I
ARBONAND NITROGEN METABOLISMOFFREE-LIVING FRANKIASPP.ANDOF
RANKIA-ALNUSSYMBIOSES
Coverdesign:Greet Zandstra
ELANDBOUWCATALOGUS
Promotor
: dr.ir. E.G.Mulder,oud-hoogleraar in dealgemene
microbiologic en demicrobiologie van bodem en water
Co-referent: dr.A.D.L.Akkermans,wetenschappelijk hoofdmedewerker
bijde vakgroep microbiologie
b\n 6>%o\
6>^<5>
JAN BLOM
CARBON AND NITROGEN METABOLISM OF
:
REE-LIVING FRANK/A SPP. AND OF
-RANKIA-ALNUS
SYMBIOSES
'roefschrift
;erverkrijging van de graad
an doctorindeLandbouwwetenschappen,
pgezagvandeRector Magnificus,
r. C.C.Oosterlee,
loogleraarindeveeteeltwetenschap,
n het openbaarteverdedigen
)pvrijdag15januari1982
tesnamiddagstevieruurindeaula
fandeLandbouwhogeschool teWageningen
\£> tf- \5*X9t[-H- ° 3
Q
BIBUOTHEEK L.H.
0 b JAH. 1382
ONTV.TIJDSCHR. ADM.
This study wascarried outattheLaboratory of Microbiology,
Agricultural University,Wageningen,theNetherlands.
The investigations were financially supported bytheFoundation for
Fundamental Biological Research (BION),which issubsidized bythe
Netherlands Organization fortheAdvancement ofPure Research (ZWO).
nioli°\ .MX
STELLINGEN
i
Het verdient aanbeveling datauteursvanoverzichtsartikelen zich beperken
totdiegebieden waarop zijzelfwerkzaam zijn.
"Root nodule associations occur between organisms other than
legumesand Rhizobium. (....)Itappears that thesymbiont
isanactinomycete,however,theorganism hasnotbeen
isolatedandcultured inthefree-living state."
L.Beevers (1981)in:Biology ofinorganic Mitrogenand
Sulfur.H.BotheandA.Trebst,Eds.p.16.Springer Verlag,
Berlin,Heidelberg,NewYork.
II
DeconclusievanKurzenLaruedat"thecoincidenceofhigh isocitric dehydrogenase activity withthepeakofnitrogen fixation ispresumptive evidence
that itprovides reductant forfixation"wordt niet gesteund doorhun
experimenten.
W.G.W.KurzandT.A.Larue (1977)Can.J.Microbiol. 23,1197-1200.
Ill
De door-Matsumotoenmedewerkers waargenomen "repressie"vannitraatreductase inkomkommerbladeren tengevolgevancalcium-deficientieis
geen echte repressie,maar veeleer stoppen vandeenzym-syntheseten
gevolgevanhetafsterven vandeplant.
H.Matsumoto, K.Teraoka andT.Kawasaki (1980) PlantandCell
Physiology 21,183-191.
IV
DeconclusiesvanBoland enmedewerkers betreffende de(sterke)affiniteit
voor ammoniakvanglutamine-synthetaseuitwortelknollenvanvlinderbloemige planten,worden niet gesteund doorhunwaarnemingen.
M.J.Boland,A.M.FordyceandR.M.Greenwood (1978)Aust.J.
Plant Physiol. 5,553-559.
V
Ten onrechte concluderen Schubert en medewerkers u i t hun l a b e l l i n g - en
pulselabelling-experimenten met
NH», dat in elzeknollen NH. niet via de
GS/GOGAT route geassimileerd wordt.
K.R.Schubert,G.T.Coker IIIandR.B.Firestone (1981) Plant
Physiol.57,662-665.
VI
Het isaantwijfel onderhevig ofdeactinomyceet-stammendievolgens
Gauthierenmedewerkers acetyleen reduceren,werkelijk Frankia-stamnen
zijn.
D.Gauthier,H.G.DiemandY.Dommergues (1981)Appl.Env.
Microbiol. 41,306-308.
VII
Het gebruik van "radio-immuno-assays" ter bepaling van het gehalte aan
d i e t h y l s t i l b e s t r o l in de urine van mestvee kan leiden t o t "vals-positieve"
uitkomsten.
J.Blom (1981) Chem. Weekbl. 77-12, 97
J.Blom (1981) Chem. Weekbl. 77-24, 215.
VIII
In tegenstellingtotdebewering vanBrill (1981)werden bacterienvan
het geslacht Rhizobium voorheteerst ge'fsoleerd door Beijerinck (1890)
en niet door Hellriegel enWilfarth (1888).
W.J.Brill (1981) Scientific American 245(3),146-156
H.Hellriegel undH.Wilfarth (1888) Beilageheft zuderZeitschriftdesVereinsf.d.Ru'benzucker-Industrie d.D.R.p.83
M.W.Beijerinck (1918) Proceedings oftheSectionof
Sciences,Kon.AkademievanWetenschappen,Amsterdam.XXI,
183-192
Index Bergeyana (1966)p.246.TheWilliamsandWi1kins
Company,Baltimore Md. 21202,U.S.A.
IX
Aangezien belastingheffing niet meer uitsluitend wordt gezien als hetdoen
bijdragen vandebelastingplichtigenaandefinanciering vandetraditionele
overheidstaken,doch medealseenvandeinstrumenten waaroverdeoverheid
beschikt bijdedoor haar gewenste herverdeling vandebeschikbare middelen,
verdient hetaanbeveling bepaalde giften vandiebelastingplichtigenin
minderingtebrengen opdedoorhenverschuldigde belasting,inplaatsvan
op nun belastbaar inkomen,voor zover deze giften passen inhetherverdelingsbeleid vandeoverheid.
X
Door medewerking teverlenen aanhetzogenaamde "damesschaak",maaktde
Koninklijke Nederlandse Schaakbond zich medeplichtigaandiscriminatie
en achterstelling vandevrouw.
Er is geen einde aan hetmaken van
veel boeken,en veel doorvorsen is
afmatting voor het lichaam. Vanal
het gehoorde is het slotwoord:
Vrees God en onderhoud zijn geboden,
want dit geldt voor alle mensen.
(Pred. 12:12-13)
/OORWOORD
ijde voltooiing van dit proefschriftwil ik iedereen,die heeftbijledragen aan het tot stand komen ervan,hartelijk bedanken.
1ijn ouders,diemijaltijd hebben gestimuleerd tot het verzamelen en
rerwerken van kennis.
lijn promotor,professor Mulder,voor het kritisch doornemen van de
lanuscripten.
ntoon Akkermans en Wim Roelofsen voorde vruchtbare samenwerking.
levrouwC.Mbller-Mol voor het type-werk.
levrouwA.Mol-Rozenboom en mevrouw J.Gerritsen-Manten voor de dagelijkse
lereidheid de doormij geproduceerde chaos teordenen.
Deheren O.van Geffen,A.Houwers en E.van Velsen voor de zorg besteed
aan deelzekweek.
Deandere personeelsleden van de vakgroep microbiologic voor hun begeleiding.
Ruud Dickhoff,Lida van den Eijkel,Reinier van Ham,ReintHarkink,Piet
Meijer,Liekle Nijholt,Eylon Shlomi,Janny Spin,Fons Stams en Karin
van Straten voor hun inzet.
De leden van de knollenclub voor hun adviezen.
BION voor definanciele steun.
DONTENTS
Chapter I
INTRODUCTION
1.1
1.2
1.3
1.4
Symbiotic nitrogen fixation
11
Cultivationofthe rootnodule endophytes {Frankia spp.)
ofnon-leguminous plants
15
Ammonia assimilation innitrogen-fixing organisms
16
Aim and outline of the present study
17
Chapter II
THECARBON AND NITROGEN METABOLISM OF FREE-LIVING
11.1
11.2
11.3
11.4
FRANKIA SPP.
Growth of Frankia Ave11onmedia containing Tween80
as C-source
Jan Blom,Wim Roelofsen and Antoon D.L.Akkermans
FEMSMicrobiology Letters9(1980) 131-135
Utilization offatty acids and NH^ by Frankia AvgI1
Jan Blom
FEMSMicrobiology Letters10(1981) 143-145
Metabolic pathways forgluconeogenesis and energy
generation in Frankia AvcI1
Jan Blom and Reint Harkink
FEMS Microbiology Letters11(1981)221-224
Carbon and nitrogen source requirementsof
11
21
23
35
43
Frankia Strains
Jan Blom
FEMSMicrobiology Letters13(1)January 1982 (in press) 51
Chapter III
ASSIMILATION OFAMMONIA IN ROOT NODULES OF
111.1
ALNUS GLUTINOSA
Assimilationof nitrogeninroot nodules ofalder
111.2
Jan Blom,Wim Roelofsen and Antoon D.L. Akkermans
The New Phytologist 89 (1981) 321-326
InhibitionofGOGAT activityinlupin nodule homogenates
{Alnus
glutinosa)
by aldernodule homogenates
Chapter IV
GENERAL DISCUSSIONAND SUMMARY
SAMENVATTING
CURRICULUM VITAE
61
63
73
77
87
93
Chapter I
INTRODUCTION
I.1 Symbiotic nitrogen fixation
Although nitrogen occursonearthinlargeamounts,thegrowthofmany
organismsislimitedbyinadequate supplyofthe element.Thisisduetothe
fact thatmost organisms cannot utilize atmospheric nitrogen (N 2 ),butrequire
combined nitrogenasitisincorporated innitrate,ammonium and aminoacids.
Only very feworganismscanfix N„, i.e. form ammonium from N ? .
Industrial nitrogen fixation,often occurring accordingtothe Haber-Bosch
process,proceedsataN9-pressureof200atmospheres andatemperatureof
300°C.Itisresponsibleforthe fixationof30x10 metric tonsofnitrogen
per annum (HardyandHavelka,1975;BurnsandHardy, 1975). Biological nitrogen
fixation proceedsataN9-pressureof0.8atmospheresandatemperature usually
notexceeding 30°C;itproduces 175x10 metric tonsoffixed nitrogenannually.
Nitrogen-fixing organisms haveattheir disposalacatalytic system viz.
theenzyme nitrogenase thatdoesnotrequire theextreme conditionsofthe
Haber-Bosch process.Detailed informationonthenitrogenase system isgiven
byOrme-Johnson et al. (1977)andEmerich et al. (1981).
Up till now,nitrogen fixationhasbeen observedtooccur onlyinprokaryotic
organisms,either free-livingorlivinginsymbiosiswith eukaryotesorwith
other prokaryotes.Inthepresent section,attention will begiventosymbiotic
nitrogen fixers only.
Symbiotic nitrogen-fixing organismsare divided into three groupsof
prokaryotic organisms, viz. cyanobacteria,rhizobiaandactinomycetes. These
microorganismscanoccurinsymbiosis withanumberofeukaryotic organisms,
eitherasectosymbiontorasendosyrnbiont (Table 1).Associationsofcyanobacteria with bacteriadoalso occur.
11
Table 1.N„~fixing symbioses (from Mulder, 1981)
^-fixing symbiont
Rhizobium spp.
Actinomycetes ofthe genus Frankia
Cyanobacteria
Non-N~-fixing partner
Leguminous plants (with a few exceptions)
Non-leguminous plants
Angiospermous plants (Gunnera spp.)
Gymnospermous plants {Cyoas spp.)
Liverworts
Mosses
Small fresh water ferns {Azolla spp.)
Fungi (lichens)
Bacteria
In thecase ofectosymbioses the^-fixing symbiont lives extracellularly,
be it inclose relation to the host.Of these ectosymbioses especially lichens
are known to contribute significantly to theN-input of their ecosystem,
particularly in regions having an extremely dry or cold climate; e.g. Stereoaaulon sp., a lichen occurring in theNorth-Swedish pine forests (Huss-Danell,
1977).
In the case of endosymbioses theN«-fixing symbiont isfound within the
cells of the host. Some primitive forms of endosymbiosis include cyanobacterium
associations with phycomycetes,diatoms and some gymnospermous and angiospermous plants.However,theroot-nodule symbioses with either rhizobia oractinomycetes as N„-fixing symbiont are ofmuch wider ecological importance.
The Rhizobium symbioses have so far gained much more attention than the
actinomycete symbioses. In the following part of this section,a comparison
will bemade between some aspects of the Rhizobium-legume symbioses and those
of theactinomycete-non-legume symbioses.Attention will be paid mainly to
differences between the two types of symbiosis.
Innon-leguminous plants,similar to legumes,infection is preceded by
deformation of root hairs (Hiltner, 1903;Pizelle, 1972). The actinomycete
{Frankia sp.) in the form of hyphae passes through a deformed root hairand
penetrates into thecells of thecortex (Angulo,van Dijk and Quispel,1976;
Pommer, 1956;Taubert, 1956). Cortical cells close to the infected cells start
to divide,causing a slight swelling of the root.The hyphae penetrate the
dividing cells and form clusters of hyphae within the host cells.The hyphae
tips at the periphery of the clusters are swelling and develop into spherical
vesicles. By invading new dividing cells,acluster of infected host cells is
formed which iscalled primary nodule. In the neighbourhood of the primary
12
nodule,a root primordium,initiated within the pericycle,grows into the
cortex where it is invaded by the endophyte.This infected root primordium
continues to grow and develops into a visible root nodule. Due todichotomous
division of the topmeristem,acluster of lobes isformed (Angulo,1974;
Angulo et at. , 1976).
Innon-leguminous plants themeristematicactivity isthus initially restricted to the immediate vicinity of the site of infection,whereas in leguminous
nodules cell divisions spread toother cells,causing the outgrowth of the
nodule (Angulo et al. , 1976).
Itis presumed that in non-leguminous nodules nitrogen fixation takes place
in the vesicles,which therefore may becompared with the bacteroids inthe
nodules of leguminous plants.Evidence as tothis presumption isasfollows:
(a)When alder root nodules are homogenized and the homogenate is filtered
through a 20ym filter,the distribution between theresidueand filtrate ofthe
N„-ase activity corresponds with the distribution ofthe numbers of vesicles
and of theamount of diamino pimelic acid (an endophyte cell-wall marker)
(Akkermans,1978;Akkermans,Roelofsen and Blom, 1979).
(b) In ineffective nodules {i.e. nodules not capable offixing nitrogen)no
vesicle clusters are found (Mian,Bond and Rodriguez-Barrueco, 1976;Baker,
Newcomb and Torrey, 1980).
(c) Infree-living Frankia CpI1 {of Chapter 1.2)nitrogen fixation is reported
to beattended with the development of vesicles (Tjepkema,Ormerod and Torrey,
1980and 1981).
Inboth the Rhizobium and theactinomycete symbioses,themicrosymbiont
obtains sources ofcarbon and energy from the host.The transport of newly
formed photosynt'natesto the root nodules of visum sativum plants has been
shown by using 'CO- (Bach,Mageeand Burris,1958;Lawrie and Wheeler, 1973).
The chemical nature of the carbon and energy source for the nitrogen-fixing
microsymbiont isthe subject of research for both types of symbiosis.
Ithas been shown that some citric acid cycle intermediates, viz. succinate,
fumarate,malateand oxaloacetate,can support respiration of Rhizobium
leguminosarum bacteroids isolated from root nodules of Visum sativum (Tuzimuraand
Meguro, 1960;Houwaard, 1979),whereas citrate,glucose and pyruvate did not
stimulate respiration (Houwaard, 1979). The uptake of succinate by free-living
and bacteroidal forms of Rhizobium spp.has been shown by Glenn,Poole and
Hudman (1980). Succinate-dependent N^-fixation was found tooccur in bacteroids
13
from soybean (Bergersen and Turner,1967)and in bacteroids from pea plants
(Houwaard, 1979).
Bacteroids of Rhizobium japoniaum contain a functional citric acid cycle
(Stovall and Cole, 1978)and an Entner-Doudoroff pathway (Keele et al. ,1969;
Martinez-de Drets and Arias, 1972;Ronson and Primrose, 1979). The presence of
the Embden-Meyerhof pathway isdisputed (Mulongoy and Elkan,1977;Martinezde Drets and Arias, 1972;Ronson and Primrose, 1979).
From the data known sofar itis generallyassumed that themicrosymbiontof
the Rhizobium-legume symbiosis obtains some dicarboxylic acid, e.g. succinic
acid from the plant.
Less is known about the physiology of the vesicle clusters inactinomycete
symbioses.Akkermansand Roelofsen (1980)found that NADH supports the respiration ofvesicle clusters isolated from rootnodules of Alnue glutinosa. This
suggests the presence ofanNADH dehydrogenase on theouter sideof themembrane
surrounding the vesicle clusters,or partial damage of thismembrane during the
homogenization of thenodules.
Recently, ithas been found inour laboratory that vesicle clusters ofalder
rootnodules contain the enzymes isocitrate dehydrogenase,succinate dehydrogenase, fumaraseand malate dehydrogenase (Akkermans,Huss-Danell and Roelofsen,
Physiol. Plant.»inpress). The same authors found thataddition ofmalate plus
glutamateplus NAD toa suspension of vesicle clusters resulted in increased
oxygen consumption,whereas the addition of only one or two of these compounds
was ineffective (Huss-Danell,Roelofsen,Akkermans and Meyer,submitted). In
addition,they found activity of glutamate-oxaloacetate transaminase inboth
vesicle clusters of theendophyte and the cytoplasm of the host cells. They
suggest that this enzyme,incooperation with malate dehydrogenase,may function
intransporting excess reducing equivalents from thecytoplasm of the host into
thevesicle clusters,analogous to themitochondrial-cytoplasmatic malateaspartate shuttle {cf Lehninger, 1975).
