Carbohydrates and Carbohydrates and RhizobiumRhizobium--Legume Legume SymbiosisSymbiosis

Why The Interest in Symbiosis???Why The Interest in Symbiosis???

Recognition between a bacterium and its eukaryote host.Recognition between a bacterium and its eukaryote host.Adaptation of the bacterium to the host defense response.Adaptation of the bacterium to the host defense response.

Regulation of the host defense response.Regulation of the host defense response.

Differentiation of the bacterium.Differentiation of the bacterium.

Differentiation of the eukaryote host.Differentiation of the eukaryote host.

Bacterial Components Bacterial Components Plant ComponentsPlant Components

Gibson, K. E., H. Kobayashi, and G. C. Walker. 2008. Molecular Determinants of a Symbiotic Chronic Infection. Annual Review of Genetics 42:413.

Oldroyd, G. E. D., and J. A. Downie. 2008. Coordinating Nodule Morphogenesis with Rhizobial

Infection in Legumes. Annual Review of Plant Biology 59:519-546.

Bacterial CarbohydratesBacterial Carbohydrates

EPS

CPSCPS

Adaptation of the Adaptation of the Bacterial Cell SurfaceBacterial Cell Surface

•• Cessation of a carbohydrate.Cessation of a carbohydrate.•• Production of a carbohydrateProduction of a carbohydrate•• Replacement of a carbohydrate.Replacement of a carbohydrate.•• Modification of a carbohydrate.Modification of a carbohydrate.

Masking.Masking.Camouflage.Camouflage.SignalingSignaling

Avoidance of the host Avoidance of the host defense responsedefense response

Cell envelope carbohydrates are Cell envelope carbohydrates are virulence factorsvirulence factors..

Cyclic glucans

Provided by Dr. Dale Noel, Department of Provided by Dr. Dale Noel, Department of Biology, Marquette University.Biology, Marquette University.

Indeterminate (pea nodules)Indeterminate (pea nodules) Determinate (bean nodules)Determinate (bean nodules)

What DoesWhat Does

RhizobiumRhizobium--Symbiosis Look Symbiosis Look Like?Like?

The Symbiotic Infection ProcessThe Symbiotic Infection Process

X. Perret, C. Staehelin, and W. J. Broughton. 2000. Molecular Basis of Symbiotic Promiscuity Microbiol

Mol Biol

Rev. 2000 March; 64(1): 180–201.

A A -- DD: legume : legume flavonoidflavonoid; ; lipochitinlipochitin oligosaccharideoligosaccharide

C C -- EE: : RhizobialRhizobial EPS, CPS, LPS, EPS, CPS, LPS, cyclic cyclic glucansglucans

DeterminateDeterminate

IndeterminateIndeterminate

B. N. Kaiser et al. 2003. The Plant Journal 35:296

BacteroidsBacteroidsDeterminateDeterminateIndeterminateIndeterminate

RhizobiumRhizobium ––

“Nod factors”“Nod factors”

n n = 2 or 3= 2 or 3

NodANodA

NodBNodB

NodCNodC(Common (Common nodnod genes)genes)

R O O

HNHAc

HH

H

HOHOH

OH

S OO-

R

O

CH3O

R

OR

H

OH

HH CH3

HOHO H

CH3

CH2CHR CH2OHOH

O

RH

OHH

H

OH

HHOH

H

NHO

CH3

O

H

HH

H

HOOH

OH

OH

R

O

NH2 R

O

NH2

R

CH3

O

R

R

CH3

NodLNodL

NodUNodUNodSNodS

NodENodENodFNodF NodPNodP

NodQNodQNodHNodH

NodXNodX

NodZNodZ

NoeCHOPNoeCHOP

(Specific (Specific nodnod genes)genes) (Specific (Specific nodnod genes)genes)

W. D’Haeze, M. Holsters. 2002. Nod factor structures, responsesW. D’Haeze, M. Holsters. 2002. Nod factor structures, responses, and perception during initiation of , and perception during initiation of nodule development. nodule development. GlycbiologyGlycbiology

12:79R12:79R--105R.105R.

pSympSym

(Nodulation genes: (Nodulation genes: nodnod, , noenoe, , nolnol. . NitrogenaseNitrogenase

genes; genes; nifnif, and , and fixfix))

NHO

CH3

O

H

HH

H

HOOH

OH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOHOH

OH

Nod Factors and Host DefenseNod Factors and Host Defense((BradyrhizobiumBradyrhizobium japonicumjaponicum –– soybean)soybean)

PathogenPathogen SymbiontSymbiontPlant Host Plant Host (soybean)(soybean)

PhenylalaninePhenylalaninePhe ammonia lyaseCinnabmate-4-hydroxylase4-courmarate-CoA ligaseChalcone reductaseChalcone isomeraseIsoflavone synthase

Eight steps catalyzed by a series of cytochrome P- 450 monooxygenases

O

O

OH

OH

O

OOH

OH

H

O

CH3

CH3

daidzeindaidzein

glyceollinglyceollin

((www.agron.iastate.edu/www.agron.iastate.edu/

soybean/beangrows.htmsoybean/beangrows.htm

l#illustrationsl#illustrations))

