1. Living Large: Elucidation of the Frankia EAN1pec Genome Sequence Shows Gene Expansion and Metabolic Versatility Louis S Tisa 1, David R Benson 2, Gary B. Smejkal 4, Pascal Lapierre 2, J. Peter Gogarten 2, Philippe Normand 5, M. Pilar Francino 3, and Paul Richardson 3 1 Dept. Microbiology, U New Hampshire, Durham, NH, USA; 2 Dept. Mol. Cell Biol., U Connecticut, Storrs, CT, USA; 3 JGI, Walnut Creek, CA, USA, 4 Pressure Biosciences, Inc, Bridgewater, MA, USA, 5 Ecologie Microbienne UMR CNRS 5557, Université Lyon, Villeurbanne, France

2. Actinorhizal Symbiosis Symbiotic association between Frankia and woody dicotyledonous plants –results in formation of root nodules over 250 species of Actinorhizal plants

3. Frankia Member of the Actinomycetales Hyphal bacteria –67-72% GC –generation time 24-48 h Structures –vesicles –spores in planta vesicle clusters Bar = 10 µm

4. Three Frankia genotypes Betulaceae Myricaceae Casuarinacee Elaeagnuaceae Rhamnaceae Myricaceae Gymnostoma Coriaiaceae Datiscaceae Rosaceae Ceanothus

5. Why Sequence CcI3 and EAN1pec? CcI3 Metabolism Member of Group I Narrow Host range Markers: Kan R,Gen R,Kas R,Nal R AsO 4 3- EAN1pec Diverse metabolism Member of Group III (globally distributed) Broader Host range Markers: Nov R, Lin R,Kas R,Nal R, AsO 4 3-,Pb 2+ and CrO 4 2+ Limited genetics

6. Surprise One: Three different genome sizes ACN 6783 CDS 2 rRNA 72.8% GC CcI3 4515 CDS 2 rRNA 70.1% GC EAN 7492 CDS 3 rRNA 71.0% GC Circular Topology

7. Comparison of the CDS Frankia ACN14a Frankia CcI3 Frankia Ean1pec 2291 630 587 1190 2730 1333 3725 reciprocal blast search with a cutoff of 10 -4.

8. Comparative distribution of ORF function COG Functional Groups

9. Gene Duplication Level is higher in EAN (18.5% of the ORFs) than ACN (7.5 %) or CcI3 (9.8 %) 1355 The EAN Genome is Expanding 1054 CcI3 has an accelerated rate of gene loss compared to EAN and ACN

10. What are the Major families of duplicated genes in these Frankia strains? BlastClust (NCBI) analysis 25% identity over at least 40% of the length (30% identity /52% length same result) An analysis of the Top 20 duplicated gene families showed major differences in functional groups

11. CcI3 (165/444) 116 out 165 (70%) duplicated genes belonged to several classes of transposases and genes associated with prophage and plasmids EAN (406/1355) Transport proteins, Dioxygenases, Short chain dehydrogenases/reductases (SDR), Regulatory proteins, cytochrome P450, monooxygenases also like CcI3 132 out of 406 (32.5%) genes associated with integrases, transposases ACN (151/512) Transport proteins, SDR,serine-threonine protein kinases, methyltransferases, endonucleases, & a variety of dehydrogenases no transposases in 151 genes of the top 20 families

12. CcI3 loss of genes associated with transport and metabolism

13. b, EAN :Elaeagnaceae (pink), Myricaceae (green) Rhamnaceae (blue, Tribe Colletieae in South America, Australia and New Zealand). Areas of overlap are brown and dark blue). Present day native distribution of actinorhizal plant hosts. a, ACN: Betulaceae (orange) Myricaceae (green) and their overlap (khaki). c, CcI3: Casuarina and Allocasuarina of the Casuarinaceae (light blue).