Vesicle clusters probably donotcontain a functional glycolysis. Huss-Danell
et al. (manuscript submitted)found in vesicle clusters only glycolytic enzymes
catalyzing reversible reactions {i.e. enzymes also functioning in gluconeogenesis) viz. aldolase,enolase,3-phosphoglycerate kinase and glyceraldehyde-3phosphate dehydrogenase,butno enzymes catalyzing the irreversible glycolytic
reactions, viz. hexokinase,pyruvate kinase and pyruvate dehydrogenase.This
suggests that the source of carbon and energywhichthemicrosymbiont innonleguminous nodules obtains from the plant cannot be acompound which isbroken
14
lown through glycolysis.Nopositive indications with respecttothechemical
latureofthis compound havesofararisen from investigations with intact
lodulesorwith themicrosymbiont (vesicles) isolated from nodules.Apossible
rayto tackle this problem wastoinvestigate the growth requirementsof
?
rankia spp.grown in vitro {of Chapters 1.2and II).
1.2 Cultivation
of the root nodule endophytes (Frankia spp.J of
non-leguminous
slants.
Researchontheactinomycete symbiosesinrootnodulesofnon-leguminous
plantswas adversely affected until recentlybythe lackofactively growing
pureculturesofthemicrosymbiont.Though some isolationsoftheseactinonycetes {Frankia spp.)have been reported inthepast,noreproducible tests
weremadetoconfirm their identity with themicrosymbiont (Quispeland
Burggraaf, 1981);inoneinstancetheisolated strains were lost rapidly after
isolation (Pommer, 1959). Only since 1978thereproducible isolationand
cultivationofFrankia spp.from rootnodulesofnon-leguminous plants have been
reported (Callaham,Torreyanddel Tredici,1978;Lalonde, 1978;Quispeland
Tak, 1978;Bakerand Torrey, 1979;Baker,TorreyandKid,1979;BerryandTorrey,
1979; Lechevalierand Lechevalier,1979;Quispel,1979;Bakerand Torrey,1980;
Baker,Newcomb andTorrey, 1980). Various isolation techniques have recently
been reviewedbyBackerand Torrey (1979)andQuispelandBurggraaf (1981).
Themedia used forcultivationofFrankia spp.bythe above-mentioned authors
all haveincommonamarked complexity. The Frankia broth medium (Bakerand
Torrey, 1979)contains,among other things,yeastextract,dextroseandCasamino
acids. ThemediumofQuispel andTak(1978)contains peptone,glucoseand
alcoholic root extracts.TheQMODmedium (LalondeandCalvert,1979)contains
yeast extract,peptone,glucoseandalipid supplement,consistingof22%pure
phosphatidyl choline.
Cell yieldsandgrowth ratesofthe Frankia spp.onthesemedia have originallynot been determinedoratbestmeasured indirectly,usingtheinfectivity
ofacultureasadevice for estimating the densityofFrankia cells (Quispel
and Tak, 1978). However,thecultivationofanorganismondefined mediaandthe
definite determinationofitscell yieldandgrowth rateare prerequisitesfor
ascertaining itsgrowth requirements {of ChapterIIofthis thesis).
Frankia spp.grown inpureculture are slow-growing {of ChapterII.1),aerobic
tomicroaerophilic actinomycetes,with long,branching,septate hyphae. The
hyphal wall isprimarily Gram-positive,although short regionsofGram-negative
15
wall dooccur (BerryandTorrey, 1979). Thehyphal diameter isvariable,ranging
from0.3to1.0um. Sporangia develop terminally onmain hyphae,terminallyon
short side branches orinanintercalary fashion (BerryandTorrey, 1979).
Up till nowlittle information isavailable onthephysiologyandbiochemistryof Frankia spp..In ChapterLIof this thesis research concerning thecarbon
and nitrogen requirements,thegrowth rateandgrowth yield,andsome enzyme
activities of Frankia spp.will bereported.
1.3 Ammonia assimilation
in nitrogen-fixing
organisms
Theammonia formed from nitrogen fixation isultimately assimilated into
proteins,nucleotidesandother nitrogen-containing compounds.Thepathwaysof
this assimilation differforthevarious nitrogen fixers.Nagatini, Shimizu
and Valentine (1971) showed that inthefree-living nitrogen fixers
pneumoniae, Azotobaoter vinelandii
, Clostridium
Klebsiella
pasteurianum and
Rhodospirillum
rubrwn theprimary ammonia assimilation proceeds via theGS/GOGAT pathway:
pc
glutamate +NHL+ATP
>glutamine+ADP+P.
glutamine+a-ketoglutarate+NAD(P)H +H +G 0 G A T>2glutamate +NAD(P) +
+
a-ketoglutarate+NH.,+ATP+NAD(P)H +H +— > glutamate+ADP+P+
iNAu(P)
From glutamate,thefixed nitrogen canbeassimilatedinto other amino acids
through transamination reactions {e.g. Meister, 1957).
In free-living nitrogen-fixing blue-green algae (cyanobacteria)anoperative
GS/GOGAT pathwaywasalso shown (LeaandMiflin, 1975). Inthese organisms
GOGAT isnotNAD(P)H-dependentbutrequires reduced ferredoxin asasourceof
reducing equivalents.
In Rhizobium symbioses,theammonia formed bythenitrogenase reactionin
the bacteroids isexcreted intothecytoplasm oftheplant cell (Brownand
Dilworth, 1975;Robertson,Warburton andFarnden,1975;U a n e et al., 1980)
and assimilated into glutamate bytheaction oftheGS/GOGAT pathway (Nagatini
et al. ,1971;Sloger, 1973;Kennedy, 1973;Dunn andKlucass,1973;Ryanand
Fottrel, 1974). Thenodules ofmost leguminous plants contain glutamineor
asparagineasprominent amino acid [e.g. vanEgeraat, 1972). These mono-amides
serveasstorage components fornitrogen andinaddition represent thecompounds
inwhich fixed nitrogen istranslocated fromthenodules toother partsofthe
leguminous plants.
Inthecyanobacterial symbiosis ofalichen belonging tothegenus
Feltigera,
ammonia assimilation isreported toproceed via theGDHreaction (Stewartand
owell, 1977;Pai,Rowel1andStewart, 1980):
•ketoglutarate +NH^+NAD(P)H J glutamate +NADP +
Less information isavailableasyet with respect tothepathwaysof
mmonia assimilation inactinoinycetous root nodules.Thepool offree amino
cidsofalder noduleswasshown todiffer from thatofother typesofroot
odules (Miettienen andVirtanen, 1952;Leaf,GardnerandBond, 1958;Wheeler
nd Bond, 1970). Theroot nodules ofthis non-legume contain citrullineasthe
lostabundant free amino acid; itcontributes 201onmolar base {i.e.
50%on
l-base)totheamino acid pool,while glutamine contributes about 10%, and
sparagine isnotdetectable. Other types ofnon-legume root nodulesmaypossess
lignificant quantities ofspecific amino acids, e.g. arginine ,ornithine,
llutamateorglutamine (WheelerandBond, 1970).
No information isasyetavailable with respect toactivities ofenzymes
esponsible fortheformation ofthese amino acids from NH. inaldernodules.
-13 - +
Schubert et al. (1981)exposed alder nodules to|_ NJ-NH.andfound incorportion ofmost ofthelabel into glutamineandglutamate. These observations
nd theresults ofpulse-labellingandinhibitor studies indicate that inalder
odules theGS/GOGAT pathway isnotoperative andthatGDHplaysarole inthe
ssimilationofexogenously supplied NH..Confirmation ofthis hypothesisby
neansofenzyme studies isrequired. InChapterIIIofthis thesisthe
activities ofsomeoftheammonia-assimilating enzymes will bereported.
T.4 Aim and outline
oj' the present
study
Theaimoftheresearch reported inthis thesiswastoclarify some aspects
Dftherelation ofthehostandtheN?-fixing microsymbiont inthe
Frankia-
Inus symbiosis.
ChapterIIdeals with thefree-living stateof Frankia AvcI1,theendophyte
(microsymbiont)oftheroot nodules of Alnus virid,is
ssp. arispa.
Thegrowth
equirementsof Fi-ankia AvcI1 andsome physiological research onthis organism
re reported.
In Chapter IIItheassimilation ofammonia inroot nodulesof Alnus
glutinosa.
A/illbedealt with.Theactivities andlocalization ofsome ammonia-assimilating
enzymes arereported.
ChapterIVconsists ofasummaryandgeneral discussion oftheresults
reported inthis thesis.
17
REFERENCES
1.Akkermans,A.D.L. (1978). In Interactions between non-pathogenic soil
microorganisms and plants (Y.R. Dommerguesand S.V. Krupa,Eds)pp.335-372,
Elsevier Scientific Pub!. Comp. Amsterdam,Oxford,NewYork.
2. Akkermans,A.D.L.and Houwers,A. (1979).In Proc.Workshop Symbiotic Nitrogen
Fixation in themanagement of temperate forests (J.C. Gordon,C.T.Wheeler
and D.A. Perry,Eds)pp.23-35,Forest Res.Laboratory,Oregon State University.
3.Akkermans,A.D.L.,Roelofsen,W. and Blom,J. (1979). In Proc.Workshop
Symbiotic Nitrogen Fixation in themanagement of temperate forests (J.C.
Gordon,C.T.Wheelerand D.A. Perry,Eds)pp. 160-170, Forest Research
Laboratory,Oregon State University.
4. Akkermans,A.D.L.and Roelofsen,W. (1980). Symbiotic nitrogen fixation by
actinomycetes in Alnus-type rootnodules. InNitrogen Fixation,Proc.Phytochemical Society of Europe (W.D.P.Stewart and J.R. Gallon,Eds)pp.279-299,
Academic Press,London.
5.Angulo,A.F. (1974)Ph.D.Thesis,University ofLeiden.
6.Angulo,A.F.,Dijk,C. van,and Quispel,A. (1976). In Symbiotic Nitrogen
Fixation in Plants (P.S.Nutman,Ed.)pp.475-484,Cambridge University
Press,Cambridge,London,New York,Melbourne.
7. Appleby,C.A. (1969)BBA 172,71-87.
8. Bach,M.K.,Magee,W.E.and Burris,R.H. (1958). Plant Physiol.33,118-124.
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management of temperate forests (J.C.Gordon,C.T.Wheeler and D.A. Perry,
Eds)pp.38-56,Forest Research Laboratory,Oregon State University.
10. Baker,D.,Torrey,J.G. and Kidd,G.H. (1979). Nature 281,76-78.
11. Baker,D.and Torrey,J.G. (1980). Can.J. Microbiol.26,1066-1071.
12. Baker,D.,Newcomb,W.and Torrey,J.G. (1980). Can.J. Microbiol.26,
1072-1089.
13. Bergersen,F.J. and Turner,G.L. (1967). BBA 141,507-515.
14. Berry,A. and Torrey,J.G. (1979) In Symbiotic Nitrogen Fixation inthe
management of temperate forests (J.C.Gordon,C.T. Wheelerand D.A. Perry,
Eds)pp.69-83,Forest Research Laboratory,Oregon State University.
15. Brown,C M . and Dilworth,M.J. (1975). J.*Gen.Microbiol.86,39-48.
16. Burns,R.C.and Hardy,R.W.F. (1975). Nitrogen Fixation in bacteria and
higher plants.Springer Verlag,Berlin.
17. Callaham,D., Torrey,J.G. and del Tredici,P. (1978). Science 199,899-902.
18. Dunn,S.D. and Klucas,R.V. (1973). Can.J. Microbiol. 19,1493-1499.
19. Egeraat,A.W.S.M. van (1972). Comm. Agric. Univ.72-27.
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52, 1-22.
21. Glenn,A.R.,Poole,P.S.and Hudman,J.F. (1980). J. Gen.Microbiol.119,
267-271.
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Forstwirtschaft 17,9-25.
24. Houwaard,F. (1979). ThesisAgricultural University Wageningen.
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1184-1191.
27. Kennedy,I.R. (1973). Proc.Aust.Biochem. Soc.6,33
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Biochem. 103,39-46.
Lalonde, M. (1978). Can. J. Bot. 56,2521-2535.
Lalonde, M. and Calvert, H.E. (1979). In Proc.Workshop Symbiotic Nitrogen
Fixation in the management of temperate forests (J.C. Gordon, C.T. Wheeler
and D.A. Perry, Eds) pp. 95-110,Forest Research Laboratory, Oregon State
University.
Lawrie,A.C. and Wheeler, C.T. (1973). New Phytol. 72, 1341-1348.
Lea, P.J. and Miflin,B.J. (1975). Biochem. Soc. Trans. 3,381.
Leaf,G., Gardner, I.C.and Bond, G. (1958). J. Exp. Bot. 9, 320-331.
Lechevalier, M.P. and Lechevalier, H.A. (1979). In Symbiotic Nitrogen
Fixation in the menagement of temperate forests (J.C. Gordon, C.T. Wheeler
and D.A. Perry, Eds) pp. 111-122,Forest Research Laboratory, Oregon State
University.
Lehninger, A.L. (1975). Biochemistry 2nd ed. Worth Publishers, Inc.New York
ISBN 0-87901-047-9.
Martinez- dePrets, G. and Arias,A. (1972). J. Bacteriol. 109,467-470.
Meister, A. (1957). Biochemistry of the amino acids.Academic Press Inc.,
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Mian, S., Bond, G. and Rodriguez-Barrueco,C. (1976). Proc. Royal Soc.B
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Miettienen, J.K. and Virtanen, A.I. (1952). Physiol.Plantarum 5, 540-557.
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Microbiology, Agricultural University Wageninyen,The Netherlands.
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Nagatani, H., Shimizu,M. and Valentine, R.C. (1971). Arch. Mikrobiol.
79, 164-175.
Orme-Johnson,W.H., Davis,L.C., Henzl,M.T.,Averill, B.A., Orme-Johnson,
N.R., Munck, E.and Zimmerman, R. (1977). In Recent Developments in Nitrogen
Fixation (W. Newton,J.R. Postgate and C. Rodriguez-Barrueco, Eds) pp.131-178.
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Pommer, E.H. (1956). Flora,Jena,143,603-634.
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temperate forests (J.C.Gordon,C.T. Wheeler and D.A. Perry, Eds) pp. 57-68,
Forest Research Laboratory, Oregon State University.
Quispel,A. and Burggraaf, A.J.P. (1981). In Proc. 4th Int. Symposium on
Nitrogen Fixation (A.H. Gibson and W.E. Newton, Eds) pp. 229-236, Australian
Academy of Science, Canberra.
Quispel,A. and Tak,T. (1978). New Phytol. 81,587-600.
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67, 662-665.
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19
Chapter II
'HE CARBON AND NITROGEN METABOLISM OF
REE-LIVING FRANK/A SPP.
:
3HAPTER 11.1
3R0WTH OF FRANKIAAVCI1 ON MEDIA CONTAINING TWEEN 80AS C-SOURCE
FEMS Microbiology Letters 9 (1980) 131-135
Jan Blom,Wim Roelofsen and Antoon D.L. Akkermans
.aboratory of Microbiology,Agricultural University,
Hesselink van Suchtelenweg 4,6703CT Wageningen,
The Netherlands
23
I.INTRODUCTION
Research onthenitrogen-fixing system inactinomycete-infected nodulesof
non-leguminous plantshasbeen hindered until recentlybythelackofpure
culturesofthemicrosymbiont (1).Only since 1978theisolationandcultivation
in vitro ofendophytesofsome non-legumes have been reported, vis. theendophytes of Comptonia peregrina
A. viridis
ssp. arispa
(2,3), Alnus rubra (4),Elaeagnus
(5), A. glutinosa
(6),and Myrica
umbellata (5),
pennsyIvanica
(7).
Until now,however,growth ofthese endophytes hasonly been achieved incomplex
media,containing peptoneoryeast extract (2-8).Toobtainaclear insight
intothephysiologyofthese endophytes,itisnecessary togrow themonawelldefined medium.
Quispel (6)addedapetrol-ether lipid extract from plant rootstothegrowth
mediumoftheendophyteofA. glutinosa.
LalondeandCalvert (8)replaced this
petrol-ether extractbyalipid supplement containing soybean lecithin. In the
present paper itisshown thatthecomplex lipid supplementcanbereplacedby
Tween80(polyoxyethylene (20) sorbitan monooleate). Itappeared that
AvcI1,thenodule endophyteof A. viridis
ssp. arispa,
Frankia
isa'bletogrow ona
medium containing Tween80assole carbon sourceandeither NH.orglutamic acid
as sole nitrogen source.Thegrowth rateandgrowth yieldofAvcI1 onvarious
media arereported.
2.MATERIALSANDMETHODS
2.1.
Organism and growth
conditions
Frankia AvcI1, isolated from nodules of Alnus viridis
ssp. arispa wasobtained
from D.BakerandJ.G. Torrey (Harvard University,Petersham,MA01366). Itwas
grown aerobically at25°C innon-shaking 100-ml Erlenmeyer flasks containing40ml
ofculture medium. Inoneexperiment anaerobic growthwasobtainedbyplacingthe
Erlenmeyer flasks inanexsiccator containing aGasPak hydrogen +carbondioxide
generator envelope (BBL,Cockeysville,MD21030,USA).Thefollowing culture media
were used: Frankia
broth (1),containing yeast extract,glucose,Casaminoacids,
vitamin B12andsalts, QMOD medium (8),containing yeast extract,peptone,glucose,
saltsandlipid supplement.
