Lipopolysaccharides
of Pseudomonas
spp.
That
Stimulate
Plant
Growth:
Composition and
Use
for Strain
Identification
LETTY A. DE WEGER,L* B. JANN,2 K. JANN,2AND BEN LUGTENBERG'
Department of Plant Molecular Biology, Botanical Laboratory, 2311VJLeiden, TheNetherlands,' and Max-Planck-Institutfur Immunobiologie, D-7800Freiburg-Zahringen, Federal Republic of Germany2
Received 14October 1986/Accepted 31 December 1986
Theoutermembraneproteins ofaseries offluorescent, root-colonizing,plant-growth-stimulating
Pseudomo-nasspp. having been characterized (L. A.de Wegeretal., J. Bacteriol. 165:585-594, 1986), the
lipopolysac-charides(LPSs) of these strainswereexamined. The chemical composition of the LPSs of the three best-studied
plant-growth-stimulating PseudomonasstrainsWCS358, WCS361,andWCS374andofP.aeruginosaPAO1as areference strainwasdeterminedand appearedtodifferfrom straintostrain. The2,6-dideoxy-2-aminosugar quinovosamine wasthemostabundantcompound in theLPS of strainWCS358. Analysis by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis of purifiedLPSandof proteinase K-treated cell envelopes revealed ladderlikepatternsformostofthese strains. Thesepatternswerenotsubstantiallyinfluencedbydifferencesin culture conditions. Analysis of proteinase K-treated cell envelopes of24 root-colonizing Pseudomonas spp.
revealedaunique bandpatternforeach strain,suggestingagreatvarietyintheLPSstructurespresentin these
rootcolonizers. Therefore, electrophoreticanalysisofLPScanbe usedfor characterization and identification
ofthe fluorescent root-colonizing Pseudomonas strains.
InDutchfieldsfrequentlyplanted withpotatoes,yieldsare
reduced by the accumulation ofdeleterious microorganisms
or their products (28). In pot and field experiments it was
shown that bacterization of potato tubers with selected root-colonizing, fluorescent Pseudomonas spp. diminishes or even abolishes yield reductions (9, 18), presumably in a
siderophore-mediated way (5, 23). Efficient colonization of
thepotato root by the plant-growth-stimulating
Pseudomo-nasstrain is thoughttobe veryimportant for yield increase
in fields (5). Our selected Pseudomonas spp. are efficient
root colonizers, as deduced from the fact that they were
isolated from the surface ofthoroughly washed roots. It is likelythat thebacterial surfaceplaysanimportant role in the
interaction between plant and bacterium. For this and other
reasons(7),we areinterested in the characteristics of the cell
surface of these plant-beneficial Pseudomonas strains. In a previous paper we reported our analysis of the
membrane proteins of 30 fluorescent root-colonizing
Pseu-domonas spp. by sodium dodecyl sulfate
(SDS)-polyacry-lamidegel electrophoresis (7). Asjudged from theirpatterns, includingtheproteins induced by
Fe3"-limited
growth,most strainsweremutually distinguishable. Of these30 strains,24were chosen foruse inthe presentstudy, which is focused
onthelipopolysaccharide (LPS)of these strains.
Research on the bacterial LPS structure in correlation
withthe interaction ofabacterium with plant tissue has been
performed preferentially for interactions of plants with
pathogenic bacteria (3, 11, 33, 35). However, a recent publication on the composition of the LPS of saprophytic
bacteria(4) mightreflectincreasinginterest in thisimportant
group ofsoil bacteria. Our interest in factors that may be
involved in the colonization of the plantroot by the plant-growth-promoting Pseudomonas spp. promptedus to study
inmoredetail the LPSstructureof the three root-colonizing
strains WCS358, WCS361, and WCS374. Therefore the
compositions of the LPSs ofthese three strains and of the
well-studied Pseudomonas aeruginosa strain PAO1 were
* Corresponding author.
compared. Furthermore, the LPS of the 24 strains was
analyzed by SDS-polyacrylamide gel electrophoresis to
study whether the LPS of these Pseudomonas strains is a
well-preserved structure common to root-colonizing
Pseu-domonasspp. orvariesamongthedifferent strains. TheLPS
patterns of all these strains appeared to differ from each other. For this reason analysis of LPS by
SDS-polyacry-lamide gel electrophoresis canbe used for characterization
and identification of these root-colonizing Pseudomonas strains.
MATERIALS AND METHODS
Strains andgrowthconditions. The relevant characteristics of the 24 Pseudomonas strains used in this studyhave been
published (7).Of thesevenstrains thatareprobablyidentical
(7), only strain WCSS358 was used in this study. After
diluting stationary-phasecultures 100-fold into fresh culture
medium, cells weregrownfor64 h undervigorous aeration at 28°C. The following culture mediawere used. The
com-positionofKing B medium, an Fe3+-deficientmedium, has been described previously (17). When required, 100 ,uM FeCl3 was added. Minimal salts medium (30) was
supple-mented either with1%glucoseasthe carbonsource orwith root exudate from axenically cultivated potato plants. For the isolation ofLPS, astationary-phase culture was diluted 100-fold in fresh King B medium and cultivated for 24 hat 280C.