((www.agron.iastate.edu/www.agron.iastate.edu/

soybean/beangrows.htmsoybean/beangrows.htm

l#illustrationsl#illustrations))

((www.extension.umn.eduwww.extension.umn.edu/.../ /.../ images/images/phytophthora.jpgphytophthora.jpg) )

((www.soils.umn.www.soils.umn.

eduedu/.../ /.../ History.htmHistory.htm

))

NHO

CH3

O

H

HH

H

HOOH

OH

O

CH3

O

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOHOH

OOH

OH

HH CH3

HOHO H

CH3

NH2

O

H

HH

H

HOOH

OH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOHOH

OH

NodANodANodBNodBNodCNodCNodLNodLNodZNodZ

NodDNodD

nodulationnodulation

Plant derived Plant derived oligosaccharidesoligosaccharides

Nod Factor FunctionNod Factor Function

NHO

CH3

O

H

HH

H

HOOH

OH

OH

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOHOH

OH

CH3

O

R

2

S OO-

.

O

NodDNodD--flavonoidflavonoid

→→

NodABCNodABC

+ + NodEFLNodEFL

+ + NodPQCNodPQC(A LysR

transcription factor)

HostHost--specificityspecificityDecoration of NF; speciesDecoration of NF; species--host specific host specific NodDNodD--flavonoidflavonoid interaction. interaction.

Root hair curlingRoot hair curlingNanomolarNanomolar concentration of the specific NF.concentration of the specific NF.

Infection thread formationInfection thread formationNF leads expression of stress and diseaseNF leads expression of stress and diseaseresistance genes at early times and downresistance genes at early times and downregulation at later times.regulation at later times.Effect on ROS.Effect on ROS.

Calcium spikingCalcium spikingCortical cell divisionCortical cell division

NF can cause nodule formation on its own, NF can cause nodule formation on its own, it is a it is a mitogenmitogen..

Induction of early Induction of early nodulinnodulin genes (ENOD).genes (ENOD).Induction of genes involved in cell wall synthesisInduction of genes involved in cell wall synthesis

cell growth and division, etc.cell growth and division, etc.HostHost--defense regulationdefense regulation

NHO

CH3

O

H

HH

H

HOOH

OH

OCH3

O

O

HNHAc

HH

H

HOOH

OH

O

HNHAc

HH

H

HOHOH

OO H

OH

HH CH3

HOHO H

CH3

33

BradyrhizobiumBradyrhizobium japonicumjaponicum NF (LCO)NF (LCO)

A Nod FactorA Nod Factor-- Based ProductBased Product

RhizobialRhizobial

ExtracellularExtracellular

Polysaccharides (some Polysaccharides (some examples)examples)

→→4)4)--ββ--DD--GlcGlcppAA--(1(1→→4)4)--33--OAcOAc--ββ--DD--GlcGlcppAA--(1(1→→4)4)--2(or 3)2(or 3)--OAcOAc--ββ--DD--GlcGlcpp--(1(1→→4,64,6--OO--PyrPyr--ββ--DD--GalGalpp--(1(1→→3)3)--4,64,6--OO--PyrPyr--ββ--DD--GlcGlcpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→66┘┘

→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4)4)--66--OAcOAc--ββ--DD--GlcGlcpp--(1(1→→3)3)--ββ--DD--GalGalpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4,64,6--OO--PyrPyr--ββ--DD--GlcGlcpp--(1(1→→3)3)--66--OO--SuccSucc--ββ--DD--GlcGlcpp--(1(1→→3)3)--ββ--DD--GlcGlcpp--(1(1→→6)6)--ββ--DD--GlcGlcpp--(1(1→→66┘┘

→→3)3)--66--OAcOAc--ββ--DD--GlcGlcpp--(1(1→→3)3)--4,64,6--OO--PyrPyr--ββ--DD--GalGalpp--(1(1→→

→→6)6)--ββ--DD--GlcGlcpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→3)3)--ββ--DD--GalGalpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→6)6)--ββ--DD--GlcpGlcp--(1(1→→22--(or 3)OAc(or 3)OAc--4,64,6--OO--PyrPyr--ββ--DD--GalGalpp--(1(1→→4)4)--ββ--DD--GlcGlcppAA--(1(1→→3)3)--ββ--DD--GlcGlcppAA--(1(1→→44┘┘

→→3)3)--ββ--DD--GlcGlcpp--(1(1→→3)3)--αα--DD--GalGalppAA--(1(1→→3)3)--αα--DD--ManManpp--(1(1→→3)3)--αα--DD--GlcGlcpp--(1(1→→44--OMeOMe--αα--DD--GalGalpp--(1(1→→66┘┘

→→4)4)--ββ--LL--RhapRhap--(1(1→→3)3)--αα--DD--GalpGalp--(1(1→→3)3)--

ββ--LL--RhapRhap--(1(1→→4)4)--ββ--LL--RhaRhapp--(1(1→→22--OMeOMe--ββ--DD--GlcpAGlcpA--(1(1→→33┘┘

R. R. LeguminosarumLeguminosarum or or R. R. etlietli

S. S. melilotimeliloti

EPS IEPS I

EPS IIEPS II

NGR234NGR234

B. B. japonicumjaponicum

EPSEPS

NPSNPS

(Review: Carlson et al. 1999. (Review: Carlson et al. 1999. RhizobialRhizobial

cell surface carbohydrates: Their structures, biosynthesis, ancell surface carbohydrates: Their structures, biosynthesis, and functions. d functions. In: In: Genetics of Bacterial PolysaccharidesGenetics of Bacterial Polysaccharides; ed. J.B. Goldberg; CRC Press; Washington DC; pp. 53; ed. J.B. Goldberg; CRC Press; Washington DC; pp. 53--90.)90.)