QMOD/Tweenmedium. FromtheQMOD medium,thelipid supplement isomittedand
Tween 80(Sigma P-1754)isadded toafinal concentration of2g/1.
25
Tween/cas medium. In the QMODmedium (8) the l i p i d supplement, glucose, peptone
and yeast extract are replaced by Tween 80 (varying concentration), Casamino
acids (Difco 479744, varying concentration) and a vitamin mixture containing
b i o t i n , f o l i c a c i d , n i c o t i n i c a c i d , Ca-pantothenate, pyridoxine-HCl, r i b o f l a v i n
and thiaminiumdichloride ( f i n a l concentration of each vitamin 0.1 mg/1).
Tween/glu medium. In the Tween/cas medium the Casamino acids are replaced by
L-glutamic acid (10 mg/1). The f i n a l concentration of Tween 80 is 2 g / 1 .
Tween/M medium. In the Tween/glu medium the glutamic acid is replaced by
NH4C1 (20 mg/1).
2.2.
2.2.1.
Methods
Inoculation
A 2-week-old cultureofFrankia Avcllwasusedasinoculum.
Erlenmeyerflasks containing40mlofculturemedium were inoculated with
2mlofawashed Frankia culture,containing 50or100yg(TOC)cells.Thecells
werewashed4timesbycentrifugation (5min 11OOxg)with sterile phosphate
buffer (50mM K-phosphate,pH 7.0).
2.2.2.
Determination
of the growth
yield
Cells were harvestedbycentrifugation (5min11OOxg)andwashed4times with
phosphate buffer (50mM K-phosphate,pH7.0).Thegrowth yieldofthecellswas
determined byassaying thecontentofbase-soluble proteins (9)and/or the total
organic carbon content,usingaBeckman 915A total organic carbon analyzer.Cell
suspensions were homogenizedbysonication before T0Canalysis.
2.2.6.
Analysis
of the culture
supernatants
Theamino acidandammonia contentsofculture supernatants were determined
by usingaBiotronik LC6000E amino acid analyzer.The Tween 80-oleate contentof
theculture supernatants was determined according tothemethodofSlijkhuis
(tobepublished).Toaproper amountofculture supernatant NaOH wasaddedto
a final concentrationof1N.The suspensionwasheatedfor10mininaboiling
water bathtohydrolyzethe ester bonds betweenthesorbitol-andthe oleateresiduesoftheTween80molecules.Thesuspension was cooledtoroom temperature
and acidifiedbyadding H ? S0.toafinal concentrationof2N.TheE, ? n ofthe
suspension isproportional tothecontentoffree oleateupto150mg/1.
2.2.4.
Examination
of the
organism
Duringandafter the experimentsthe strainweused was examinedforknown
Frankia-characteristics inthreeways: (1)Macroscopically:the organism grows
26
inflocks,leavingtheculturemedium clear (1).(2)Microscopically:the
organism forms typical sporangia (1,10). (3)Byinfectivity tests:after
inoculating A. glutinosa seedlings withthecultures usedintheexperiments
mentioned inChapter 3,nitrogen-fixing nodules were formed (quantitativedata
tobepublished elsewhere).Sothere seems little doubt thattheorganismhas
maintained its Frankia characteristics duringtheexperiments.Thecultures
were testedforcontamination inthreeways: (1)Macroscopically (acontaminant
will usually cause turbidityofthemedium). (2)Microscopically. (3)Byplating
onyeast-glucose agar.
3.RESULTS
2.1. Course of growth of Frankia Avail
Erlenmeyerflasks containing 40mlQMOD/Tween medium were inoculated withlOyg
(TOC)cells.Thecells were cultivatedat25°C eitheraerobicallyoranaerobically.Atdifferent periodsofgrowth,theyield in4flaskswasdetermined
(Fig.1).
Growth wasobservedtocontinuefor12days.Aerobic conditions were required
forgrowth.
150
£
o
73"
o
i—
o>
5.100
•o
50
8
10
Fig.1.Growth curveofFrankia AvcH onQMOD/Tween medium
o-o Aerobic,x-xanaerobic cultivation.Allvalues shown
aretheaveragesof4determinations.
12
K
Days
27
3.2. Comparison of growth yield
on various
media
Fiftyyg(TOC) Frankia Ave11 cells were usedtoinoculate Erlenmeyer flasks
containing 40mlofthe following media: Frankia broth,QMOD,QMOD/Tween,
Tween/cas (concentration ofboth Tween80andCasamino acids 1g/1), Tween/glu
and Tween/NH».
After cultivation thegrowth yield (proteinandTOC content)ofthe cellswas
determined. Since after 12daysofcultivation nofurther growth was observed
(byTOCanalysisofthe QMOD/Tween medium,visuallyoftheother media),the
differences inyield after 14daysofcultivation have not beenduetodifferences ingrowing timeinsome media.
Intheculture supernatantofcells cultivated onTween/cas,Tween/gluand
Tween/NH.media,theamountofoleate utilized during growth was determined.In
the culture supernatantofcells grownonTween/gluandTween/NH,media, the
amountofglutamic acidorNH. remaining after growth was measured.Theresults
are showninTable 1.
'6.3. Growth of Frankia Avail on Tween/cas medium with varying concentrations
Tween 80 and Casamino
acids
Erlenmeyer flasks containing40mlTween/cas medium with varying concentrationsofTween80andCasamino acids were inoculated with 100yg(TOC) cells.
After cultivation for14daysat25°Cthetotal organic carbon contentofthe
cells was measured. Intheculture supernatants theamountsofoleate,glutamic
acidandaspartic acid utilized during growth were determined.Theresultsare
shown inTable2.
The amino acid contentofthemedia beforeandafter growth was determined.
The resultsareshown inTable3for growthonamedium containing 0.2gTween
80/1and0.2gCasamino acids/1.Similar results were obtained with other
combinationsofTween80andCasamino acids (not shown).
3.4. Carbon sources not used by Frankia Avail
We have not observedanygrowthofFrankia AvcI1 onmedia containingthe
same components (except Tween 80)astheTween/cas medium with 1g/1Casamino
acids,after additionofoneofthe following compoundsasC-source: glucose
(10g/1or1g/1), malate(1g/1),succinate (1g/1),acetate (1g/1),sorbitol
(1 g/1),polyethylene glycol (1g/1)orK-oleate(1 g/1).
28
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29
Table 2.Growth of Frankia Ave11onTween/casmedium withvarying concentrations
ofTween 80and Casamino acids^)
Concentration inmed
urn
Tween 80 2 )
(mg/1)
Casamino aci ds3>
(mg/1)
0
0
0
200
200
200
1000
1000
1000
0
200
1000
0
200
1000
0
200
1000
Yield
TOC
(mg/1)
Oleate
utilized
(mg/1)
Glutamic acid
utilized
(mg/1)
Asparticacid
utilized
(mg/1)
3.3
5.5
4.8
7.5
17.0
21.8
9.0
21.0
42.5
ND
ND
ND
40
120
140
60
125
175
ND
1.9
11.0
ND
29
64
ND
74
ND
1.0
4.5
ND
11
28
ND
36
All values shown are theaverages of 3determinations.
2)
Tween 80contains 0,21 goleate/g Tween80.
3)'Casaminoacids contain 152mg glu/g Casaminoacids and 61 mg asp/g Casamino
acids.
ND,not detected, -,not determined.
Fordetails seetext.
Table 3.Amino acid contentofTween/cas medium (0.2gTween 80/1 and 0.2g
Casamino acids/1)beforeand after growth for 14days of Frankia AvcI1
Amino acid
Glu
Lys
Leu
Asp
Val
Ser
Ala
Thr
He
Gly
Arg
Phe
His
Met
Tyr
Sum
30
Concentration before
growth (nmol/ml)
200
98
95
89
84
80
74
59
52
47
36
35
25
16
6
996
Concentration after
growth (nmol/ml)
3
102
91
5
91
77
72
59
51
44
36
35
26
21
6
719
DISCUSSION
From the growth curve presented in section 3.1 itcan be concluded that the
ndophyte grows on QMOD/Tween medium with a doubling time of about 2days,which
s slow,even for an actinomycete. Itmust be pointed out,however,that growth
f the endophyte in the alder nodule is slow as well (van Ham, personal communiation), so that it is likely that the slow growth isa feature of the organism
tself,rather than that it is due toa suboptimal medium.
Until now,growth yield of the isolatedmicrosymbiont of actinomycetenfected nodules has only been measured indirectly, i.e.
visually (1-5,7,8) or
ased on the infective capacity of endophyte suspensions (6,10). The experiments
eported in the present paper give a direct measurement of the growth yield,
ased on the total organic carbon yield of the endophyte. Using this method one
an compare the growth on various media quantitatively.
Growth of the endophyte has until nowonly been brought about on poorly der
ined media,-i.e.media containing yeast extract or peptone.The growth yield re-
ported hereon awell-defined medium like the Tween/cas medium or the Tween/NH.
nedium isnot outdistanced by the growth yield on a poorly-defined medium like
Frankia broth or QMOD (section3.2).
The poor growth on the Tween/glu medium is due to nitrogen limitation,as can
>eeasily concluded from comparing theyield on Tween/glu and Tween/cas media
sections 3.2 and 3.2).Itmust be pointed out that after this nitrogen-limited
jrowth the ratio of the protein yield to the TOC yield is low compared to
this ratio after growth on othermedia (Table1 ) .
From the results presented here itcan be concluded that free-living
Frankia
AvcI1 can use NH.as well as glutamic acid as sole nitrogen source.The uptake of NH.by Frankia AvcI1,growing on the Tween/NH^medium was confirmed in
inadditional experiment (to be published), inwhich ^5|sj_enr-jchmentof the
15
:ellswas observed after cultivation ina medium containing N-NH.C1.
During growth on the Tween/cas medium (section 3.3) the endophyte uses not
only oleate,but glutamic acid and aspartic acid as well.The possibility cannot
be ruled out that these amino acids are used as carbon source,although this is
ess likely since Frankia AvcI1 is able to grow on amedium containing Tween 80
as sole carbon source and NH.as sole nitrogen source.Comparison of theyield
on this medium with theyield on the Tween/cas medium shows that theyield in
both media is proportional to the quantity of oleate utilized during growth
(Table1 ) .
31
Itcan.beconcludedfrom Table3that Frankia Ave11 growing on the Tween/ca-'
medium,of all the available amino acids,utilizes only glutamic acid and
aspartic acid, leaving the concentrations of the other detectable amino acids
unchanged.
Since it is clear from the experiments reported here that Frankia Avcll
isable to grow on a medium containing Tween 80 as sole carbon source,the
question arises,which part of the Tween 80 molecule is degraded by this
organism. Although there isa significant drop in the amount of oleate residues in themedium during growth,it cannot be excluded that other parts of
themolecule {i.e.
the polyethyleneglycol or the sorbitan residues)are de-
graded as well. This is not likely,however,sincewe have not been able to
observe any growth on media containing polyethyleneglycol or sorbitan as sole
carbon source (section 3.4).The failure to observe any growth on a medium
containing K-oleate as sole carbon source (section 3.4) might be due toa
poisoning effect of free oleate (to be published elsewhere).
It remains an open question what is the carbon source for the endophyte
growing in the nodule. Based on the results presented in this paper it seems
opportuneto look for this compound in theclass of lipids.
ACKNOWLEDGEMENTS
The skillful assistance of Lida van den Eykel,Reinier van Ham and Karin
van Straten is gratefully acknowledged. These investigations were supported by
the Foundation for Fundamental Biological Research (BION),which is subsidized
by the Netherlands Organization for the Advancement of Pure Research (ZWO).
REFERENCES
1. Baker,D. and Torrey,J.G. (1979) In Symbiotic Nitrogen Fixation in the
Management of Temperate Forests (J.C. Gordon,C.T. Wheeler and D.A. Perry,
Eds )p. 38,Forest Research Laboratory, Oregon State University, Corvallis
OR 97331.
2. Callaham,D.,.Torrey, J.G. and del Tredici,P. (1978) Science 199,899-902.
3. Lalonde,M. (1978)Can. J. Botany 56,2621-2635.
4. Berry,A. and Torrey,J.G. (1979) In Symbiotic Nitrogen Fixation in the
Management of Temperate Forests (J.C.Gordon,C.T. Wheeler and D.A. Perry,
Eds )p.69,Forest Research Laboratory, Oregon State University,Corvallis
OR97331.
5. Baker,D.,Torrey,J.G. and Kidd,G.H. (1979)Nature 281,76-78.
6. Quispel,A. and Tak,T. (1978)New Phytol. 81,587-600
32
7.Lechevalier,M.P.and Lechevalier,H.A. (1979) In Symbiotic Nitrogen Fixation
in theManagement ofTemperate Forests (J.C.Gordon,C.T.Wheelerand D.A.
Perry,Eds )p. 111,Forest Research Laboratory,Oregon State University,
Corvallis OR 97331.
8. Lalonde,M.and Calvert,H,E. (1979) InSymbiotic Nitrogen Fixation inthe
Managementof Temperate Forests (J.C.Gordon,C.T.Wheeler and D.A. Perry,
Eds)p. 95,Forest Research Laboratory,Oregon State University,Corvallis
OR 97331.
9. Demoss,R.D.and Bard,R.C. (1957)Physiological and Biochemical Technics.
InManual ofmicrobiological methods.The Society ofAmerican Bacteriologists
(Ed.)p. 169,McGraw-Hill New York.
0.Quispel,A. (1979) In Symbiotic Nitrogen Fixation in the Management of
Temperate Forests (J.C.Gordon,C.T.Wheeler and D.A. Perry,Eds)p.57,
Forest Research Laboratory,Oregon State University,Corvallis OR 97331.
33
IAPTER 11.2
riLIZATION OF FATTY ACIDS AND NH" BY FRANKIA AVCI1
IMS Microbiology Letters 10 (1981) 143-145
in Blom
iboratory of Microbiology, A g r i c u l t u r a l U n i v e r s i t y ,
sselink van Suchtelenweg 4 , 6703 CT Wageningen,
he Netherlands
35
. INTRODUCTION
Inaprevious communication (1),adescription was givenofthe in vitro
ultivationofthenodule endophyte {Frankia AvcI1)of AInus viridis spp. crispa
nawell-defined medium. Itwas shown that Frankia Ave11isabletogrowona
edium containing Tween80assole carbon sourceandeither Casaminoacids,
lutamic acidorNH.C1assole nitrogen source.Itwas notclear,however,which
artoftheTween80molecule was utilized byFrankia AvcI1.Inthepresentpaper
tisshown that Frankia AvcI1canuse other Tweensandfatty acids.Theuptake
15 +
y thecellsof NH.from themediumisdemonstrated,andthe dependenceofthe
rowth yieldonthe concentration ofNH.C1 inthemediumisshown.
MATERIALSANDMETHODS
The Frankia strain AvcI1,was obtained fromD.BakerandJ.G. Torrey (Harvard
Iniversity,Petersham,MA01366). The organism was grown inamedium containing
:arbonandnitrogen sourcesasindicated,andsalts,trace elementsandvitamins
s described earlier(1).
Inoculationofthemedia,cultivation oftheorganism,washingandcollecting
)fthecellsanddetermination ofthe total organic carbonandprotein contents
<eredescribed previously(1).
15
N-enrichment ofthe cellswas determined according toAkkermans(2);
:alculations were performed according toFerrarisandProksch (3).Fordetermilationofthe total-nitrogen content thecells were digested according tothe
Cjeldahl method,followed bydistillation ofthe NH. formedanddetermination
Dfammonia with Nessler's reagent.
3. RESULTS
3.1.
Comparison of growth yield
carbon
of Frankia Avail on media containing
various
sources
Fifty yg (TOC) Frankia AvcI1 cells were usedtoinoculate Erlenmeyer flasks
containing40mlofculture medium with various carbon sourcesand NH.C1
(60mg/1)asnitrogen source.After20daysofcultivation theTOCandprotein
contentsofthecells were determined (Table 1).Nomore growth was observed
after cultivation formore than20days (not shown).
37
Table1.Growth yieldofFrankia cells after cultivation for20daysonmedia
containing various carbon sources 1)
C source
mg/1
Yield
TOC (mg/1)
Na-acetate
Na-propionate
Na-butyrate
Valeric acid
Caproic acid
Caprylic acid
Capric acid
Palmitic acid
Stearic acid
Oleic acid
Tween 203>
C
2
3
C
4
C
C5
C
c 68
c
p10
p16
p18
L
18-9
Tween 403N
Tween 60J,
Tween 80J
Tween 85
200
200
200
200
200
200
200
200
200
200
5.1
12.5
8.8
3.0
4.9
13.1
7.5
- 6.1
- 16.2
- 9.5
- 4.2
- 5.9
- 14.4
- 9.0
+ 2)
0
1000
1000
1000
1000
1000
2.9
14.0
7.6
10.3
11.6
Proteir i (mg/1)
2.3
7.0
3.9
1.0
2.6
9.5
4.9
- 2.8
- 9.9
- 5.6
- 2.1
- 3.0
- 10.5
- 5.9
+ 2)
0
- 4.0
- 15.9
- 8.8
- 11.9
- 12.6
1.5
10.8
5.0
5.8
9.9
- 2.3
- 12.9
- 5.9
- 6.6
- 10.6
1)
Values shownaretheextremesof4determinations
2)
+:Growth observed visually.Seediscussion
3)
Tween 20,40,60and80are thepolyethyleneglycol sorbitan estersoflaunc,
palmitic,stearicandoleic acids,respectively.Tween85consistsof3oleic
acid residues esterified with polyethyleneglycol sorbitan.
