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
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