RhizobialRhizobial EPSEPS--Minus Mutants and SymbiosisMinus Mutants and Symbiosis

R. R. LeguminosarumLeguminosarum and and S. S. melilotimeliloti EPS minus EPS minus mutants: mutants: Nodules, no nitrogen fixation.Nodules, no nitrogen fixation.

K. K. NiehausNiehaus, D. , D. KappKapp, A. , A. PuhlerPuhler

(1993) Plant (1993) Plant defencedefence

and delayed infection of alfalfa and delayed infection of alfalfa pseudonodulespseudonodules

induced by an induced by an exopolysaccharideexopolysaccharide

(EPS I)(EPS I)--deficient deficient RhizobiumRhizobium melilotimeliloti mutant” mutant” PlantaPlanta 190:415190:415--425.425.

Thick cell walls with Thick cell walls with callosecallose..Increased production of Increased production of phenolicsphenolics; e.g. ; e.g. pp--coumariccoumaric acid.acid.

(Invasion of nodule cells after prolonged exposure.)(Invasion of nodule cells after prolonged exposure.)

RhizobialRhizobial EPS as A Signal MoleculeEPS as A Signal Molecule

→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4)4)--66--OAcOAc--ββ--DD--GlcGlcpp--(1(1→→3)3)--ββ--DD--GalGalpp--(1(1→→4)4)--ββ--DD--GlcGlcpp--(1(1→→4,64,6--OO--PyrPyr--ββ--DD--GlcGlcpp--(1(1→→3)3)--66--OO--SuccSucc--ββ--DD--GlcGlcpp--(1(1→→3)3)--ββ--DD--GlcGlcpp--(1(1→→6)6)--ββ--DD--GlcGlcpp--(1(1→→66┘┘

→→3)3)--66--OAcOAc--ββ--DD--GlcGlcpp--(1(1→→3)3)--4,64,6--OO--PyrPyr--ββ--DD--GalGalpp--(1(1→→

SinorhizobiumSinorhizobium melilotimeliloti

““Specific oligosaccharide form of the Specific oligosaccharide form of the RhizobiumRhizobium melilotimeliloti exopolysaccharideexopolysaccharide

promotes nodule invasion in alfalfa”promotes nodule invasion in alfalfa”L. L. BattistiBattisti, J.C. Lara, and J.A. Leigh. PNAS (1992) 89:5625, J.C. Lara, and J.A. Leigh. PNAS (1992) 89:5625--5629.5629.

““Low molecular weight EPS II of Low molecular weight EPS II of RhizobiumRhizobium melilotimeliloti allows nodule invasion in allows nodule invasion in MedicagoMedicago sativasativa.” .” J.E. Gonzales, B.L. J.E. Gonzales, B.L. ReuhsReuhs, and G.C. Walker. PNAS (1996) 89:5625, and G.C. Walker. PNAS (1996) 89:5625--5629.5629.““Nitrogen fixation ability of Nitrogen fixation ability of exopolysaccharideexopolysaccharide

mutants of mutants of RhizobiumRhizobium sp. Strain NGR234 and sp. Strain NGR234 and RhizobiumRhizobium trifoliitrifolii is restored by the is restored by the addition of homologous addition of homologous exopolysaccharidesexopolysaccharides.”.”S.P. S.P. DjordjevicDjordjevic, H. Chen, M. , H. Chen, M. BatleyBatley, J.W. Redmond, and B.G. , J.W. Redmond, and B.G. RolfeRolfe. J. . J. BacteriolBacteriol. (1987) 169:53. (1987) 169:53--60.60.

EPS IEPS I

EPS IIEPS II

Modulation of defense responseModulation of defense response::SmSm EPS mutants result in host defense response.EPS mutants result in host defense response.SmSm EPS mutants results in EPS mutants results in upregulationupregulation of plant defense proteins.of plant defense proteins.SmSm Wild type inoculation results in transient Wild type inoculation results in transient upregulationupregulation (1h) followed by decrease (6h).(1h) followed by decrease (6h).ConclusionConclusion: EPS suppresses a potentially lethal host defense response.: EPS suppresses a potentially lethal host defense response.

Required for initiation and elongation of infection Required for initiation and elongation of infection threadsChengthreadsCheng

& Walker. 1998. J. & Walker. 1998. J. BacteriolBacteriol. . 180:5183180:5183--51915191

““Extensibility of the infection thread is apparently controlled bExtensibility of the infection thread is apparently controlled by peroxidey peroxide--driven protein crossdriven protein cross--linking and perhaps also linking and perhaps also by modification of the by modification of the pecticpectic

matrix” N.J. matrix” N.J. BrewinBrewin. 2004. Plant cell wall . 2004. Plant cell wall remodellingremodelling

in the in the RhizobiumRhizobium--legume symbiosis. legume symbiosis. CritCrit. Rev. . Rev. MicrobiolMicrobiol. 4:293. 4:293--316.316.

