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

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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,
  5 Ecologie Microbienne UMR CNRS 5557, Université
  Lyon, Villeurbanne, France

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Actinorhizal Symbiosis

• Symbiotic association between Frankia and woody
  dicotyledonous plants
• results in formation of root nodules
• over 250 species of Actinorhizal plants

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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
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Three Frankia genotypes
Betulaceae Myricaceae Casuarinacee
Elaeagnuaceae Rhamnaceae Myricaceae Gymnostoma
Coriaiaceae Datiscaceae Rosaceae Ceanothus
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Why Sequence CcI3 and EAN1pec?

• 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

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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
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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
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Comparative distribution of ORF function
COG Functional Groups
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The EAN Genome is Expanding
Gene Duplication Level is higher in EAN (18.5 of
the ORFs) than ACN (7.5 ) or CcI3 (9.8 )
1355
CcI3 has an accelerated rate of gene loss
compared to EAN and ACN
1054
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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
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• CcI3 (165/444)
• 116 out 165 (70) duplicated genes belonged to
  several classes of transposases and genes
  associated with prophage and plasmids

• 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

• 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

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• CcI3
• loss of genes associated with transport and
  metabolism

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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).
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What about genes identified as potentially
involved in Symbiosis?

• Nitrogenase components
• Hopanoid biosynthesis
• Uptake Hydrogenase biosynthesis
• Hemoglobin
• Nodulation

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Nitrogenase Cluster for EAN1pec
Synteny
hypothetical proteins
3 Fd genes
NifS
NifX, NifN, NifE
NifK, NifD, NifH
NifB NifZ NifW
NifV (homocitrate synthase) is located in another
region of the chromosome
NifV
MaGe site
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Frankia Vesicles
Parsons et al 1987

• Laminated hopanoid lipids
• Restrict oxygen diffusion
• N2 fixation can occur free-living

Berry et al. PNAS 1993
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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
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Surprise Two Potential symbiosis genes are not
clustered
katA

• 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

katG
HbO
shc2
sodF
nifV
HbN
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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
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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

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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

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• 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

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Is Frankia EAN1pec versatile?
Quercetin
Catechol
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DNA Regulatory Proteins
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• 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.
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Search for Vesicle-Specific Proteins
Two-dimensional gel electrophoresis of vesicle
proteins isolated by Pressure Cycling Technology
Purified Vesicles
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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

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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) UConn JGI Da
vid Benson Pilar Francino, Alla Lapidus Peter
Gogarten Paul Richardson, Chris Detter,
UMaine UNH CSB John Tjepkema Vern
Rienhold ULyon PCT All of the Frankia
Philippe Normand Gary Smejkal community
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The PULSE Tube used in Pressure Cycling
Technology facilitates high efficiency lysis of
cells and subcellular components
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Isolation of proteins from Frankia mycelium and
vesicles by PCT