NH.C1 (60mg/1)wasusedasnitrogen source.
3.2. Carbon sources not utilized
by Frankia Avail
NogrowthofFrankia AvcI1wasobservedonmedia containing NH.C1(60mg/1)
asnitrogen source,andthe following compoundsascarbon source:methyl-oleate
(200 mg/1), tributyrin (500mg/1), tricaprin (500mg/1), triundecylin (500mg/1),
tritridecylin (500mg/1), gallic acid (250mg/1), protocatechuic acid (250mg/1),
vanillic acid (250mg/1), cholesterol (1000mg/1)andtannin (1000mg/1).
3.3. Uptake of
15
N-enriched
NH+
Frankia Avail
Three Erlenmeyer flasks containing 250mlofculturemedium with Tween80
1^
(1000mg/1)andNH.C1 (10mg/1, N-enrichment51atom- %excess)were inoculated withasuspensionofFrankia AvcI1 cells (50pgTOC,7ug total nitrogen).
After cultivationfor3weeks thecells werecollectedandwashed5timeswith
phosphate buffer (50mM K-phosphate;pH7.0).Thetotal organic carbon content,
15
the total nitrogen content,andthe N-enrichmentofthecellswere determined
38
Table 2 ) .Nomore growthwasobserved after cultivation formore than 3weeks
not shown).
_
15
'able2.Uptakeof N-NH.by Frankia cells during growth for3weeks
loofbatch
Yield (TOC)
(mg/1)
Total Ncontent
Atom-% Nexcess
ofcells(mg/l)
5.8
7.0
6.2
0.480
0.680
0.630
35.1
38.9
36.3
Tween80(1g/1)wasusedascarbon source
3.4. Comparison of growth yield
of Frankia Avail on media varying in NH CI
loncentration
Fifty yg (TOC)Frankia AvcI1 cells were used toinoculate Erlenmeyer flasks
:ontaining 40mlofculture medium with Tween 80(1000mg/1)andNH.C1 (varying
concentrations).After20daysofcultivation thecells were collectedand
washed,andthetotal organic carbon andthetotal nitrogen contents ofthe
cells were determined (Table 3 ) .ThepHofthemedia didnotdecrease below6.5
during growth.Nomore growthwasobserved after cultivation formore than
20days (notshown).
Table 3.Dependence ofyield of Frankia ontheNH.concentration ofthe
culture medium after cultivation for20days 1)
NH4CI concentration
ofculture medium
(mg/1)
0
10
20
50
100
200
500
1000
Yield
C/Nratioofcells
TOC
(mg/1)
5.3
5.3
6.8
6.8
9.6
9.6
10.6
10.6
15.6
15.6
18.1
18.1
13.4
10.9
Total N
(mg/1)
0.6
0.6
0.70.7
1.11.1
1.71.7
2.32.3
2.72.7
2.3
1.4
9
10
9
6
7
7
1)
The values shown aretheaverages of4 determinations
Tween 80(1g/1)wasusedascarbon source.
39
4. DISCUSSION
From the data shown in Table 1, itcan beconcluded that Frankia can useanumberoffreefatty acids and someTweens as solecarbon source.This conclusion is
inagreement with theassumption (1)that Frankia can use theoleate residues of
Tween 80molecules as sole carbon source.Obviously,valeric acid and Tween 20
arevery poorcarbon sources,whereas Na-propionate,caproic acid and Tween 40
are relatively good carbon sources.The differences inyield on thesemedia
indicate differences intransport and/ormetabolism of the carbon sources.
Inall media,theTOCyield of the cells issmall compared to theamount of
carbon offered in themedium. This indicates that some othercomponent of the
medium ispresent in limiting concentration.
Theyield onmedia containing palmitic acid and stearic acid was notdetermined quantitatively because of the poor solubility of these compounds.However,
growth could beobserved visually. No growth was observed onmedia containing
oleic acid.Thismight be due toa poisoning effect of free oleate(1).
By comparing Table 1with Chapter 3.2 itappears that Frankia can use simple
fatty acids esterified with polyoxyethylene sorbitan as sole carbon source,but
no complex organic acids (like gallic acid,protocatechuic acid and vanillic
acid)orfatty acids esterified with glycerol (like tributyrin,tricaprin,
triundecylin and tritridecylin).
It isnot clearwhich carbon source Frankia, when living in thenodule,
obtains from the plant. Ifthe requirements oftheendophyte are comparable
to thoseof Frankia in pure culture,this carbon sourcemight bea fatty acid,
either free or esterified.
From thedata shown inTable 2itcan beconcluded that NH. istaken upby
Frankia cells.That the enrichment of the nitrogen in thecells does not reach
51%,can in part beascribed to dilution of the label by the nitrogen of the
inoculum (28pg N/1)and by the nitrogen in the vitamins of themedium (700ug
vitamins/1,approx. 12% nitrogen).
From the data shown inTable 3, itcan beconcluded that theyield of Frankia
isdependent on the concentration of the NH.C1 in themedium. There isan
optimum concentration between 100and 500mg NH.C1/1. Ifthe NH.C1 concentration
exceeds this optimum,theyield decreases,probably due to some toxic effect of
excess NH..The C/N ratio of the cells seems to be little affected by the C/N
ratio of the medium.
40
Theamount ofNHL taken up is small compared to theamount of NH.offered in
themedium. This suggests that some other component of themedium ispresent
in limiting concentration.The nature ofthis component remains tobe clarified.
ACKNOWLEDGEMENT
These investigations were supported by the Foundation for Fundamental
Biological Research (BION),which is subsidized by theNetherlands Organization
for theAdvancement of Pure Research (ZWO).
REFERENCES
Blom,J., Roelofsen,W.and Akkermans,A.D.L. (1980) FEMSMicrobiol.Letters
9,131-135.
Akkermans,A.D.L. (1971)Ph.D.thesis,University of Leiden,TheNetherlands.
Ferraris,M.M.and Proksch,G. (1972)Anal.Chim.Acta 59,177-185.
41
CHAPTER 11.3
METABOLIC PATHWAYS FOR GLUCONEOGENESISAND ENERGY GENERATION IN FMNKIA AVCI1
FEMS Microbiology Letters 11 (1981)221-224
Jan Blom and Reint Harkink
Laboratory ofMicrobiology,Agricultural University,
Hesselink van Suchtelenweg 4,6703CTWageningen,
The Netherlands
43
INTRODUCTION
In previouscommunications (1,2),adescription was given of the cultivation
Dndefined media of Frankia Avcll,an actinomycetegiving rise totheformation
ofnitrogen-fixing nodules on roots of Alnus viridis spp. crispa. Itwas shown
that Frankia Avcll can usesome freefatty acidsand the fattyacid residues of
someTweens as solesourceofcarbon and energy,and N.H.C1as sole sourceof
nitrogen (1,2). Nogrowth was observed onmedia containing glucose as sole
carbon source (1).However,it isnot excluded that in theQMOD/Tween medium,
which contains both glucose (10g/1)and Tween 80 (1 g/1), Frankia does utilize
glucose. Inthe present investigation the uptake ofglucose from amediumcontaining Tween 80and glucose hasbeen studied.
Noquantitative information isyet availableabout themetabolic pathways for
biosynthesis and energy generation used by Frankia when growing in pure culture.
Inthis paper,studies ofthemetabolism of Frankia on theenzyme level will be
reported,
MATERIALSAND METHODS
The Frankia strainAvcll,was obtained from D.Bakerand J.G. Torrey (Harvard
University,Petersham MA 01366). Theorganism was cultivated for 10dayson
eitherQMOD/Tween orTween/NH^ (60mg NH4C1/1 medium)at 25°C (1).The Arthrobaoter simplex strainAC4was obtained from J. Antheunisse (this laboratory)
and grown onyeast/glucose medium,containing 7gyeast extractand 10g
14-
glucose/1. [ Cj-glucose (uniformly labelled,336mCi/mmole)was purchased from
the Radiochemical Centre,Amersham,England.Cell-freeextracts ofFrankia cells
were prepared bywashing cells 3times with an appropriate buffer,followed by
sonication for 2min (Branson sonifier B-12),and centrifugation for 10min at
10,000xgto removethecell debris.Inthecell-free extractsactivities of
thefollowing enzymeswereassayed: isocitrate lyase EC4.1.3.1 (3); malate
synthase EC4.1.3.2 (3);malic enzyme EC 1.1.1.40 (4);pyruvate orthophosphate
dikinase EC2.7.9.1 (5);phosphoglycerate kinase EC2.7.2.3 (6);3-phosphoglyceraldehydedehydrogenase EC 1.2.1.12 (7);isocitrate dehydrogenase
EC 1.1.1.42 (8);a-ketoglutaratedehydrogenase analogous toother dehydrogenase
assays (9)modified asfollows: Theassay mixturecontained 150ymolesK-phosphate pH 7.1,0.9 umoles phenazinemetasulphate, 15ymoles MTT (3-(4,5-dimethyl
thiazolyl-2)-2s4-diphenyltetrazolium bromide),0.9ymolesNAD,0.5 ymoles thiamine
45
pyrophosphate,10ymoles MgCl ? ,150ymoles a-ketoglutarate,cell-free extract
and water toafinal volumeof3ml. After incubation,theformazan formedwas
precipitated bycentrigugation (5min 3000xg)anddissolved in3mldimethylformamide.Theextinction at550nmofthis solution isproportional tothe
-4 -1 -1
amountofformazan formed (f.5501.6x10 M .cm ) .Succinyl-CoA synthetase
EC6.2.1.5 (modified from Cha, 10).Theassay mixture contained 25ymolesTris
HCl,pH 8.0,12.5ymoles MgCl 9 ,1ymole CoASH,4ymoles ATP,50ymoles K-succinate,cell-free extractandwatertoafinal volumeof1ml.After incubation
thefreeCoASHwasdetermined according toEllman (11).Succinate dehydrogenase
EC 1.3.99.1 (ref.12,spectrophotometric adaptation with DCPIP);fumarase
EC4.2.1.2 (13);malate dehydrogenase EC1.1.1.37 (14).Protein contentof
cell-free extractswasdetermined according toTsuyoshi Ohnishi andBarr(15).
RESULTS
Comparison of glucose uptake by Frankia Avail and Arthrobactersimplex.Fifty
mg (total organic carbon)ofcellsof Frankia Ave11 grown onQMOD/Tween medium
and 10mg(T0C)of cellsof Arthrobacter
+
simplex AC4were incubated separately
-14-
in 10mlTween/NH.medium towhich |_ CJ-glucosewasadded (final concentration
of glucose 1mM). After incubation for6hthesuspensions were centrifuged
for20minat10,000xg,andtheradioactivity inthesupernatants wasdetermined (Table1 ) .
Table 1.Comparison ofglucose uptakeby Frankia Ave11cellsand
simplex cells
Organism
Label added to
incubation mixture
(cpm)1)
Frankia Ave11
Arthrobacter
simplex
strainAC4
1,66x10 6
Arthrobacter
Label recovered insupernatant
after incubation for6h
(cpm)')
1,65x10 6
c
1,33x10°
c
0,08x 10 D
1)
Values denoted aretheaveragesof5determinations which didnotdeviate
more than1%.
Activities
of biosynthetic
enzymes in cell-free
extracts
of Frankia
Avail.
Cell-free extracts weremadeof Frankia cells grown onQMOD/Tween andTween/NHt
media,respectively. These extracts,adjusted to1.0mgprotein/ml,were tested
fortheactivities ofisocitrate lyase,malate synthase,malic enzyme,pyruvate
46
rthophosphate dikinase, phosphoglycerate kinase and3-phosphoglyceraldehyde
ehydrogenase (Table2 )
able 2.Activities ofenzymes involved ingluconeogenesis of Frankia A v c l l ,
issayed incell-free extracts ofthis organism, grown onQMOD/Tweenand
ween/NH^ media')
QMOD/Tween
(containing both
glucose and Tween 80)
Growth medium
.nzyme
isocitrate lyase
lalate synthase
l a l i c enzyme
'yruvate orthophosphate dikinase
'hosphoglycerate kinase
i-Phosphoglyceraldehyde
lehydrogenase
Tween/NH^
(containing Tween80
as sole C-source)
70
70
250
10
80
100
45
250
15
50
50
30
I)
-1
-1
'Values, expressed asnmoles.min .mg protein ,areaverages ofatleast
3 determinations.
Activities
ivall.
of citric
acid cycle enzymesin cell-free
Cell-free extracts were made ofFrankia
extracts
of Frankia
Ave11 cells grown onQMOD/Tween
and Tween/NH. media. These cell-free extracts, adjusted to1.0mgprotein/ml,
were tested forthe activities ofcitric acid cycle enzymes (Table3 ) .
Table 3.Activities ofcitric acid cycle enzymes incell-free extractsof
Frankia Avcll grown onQMOD/Tween andTween/NHt media1)
Growth medium
Enzyme
QMOD/Tween
(containing both
glucose andTween
Isocitrate dehydrogenase
-Ketoglutarate dehydrogenase
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumarase
Malate dehydrogenase
195
20
1
20
700
2500
1)
-1
.mg protein
Values, expressed as nmoles.min
3 determinations.
Tween/NH^
(containing Tween80
80) as sole C-source)
210
15
1
20
650
3000
-1
, are averages of at least
47
DISCUSSION
Frontthedata shown inTable 1it isclear thatglucose uptake by Frankia
cells is less than thedetection level oftheassay,while theglucose uptake
by Arthrobaater cells demonstrates that this isnotduetoatechnical failure.
Ifthedetection level of thecounting is 1%,and thedoubling time of Frankia
Ave11 is48hours (1),itappears that Frankia Ave11 forms less than 0.1%of
itscell material from glucose. Itmay thusbeconcluded that theuptakeof
glucosefrom theQMOD/Tween medium by Frankia Ave11can beneglected.
Inagreementwith thisconclusion,noactivity could bedetected of the
glycolytic enzymes hexokinase,pyruvate kinaseand pyruvate dehydrogenase in
cell-freeextracts of Frankia cells grown onQMOD/Tween medium (not shown).
Furthermore,itwasfound that theactivities of isocitrate lyaseandmalate
synthase,being enzymes involved ingluconeogenesis,are ofthe sameorderof
magnitude incells grown onQMOD/Tween medium as incells grown onTween/NH.
medium (Table 2). Inmost organisms studied sofar,theexpression of isocitrate
lyaseand malate synthase is inhibited by thepresence ofglucose inthegrowth
medium (16,17). Itisnot surprising that this effectdid not occur in Frankia
(Table 2)sincenoglucose uptakeby Frankia was found (Table1).
A similar preference ofafattyacid overglucosewas shown to occur inthe
fungus Aspergillus
nidulans (18).This organism selectively usesacetatewhen
growing onamedium containing both this fattyacid and glucose.When A. nidulans
was grown onamedium containing acetateas solecarbon source,the isocitrate
lyaseactivity ofthefunguswas reported to beequal tothatofcells grown on
a medium containing both acetate and glucose (19).Mostofthe known bacteria
ofthegenus Aainetobaater alsocan utilize fatty acids,butnotglucose(20).
Organisms growing on fatty acids,and degrading these compounds toacetyl-CoA,
startgluconeogenesiswith theaction oftheglyoxylatecycle (21).Frankia AvcI1
contains the two key enzymes ofthis cycle, i.e. isocitrate lyaseandmalate
synthase (Table 2). The succinate formed in theglyoxylate cyclecan beconverted
to pyruvate by the subsequentaction of succinate dehydrogenase,fumaraseand
malic enzyme.Pyruvate can beconverted to phosphoenolpyruvateby theaction of
pyruvateorthophosphate dikinase.Theenzymesphosphoglycerate kinaseand
3-phosphoglyceraldehyde dehydrogenase are thought to haveabiosynthetic function,
sinceglucose isnot taken upby Frankia (Table1).
For energy generation, Frankia can oxidizeacetyl-CoA,formed from fatty
acids,inthecitric acid cycle (Table 3 ) .Oftheenzymes ofthis cycleonly
48
citrate synthaseandaconitase havenot been determinedincell-free extracts
of Frankia, but thesetwoenzymesareassumedtobefunctioning inanorganism
containing isocitrate lyaseandisocitrate dehydrogenase.Theactivityof
succinyl-CoA synthetaseislowascomparedtotheactivityoftheother citric
acid cycleenzymes.
Themetabolic activitiesofFrankia spp.growing inpureculture are likely
todiffer from theactivitiesofFrankia spp.occurringasvesicle clusters
inrootnodulesofalder.Intheformer case,much biosynthetic activityis
required, whereasinthelattercase thereisalmostnogrowth,butenergy
generation isrequired,among others,fornitrogen fixation.Inaccordance
with this assumption,Akkermans et at. detected activityofcitric acid cycle
enzymesintheendophyte fraction [i.e. thevesicle clusters)ofalder root
nodules (22),butnoactivityofglyoxylatecycle enzymes(23).
Thenatureofthecarbon source(s)thattheendophyte obtains fromthe
plant still needstobeclarified. Basedontheresults presented inthis paper,
itisunlikely that glucoseisoneofthese carbon sources.
ACKNOWLEDGEMENT
These investigations were supportedbytheFoundationforFundamental
Biological Research (BION),which issubsidizedbytheNetherlands OrganizationfortheAdvancementofPure Research (ZWO).
REFERENCES
Blom, J . , Roelofsen, W. and Akkermans, A.D.L. (1980). FEMS Microbiol. L e t t .
9, 131-135.