14. What about genes identified as potentially involved in Symbiosis? Nitrogenase components Hopanoid biosynthesis Uptake Hydrogenase biosynthesis Hemoglobin Nodulation

15. Nitrogenase Cluster for EAN1pec NifK, NifD, NifH NifX, NifN, NifE hypothetical proteins NifB NifZ NifW 3 Fd genes NifS NifV (homocitrate synthase) is located in another region of the chromosome MaGe site NifV Synteny

16. Frankia Vesicles Laminated hopanoid lipids Restrict oxygen diffusion N 2 fixation can occur “free- living” Berry et al. PNAS 1993 Parsons et al 1987

17. Cluster I: shcI Polyprenyl synthetase Squalene/phytoene synthase TetR amine oxidase putative phytoene DH http://img.jgi.doe.gov/ cgi-bin/pub/main.cgi EAN & ACN extra shcI gene shcI

18. Surprise Two: Potential symbiosis genes are not clustered cluster I: shc1 FRAEA6946-6954 cluster II: hup2* FRAEA4081-4086 cluster III: hup1 FRAEA2955-2965 cluster IV: nif, FRAEA8447-8463 HbO, FRAEA6420 HbN, FRAEA4419 shc2, FRAEA5736 katA,FRAEA8358 sodF,FRAEA4204 nodB-like FRAEA6279, NifV, FRAEA4890 shc2 HbO nifV HbN katA katG sodF

19. Transcription Analysis of Two Frankia hemoglobins HboN expression is up-regulated by NO release HboO expression is up-regulated under hypoxic conditions Nitrogen status did not significantly affect expression

20. Why the large genome (9.1 Mb) for Frankia EAN1pec? many soil dwellers have large genomes (Streptomyces, Bradyrhizobium, Burkholderia, etc. these “boy-scouts” are always prepared for changing conditions of the soil environment –wide array of substrates (uptake systems) –need for tight regulation

21. Why the large genome (9.1 Mb) for Frankia EAN1pec? many soil dwellers have large genomes (Streptomyces, Bradyrhizobium, Burkholderia, etc. these “boy-scouts” are always prepared for changing conditions of the soil environment –wide array of substrates (uptake systems) –need for tight regulation

22. Metabolism Complete Embden-Meyerhof, TCA and Pentose Phosphate pathways wide arsenal of transport genes large numbers of genes for short chain dehydrogenase/reductase, dioxygenase, etc. Regulatory mechanisms Large number of DNA binding proteins Two-component systems Sigma Factors Anti-sigma Factors Anti-sigma Factor Antagonists

23. Is Frankia EAN1pec versatile? Quercetin Catechol

24. DNA Regulatory Proteins

25. Proteome profiles of Frankia CcI3 grown under N 2 or NH 4 Cl conditions. Arrow point out N 2 -grown specific proteins. Vesicle development is influenced by: a. N status b. Oxygen c. Mo & Fe d. Calcium e. Temperature f. host plant

26. Search for Vesicle- Specific Proteins Purified Vesicles Two-dimensional gel electrophoresis of vesicle proteins isolated by Pressure Cycling Technology

28. Perspectives Frankia genome expansion and contraction reflects biogeographic history of symbioses No “symbiosis islands” The time is right for functional genetics –Proteomic Profiles –Transcriptome Profiles (DNA arrays) –Genetics

29. Acknowledgements This work was supported by: USDA Hatch grant 486; USDA 2003-01127; NSF EF-0333173; DOE Microbial Genome Program TISA LAB: Tania Rawnsley, James Niemann, Teal Furnholm, Nick Beauchemin, Joanne Coulburn, Anna Myers Arnab Sen (U. North Bengal) UConnJGI David Benson Pilar Francino, Alla Lapidus Peter GogartenPaul Richardson, Chris Detter, UMaineUNH CSB John TjepkemaVern Rienhold ULyon PCT All of the Frankia Philippe Normand Gary Smejkalcommunity

30. The PULSE Tube used in Pressure Cycling Technology facilitates high efficiency lysis of cells and subcellular components

31. Isolation of proteins from Frankia mycelium and vesicles by PCT