α-D-GalA-(1→4)-β-D-GlcN-(1→6)-GlcNonateα-Kdo(III)-(2→6)-α-D-Gal-(1→6)-α-D-Man-(1→5)-α-Kdo(I)-(2→6)┐

α-D-GalA-(1→4)-α-Kdo(II)-(2→4)┐α-D-GalA-(1→5)┐

α-D-GalA-(1→4)┘

OPS-(→4)┐

OC

CH3

.

OH

CO

CH3

.

OCO

CH3

OH

OC

CH3

.

OH

OC

CH3

.

OH

β-D-Glc3NA-(1→4)-α-L-Fuc-(1→3)-α-L-QuiNAc-(1→4)-β-D-Glc3NA-(1-.R3

-(→4)┐

R1

-(→3)┘

3-OMe-α-D-6dTal-(1→3)┐

CORE-Lipid AR2

-(→2)┘R1 = CH3

CNH-R2 = CH3

CO-R3 = 3-OMe-α-6dHex4NFo-(1-

n = 3

2,3,4-TOMe-α-L-Fuc-(1→4)-β-D-GlcAMe-(1→4)-α-L-Fuc-(1→3-OMe-α-6dTal-(1→3)┘

CORE-Lipid An = 53)-α-L-Fuc-(1→4)-β-D-Man-(1→4)-α-QuiNAc-

RhizobiumRhizobium LipopolysaccharidesLipopolysaccharides

R. R. etlietli CE3 OCE3 O--chain polysaccharide (OPS)chain polysaccharide (OPS)

R. R. leguminosarumleguminosarum biovarbiovar

viciaeviciae

3841 O3841 O--chain polysaccharidechain polysaccharide

Structural Analysis:Bhat, U. R., L. S. Forsberg, et al. (1994). JBC

269: 14402-14410.Forsberg, L. S. and R. W. Carlson (1998). JBC

273(5): 2747-2757.Forsberg, L. S., U. R. Bhat, et al. (2000). JBC

275: 18851-18863.Que, N. L. S., S. H. Lin, et al. (2000). JBC

275(36): 28006-28016.Que, N. L. S., A. A. Ribeiro, et al. (2000). JBC 275(36): 28017-28027.Forsberg, L.S. and R.W. Carlson (2008). JBC 283(23):16037-16050.

Hydrophobicity changes in bacterial cell.

Hydrophobicity changes in LPS.The O-chain PS is required.Modifications occur to the O-chain PS.

Modifications occur to the lipid-A.The core region is unchanged.

Common to theCommon to the

RhizobiaceaeRhizobiaceae and and several pathogen several pathogen bacterial speciesbacterial species

GlcNonateGlcNonate

(possibly (possibly common to common to RhizobiumRhizobium species.)species.)

Common to Common to R. R. etlietli and and R. R. leguminosarumleguminosarum

CoreCore--LipidLipid--AA

Perotto, S., N. J. Brewin, and E. L. Kannenberg. 1994. Cytological evidence for a host defense response that reduces cell and tissue invasion in

pea nodules by lipopolysaccharide-defective mutants of Rhizobium

leguminosarum

strain 3841. Mol.Plant

Microbe Interact. 7:99-112.

Carlson, R. W., S. Kalembasa, D. Turowski, P. Pachori, and K. D. Noel. 1987. Characterization of the lipopolysaccharide

from a Rhizobium

phaseoli

mutant that is defective in infection thread development. The J. Bacteriol. 169:4923-4928.

Replacement of the O-Chain Polysaccharide

3)-α-L-Rha-(1 3)-α-L-Rha-(1 2)-α-L-(3-OMe)Rha-(1A B C

Present in LPS from bacteria isolated from host nodules.Present in LPS from bacteria isolated from host nodules.Present in LPS from bacteria grown in the presence of Present in LPS from bacteria grown in the presence of flavonoidflavonoid..Not induced in Not induced in fixFfixF mutant which is defective in infection of root nodule mutant which is defective in infection of root nodule

cells.cells.((FixFFixF is similar to proteins involved in capsule production, export, is similar to proteins involved in capsule production, export, such as such as KpsSKpsS in in E. E. colicoli or or RkpJRkpJ in in R. R. melilotimeliloti.).)

((ReuhsReuhs

et. al. 2005. J. et. al. 2005. J. BacteriolBacteriol. 187:6479. 187:6479--6487.)6487.)