Blom,J.(1981). FEMSMicrobiol.Lett. 10,143-146.
Dixon,G.H.andKornberg,H.L. (1959). Biochem. Journ.72,3p.
Ochoa,S.(1955). InMethodsinEnzymologyI,739-753.Colowick, S.P.,
Kaplan,N.O.Eds.Academic Press,New York.
5.South,D.J.andReeves,R.E. (1975). InMethodsinEnzymology XLII,187-192.
W.A. Wood ed. Academic Press,NewYork.
6. Blicher,T.(1955). InMethodsinEnzymology I,415-422.Colowick, S.P.,
Kaplan,N.O.eds.Academic Press,NewYork.
Velick,S.F. (1955). InMethodsinEnzymology I,401-406.Colowick, S.P.,
Kaplan,N.O.eds.Academic Press,NewYork.
Ochoa,S.(1955). InMethodsinEnzymology I,699-704.Colowick,S.P.,
Kaplan,N.O.eds.Academic Press,New York.
Altman,F.P. (1972).Anintroductiontotheuseoftetrazolium saltsin
quantitative enzyme cytochemistry. Koch Light Laboratories Ltd.,Colmbrook,
Bucks.,England.
49
10. Cha,S. (1969). In Methods in Enzymology XIII,62-69.Lowenstein, J.M.ed.
Academic Press,New York and London.
11. Ellman,G.L. (1959). Arch. Biochem.Biophys. 82,70-77.
12. Veeger, C , Dervartanian, D.V. and Zeylemaker,W.P. (1969). In Methods in
Enzymology XIII,81-90.Lowenstein, J.M. ed.Academic Press,New York and
London.
13. Hill,R.L. and Bradshaw, R.A. (1969). In Methods in Enzymology XIII,91-99.
Lowenstein, J.M. ed. Academic Press,New York and London.
14. Ochoa,S. (1955). In Methods in Enzymology I,735-739.Colowick, S.P.,
Kaplan,N.O. eds.Academic Press,New York.
15.Tsuyoshi Ohnishi,S.and Barr,J.K. (1978).Analytical Biochem. 86, 193-200.
16. Beck,C. and Kaspar von Meyenburg,H. (1968). J. Bacterid. 96,479-486.
17. Witt, I.,Kronau,R. and Holzer,H. (1966). Biochem. Biophys.Acta 118,
522-537.
18. Romano,A.H. and Kornberg,H.L. (1969). Proc. Royal Soc.London Ser. B 173,
475-490.
19.Armitt,S., Roberts,C.F. and Kornberg,H.L. (1970) FEBS Lett. 7,231.234.
20. Baumann,P., Doudoroff,M. and Stanier, R.Y. (1968). J. Bacteriol. 95,
1520-1541.
21. Kornberg, H.L. and Krebs,H.A. (1957). Nature 179,988-991.
22.Akkermans,A.D.i .Huss-Danell,K.and Roelofsen, W. (1981). Physiologia
Plantarum, in press.
23. Roelofsen, W.,Huss-Danell, K.,Akkermans,A.D.L. and Meyer, P. Manuscript
submitted.
50
CHAPTER II.4
CARBON AND NITROGEN SOURCE REQUIREMENTS OF FRANKIA STRAINS
r
EMS Microbiology Letters 13 (1)January 1982 (in press)
Jan Blom
.aboratory of Microbiology,Agricultural University,
lesselink van Suchtelenweg 4,6703 CT Wageningen,
The Netherlands
51
INTRODUCTION
The isolationandin vitro cultivation ofa Frankia sp. viz. Frankia C P U ,an
ctinomycete that gives risetotheformationofnitrogen-fixing root nodules
in Comptonia peregrina wasreportedbyCallaham,TorreyanddelTrediciin
978 (1).Since that timeanumberofother Frankia strainshasbeen isolated
rom root nodulesofvarious non-leguminous plants, viz. Alnus glutinosa (2,3),
Inus rubra (4), Alnus viridis
ssp. arispa (5,6), Myriaa penssylvanioa (7),
Zlaeagnus umbellata (8,9), Casuarina equisetifolia
(10), Hippophaespp.(11)and
'hepherdia spp. (11).Some research hasbeen doneinthis laboratoryontheCnd N-metabolismof Frankia AvcI1,isolated from root nodulesofAlnus
viridis
sp. crispa (12,13,14).Itwasshown that Frankia AvcI1canutilizeanumberof
fatty acidsandthefatty acid residuesofsome Tweensassole carbon source
(13, 12,respectively).Nogrowthof Frankia AvcI1wasobservedonmedia containing glucoseassole carbon source,whiletheorganism doesnottakeup
glucose fromamedium containing both Tween80andglucose (12,14). In the
present contribution itwasinvestigated whether other Frankia strains behave
similarly toFrankia AvcI1 regarding their nutrional demands.
Further investigations concerning someoftheabove-mentioned Frankia strains
includetheabilityoftheseactinomycetestoform rootnoduleson Alnus
glutinosa.
Earlieritwasshown (12)that Frankia AvcI1 cannot utilize succinateas
solecarbon source.This phenomenon isduetothefact thatnouptakeof
this compound occursasshown inthepresent paper.Also someadditional work
has been doneonthenitrogen metabolismofFrankia AvcIL Inearlier papers
itwasshown that free-living Frankia AvcI1 canutilize either NH. (13)or
Casaminoacids (12)asN-source.Glutamic acidandaspartic acid were foundto
bepreferably takenupfromamixtureofamino acids,whereastheconcentrations
oftheotheramino acids remained unchanged duringthegrowthoftheorganism
(12). Some more detailsontheutilization ofamino acidsby Frankia AvcI1are
reported inthepresent work.
MATERIALSANDMETHODS
The Frankia strains AvcI1(isolated from root nodulesofAlnus viridis ssp.
arispa)(5,6) andCpI1 (isolated from root nodulesof Comptonia
peregrina)(1)
were obtained fromD.BakerandJ.G. Torrey (PetershamMA01366).The Frankia
strain AgSp+1 (isolated from spore(+)-type nodulesofAlnus glutinosa)(15) was
53
obtained from A.J.P. Burggraaf (Leiden,theNetherlands).Themediafor
cultivation ofthestrains contained C-andN-sources asindicated andthe
following basal nutrients (mg/1)K 2 HP0 4 ,300;NaH 2 P0 4 , 200;KC1,200;MgS0 4 .7H 2 0,
200; CaC0 3 , 100;Fe-citrate,10;ZnS0 4 , 1;riboflavin,0.1;ihiaminium dichloride, 0.1;biotin,0.1;nicotinic acid,0.1;pyridoxine HC1,0.1;Ca-pantothenate,
0.1;folic acid,0.1;H 3 B0 3 , 1.5;MnS0 4 .7H 2 0,0.8;CuS0 4 .7H 2 0,0.1;(NH 4 ) 6 Mo0 2 4 ,
0.2;CoS0 4 .7H„0,0.01.Thecells were grown andtheyieldwasdeterminedas
described earlier (12,13). Infectivity ofthestrains wastested bycountingthe
nodules formed ontherootsof Alnus
glutinosa
4weeks after inoculationby
immersing therootsfor1min ina Frankia culture (20mg T0Ccells/1). Roots
of control plants were immersed insterile water. Plants were grown inperlite
(Houwers,Roelofsen andAkkermans,inpreparation).
Succinateandacetate were determined gaschromatographicallyasdescribed
in (16)and(17),respectively. Cell-free extracts were prepared asreportedin
(10). Isocitrate lyase (ICL,EC4.1.3.1)andmalate synthase (MS,EC4.1.3.2)
activities were determined according to (18);isocitrate dehydrogenase
(IDH,EC1.1.1.42)according to (19),andmalate dehydrogenase (MDH,EC1.1.1.37)
according to(20).Theamino acid contentofthegrowth media beforeandafter
growthwasdetermined asdescribed previously(12).
RESULTS
Growth yield
of some Frankia strains
on media containing
various
C- and. N-sources
Media containing various C-andN-sources were inoculated with three
Frankia
strains.Theinoculation density was0.8mgTOCcells/1.After20daysof
cultivation thecells were harvested andtheTOCcontentofthecellswas
determined (Table 1).Nofurther growth wasobserved after cultivation formore
than 20days (notshown).
Infectivity
of various Frankia strains
onAlnusglutinosa
plants
The infectivity of Frankia AvcI1, Frankia CpI1and Frankia AgSp+1 on
Alnus
glutinosa
plantswastested byinoculating fifteen plants with each
Frankia
strain. Fourweeks after inoculation theroot nodules ontheplants were counted
(Table2 ) .
The effect
of acetate,
propionate
and succinate
on Frankia Avail
cells
Media containing either acetate (60mg/1), propionate (60mg/1), succinate
(50mg/1)oracetate plus succinateasC-sources andNhLCl (60mg/1)asN-source
54
Table 1.Growthyield of Frankia AvcI1, Frankia CpI1,and Frankia AgSp+1 after
20-day cultivation period onmedia with variousC-andN-sourcesU
;-source (mg/1)
N-source (mg/1 )
Yield (mgTOC/1)
Frankia Aveil
"ween80
Slucose
la-acetate
la-lactate
la-succinate
ithanol
'ween80
Tween80
Tween80
1000
200
200
200
200
200
1000
1000
1000
NH4C1
100 13.5-17.0
NH4C1
100 0.2- 0.9
NH4C1
100 4.0- 5.0
NH4C1
100 0.1- 0.5
NH4C1
100 0.2- 0.6
NH4C1
100 -0.3- 0.2
Casamino acids 100 13.5-15.0
aspartic acid 100 19.5-21.2
alanine
100 13.0-13.9
Frankia Cp11
Frankia AgSp+1
18.2- 21.2
0.2- 0.5
6.9- 7.2
0.0- 0.5
0.3- 0.6
-0.1 - 0.3
22.2- 25.0
21.2- 23.0
12.7-13.5
3.0-5.2
0.3-0.4
2.9 -3.2
0.2-0.4
0.1-0.7
0.2-0.4
3.9 -4.5
3.7-4.5
6.0-6.9
Values denoted aretheextremesof4determinations.
Table2.Infectivity of Frankia AvcI1, Frankia CpI1and Frankia AgSp+1
on Alnus glutivcca
plants grown inperlite
Numberofnodulesper15plants
Frankia Ave11
Frankia CpI1
Frankia Ag+1
Control
225
248
2
0
were inoculated with Frankia AvcI1 cells. Inoculation densitywas0.5mgTOC
cells/1.Aftercultivation for12days,cell yields were determined andacetate
and succinate assayed intheused media.Cell-free extractsofthecells were
usedformeasuring theactivitiesofICL,MS,IDHandMDH(Table3 ) .
Amino acids as nitrogen
source for Frankia Avail
Media containing Tween80(1 g/1)asC-source andvarious amino acidsas
N-sourcewere inoculated with Frankia AvcI1 cells. Inoculation densitywas1mg
TOC cells/1.Aftercultivation for20days cell yieldsandamino acid contents
oftheused media were determined (Table 4 ) .
55
Table 3.Comparison of Frankia AvcI1 cells grown with eitheracetate,
propionate,succinate oracetate plus succinate aftercultivation for 12days
C-sourceof
medium
(mg/1)
Yield1J
(mgTOC/1)
Succinate
Acetate
concentration 1) concentration 1)
of used medium
ofused medium
(mg/1)
(mg/1)
Enzyme activities
incell-free
extracts ,
(nmol.min
mg protein"^)
ICL MS IDHMDH
Acetate
60
3.0 - 3.9
Propionate
60
4.0 -5.0
Succinate
50
0
Acetate+
succinate
60+50
3.6 - 4.2
30-33
- 0.2
72
60 1702800
<5
<5 2302100
68
75 2102500
47 -50
49 -52
30 -32
1)Values denoted are theextremes of4determinations.
NH.C1 (60mg/1)was used asN-source.
Table 4.Cell yields of Frankia AvcI1 aftera20-day cultivation period on
media with Tween 80 (1g/1)asC-sourceand various aminoacids as N-source
N-source ofmedium
Alanine
Y-Amino-butyrate
Aspartic acid
Glycine
Glutamic acid
Leucine
Phenylalanine
Serine
Threonine
Tyrosine
Valine
1)
before growth 1) after growth '
Yield
7s
(mgTOC/1)^'
400
400
400
400
400
400
400
400
400
400
400
12.4 -13.4
8.9 - 9.4
22.3 -26.0
10.2 -10.6
22.9 -24.1
12.6 - 13.9
19.0 -20.3
9.0 -11.0
11.5 - 12.2
13.6 -14.5
10.0 - 11.2
Amino acids( Mmol/1)
260
300
100
325
100
320
230
340
300
340
330
1) Averages ofvalues of4samples.
2)Values denoted are the extremes of4determinations.
56
DISCUSSION
Fromthedata presented inTable 1itcanbeconcluded that neitheroneof
\he three Frankia strains tested isabletoutilize glucose,lactate,succinate
r ethanol assole carbon source.Theinability oftheAvcI1 strain andthe
pi1 strain toqrowwith glucoseassoleC-source isaconfirmation ofthe
esultsofBakerandTorrey (6)andBlomandHarkink (14).Allthree strains
:angrowonmedia containing either Tween 80oracetateasC-source andNH-,
lasaminoacids,aspartic acid oralanineasN-source.Apart fromthesimilarity
nC-andN-source requirement,asignificant difference ingrowth yield exists
etween theAgSp+1 strain ontheonehand,andtheAvcI1 andCpI1strains onthe
ther.Thereason forthis difference isnotclear. Sincenodata haveasyet
>een published ongrowthyieldsofother Frankia strains,itisnotpossible
todetermine whether ornotthestrains tested inthis study behave likenormal
?rankia strains.
Fromthedata shown inTable2,itisclear thattheAvcI1 andCpl1 strains
ireinfectiveon Alnus glutinosa, whereas no infectivity oftheAg+1strain
*asobserved. Thismightbeduetothefact that theplants were grownin
perlite,since Frankia AgSp+1 isable toinfect sterile A. glutinosa plants
grown inagar (15;A.J.P. Burggraaf,personal communication).Becausethethree
strains tested thus belong tothesame cross-inoculation group (5,9),the
marked similarity inC-andN-source requirement (Table 1)isnotsurprising.
The data given inTable3showthat thefailure ofthe Frankia strains
tested togrowwith succinateassolecarbon source isduetothefact thatno
uptakeofthis compound occurs.This isalso trueofmedia containing acetate
and succinate.Under such conditions,theglyoxylatecycle enzymes isocitrate
lyaseandmalate synthasearenotrepressed asitisthecase incells grown
with propionate (cfTable 3). Therepressing effect ofpropionate onthe
glyoxylate cycle enzymes suggests that in Frankia AvcI1 propionate ismetabolized
via succinateasitwasfound tobethecase in E. eoli (21).
The activities ofthecitric acid cycle enzymes isocitrate dehydrogenaseand
malate dehydrogenase seemnottobeaffected bythenatureofthecarbon sources
tested inthisexperiment.
Vesicle clusters of Frankia spp.as they occur innitrogen-fixing root nodules
of Alnus glutinosa, areable torespire succinate (AkkermansandRoelofsen,
personal communication').This suggests an interesting alteration ofthe
capacity totakeupsuccinate during thetransition of Frankia spp.fromthe
57
free-living to the symbiotic state. In this respect Frankia spp.show marked
differences with Rhizobiwn spp.,since the latter were shown to take up
succinate in both the free-living and the symbiotic (bacteroid) state(22).
Inan earlier study itwas shown (12)that Frankia AvcI1 selectively takes
upaspartic acid and glutamic acid from amixture ofamino acids,leaving
the concentrations of the other amino acids unchanged during growth.From the
data shown inTable 4 itcan beconcluded that Frankia AvcI1 can utilize other
amino acids as sole nitrogen source as well. The mechanism of selection of
aspartic acid and glutamic acid from amixture of amino acids isnotyet clear
Incell-free extracts of Frankia Ave11 grown with either NH* glutamate or
Casamino acids as N-source,activity was observed of glutamine synthetase and
glutamate-oxaloacetate transaminase,but not of glutamate dehydrogenase,
glutamate synthase,L-amino acid oxidase and branched-chain amino acid aminotransferase (Blom,unpublished results). The pathways of assimilation of
nitrogen in Frankia spp. still remain obscure.
ACKNOWLEDGEMENT
These investigations were supported by the Foundation for Fundamental
Biological Research (BION),which is subsidized by theNetherlands Organizatior
for theAdvancement of Pure Research (ZWO).
REFERENCES
1.Callaham,D.,Torrey,J.G. and del Tredici, P. (1978). Science 199,899-902.
2. Quispel,A. and Tak, .T. (1978). New Phytol. 81,587-600.
3. Quispel,A. (1979). In Symbiotic Nitrogen Fixation inthemanagement of
temperate forests (J.C. Gordon and C.T. Wheeler,Eds)pp. 57-68,Forest Researc
Laboratory,Oregon State University.
4. Berry,A. and Torrey,J.G. (1979). In Symbiotic Nitrogen Fixation in the
management of temperate forests (J.C.Gordon and C.T. Wheeler,Eds) pp.69-83
Forest Research Laboratory,Oregon State University.
5. Baker,D. and Torrey, J.G.(1979). In Symbiotic Nitrogen Fixation in the
management of temperate forests (J.C.Gordon and C.T.Wheeler,Eds)pp. 38-56,
Forest Research Laboratory,Oregon State University.
