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Living Large: Elucidation of the Frankia EAN1pec Genome
Sequence Shows Gene Expansion and Metabolic
VersatilityLouis 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
Actinorhizal Symbiosis
• Symbiotic association between Frankia and woody dicotyledonous plants – results in formation of
root nodules
• over 250 species of Actinorhizal plants
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
Three Frankia genotypes
Betulaceae
Myricaceae
Casuarinacee
Elaeagnuaceae
Rhamnaceae
Myricaceae
Gymnostoma
CoriaiaceaeDatiscaceaeRosaceaeCeanothus
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, AsO4
3-,Pb2+ and CrO4
2+
• Limited genetics
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
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.
Comparative distribution of ORF function
0
200
400
600
800
1000
1200
1400 Frankia sp. CcI3
Frankia sp. EAN1pec
Acidothermus cellulolyticus 11B
Arthrobacter sp. FB24
Kineococcus radiotolerans SRS30216
Streptomyces coelicolor A3(2)
COG Functional Groups
-1500
-1000
-500
0
500
1000
1500
T + I Duplicates ORFans
CcI3
ACN
EaNGene 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
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
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
CcI3
•loss of genes associated with transport and metabolism
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).
What about genes identified as potentially involved in Symbiosis?
• Nitrogenase components
• Hopanoid biosynthesis
• Uptake Hydrogenase biosynthesis
• Hemoglobin
• Nodulation
Nitrogenase Cluster for EAN1pec
NifK, NifD, NifHNifX, NifN, NifE
hypothetical proteins
NifB NifZ NifW
3 Fd genesNifS
NifV (homocitrate synthase) is located in another region of the chromosome
MaGe site
NifV
Synteny
Frankia Vesicles• Laminated hopanoid lipids• Restrict oxygen diffusion
• N2 fixation can occur “free-living”
Berry et al. PNAS 1993
Parsons et al 1987
Cluster I: shcI
Polyprenylsynthetase
Squalene/phytoene synthase
TetR
amine oxidase
putative phytoene DH
http://img.jgi.doe.gov/cgi-bin/pub/main.cgi
EAN & ACN extra shcI geneshcI
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
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
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
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
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
Is Frankia EAN1pec versatile?
Quercetin Catechol
DNA Regulatory Proteins
0
20
40
60
80
100
120
140
160
ArsR DeoR AraC AsnC GntR IclR LacI LuxR LysR Lrp MerR TetR
Frankia sp. EAN1pecFrankia sp. CcI3Acidothermus cellulolyticus 11BArthrobacter sp. FB24Kineococcus radiotolerans SRS30216Streptomyces coelicolor A3(2)
Proteome profiles of Frankia CcI3 grown under N2 or NH4Cl conditions. Arrow point out N2-grown specific proteins.
Vesicle development is influenced by:
a. N statusb. Oxygen c. Mo & Fed. Calcium e. Temperaturef. host plant
Search for Vesicle-Specific Proteins
Purified Vesicles
Two-dimensional gel electrophoresis of vesicle proteins isolated by Pressure Cycling Technology
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
AcknowledgementsThis 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 MyersArnab Sen (U. North Bengal)
UConn JGIDavid Benson Pilar Francino, Alla LapidusPeter Gogarten Paul Richardson, Chris Detter,
UMaine UNH CSBJohn Tjepkema Vern Rienhold
ULyon PCT All of the Frankia Philippe Normand Gary Smejkal community
The PULSE Tube used in Pressure Cycling Technology facilitateshigh efficiency lysis of cells and subcellular components
Isolation of proteins from Frankia mycelium and vesicles by PCT