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Living Large: Elucidation of the Frankia EAN1pec Genome Sequence Shows Gene Expansion and Metabolic Versatility

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

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Living Large: Elucidation of the Frankia EAN1pec Genome Sequence Shows Gene Expansion and Metabolic Versatility

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  1. Living Large: Elucidation of the Frankia EAN1pec Genome Sequence Shows Gene Expansion and Metabolic Versatility Louis S Tisa1, David R Benson2, Gary B. Smejkal4, Pascal Lapierre2, J. Peter Gogarten2, Philippe Normand5, M. Pilar Francino3, and Paul Richardson3 1Dept. Microbiology , U New Hampshire, Durham, NH, USA; 2Dept. Mol. Cell Biol., U Connecticut , Storrs, CT, USA; 3 JGI, Walnut Creek, CA, USA, 4 Pressure Biosciences, Inc, Bridgewater, MA, USA, 5Ecologie 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. CcI3 Metabolism Member of Group I Narrow Host range Markers: KanR,GenR,KasR,NalR AsO43- EAN1pec Diverse metabolism Member of Group III (globally distributed) Broader Host range Markers: NovR, LinR,KasR,NalR, AsO43-,Pb2+ and CrO42+ Limited genetics Why Sequence CcI3 and EAN1pec?

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

  8. Comparative distribution of ORF function COG Functional Groups

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

  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. 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). 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).

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

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

  16. Frankia Vesicles Parsons et al 1987 • Laminated hopanoid lipids • Restrict oxygen diffusion • N2 fixation can occur “free-living” Berry et al. PNAS 1993

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

  18. Surprise Two: Potentialsymbiosis 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 katA katG HbO shc2 sodF nifV HbN

  19. Transcription Analysis of Two Frankia hemoglobins HboO expression is up-regulated under hypoxic conditions HboN expression is up-regulated by NO release 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. Vesicle development is influenced by: a. N status b. Oxygen c. Mo & Fe d. Calcium e. Temperature f. host plant Proteome profiles of Frankia CcI3 grown under N2 or NH4Cl conditions. Arrow point out N2-grown specific proteins.

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

  27. 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

  28. 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,TealFurnholm, NickBeauchemin,Joanne Coulburn, Anna Myers Arnab Sen (U. North Bengal) UConnJGI David Benson Pilar Francino, Alla Lapidus Peter Gogarten Paul Richardson, Chris Detter, UMaineUNH CSB John Tjepkema Vern Rienhold ULyonPCTAll of the Frankia Philippe Normand Gary Smejkal community

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

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

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