6. Baker,D. and Torrey,J.G. (1980). Can.J. Microbiol.26,1066-1071.
7. Lechevalier,M. and Lechevalier,H. (1979). InSymbiotic Nitrogen Fixation in
the management of temperate forests (J.C. Gordon and C.T. Wheeler,Eds)
pp. 111-122,Forest Research Laboratory,Oregon State University.
8. Baker,D. and Torrey,J.G.and Kidd,G.M. (1979). Nature 281,76-78.
9. Baker,D., Newcomb,W. and Torrey,J.G. (1980). Can.J. Microbiol.26,
1072-1089.
58
Gauthier,D., Diem. H.G. and Dommergues,Y. (1981). Appl. Env. Microbiol.41,
306-308.
Lalonde,M.,Calvert,H.E. and Pine,S. (1981). In Current Perspectives in
Nitrogen Fixation (A.H. Gibson and W.E. Newton, Eds) pp. 296-299,Australian
Academy of Science,Canberra.
2. Blom,J., Roelofsen,W. and Akkermans,A.D.L. (1980). FEMS Microbiol. Letters
9, 131-135.
3. Blom,J. (1981). FEMS Microbiol. Letters 10,143-146.
4. Blom,J. and Harkink, R. (1981). FEMS Microbiol. Letters 11,221-224.
5. Quispel,A. and Burggraaf,A.J.P.(1981).In Current Perspectives in Nitrogen
Fixation (A.H. Gibson and W.E.Newton, Eds) pp. 229-236, Australian
Academy of Science,Canberra.
Laanbroek, H.J., Kingma,W. and Veltkamp,H. (1977). FEMS Letters 1,99-102.
7. Adamse,A.D. (1980). Antonie van Leeuwenhoek 46,523-531.
Dixon,G.H. and Kornberg, H.L. (1959). Biochem. J. 72,3p.
9. Ochoa, S. (1955). In Methods in Enzymology I (S.P. Colowick and N.O. Kaplan,
Eds) pp.699-704,Academic Press New York.
0. Boehringer (1973). Biochemica information. Boehringer Mannheim GmbH, Mannheim.
1.Wegener,W.S.,Reeves,H.C., Rabin,R. and Ajl,S.J. (1968). Bact. Rev.
32, 1-36.
Glenn,A.R., Poole, P.S. and Hudman, J.F. (1980). J. Gen. Microbiol. 119,
267-271.
59
Chapter III
•ASSIMILATION OF AMMONIA IN ROOT NODULES
| 0 F ALNUSGLUTINOSA
61
JHAPTER III.1
"iSSIMILATION OF NITROGEN IN ROOT NODULES OF ALDER (ALNUS GLUTINOSA)
l'heNew Phytologist 89 (1981)321-326
Jan Blom,Wim Roelofsen and Antoon D.L. Akkermans
.aboratory of Microbiology,Agricultural University,
lesselink van Suchtelenweg 4,6703 CT Wageningen,
'heNetherlands
63
JUMMARY
Citrulline is shown to be the predominant freeamino acid inalder root
dules,while serine also occurs in relatively large amounts.Citrulline and
lutamic acid are prominent free amino acids inalderxylem tissue.Theactiviies of Np-ase,GS,GDH and OCT in root nodule homogenatesare reported. From
he Km values ofGDH forNHt (16mM)and glutamate (0.9mM)and theconcenrations in thenodule ofNH» (1.5mM)and glutamate (0.5mM) it is concluded
,hatGDH inalder nodules is responsible for the deamination of glutamate. The
mportant function of GDH inthe nitrogen metabolism ofalder nodules isconirmed by themuch higher activity of this enzyme in homogenates of nodules as
ompared to that in homogenates of root-tips and leaves.
The vesicle clusters,which contain the Ng-aseactivity,do not show activity
f GS,GDH and OCT.The cytoplasm of the host cells was shown to possess the GS
ctivity,while GDH and OCT probably are localized in the organelles of the
lostcells.
Ibbreviations:Cfraction,plant cytoplasm; Efraction,endophyte vesicle
:lusters;GDH,glutamate dehydrogenaseEC1.4.1.3;GOGAT,glutamine:a-ketolutarateaminotransferase (NAD(P)H-oxidizing) EC 2.6.1.53; GS,glutamine
synthetase EC 6.3.1.2; N^-ase.nitrogenase;OCT,ornithine carbamyl transferase
EC 2.1.3.3;0fraction,plant organelles;Pfraction,plant fraction;
TNfraction,total nodule homogenate.
INTRODUCTION
At least two pathways for assimilation ofammonia occur in nitrogen-fixing
organisms.The first,the reductive amination ofa-ketoglutarate,is catalyzed
byGDH.
a-ketoglutarate +NH* +NADHJ glutamate +NAD + +H 2 0
The second pathway includes the combined action of GSand GOGAT
glutamate +NH,+ATP•+glutamine +ADP + P.
+
glutamine +a-ketoglutarate +NAD(P)H +H + •+2 glutamate +NAD(P).
65
Recent studies (reviewed by Scott,1978)have demonstrated that the second
pathway functions inmany free-living nitrogen-fixing microorganisms and in
Rhizobium-~\egume symbioses,whereas the former ispredominant in the Nostoocontaining lichen ofthe genus Feltigera (Stewart and Rowel1,1977;Rai,Rowell and
Stewart, 1980). No information has been reported so far on the ammonia assimilation inactinomycete root nodule symbioses.
Ithas been shown (Miettienen and Virtanen, 1952;Leaf,Gardner and Bond,
1958; Wheeler and Bond, 1970)that the pool of free amino acids of^Z-nws-type
rootnodules differs from that of other types of root nodules. In rootnodulesof
Alnus glutinosa , citrulline is themost prominent free amino acid; itcontributed
about 20%(onmolar base , i.e. about 50%on N-base)tothe amino acid pool,
whereas asparagine was not detectable and glutamine contributed about10%.
Different types of non-legume rootnodules may possess significant quantities
of specific other amino acids, e.g. arginine and ornithine.
Incontrast,rootnodules ofmost leguminous plants contain glutamine and
asparagine as dominant amino acids (van Egeraat, 1972).
Inthe present paper thecomposition of the pool offreeamino acids inalder
rootnodules is shown.Activities ofenzymes concerned with theassimilation of
ammonia and with the production ofcitrulline in the noduleswill be reported
with special attention to the distribution of these enzymes between different
fractions of thenodule.
MATERIALS AND METHODS
Nodules,root-tips,stems and leaves were collected from 0.5-year-old greenhouse-grown A. glutinosa plants,which had been inoculated with nodule brei
(final concentration 750yg nodule fresh weigt/1)4weeks after germination. Per
plant 20-50 nodules developed. Samples of 2-10 g freshweight ofnodules were
obtained from each plant and assayed for acetylene-reducing activity (Akkermans,
van Straten and Roelofsen, 1977),and those which produced 500-1500 nmoles
-1
-1
CpHL.g nodule fresh weight.h (assayed afterdetaching)were used for homogenization and fractionation. They were homogenized ina Virtis mixer (45-Hispeed)for 2min at5,000rpm in the following buffer: K-phosphate (50 mM),
sucrose (300mM), dithioerythritol (2.5 mM), MgCl„ (1 mM), polyvinyl pyrrolidone
(4%), pH 7.6.
The homogenatewas filtered through a 100-ymfilter to remove large plant
cell debris.Thefiltrate, denoted as total nodule (TN)fraction.
66
lisfiltered througha20urnfilter,andtheresiduewas takenupinhomoimization buffer.The20-um residue(Efraction)contained intact vesicle
lustersoftheendophyte (Akkermans, 1978),whereasthe 20-um filtrate
fraction)contained plantcell material,distorted vesicle clustersand
Vphae.ThePfraction was centrifugedfor15minat10,000xg.Thepellet
i)fraction)contained plantcell organellesanddistorted vesicle clusters
idhyphae,whilethesupernatant (Cfraction)containedtheplant cytoplasm
idthecontentsofdistorted vesicles.Allfractions were sonicatedfor1min
It4 0 W .
Root-tipsandleaveswere homogenized inthesamewayasthe nodules,and
hehomogenateswere filtered througha100-ym filter.Amino acidandammonium
nalysesof80%-ethanolic extractsofmortar-homogenized nodulesandxylem
issueofstemswere made usingaBiotronic LC6000E amino acid analyzer.
For the determinationofthenitrogenase activity (Akkermans et at. , 1977)
hehomogenizationandfractionationofthenodules werecarried outanaerbicallybyflushingthebufferwith argonandadding sodium dithionitetoa
inal concentrationof10mM.
GSactivity was determined usingthetransferase assay (ShapiroandStadtman,
970).Thebiosynthetic assay could notbeused becauseofthecolourofthe
odulefractions.
GDH activity was determined accordingtoFahienandCohen (1970).TheKm
aluesofGDH for NH.andglutamatewere determined accordingtoAhmad,Larker,
hodesandStewart (1979);KCN(0.1 mM)wasaddedtoprevent NADH oxidase
ctivityinassaying GDH activity inthedirectionofNH. formation.
TheactivityofOCTwas determinedbyincubatingthe fractions with ornithine
5 mM)andcarbamyl phosphate (5mM)at30°Candassaying theamountof
itrulline formed usingaBiotronic LC6000E amino acid analayzer.Acolorimetric
ssay could notbeused duetotheintensecolourofthe nodulefractions.
RESULTSAND DISCUSSION
?ree amino ao-ids in alder nodules and stems
Rootnodulesandthexylem tissueofthestemsof4plantsof A. glutinosa
<erehomogenized in80%ethanoltoextract the freeamino acids.Theresultsof
heamino acidanalyses (Table1)confirm the observation (Miettienenand
irtanen, 1952)thatcitrullineisthepredominant free aminoacid inroot
lodulesof A. glutinosa. Serineisalso found inrelatively high amountsinthe
67
Table1.Thefree-amino acid contentofroot nodulesandthexylem tissueof
stemsof Alnus glutinosa (ymolesofamino acid/goftissue,dryweight)
Amino acid
Nodules
Citrulline
Serine
Glycine
Aspartic acid
Alanine
Ornithine
y-Amino butyric acid
Threonine
Arginine
Valine
Glutamic acid
Leucine
Glutamine
Histidine
Isoleucine
Tyrosine
Proline
Phenylalanine
Lysine
Tryptophan
Asparagine
Cystine
Methionine
13.5
13.2
7.6
5.5
5.0
3.3
3.1
3.1
2.9
2.0
1.9
1.6
1.5
1.2
1.2
1.1
1.1
1.0
0.9
0.9
0.6
0.6
0.2
Xylem
10.2
4.8
2.1
3.4
2.0
1.0
0.8
1.4
0.5
0.9
8.1
0.6
2.1
0.3
0.5
1.0
0
1.3
0.5
0
0
0.4
0.1
nodules,whereastheother amino acids occurinrelatively small quantities.
Comparisonoftheamino acid contentofnodulesandxylem tissue supportsthe
hypothesis thatcitrullineisthemain transport vehicleoffixed nitrogenin
alder (Miettienen andVirtanen, 1952;Leaf et al.; WheelerandBond, 1970).
Activities of some nitrogen-assimilating
enzymes in the total nodule
The activitiesofN ? -ase,GS,GDHandOCTinthetotal nodule fractionof
nodulesof4plantsofA . glutinosa were determined (Table2 ) .
fraction
Table 2.Activitiesofsomenitrogen-assimilating enzymesinthetotal nodule
fractionofaldeH)
N2-ase
GS
GDH
OCT
-1
ymoles.min .gnoduledryweight
0.0-9- 0.25
100 -130
8.8- 10.0
0.11- 0.12
TheactivitiesofN 2 -ase,GSandGDHhave been determined four times,that
ofOCTtwice.Thevalues reported inthe Table arethe extremes.
68
-1
the relatively highGSactivity maybeascribed tothe fact that the transsraseassay (which inthepresenceofMn+ doesnotrequire deadenylationof
heenzyme)gives higher values for the enzyme activity than the biosynthetic
>say,sothat theGSactivity in vivo islikelynotashighassuggestedby
hetransferase assay (ShapiroandStadtman, 1970).
We havenot been abletodetectany activity ofGOGAT,using the assay
sthoddescribed byMeers,TempestandBrown (1979).Inthis method NAD(P)H
s usedasdonorofreduction equivalents.
The absenceofGOGAT activitymaybeexplained intwo ways: eitherthe
oncentration ofthe enzymeinaldernodules istoolowtobeobserved,or
iOGATinthese nodules doesnot function with NADHorNADPH,butrequires
•educed ferredoxinasdonorofreduction equivalents.Thisisthecase with
iOGATine.g. peachloroplasts (LeaandMiflin, 1974)andVioia faha leaves
Wallsgrove,Harel,LeaandMiflin, 1977).Thelatter possibility isbeing
tudiedatthemoment.
GDH activity was dependentonNADHascofactor;NADPH gavenoactivity.
%e direction
of the reaction
catalyzed
by GDH
TheKmofGDH forNHt (assayed intheTNfraction)wasfoundtobe16mM,
Khilethe NH.concentration inthe nodule was only 1.5mM. Since toxic effects
)fNH.inplant tissueatconcentrationsof1mM have been reported (Krogman,
Jagendorf and Avron, 1959;Chibnal, 1939;Prianishnikov, 1951),the existence
)fsub-cellular poolsofhigher NH*concentrations isunlikely.TheKmofGDH
forglutamate (assayed intheTNfracton)wasfoundtobe0.9mM,whilethe
glutamate concentration inthe nodule was0.5mM.
These data suggest thatGDHinaldernodules catalyzes the deamination
rather thanthesynthesisofglutamate.Apossible function ofGDHmightbe
the productionofNH. required for the formation ofcarbamyl-phosphate,which
isinturn needed for the formation ofcitrulline.
GDH activities
in homogenates of nodules,
root-tips
and
leaves
The activityofGDHinahomogenateofalder nodules isabout 5-10 times
higher than thatinhomogenatesofalder root-tipsandleaves (activities based
on tissuedry weight((Table 3 ) .Thisisthemore significant since nodules
contain morewoody material than leavesorroot-tips.Therelatively highGDH
activity innodules suggests that this enzyme playsanimportant roleinthe
nitrogen metabolism ofnodules.
69
Table3.ActivityofGDHinhomogenatesofnodules,root-tipsandleaves
ofalder^)
-1
ymoles.min .gtissuedryweight
8.8- 10.0
1.1-1.5
1.1-2.1
Nodules
Root-tips
Leaves
-1
1)
The values reportedare theextremesoffourdeterminations.
Localization of nitrogen-assimilating
enzymes within the nodule
ThedistributionofN^-ase,GS,GDHandOCTactivities over the different
fractionsofthenoduleisshowninTable 4.The distributionofN^-aseactivityovertheEfractionandthePfraction isinaccordance with earlier investigations (Akkermans,1978;Akkermans,RoelofsenandBlom, 1979)inwhich diaminopimelic acid was usedasanendophyte cell wall marker.Thelarge proportionof
N„-aseinthePfraction dependsonthepresence cf smallandbroken vesicle
clusters.Therefore,itisconcluded thatN^-aseisanendophyte vesicle cluster
enzyme.
Table 4.Distributionofsome nitrogen-assimilating enzymes over different
fractionsofthe nodule
Fraction
N2-ase
TN (total nodule) 100%
E (endophyte
vesicle clusters) 20%
P (plant fraction) 70%
0 (plant organelles)ND1 )
C (plant cytoplasm) ND1 )
GS
GDH
100%
1%
02%
3%
98%
OCT
100%
100%
0%
91%
46%
63%
0%
100%
70%
20%
1)
ND,notdetermined.
From thedistributionofGSactivityitisconcludedthatGSisaplantcytoplasm enzyme.ThedistributionofGDHactivity overthedifferent fractions
suggeststhatGDHislocalized inplantcell organelles.Thisobservation,which
isincontrasttoearlier results (Akkermans et al., 1979),hasfurther been
supportedbytheabsenceofGDHactivity incell-free extractsofFrankia AvcI1
(thenodule endophyteofalder),growninpureculture (Blom,inpreparation).
The activityofOCT also seemstobeassociated with plant cell organelles.
Thedifference indistributionofGDHandOCTsuggestsadifferent degreeof
70
disintegration of plant cell organelles during homogenization of the nodules
(GDH and OCTactivities were determined in different samples).
Thenature of these organelles remains to beclarified,although GDH is
generally accepted as amarker enzyme for plantmitochondria {e.g. Lehninger,
1975;Nauen and Hartmann,1980). Gardner (1976),using acytochemical assay,
demonstrated OCT inaldernodules inthemitochondria and the endoplasmic
reticulum ofthe host cell;after longer incubation periods staining was also
observed inthe endophyte.
ACKNOWLEDGEMENT
These investigations were supported by the Foundation for Fundamental
Biological Research (BION),which is subsidized by the Netherlands
Organization for theAdvancement of Pure Research (ZWO).
REFERENCES
1.Ahmad, I.,Larher,E.,Rhodes,D.and Stewart,G.R. (1979). Some aspects of
ammonia assimilation in higher plants Halophytes. InNitrogen assimilation
of plants (E.J.Hewitt and E.V. Cutting,Eds)pp.653-657,Academic Press,
London,NewYork,San Francisco.
2.Akkermans,A.D.L. (1978). Rootnodule symbioses in non-leguminous 1,-fixing
plants.In Interactions between non-pathogenic soil microorganisms ahd plants
(Y.R. Dommergues and S.V. Krupa,Eds) pp.335-372,Elsevier Scientific Publ.
Comp.,Amsterdam, Oxford,New York.