SinorhizobiumSinorhizobium NGR234NGR234

Isolation of Isolation of RhizobiumRhizobium etlietli CE3 CE3 BacteroidsBacteroids from from PhaseolusPhaseolus vulgarisvulgaris (bean) Root (determinate) Nodules(bean) Root (determinate) Nodules

AeroponicsAeroponicsGrowth System Growth System

500 500 nodules/plant.nodules/plant.65 plants per 65 plants per

growthgrowth

BacteroidBacteroid Isolation Isolation

RhizobiumRhizobium etlietli CE3 CE3 BacteroidsBacteroids are Increased in 2are Increased in 2--OO-- Methylation of Methylation of FucoseFucose

0

100000

200000

300000

400000

500000

600000

700000

800000

11 12 13 14 15 16 17 18

0

50000

100000

150000

200000

250000

300000

350000

400000

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00

Bacteria

1

1

4

Time (min)

TOM

Fuc

TOM

Fuc

DO

MFu

cD

OM

Fuc

22 --O

MFu

cO

MFu

c33 --

OM

6dTa

lO

M6d

Tal

Fuc

Fuc

BacteroidsBacteroids have an increase in one have an increase in one 22--methyl group per OPS.methyl group per OPS.

Bacteriods

D'Haeze, W., C. Leoff, G. Freshour, K. D. Noel, and R. W. Carlson. 2007. Rhizobium

etli

CE3 Bacteroid

Lipopolysaccharides

Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria. J. Biol. Chem. 282:17101-17113.

Table 3. The Table 3. The glycosylglycosyl

linkages* of the Olinkages* of the O--chain polysaccharide chain polysaccharide fucosylfucosyl

residues: The residues: The location of the endogenous methyl groups on the Olocation of the endogenous methyl groups on the O--chain polysaccharide.chain polysaccharide.

Glycosyl

Residue

Laboratory-

cultured CE3water phase

CE3 Bacteroid

water phaseLaboratory-

cultured CE3phenol phase

CE3 Bacteroid

phenol phase

Terminal TOM or DOMFuc

23 24 19 20

3-Linked Fuc 23 12 19 13

2-O-Me-3-

Linked Fuc2.7 6.7 2.9 3.2

3,4-Linked Fuc 37 22 41 25

Fuc

2-O-Me-

3,4-Linked 13 34 18 39

*Calculated as relative percent of total fucosyl

residue partially methylated

alditol

acetate peak areas.

2,3,4-TOMe-α-L-Fuc-(1→4)-β-D-GlcAMe-(1→4)-α-L-Fuc-(1→3-OMe-α-6dTal-(1→3)┘

CORE-Lipid An = 53)-α-L-Fuc-(1→4)-β-D-Man-(1→4)-α-QuiNAc-

1122--66

ABCDEcap outer core

123456

= Kdo= QuiNAc= Man

= 6dTal= GlcAMe= Fuc = Me

five repeat units

Bacteria = one 2MeFuc Bacteroids = two 2MeFuc

= one of these five Fuc

residues is 2-O-methylated in bacteria.

= proposed Fuc

residue that is 2-O-methylated in bacteroids.

Which Which FucosylFucosyl Residue is Residue is MethylatedMethylated During During Symbiosis?Symbiosis?

2,3,4-TOMe-α-L-Fuc-(1→4)-β-D-GlcAMe-(1→4)-α-L-Fuc-(1→3-OMe-α-6dTal-(1→3)┘

CORE-Lipid An = 53)-α-L-Fuc-(1→4)-β-D-Man-(1→4)-α-QuiNAc-

1122--66

Noel, K. D., J. M. Box, and V. J. Bonne.Noel, K. D., J. M. Box, and V. J. Bonne. 2004. 22004. 2--OO--Methylation of Methylation of FucosylFucosyl

Residues of a Residues of a RhizobialRhizobial

LipopolysaccharideLipopolysaccharide

Is Increased in Response to Host Is Increased in Response to Host ExudateExudate

and Is Eliminated in a Symbiotically Defective and Is Eliminated in a Symbiotically Defective Mutant. Applied and Environmental Microbiology Mutant. Applied and Environmental Microbiology 70:70:15371537--1544.1544.

Changes to the LipidChanges to the Lipid--A During SymbiosisA During Symbiosis

O

OHHO

HO C O2 HCore

OHO

O

O

NHO

O O

O NHC O2 H

OHHO

CH2 O

OHO

HOOO

O

O

HO O

Changes in LPS Changes in LPS hydrophobicityhydrophobicity durngdurng symbiosis.symbiosis.

Determinate: transient increase.Determinate: transient increase.Indeterminate: permanent increase.Indeterminate: permanent increase.

Indeterminate:Indeterminate:Increase in VLCFA during growth at Increase in VLCFA during growth at

low Olow O22 ..

(E.L. Kannenberg & R.W. Carlson. 2001. (E.L. Kannenberg & R.W. Carlson. 2001. Mol. Mol. MicrobiolMicrobiol. 39:379. 39:379--391)391)

The LipidThe Lipid--A from A from RhizobiumRhizobium etlietli BacteroidsBacteroids

0

20

40

60

80

100

120

1500 1600 1700 1800 1900 2000 2100

0

50

100

150

200

250

300

350

1500 1600 1700 1800 1900 2000 2100

C1730.03

C

C

C

1974.45

0

20

40

60

80

100

120

140

160

1500 1600 1700 1800 1900 2000 2100

BacteriaBacteria

BacteriodsBacteriods

Bacteria + Bacteria + AnthocyaninAnthocyanin

NH

HH

H

H

OHOH

O

O

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

O

CH3

O

OH

O

CH3

O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

HOH

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

CH3

O

OH

NH

HH

H

H

OOH

O

O

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

HOH

CH3

O

OH

C1758.96

1738.03

B1985.73

D12002.45

BB D1D1

CC

In In plantaplanta: Addition of one methyl group to : Addition of one methyl group to FucFuc residue + lack of 3OHC14residue + lack of 3OHC14:0 in lipid:0 in lipid--AAIn In anthocyaninanthocyanin: Addition of one methyl group to : Addition of one methyl group to FucFuc but no change in lipidbut no change in lipid--A.A.

