galactanase (EC 3.2.1.164);3.2.1.164 endoglycoceramidase (EC 3.2.1.123)3.2.1.123 GH6 endoglucanase (EC 3.2.1.4); cellobiohydrolase (EC 3.2.1.91)3.2.1.43.2.1.91 GH6 = « cellulase »GH5 = Multifunction Free cellulases from the GH6 are Associated to the cellulose production In actynomycetes ! ?

18. Is there an efficient combination of enzymes ?

19. 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 ?

20. The keys of the succes in Fibrobacteria

21. 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…

22. What’s next Looking at the GHx-distribution in subgroups (e.g Proteobacteria, Firmicutes, …)  Detailed table of the GHx distribution amongst (sub)-taxa

23. 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

24. Do the cellulose degradation potential vary in environment ?

25. Some cases studies … GHx distribution in metagenomes % of CBM linked GHx Warnecke 2007 Hess 2011 Bacteroidetes, Fibrobacteria, Clostridia, … Spirochaetes, Fibrobacter, Bacteroidetes, …

26. …Vs. Our study Using the SSU…

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

28. Metagenomes Clustrering Environment selects for different populations (with different GHx) 16S rRNAGHx ?

29. 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…

30. 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

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

34. 24 samples TruSeq TM DNA (Illumina) 24 samples 22 samples ready to be sequenced

35. Complex architectures Cel5 CBM2 Xyl8Cel5 CBM2 Cel5 Amount of FigFam IDs corresponding to a 2-domain protein Plant Cell Wall Amount of FigFam IDs ≠ Amount of genes

36. Metagenomes Clustrering Environment selects for different GHx potential 16S rRNAGHx