3.Akkermans,A.D.L.,Straten,J. van,and Roelofsen,W. (1977). Nitrogenase
activity ofnodule homogenates of Alnus glutinosa: acomparison with the
Rhizobium-pea system. In Proc. Second Int.Symp.Nitrogen Fixation (W.E.Newton,
J.R. Postgate and C. Rodriguez-Barrueco, Eds)pp.591-603,Academic Press,
London.
4.Akkermans,A.D.L.,Roelofsen,W.and Blom,J. (1979). Dinitrogen fixation and
ammonia assimilation inactinomycetous root nodules of Alnus
glutinosa.
In Proc.Workshop Symbiotic Nitrogen Fixation in themanagement of temperate
forests (J.C.Gordon,C.T. Wheeler and D.A. Perry,Eds) pp. 160-170,Oregon
State University.
5.Chibnal,A.C. (1939). Protein metabolism in the plant, pp. 1-306, Yale
University Press,NewHaven.
6. Egeraat,A.W.S.M. van (1972). Pea-root exudates and theireffect upon rootnodule bacteria.Communications Agricultural University Wageningen,The
Netherlands,72-27.
7. Fahien,A.and Cohen,P.P. (1970). L-glutamatedehydrogenase. InMethods in
Enzymology XVIla:metabolism ofamino acids and amines (H.Tabor and
C. White Tabor,Eds)pp839-844,Academic Press,New York and London.
8. Gardner, I.C. (1976). Ultrastructural studies of non-leguminous rootnodules.
InSymbiotic nitrogen fixation in plants. International Biological Programme 7
(P.S.Nutman,Ed.)pp.485-495,Cambridge University Press.
71
9. Krogman,D.W., Jagendorf,A.T.andAvron,M. (1959). Uncouplers of spinach
chloroplast photosynthetic phosphorylation. Plant Physiol.34,272-277.
10. Lea,P.J. and Miflin,B.J. (1974).Alternative route for nitrogen assimilation in higher plants.Nature 251,614-616.
11.Leaf,G.,Gardner,I.C.and Bond,G. (1958).Observations on thecomposition
and metabolism of the nitrogen-fixing root nodules of Alnus. J. Exp.Bot.
9,320-331.
12. Lehninger,A.L. (1975). Biochemistry, p.512,Worth Publishers Inc.,New York,
2nd ed.
13. Meers,J.L.,Tempest,D.W. and Brown,C M . (1970).Glutamine (amide):
2-oxoglutarateamino transferase oxido-reductase NADP,an enzyme involved
in the synthesis of glutamateby some bacteria.J. Gen.Microbiol.64,
187-194.
14. Miettienen,J.K. and Virtanen,A.I. (1952). The freeaminoacids in the
leaves,roots and root nodules of thealder {Alnus). Physiologica Plantarum
5,540-557.
15. Nauen,W.and Hartmann,T. (1980). Glutamate dehydrogenase from Pisum
sativum L.Localization of themultiple forms of glutamate formation in
isolated mitochondria. Planta 148,7-16.
16. Prianishnikov, D.N. (1951).Nitrogen in the life of plants, pp. 1-109,
Kramer Business Service,Madison.
17. Rai,A.N.,Rowell,P.and Stewart,W.D.P. (1980). NH^assimilation and
nitrogenase regulation in the lichen Peltigera
aphtftosa Willd.New Phytol.
85,545-555.
18. Scott,D.B. (1978).Ammonia assimilation innitrogen-fixing systems.
In Limitations and potentials for biological nitrogen fixation in the tropics
(J.DSbereiner,R.H. Burris,A. Hollaender,A.A. Franco,C.A. Neyta and
D.B. Scott,Eds)pp.223-235.Plenum Press,NewYork and London.
19. Shapiro,B.M. and Stadtman,E.R. (1970).Glutamine synthetase. In Methods
in Enzymology XVIla:metabolism of amino acids and amines (H.Taborand
C.White Tabor,Eds) pp.910-922,Academic Press,NewYork and London.
20. Stewart,W.D.P.and Rowell,P. (1977). Modifications of nitrogen-fixing
algae in lichen symbioses.Nature 265,371-372.
21. Wallsgrove,R.M., Harel,E.,Lea,P.J. and Miflin,B.J. (1977). Studies on
glutamate synthase from the leaves of higher plants.J. Exp.Bot.28,
588-596.
22. Wheeler,C.T.and Bond,G. (1970). Theaminoacids ofnon-legume root
nodules. Phytochemistry 9,705-708.
72
CHAPTER 111.2
INHIBITION OF GOGAT ACTIVITY IN LUPIN NODULE HOMOGENATES BY ALDER NODULE
HOMOGENATES
73
INTRODUCTION
In a previous contribution (1),the activities of some enzymes concerned
with the assimilation of nitrogen in root nodules of alder {Alnus glutinosa)
were reported. Itwas shown that nitrogenase (N„-ase) is localized in the
vesicle clusters of the endophyte,whereas glutamine synthetase (GS)is
found in the cytoplasm of the host cells,and glutamate dehydrogenase (GDH)
and ornithine carbamyl transferase (OCT) are localized in the organelles
of the host cells. No activity of glutamate synthase (GOGAT)was observed.
Two pathways are known for the assimilation of ammonia in nitrogenfixing organisms. The first, the reductive amination of a-ketoglutarate
catalyzed by GDH, is found in theAfostoe-containing lichen of the genus
Peltigera (2,3). The second, the combined action of GS and GOGAT, occurs
inmany free-living nitrogen-fixing organisms and in Rhizob-ium-]egume
symbioses (reviewed in ref.4 ) .
The K mof GDH for NH. in4alder nodules was found to be 16 mMand the
concentration of NH. to be 1.5 mM,whereas the K of GDH for glutamate
was 0.9 mM and the concentration of glutamate 0.5 mM (1).These data
render it unlikely that in root nodules ofalder the GDH pathway for
assimilation of ammonia is prominent. They suggest that ammonia is
assimilated via the GS/GOGAT pathway.
In the present contribution itwill be shown that our failure to
detect any GOGAT activity in alder nodule homogenatesmight be ascribed
to the presence of an inhibiting compound in this homogenate.
MATERIALS AND METHODS
Lupin nodules were collected from non-inoculated field-grown Lupinus luteus
(yellow lupin). The crude bacteroid fraction was obtained from the nodules
as described by Robertson et al. (5).This fraction was sonicated for 2min
at 40 Wand centrifuged for 10min at20,000 gyielding a cell-free preparation
containing GOGAT activity inwhich alder nodules were homogenized. Alder
nodules were collected as described earlier (1).Varying amounts of
nodules were homogenized in a Virtis mixer (45-Hi-speed) for 2min at
5000 r.min in 30ml of the GOGAT-containing lupin extract. The homogenate was filtered through a 100ym filter to remove large plant cell debris.
75
NADH-dependent GOGAT activity was determined inthisfraction according to
Planque et at. (6).
RESULTSAND DISCUSSION
Theeffect ofadded aldernodule homogenate on theGOGAT activity ofthe
lupin nodule extract isshown inTable 1.From the data shown inthistable
itcan beconcluded that during thehomogenization ofaldernodules some
Table 1. Inhibitory effect of homogenized aldernodules on theGOGAT activity
of lupin nodule extract
Concentration of homogenized
aldernodules inthe lupin
nodule extract
(mg/ml)
Inhibition of NADH-dependent
GOGAT activity
0
200
250
400
0
35
40
80
QD
2)
I)
(%)
.1
'TheGOGAT activity varied between 50and 100nmol.min .glupin nodule
freshweight-1,
2)Aldernodules homogenized in buffercontaining no lupin nodule extract(ref. 1)
3)NoGOGAT activity detected
compound isreleased which is responsible forthe inhibition of GOGAT activity
inthe lupin nodule extract.Thismay explain why no GOGAT activity was observed
in homogenatesofaldernodules (1).Therefore thefailure to detect any GOGAT
activity inhomogenates does not exclude theoperation of theGS/GOGAT pathway
intheassimilation ofammonia inalder rootnodules in vivo.
REFERENCES
LBlom.J., Roelofsen,W.and Akkermans,A.D.L. (1981). New Phytol.89,321-326.
2.Stewart,W.D.P.and Rowel1,P. (1977). Nature 265,371-372.
3.Rai,A.N., Rowell,P.and Stewart,W.D.P. (1980). New Phytol.85,545-555.
4.Scott,D.B. (1978). InLimitations and potentials forBiological Nitrogen
FixationintheTropics (J.DSbereiner,R.H.Burris.A. Hollaender,A.A.Franco,
C.A.Neyta and D.B.Scott,Eds)pp.223-235.Plenum Press,NewYork &London.
5.Robertson, J.G., Warburton,M.P.and Farnden,K.J.F. (1975). FEBSLetters
55,33-37.
6.Planqu6,K.,Kennedy, I.R., Vries,G.E.de,Quispel, A.and Brussel,A.A.N,van
(1977). J. Gen.Microbiol. 102,95-104.
76
Chapter IV
GENERAL DISCUSSION AND SUMMARY
77
The research reported in this thesis dealswith the symbiosis of Frankia spp.
bnd Alnus glutinosa.
Frankia spp.areactinomycetes giving rise tothe formation
Dfnitrogen-fixing nodules on the roots ofanumber ofnon-leguminous plants.
Inthese nodules Frankia spp.livewithin the plantcells and obtain all sources
Dfcarbon and energy from the plant,giving fixed nitrogen inexchange.
To answer thequestion what compounds Frankia spp.obtain from the plant,
insight into themetabolism of thismicroorganism is required.Toobtain this
insight,researches havebeenmadewith thefree-livi)ng Frankia AvcI1, isolated
from rootnodules of Alnus viridis ssp orispa byBaker et al. (1979).Thisorqanism
has been isolated and cultivated oncomplex media. Inorder toobtain insight into
theC-and N-source requirements of Frankia AvcI1,asimple andweTl-defined
growth medium isneeded.Thecomposition of this medium and theC-and Nmetabolismof Frankia AvcI1 are thesubjects ofChapter IIofthisthesis.
InChapter II.1 it is shown that Frankia Ave11 isable togrowon amedium
containing Tween 80 (an oleateesterof polyethyleneglycol sorbitan)as sole
C-source and either glutamic acid orNH.C1as soleN-source.The growth yield of
Frankia AvcI1 on various media isreported. It isshown that thedoubling time
of Frankia Ave11growing on QMOD/Tween medium isabout 2days,which isslow,
even for anactinomycete.When growingwith Casaminoacids asnitrogen source,
Frankia AvcI1 selectively takes up glutamic acid and aspartic acid,leaving the
concentrations of theother detectable amino acids unchanged. Themechanism of
this selection isstill unclear.
Chapter II.2contains further data on theC-sources utilized by Frankia AvcI1.
Itisshown that Frankia AvcI1 can utilize asC-source also other Tweens, viz.
Tweens 20,40,60and 85,and inaddition several fatty acids, viz. acetic,
propionic,butyric,valeric,caproic,caprylic,capric,palmitic and stearic
acids.No growth of Frankia AvcI1 was observed onmedia containing triglycerides
as C-sources.
79
The dependence of the growth yield on the nature of the carbon source
+
+
and theconcentration ofNH. inthemedia is shown.Utilization ofNH» as
nitrogen source of Frankia AvcI1 growing in theTween/NH,medium isconfirmed
15
by incorporation experiments with N-NH.C1.
The results reported inChapter II.3show that Frankia AvcI1 does not
take upglucose from amedium containing both glucose and Tween 80.In
agreement with thisobservation,it isdemonstrated that the activities
of isocitrate lyaseand malate synthase incells grown on theQMOD/Tween
medium (containing both glucose and Tween 80)are of the same order of
magnitude as incells grown on theTween/NH.medium (containing Tween 80
as sole C-source).
Organisms growing on fatty acids,and degrading thesecompounds toacetyl-CoA,
start gluconeogenesis with the action of the glyoxylatecycle (Kornbergand
Krebs,1957) leading to theconversion of 2acetyl-CoAmolecules to 1molecule
of succinate.The presence of the glyoxylate cycle enzymes isocitrate lyase
and malate synthase in Frankia AvcI1 cells grown with Tween 80as C-source
istherefore not surprising.The succinate formed in the glyoxylate cycle
can beconverted to phosphoenolpyruvate by the subsequent action of the
enzymes succinate dehydrogenase,fumarase,malic enzyme and pyruvate
orthophosphate dikinase,which are found incell-free extracts of Frankia
AvcI1. From phosphoenolpyruvate,gluconeogenesiscan continue with the
action of phosphoglycerate kinase and 3-phosphoglyceraldehyde dehydrogenase.
Forenergy generation, Frankia AvcI1 can oxidize theacetyl-CoA derived
from fatty acid breakdown in thecitric acid cycle,although the low activity
of the enzyme succinyl-CoA synthetase leaves room for the presumption that
thecitric acid cycle isnotvery operative in Frankia AvcI1. This isnot
impossible since theoxidation of fatty acidsyields many reducing equivalents.
The data contained in Chapter II.4show that Frankia AvcI1 does not take
up succinate from amedium containing acetate plus succinate or succinate
alone. Inaccordance,no repressing effect of succinate on theactivities of
the glyoxylate cycle enzymes was observed,whereas incells grown on propionate
these enzymeswere not found,indicating that they arehotconstitutive in
Frankia Ave11.
Frankia AvcI1 isable to utilize several amino acids as sole nitrogen
source, viz. alanine,Y~aminobutyric acid,aspartic acid,glutamic acid,
glycine,leucine,phenylalanine,serine,threonine,tyrosine and valine.
80
Nodifferences inC-and N-source requirements wereobserved for the
Airee Frankia strains AvcI1,CpI1 (isolated byCallaham et al. ,1978,from
'.omptonia peregrina root nodules)and AgSp+1 (isolated by Quispel and
lurggraaf, 1981,from Alnus glutinosa spore-(+) type root nodules). These
hree strains were shown to utilize either Tween 80oracetate,butno
ithanol,lactate,glucose or succinate as soleC-source,and either NH.C1,
asamino acids,aspartic acid or alanine asN-source.
From the data given in Chapter II itwill be clear that Frankia AvcI1
s able to grow on awell-defined medium,which isa prerequisite for
btaining aclear insight into the physiology of the organism. The alcoholic
oot extract (Quispel and Tak, 1978)or the soybean lecithin (Lalonde and
alvert, 1979),added to themedium as growth factors,can be replaced
iyTween 80 or fatty acidswhich are utilized ascarbon source,while
IH.C1oramino acids can be utilized asnitrogen source.
Itis unknown whether Frankia spp.growwith the same growth rate
inmedia with different constituents.The growth yields presented in
lhapter IIare usually small as compared to the amount of carbon present
inthemedia.This suggests that either an additional component of the
jedium ispresent in limiting concentrations or that Frankia spp.can only
)rowfor a limited time after inoculation.
Theonly group ofcompounds known so far tobe utilized asC-source by
r
ree-living Frankia AvcI1 are fatty acids,either free oresterified. The
juestionwhat carbon source Frankia spp.living symbiotically in the nodule
jbtain from the plant,still remains to beanswered. Based on the data
)btained for free-living Frankia spp.it isnot unlikely that some fatty
icidfunctions asC-source under such conditions.Other possible candidates
Forthis function are plant lipids like thealcoholic root extract
(Quispel and Tak, 1978)or the soybean lecithin (Lalonde and Calvert, 1979).
Itisunlikely that sugars like glucose or dicarboxylic acids like succinate
areplaying this role,unless theability of Frankia spp.totake upone
ormore of these compounds should alter during the transition from the
free-living tothe symbiotic stage.
The cultivation of free-living Frankia spp.isa powerful tool in
discovering symbiotic interactions of the endophyte and the host.In
Chapter II.4of this thesis it is shown that by replacing acetate as
C-source of themedium by propionate,theactivities of the glyoxylate
cycle enzymes are repressed. Other authors (Tjepkema,Ormerod and Torrey,
1980and 1981;Gauthier,Diem and Dommergues, 1981)reported amedium in
which free-living Frankia spp. showvesicle formation and N„-aseactivity.
The present knowledge thus enables one to influence regulation in freeliving Frankia spp.,which is important in studying the symbiotic
interactions mentioned above.
InChapter III attention is paid to the assimilation of the ammonia
produced by theendophyte living symbiotically in the root nodules of
Alnus glutinosa grown in the greenhouse from seeds collected inWageningen.
InChapter III.1 thecomposition of the pool of free amino acids in root
nodules and the xylem tissue of stems is reported. It isshown that
citrulline is the predominant free amino acid innodules,while serine
also occurs in relatively large amounts. Inxylem tissuecitrulline and
glutamic acid are prominent.
The activities of N^-ase,GS,GDH and OCT in root nodule homogenates
are reported. From the K values of GDH forNH. (16mM)and glutamate
(0.9mM)and theconcentrations in thenodule ofNH. (1.5mM)and glutamate
(0.5mM) it isconcluded that GDH inaldernodules probably is responsible
for thedeamination of glutamate and not for the synthesis of this key
amino acid. The important function of GDH in thenitrogen metabolism
ofaldernodules isconfirmed by themuch higheractivity ofthis enzyme
in homogenates ofnodules ascompared to that in homogenates of root-tips
and leaves.
The vesicle clusters,which contain theN^-aseactivity,did not show
activity of GS,GDH and OCT.Thecytoplasm of the hostcellswas shown
to possess theGSactivity,whileGDH and OCT are localized in the
organelles of the host cells. No activity ofNADH-dependent GOGAT was
observed.