BasuBasu, S. S., K. A. White, et al. (1999). "A , S. S., K. A. White, et al. (1999). "A deacylasedeacylase

in in RhizobiumRhizobium leguminosarumleguminosarum membranes that cleaves the 3membranes that cleaves the 3--

O O --linked linked ββ--

hydroxymyristoylhydroxymyristoyl

moiety of lipid A precursors." moiety of lipid A precursors." Journal of Biological ChemistryJournal of Biological Chemistry 274(16): 274(16): 1115011150--11158.11158.

What are the Functions of the LPS During What are the Functions of the LPS During Symbiosis?Symbiosis?

E. coliE. coli RhizobiumRhizobium

DeterminateDeterminate

IndeterminateIndeterminateOO--chain PS changes required for adherence between chain PS changes required for adherence between bacterial and plant membranes during bacterial and plant membranes during endocytosisendocytosisand synchronous division (indeterminate).and synchronous division (indeterminate).LipidLipid--A VLCFA is required to maintain bacterial A VLCFA is required to maintain bacterial membrane integrity during above processes.membrane integrity during above processes.LipidLipid--A fatty A fatty acylacyl changes required for the formation changes required for the formation of lipid rafts important in metabolite exchange.of lipid rafts important in metabolite exchange.

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

O

O

OH

CH3

CH3

O

O

CH3

O

OH

O

CH3

O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

O

OH

O

H

HH

OH

HCOOH

O H

OHOHO

H

HH

OH

HCOOHOH

H

OHOH

NH

HH

H

H

OOH

O

O

O

HNH

HH

H

H

OO

O

CH3

O

OH

CH3

O

O

CH3

O

O

O OHOH

OP

OH

O

POH

CH3

O

OH

CH3

O

CH3

O

O

H

HH

OH

HCOOH

O H

OHOHO

H

HH

OH

HCOOHOH

H

OHOH

NH

HH

H

H

OOH

O

O

O

HNH

HH

H

H

OO

O

CH3

O

OH

CH3

O

OH

CH3

O

OH

O OHOH

OP

OH

O

POH

CH3

O

OH

O

H

HH

OH

HCOOH

O H

OHOHO

H

HH

OH

HCOOHOH

H

OHOH

UDP-GlcNAc

LpxA (RL2229, UDP GlcNAc 3-acyltransferase)

LpxC (RL3297, de-N-acetylase)

LpxD (RL2232, N-acyltransferase)LpxH (RL2664, UDP-diacylGlcN pyrophosphorylase)LpxB (RL2231, lipid-A disaccharide synthase)LpxK (RL0904, lipid-A 4’-kinase)

KdtA (RL0902, lipid-A Kdo transferase)

LpxF (4’-Pase, RL1570)4’-GalA transferase(possibly RL0684 or RL4664 )LpxXL (RL2812)LpxE (RL4708, 1-phosphatase)LpxQ (RL0868, lipid-A GlcNoxidase)

LpxL (lauryl transferase)LpxM (myristyl transferase)

Common to R. leguminosarum and E. coli

Specific to R. leguminosarum

Specific to E. coli

Figure 2. A comparison of the biosynthetic pathway of E.coli and R. leguminosarum lipid-A. The identification of the genes that encode for each of the enzymes for Rlv 3841 is as indicated.

Biosynthesis of Biosynthesis of RhizobialRhizobial lipid Alipid A

U.R. U.R. BhatBhat, L.S. Forsberg, & R.W. Carlson. 1994., L.S. Forsberg, & R.W. Carlson. 1994.J. Biol. ChemJ. Biol. Chem. 269:14402. 269:14402--1441014410N. L. N. L. QueQue, S. Lin, R.J.Cotter & C.R. Raetz., S. Lin, R.J.Cotter & C.R. Raetz.2000.2000.

J. Biol. ChemJ. Biol. Chem. 275:28006. 275:28006--2801628016N.L. N.L. QueQue, A.A. Ribeiro & C.R. Raetz, A.A. Ribeiro & C.R. Raetz2000. 2000. J. Biol. ChemJ. Biol. Chem. 275:28017. 275:28017--28027)28027)

[M-H]-

= 1915.4 (-3OHC4:0)2001.4 (+3OHC4:0)

V. Vedam, E.L. Kannenberg, J.G. Haynes, D. J. Sherrier, A. DattaV. Vedam, E.L. Kannenberg, J.G. Haynes, D. J. Sherrier, A. Datta, and R.W. Carlson. 2003. J. , and R.W. Carlson. 2003. J. BacteriolBacteriol. 185:1841. 185:1841--1850.1850.

A.

B.

