Exploration of the Actinorhizal Symbiosis: What can we learn from the Frankia genomes and

where do we go from here?

Louis S Tisa1, J. Niemann1, D.R Benson2, G. Smejkal4, P. Lapierre2, J.P. Gogarten2, T. Rawnsley1,Y. Huang2, Juliana Mastronunzio2, C. Bassi2, P. Normand5, M. P. Francino3, and P.Richardson3

1U. New Hampshire, Durham, NH, USA; 2U Connecticut , Storrs, CT, USA; 3 JGI, Walnut Creek, CA, USA, 4 Pressure Biosciences, Inc, Bridgewater, MA, USA, 5 Université Lyón, 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

16S rRNA

glutamine synthetase

Clawson et al. 2003

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

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

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.

-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

Categories of Deleted GenesGeneral Category CcI3 ACN EAN• Total missing 1054 466 555• Transport 113 33 28• Regulatory 95 21 35• Short chain DH/R 32 4 4• Acetyl-CoA DH-like 19 3 1• Alkanesulfonate 17 0 0 monooxygenase

• Amidohydrolase 13 1 1• Transposase/Intergase 10 40 17• Hypothetical, Conserved 289 158 176hypothetical, unknown function

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?

Habitat or Life Style?

Habitat?

• 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

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)

Frankia Genome Sequence

Proteome

•2D-PAGE•Mass spectrometry

Transcriptome

DNA Microarrays

Bioinformatics

•Comparative•Modeling

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 Myers

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

Phylogenetic tree based on 16S rRNA gene analysis of the three

sequenced strains

OUTGROUP

Streptomyces avermitilis

Kineococcus radiodurans

Acidothermus cellulolyticus

Frankia ACN (Cluster 1)

Frankia CcI3 (Cluster 1)

Frankia EAN (Cluster 3)

Frankia (C. arborea nod, Cluster 2)

100

100

100

47

94

70

0.01

Neighbor-joining tree

Corynebacterium glutamicum, Mycobacterium tuberculosis and Nocardia farcinica