InChapter III.2 it is shown that theactivity of NADH-dependent GOGAT
from root nodules of lupins is inhibited by somecompound in the homogenate
ofaldernodules.
Simultaneously with and independently of the present research,Schubert
et al. (1981)analyzed thecomposition of the pool offree amino acids
innodules and thexylem tissue of Alnus glutinosa grown in the field in
East Lansing,Michigan.The only differencewith respect to these amino
82
acid pools between theAmerican aldersand the European alders studied
in the present research,is the relatively high amount of serine in
nodules of the latter,whereas in nodules of the former this amino acid
isnot found.The results of both Schubert et at. (1981)and the present
research confirm the hypothesis that inalder.citrulline isthemain transport
vehicle of fixed nitrogen (Miettienen and Virtanen,1952;Leaf,Gardner
and Bond, 1958;Wheeler and Bond, 1970).
From the results shown inChapter III.2 itcan beconcluded that our
failure to find any GOGAT activity may beascribed tothe presence of
an inhibiting compound in the homogenate ofaldernodules,so that it
isnot excluded that GOGAT isactive inaldernodules in vivo.
The data reported in Chapter IIIare inaccordance with the following
model: Thenitrogen fixed as ammonia in the vesicle clusters of the
endophyte,isassimilated in thecytoplasm of the host cell into glutamate
by theaction of GSand presumably GOGAT.Glutamate is in part deaminated
in theplantorganelles by theaction of GDH to supply theNH. required
for the synthesis ofcarbamyl phosphate.Another partof the glutamate
isconverted to ornithine,which inthe organelles of the hostcell
reactswith carbamyl phosphate toform citrullineaccording to the OCT
reaction.Citrulline isexcreted from the nodule and serves as nitrogen
source for theplant.
REFERENCES
Baker,D.,Torrey,J.G. and Kidd,G.H. (1979). Nature 281,76-78.
1.Callaham,D.,Torrey,J.G. and del Tredici,P. (1978). Science 199,899-902.
Gauthier,D., Diem,H.G. and Dommergues,Y. (1981). Appl. Env.Microbiol.
41,306-308.
Kornberg,H.L. and Krebs,H.A. (1957).Nature 179,988-991.
Lalonde,M.and Calvert,H.E. (1979). In Proc.Workshop on Symbiotic Nitrogen
Fixation in the Management of Temperate Forests (J.C.Gordon,C.T. Wheeler
and D.A. Perry,Eds)pp. 95-110. Forest Research Laboratory,Oregon State
University.
Leaf,G.,Gardner,I.C.and Bond,G. (1958). J. Exp.Bot.9,320-331.
Miettienen,J.K. and Virtanen,A.I. (1952). Physiol. Plant.5,540-557.
3.Quispel,A. and Burggraaf,A.J.P. (1981). In Proc.4th Int.Symposium on
Nitrogen Fixation (A.H.Gibsen and W.E.Newton,Eds)pp.229-236. Australian
Academy of Science,Canberra.
Quispel,A. and Tak,T. (1978). New Phytol.81,587-600.
83
10. Schubert,K.R.,Coker III,G.T.and Firestone,R.B. (1981). Plant Physiol.
67,662-665.
11.Tjepkema,J.D., Ormerod,W.and Torrey,J.G. (1980). Nature 287,633-635.
12. Ijepkema,J.D., Ormerod,W.and Torrey,J.G. (1981). Can.J.Microbiol.
27,815-823.
13.Wheeler,C.T.and Bond,G. (1970). Phytochemistry 9,705-708.
84
SAMENVATTING
Hetonderwerpvanditproefschriftisdesymbiosevan Frankia spp.en
ilnus glutinosa.
Frankia spp.zijn actinomycetendiedevormingvanN«bindendewortelknollen induceren bijeenaantal niet-vlinderbloemige
planten,waaronderdeels.Indezewortelknollen bevindende Frankia
cellen zichindecellenvandeplant. Frankia spp.ontvangen voornun
energievoorzieningeenofmeer koolstofverbindingenvandeplant. Zij
bindenmolekulaire stikstofdiealsammonia wordt uitgescheiden inde
gastheercel. Hier vindt vormingvanorganische N-verbindingen plaatsdie
dienen voordegroeivandeplant.
Omdevraagtekunnen beantwoorden,welke koolstofverbindingen Frankia spp.
vandeplant ontvangen,isinzichtinhetmetabolismevanditmicroorganismevereist.Voorhetverkrijgenvanditinzichtisonderzoek gedaan
metdevrijlevende Frankia AvcI1,geTsoleerd uitwortelknollen van Alnus
viridis ssp. arispa doorBakerenmedewerkers (1979). Isolatieenkweek
vandit organisme vondentothetbeginvanhethierbeschreven onderzoek
plaatsincomplexemedia.Ominzichtteverkrijgen indeC-enN-stofwisseling
van Frankia AvcI1,ishetnodig dezeactinomyceettekwekenineengoed
gedefinieerd medium.Inhoofdstuk IIvanditproefschriftwordendesamenstellingvaneendergelijk mediumenhetC-enN-metabolismevanenkele
Frankia spp.behandeld.
In hoofdstuk II.1wordtaangetoond dat Frankia AvcI1 kangroeienin
een medium metTween80(een oleaat-estervan polyethyleenglycol-sorbitan)alskoolstofbronenNH.C1ofglutaminezuurals stikstofbron.De
opbrengstenaancelmateriaal inverschillendemedia,endegroeisnelheid
in hetQMOD/Tween medium worden Vermeld.Bijgroei van Frankia Ave11 met
Casaminozurenalsstikstofbron worden glutaminezuurenasparaginezuur
selectief opgenomenenbenut,terwijldeconcentratievandeandere aminozureninhetvoedingsmedium tijdensdegroei ongewijzigd blijft.
Inhoofdstuk II.2wordt aangetoond dat Frankia AvcI1alskoolstofbron,
87
behalveTween 80,ook deTweens 20,40,60 en 85 kan gebruiken,alsmedeeen
aantal vetzuren,nl. azijn-,propion-,boter-,valeriaan-,capron-,capryl-,
caprine-,palmitine- en stearinezuur. Inmedia met triglyceriden als
koolstofbron werd geen groei waargenomen. Decelopbrengsten zijn afhankelijk van de aard van de koolstofbron en van deconcentratie van NH.C1 in
net groeimedium.Gebruik van NH.als N-bron door Frank-La AvcI1 bijgroei
+
15
in netTween/NH.medium werd bevestigd ineen experiment met N-NH.C1.
Uit deexperimenten beschreven in hoofdstuk II.3blijkt dat Frankia AvcI1
geen glucose opneemt uit een medium dat zowel Tween 80als glucose bevat.
Incellen gekweekt in hetQMOD/Tweenmedium (dat zowel Tween 80 alsglucose bevat) isde aktiviteit van de glyoxylaat-cyclus enzymen isocitraatlyaseenmalaat-synthasedan ook van dezelfde orde van grootte alsin
cellen gekweekt inhetTween/NH.medium (metTween 80 alsenige C-bron).
Het produkt van de glyoxylaat-cyclus,succinaat,kanworden omgezet in
fosfoenolpyruvaat door deenzymen succinaat-dehydrogenase,fumarase,
"malic enzyme"en pyruvaat-orthofosfaat-dikinase,die alien incelvrije
extracten van Frankia AvcI1 werden aangetroffen.Ookwerd aktiviteit
van de enzymen fosfoglyceraat-kinase en 3-fosfoglyceraldehyde-dehydrogenase gevonden. Deze enzymen spelen waarschijnlijk een rol bijde biosynthese
van glucose en andere suikers (gluconeogenese)uit fosfoenolpyruvaat.
Acetyl-CoA,verkregen bijdeafbraak van vetzuren,kan voorde
generering van energie in Frankia AvcI1 waarschijnlijk worden geoxideerd indecitroenzuur-cyclus,hoewel de lageaktiviteit van hetenzym
succinyl-CoA-synthetase incelvrije extracten van Frankia AvcI1 ruimte
laat voor deveronderstelling datdecitroenzuur-cyclus in Frankia AvcI1
niet zeeraktief is.Dit ismogelijk omdat bij deafbraak van vetzuren
totacetyl-CoA reeds veel reduktie-equivalenten vrijkomen.
In hoofdstuk II.4wordt aangetoond dat Frankia AvcI1 geen succinaat
opneemt uit een medium datals koolstofbron alleen succinaat of succinaat
plus acetaat bevat. Inovereenstemming hiermeewerd geen represserend
effekt van succinaat op deaktiviteit van de glyoxylaat-cyclus enzymen
waargenomen,terwijl propionaat wel een dergelijk effekt bleek te
hebben.
Frankia AvcI1 kan als enige stikstofbron een aantal aminozuren
gebruiken,nl. alanine,yaminoboterzuur,asparaginezuur,glutaminezuur,
glycine,leucine, fenylalanine,serine threonine,tyrosine ofvaline.
Destammen Fvankia Avcll,CpI1 (ge'fsoleerd doorCallaham en medewerkers, 1978,uitwortelknollen van Comptonia pevegvina) enAgSp+1 (ge'fsoleerd door Quispel en Burggraaf, 1981,uit spore-positieve wortelknollen
van Alnus glutinosa) vertoonden geen verschillen wat betreft hun C-en
N-bron behoeften.Alle drie de stammen kunnen Tween 80en acetaat,maar
geen ethanol,lactaat,glucose of succinaat,als C-bron gebruiken.AlsNbron kunnen alle drie de stammen NH.C1,Casaminozuren,asparaginezuur
enalanine benutten.
Uitde resultaten beschreven in hoofdstuk IIblijkt dat Fvankia Avcll
kan groeien ineen gedefinieerd medium. Hetwortel-extract (Quispel en
Tak, 1978)en de soja-lecithine (Lalonde en Calvert,1979)dieaan hetmedium werden toegevoegd als groei-faktor,kunnen worden vervangen door
Tween 80of vetzuren dieworden gebruikt als koolstofbron,terwijl NH.C1
ofsommigeaminozuren geschikt zijn als stikstofbron.
Het isniet bekend of Fvankia Avcll inalle in hoofdstuk IIbeschreven media met dezelfde snelheid groeit. Deopbrengsten aan celmateriaal
van Fvankia Avcll zijn gewoonlijk klein vergeleken bij de hoeveelheid
koolstofdie in hetmedium wordt aangeboden. Ditwijst erop dat een
anderecomponent in hetmedium beperkend is,of dat Fvankia Avcll slechts
gedurende een beperkte tijd na entinq kan groeien.
Deenige groep verbindingen waarvan totnu toebekend isdatzeals
koolstofbron door vrijlevende Fvankia spp. gebruikt kunnen worden,zijn devetzuren,hetzij vrij, hetzij veresterd. Het isnog niet duidelijkwelkekoolstofverbinding symbiontische Frankia spp. indewortelknolvan de plant ontvangen.
Geletop de resultaten die verkregen zijn met vrijlevende Fvankia spp.
is hetniet onwaarschijnlijk dat deze koolstofbron een vetzuur is.Ook
lipiden van planten zoals wortelextract (Quispel en Tak, 1978)
of soja-lecithine (Lalonde en Calvert,1979)zouden deze funktie
kunnen vervullen. Het isniet waarschijnlijk dat suikers zoals glucose
of dicarbonzuren zoals succinaat geschikt zijn als koolstofbron, tenzij
het vermogen van Fvankia spp. om deze verbindingen op tenemen verandert
tijdens de overgang van de vrijlevende in de symbiontische fase.
Hetonderwerp van hoofdstuk III betreft deassimilatie van de stikstof
diealsammoniak in deblaasjes-"clusters" van de endofyt (de symbiontische fase van de Fvankia spp.)inwortelknollen van deelswordt gebon-
89
den. Hetonderzoek isverrichtmet Alnus glutinosa opgekweekt inde kas
uitzaden verzameld inWageningen.
Inhoofdstuk III.1 wordt desamenstelling van de "pool" van vrije
aminozuren inwortelknollen en in het xyleem van de stam van deze planten
vermeld. In knollen blijkt citrulline hetmeest voorkomendeaminozuur
tezijn,terwijl ook serine in relatief grote hoeveelheden voorkomt. In
het xyleem van de stamworden citrulline en glutaminezuur in grote hoeveelheden aangetroffen.
Deaktiviteiten van deenzymen N„-ase,GS,GDH en OCT in homogenaten
vanwortelknollen worden vermeld. Uitde K van GDH voorNH. (16mM)en
voor glutamaat (0.9 mM),en deconcentratie inde knol van NH. (1.5mM)en
glutamaat (0.5mM)wordt afgeleid dat GDH inelzeknollenwaarschijnlijk
verantwoordelijk isvoor dedeaminering van glutamaat.DatGDH een
belangrijke funktie vervuld inelzeknollen blijkt uitde hogeaktiviteit
van ditenzym in knolhomogenaten vergeleken bij die in homogenaten van
bladeren enwortelpunten.
N?-aseaktiviteitwordt aangetroffen in deblaasjes-"clusters",terwijl GSzich in hetcytoplasma van de plantecellen bevindt.GDHen
OCTzijn gelokaliseerd in deorganellen van de gastheercellen.Geen
aktiviteit van NADH-afhankelijk GOGAT kon inelzeknollen worden waargenomen.
In hoofdstuk 111.2wordt aangetoond datdeaktiviteit van NADH-afhankelijk GOGAT uitwortelknollen van lupinewordt geremd dooreen component
van het homogenaat van elzeknollen.
Gelijktijdig met,maaronafhankelijk van,het indit proefschrift
beschreven onderzoek,onderzochten Schubert en medewerkers (1981)de
samenstelling van de "pool" van vrijeaminozuren inwortelknollen en
hetxyleem van de stam van A. glutinosa in East Lansing,Michigan.
Hetenige verschil tussen deelzen uit Michigan en die uit Nederland,
met betrekking totdezeaminozuren,is gelegen in de relatief grote
hoeveelheid serine inwortelknollen van deNederlandse elzen,terwijl
de elzen uitMichigan ditaminozuur niet bevatten.De resultaten van
zowel Schubert enmedewerkers (1981)als die van het indit proefschrift
beschreven onderzoek,bevestigen de hypothese dat inelzen gebonden
stikstof voornamelijk inde vorm vancitrullinewordt getransporteerd
(Miettienen en Virtanen, 1952;Leaf,Gardner en Bond, 1958;Wheeleren
90
Bond, 1970).
Uit de resultaten die in hoofdstuk 111.2worden Vermeld kan worden
geconcludeerd dat in homogenaten van elzeknollen een verbinding voorkomt
die de aktiviteit van GOGAT remt. Uit het feit dat tot nu toe in elzeknolhomogenaten geen GOGAT-aktiviteit werd aangetroffen,mag dus niet
worden geconcludeerd dat GOGAT in elzeknollen in vivo niet aktief is.
De resultaten van hoofdstuk III zijn in overeenstemming met het
volgende model: De stikstof die in de blaasjes-"clusters" van de endofyt wordt gebonden,wordt in het cytoplasma van de gastheercel geassimileerd tot glutamaat door GS en waarschijnlijk GOGAT. Een gedeelte
van het aldus gevormde glutamaat wordt in organellen van de gastheercel
gedeamineerd door GDH,waarbij de voor de vorming van carbamyl-fosfaat
vereiste NH.vrijkomt. Een ander gedeelte van het glutamaat wordt omgezet in ornithine,dat in de organellen van de gastheercel met carbamylfosfaat reageert tot citrulline in de OCT-reaktie. Citrullinewordtdoor deze
cellen uitgescheiden en dient als stikstofbron voor de plant.
LITERATUUR
1.Baker, D.,Torrey, J.G. en Kidd,G.H. (1979). Nature 281,76-78.
2. Callaham, D.,Torrey, J.G. en del Tredici, P. (1978). Science 199,899-902.
3. Lalonde,M. en Calvert, H.E. (1979). In Proc.Workshop on Symbiotic Nitrogen
Fixation inthe Managementof Temperate Forests (J.C. Gordon,C.T.Wheeler and
D.A. Perry, Eds.)pp. 95-110.Forest Research Laboratory, Oregon State
University.
4. Leaf,G., Gardner, I.C.en Bond, G. (1958). J. Exp. Bot. 9,320-331.
5.Miettienen, J.K. en Virtanen,A.I. (1952). Physiol. Plant. 5,540-557.
6. Quispel,A. en Burggraaf,A.J.P. (1981). In Proc.4th Int. Symposium on
Nitrogen Fixation (A.H.Gibson en W.E.Newton, Eds.) pp.229-236.
Australian Academy of Science, Canberra.
Quispel,A. en Tak,T. (1978). New Phytol.81,587-600.
8. Schubert, K.R., Coker III,G.T. en Firestone, R.B. (1981). Plant Physiol.
67, 662-665.
9. Wheeler,C.T. en Bond,G. (1970). Phytochemistry 9, 705-708.
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CURRICULUM VITAE
Deschrijver van dit proefschriftwerd geborenop24augustus 1952
te Rotterdam.In1970behaalde hij het gymnasium-Bdiplomaaan het
Marnix gymnasium aldaar,enbegonmetdestudie biologieaande
Vrije UniversiteitteAmsterdam.In1974werd het kandidaats examen
B4(biologie en-scheikundemet natuurkundeenwiskunde)afgelegd.
In 1977volgde het doktoraal examen (hoofdvak biochemie,bijvakken
radiobiofysicaenmoleculaire genetica).In1978werd begonnenmet
hetinditproefschrift beschreven onderzoek.
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