I

II

III III+C

16:0

0

50

100

150

200

250

1000 1200 1400 1600 1800 2000 2200

1758 1786

1493

1521

1202

1230

1258

1286

IV

0

50

100

150

200

250

300

1000 1200 1400 1600 1800 2000 2200

1970

1948

1914

1886

1652 1680

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

O

OH

CH3

NH

H

H

OH

O

O

O

HNH

HH

H

H

OO

OH

O

OH

CH3

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

OH

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

OH

CH3

O

OH

O

OH

HH

OH

H

HOHH

HOOC

CH3

O

OH

CH3

NH

H

H

OH

O

O

O

HNH

HH

H

H

OO

OH

CH3

O

OH

CH3

O

OH

CH3

O

OH

O

OH

HH

OH

H

HOHH

HOOC

OH

[M-H]-

= 1230.6[M-H]-

= 1493.2Structure III Structure IV

Structure I Structure II

[M-H]-

= 1653.5 (-3OHC4:0)1710.4 (+3OHC4:0)

ParentParent MutantMutant

MutantMutantMutantMutant

MutantMutantParentParent

ParentParent

MutantMutant

BacteroidsBacteroids from from R. R. LeguminosarumLeguminosarum bvbv. . viciaeviciae 3841 and its 3841 and its acpXLacpXL mutantmutant

The lipid A from The lipid A from R. R. leguminosarumleguminosarum bvbv. . viciaeviciae 3841 3841 acpXLacpXL mutant mutant bacteroidsbacteroids

0

50

100

150

200

250

1000 1200 1400 1600 1800 2000 2200

1758 1786

1493

1521

1202

1230

1258

1286

0

20

40

60

80

100

120

140

1000 1200 1400 1600 1800 2000 2200

1914

1942

1970

1730

1758 1786

1493152112861258

1230

III

IV

III+C

16:0

II

III+C

16:0

I

III

IV

[M-H]-

= 1915.4 (-3OHC4:0)2001.4 (+3OHC4:0)

[M-H]-

= 1653.5 (-3OHC4:0)1710.4 (+3OHC4:0)

[M-H]-

= 1230.6[M-H]-

= 1493.2

Structure I Structure II

Structure III Structure IV

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

CH3

O

OH

CH3

COOHNH

HH

H

H

OOH

O

OH

O

HNH

HH

H

H

OO

OH

O

OH

CH3

O

OH

CH3

O

OH

O

OH

HH

OH

H

HOHH

HOOC

CH3

O

OH

CH3

NH

H

H

OH

O

O

O

HNH

HH

H

H

OO

OH

O

OH

CH3

CH3

O

O

CH3

O

OH

O

CH3O

O

OH

HH

OH

H

HOHH

HOOC

CH3

OOH

OH

NH

H

H

OH

O

O

O

HNH

HH

H

H

OO

OH

CH3

O

OH

CH3

O

OH

CH3

O

OH

O

OH

HH

OH

H

HOHH

HOOC

OH

5001000 1500 2000

2500 bp

100143 100144 100145

pRL100143 = hypothetical proteinpRL100144 = acyl carrier proteinpRL100145 = acyl-CoA dehydrogenase

acp5

BamHI

NsiIPpu10I

Bpu1102

Asp718KpnI

SexAI

AatI

BsgI

EcoRV

NruI

SgfI

DsaINcoI

PvuII PmaCI

XmnI

BseRI

AccISalI

PstIAhdI

BsmI

BspHI

BsaVVI

BanII

SmaIXmaI

Possible Possible in in plantaplanta Oxygen Regulation of VLCFA Oxygen Regulation of VLCFA Synthesis In Synthesis In R. R. leguminosarumleguminosarum??

NifANifA binding site binding site (upstream activator (upstream activator sequence, UAS)sequence, UAS)

σ54-binding consensus site

Ribosome binding site

What is the Function of the LipidWhat is the Function of the Lipid--A VLCFA?A VLCFA?

O

OHHO

HO CO2H Core

OHO

O

O

NHOO O

O NHCO2H

OHHO

CH2O

OHO

HOOO

O

OHO O

Brozek, K. A., R. W. Carlson, et al. (1996). J. Biol. Chem.

271(50): 32126-32136.Basu, S. S., M. J. Karbarz, et al. (2002). J. Biol. Chem. 277(32): 28959-28971.Vedam, V., E. L. Kannenberg, et al. (2003). J. Bacteriol. 185(6): 1841-1850.Vedam, V., J. G. Haynes, et al. (2004). Mol.Plant-Microbe Interact. 17: 283-291.Vedam, V., E. Kannenberg, et al. (2006). J. Bacteriol. 188(6): 2126-2133.

1 kb

acpXLOrf 1fatty acpdehydratase

Orf 23-oxoacyl acpsynthase

Orf 33-oxoacyl acpsynthase

lpxXL Orf 4Alcoholdehydrogenase

R. leguminosarumR. leguminosarum

S. melilotiS. meliloti

M. lotiM. loti

B. japonicumB. japonicum

BartonellaBartonella henselaehenselae

Brucella Brucella melitensismelitensis

A. A. tumefacienstumefaciens

R. R. palustrispalustris

LegionellaLegionella pneumophiliapneumophilia

ee--129129

ee--9393

ee--130130

ee--5858

ee--5252

ee--9797

ee--8585

ee--1111

refref

ee--152152

ee--146146

ee--156156

ee--135135

ee--133133

ee--149149

ee--137137

ee--6060

ee--159159

ee--148148

ee--154154

ee--115115

ee--151151

ee--125125

refref

ee--104104

ee--7474

ee--7575

ee--9393

ee--8383

** 2.72.7

ee--2424

ee--2525

ee--4444

ee--4141

refrefrefref refref refref

ee--4444

ee--3737

ee--4343

ee--3535

ee--3535

ee--4040

ee--3737

ee--1010

00

00

1 kb

rgtBrgtB(RL1468)(RL1468)

rgtArgtA(RL1469)(RL1469)

orf3orf3(RL1470)(RL1470)

rgtCrgtC(RL1471)(RL1471)

Possible Possible rgtDrgtD

(RL4664)(RL4664)

(RL0684)(RL0684)

UDP- -P-Dod

Dod-P

UDP

UDP- UDP-Exo5Exo5 LpsLLpsL

Orf3 (RL1470)?Orf3 (RL1470)?

-PP-.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Rxn 1 Rxn 2 Rxn 3

2 Pi

-P-Dod4LpxELpxELpxFLpxFRxn 4,5

RgtARgtARgtBRgtBRgtCRgtCRgtDRgtD (?)(?)

4 P-Dod

Rxn 6,7,8,9

Glc GlcA GalA

Kdo

Gal

Man

GlcN

A.

B. O

OOO

O

O

OH

OO

OHCOOH

O

OH

OH

OH

HOOC

CH3

OH

O

CH3

O

O

CH3

OH

O

CH3

OH

O

CH3

O

OCH3

OOH

O

O

COOHO

OHOH

O

O

COOHO

OHOH

O

OH

OH

OH

HOOC

O

OH

OH

OH

HOOC

OOHOHO

OO

OH

OH

OH

HOOCO

OH

OH

OH

O

O

OH

COOHO

OHOH

O-ChainKdo, lpcB

Gal, lpcA

Man, lpcC

Kdo, kdtA

Kdo, kdtA

GalA,

rgtC

GalA,

rgtA or B

GalA,

rgtA or B

GalA, lpx?GlcN

GlcNonate,

lpxE, lpxQ

27OHC28, acpXL - lpxXL

The Functions of the The Functions of the RhizobiumRhizobium--Specific Structural Specific Structural FeaturesFeatures

Plant Components Plant Components –– Nod Factor PerceptionNod Factor Perception

Oldroyd, G. E. D., and J. A. Downie. 2008. Coordinating Nodule Morphogenesis with Rhizobial

Infection in Legumes. Annual Review of Plant Biology 59:519-546.

Herbert Bosshart, M. H. 2007. Targeting Bacterial Endotoxin. Annals of the New York Academy of Sciences 1096:1-17.

Recognition of Microbes by Plant & Animal CellsRecognition of Microbes by Plant & Animal CellsPlant CellPlant Cell

Animal CellAnimal Cell

Microbes (Microbes (PAMPsPAMPs

or “or “MAMPsMAMPs”)”)

Animals (Animals (PRRsPRRs))

Plants (Plants (PRRsPRRs))LPSLPS

TLRsTLRs

NFRNFRFlagellaFlagella

CLRCLR

FLSFLSPGPG

Nod proteinsNod proteins

Virulence factor receptorsVirulence factor receptors

Common featuresCommon featuresTransmembraneTransmembrane

LRRsLRRsMAPK signaling cascadesMAPK signaling cascadesROS and RNSROS and RNSCa FluxesCa FluxesTranscription factorsTranscription factorsInducible immune effectorsInducible immune effectors““Moreover, plants can detect Moreover, plants can detect phytopathogensphytopathogens

using receptors that are using receptors that are evolutionarily related to those employed by legumes to detect syevolutionarily related to those employed by legumes to detect symbiotic mbiotic rhizobiarhizobia

(144, 180). Thus, the broad importance of this symbiosis is eve(144, 180). Thus, the broad importance of this symbiosis is ever r growing.” (Gibson et al. 2008. Ann. Rev. Genet. 42:413growing.” (Gibson et al. 2008. Ann. Rev. Genet. 42:413--41)41)

Ausubel, F. M. 2005. Are innate immune signaling pathways in plants and animals conserved? Nat.Immunol. 6:973-979.

“Given the compelling case for convergent evolution of innate immune pathways, an important issue is why evolution has chosen a limited number of apparently analogous regulatory modules in disparate evolutionary lineages. Does this reflect inherent biochemical constraints that result from a similar overall ‘logic’ of how an effective immune system can be constructed?”

“Although it seems to be generally accepted that the innate immune responses of plants and animals share at least some common evolutionary origins (i.e. divergent evolution), examination of the available data fails to support that conclusion, despite similarities in the overall ‘logic’ of the innate immune response in diverse multicellular

eukaryotes.”

Comparing the Animal and Plant Innate Immune PathwaysComparing the Animal and Plant Innate Immune Pathways