Cocultivation of bacteria withsterile potatoplantlets. Ster-ile potato plantlets of the potato cultivar Bintje were ob-tained from G. S. Bokelman, ITAL Research Institute, Wageningen, The Netherlands. Plantlets were cultivated in culture vessels(typeGA7; Magenta Corp., Chicago, Ill.) on
medium as described by Murashige and Skooge (24), final
pH 5.8, supplementedwith2.0%sucroseand solidifiedwith 0.8% agar. The culture vessels were placed in a growth
chamberat28°Cwithaday lengthof14 h. Prior to cocultiva-tion of plantlets and bacteria, eight sterile plantlets were
placed on a metal grid and cultivated on 100 ml ofliquid Murashige-Skooge medium. After 1 week the mediumwas
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TABLE 1. Composition of LPS from strains WCS358, WCS361, WCS374, andP. aeruginosa PAO1
Yield Composition of LPS (% by wt, mean±SD)a
Strain
celsof
bmpg
KDOb
Heptoseb
P'
Glucosec Mannosec FucosecRhamnosec
GGlucosaluoa
mied mine Galactos-Gamined
vosamined
Quino-d
Alanined
cells) mine phosphated
PA01 55 2.4±0.2 3.1 ±0.7 1.3 ±0.3 5.0± 0.8 0 0 3.3±0.1 NDe ND ND ND ND WCS358 25 3.1 ± 0.3 2.0± 0.5 1.3 ± 0.2 9.6± 0.8 0 0 0 5.9 1.7 2.0 15-18f 0.8 WCS361 50 2.9 ±0.4 2.1 ± 0.4 1.8 ± 0.3 4.3 ± 0.2 0.6±0.1 0 0.9±0.1 7.8 2.0 2.5 0 1.2 WCS374 5 1.4 ±0.1 3.2 ± 0.5 0.8 ± 0.1 9.5± 0.2 0 1.9±0.2 0.6 ±0.0 1.7 0.2 0 0 0.79
aAt least two determinations. bDeterminedcolorimetrically.
cDetermined by gas-liquid chromatography.
dSingle determination on an amino acid analyzer.
eND, Not determined.
fEstimated from the peak integral.
gBesides alanine, glycine (0.8%) was detected in the LPS of strainWCS374 and was predominantly associated with the lipid A fraction.
replaced by bacterialminimal salts medium (30) without any
carbon source. A 100-fold-diluted stationary-phase culture of bacteriawascocultivated with thepotatoplantrootsfor3
daysat28°C undergentlerotation.Theopticaldensityofthe
resulting bacterial suspensions variedfrom 0.6 to 1.0. Isolation of LPS and cell envelopes. Cells were washed
once with PBS
(0.9%
NaCl buffered with 10 mM sodium phosphate,pH 7.2) and lyophilized.LPS wasisolated either after extraction of the cells with hot phenol-water asde-scribed by Westphal andJann (32) orby successive
Mg2+
and ethanolprecipitations after solubilizing the membranes with 2% SDS as described by Darveau and Hancock (6).
Contaminating nucleic acidsweredeterminedbyUV absor-bance. Cell envelopeswereisolated by differential centrifu-gation after disruption of the cells by ultrasonictreatment
(22).
SDS-polyacrylamide gel electrophoresis. Samples were
solubilized by incubation for 15 minat95°Cinthe standard sample mixture described previously (22). Solubilized cell envelope samples, containing approximately 1 mg of cell envelope proteinperml,weresupplementedwithproteinase
K (13)to afinal concentration of 50 ,ug/ml and incubated at
56°C
for 1 h. Fifteenmicroliters of the 10-fold-dilutedsam-ples was appliedper slot. The gel system described previ-ously (22) was used, except thatgels contained
13%
poly-acrylamide instead of11%.
Fast green (22) was used for staining proteinase K-resistant protein fragments, while LPSwas stained by the silver-staining procedure described by Tsai and Frasch (29).
Sugaranalysis of LPS. Toliberate thecarbohydrate moiety (core and
0-antigenic
sidechain) fromlipid
A,smallamounts(1to 3 mg)of LPSwerehydrolyzed in1 M
HCl
at100°C for15 min. Centrifugation at 10,000 x gfor 15minresultedin
separation of lipidA(pelletfraction) from the carbohydrate moiety
(supernatant
fraction).Foranalysis ofneutral sugarsby gas-liquid chromatogra-phy, LPS was hydrolyzed in 2 N trifluoroacetic acid by incubation for 1 h at 120°C. The sugars were converted to
their alditol acetate derivatives (1) and analyzed by
gas-liquid chromatography at 180°C with a gas chromatograph
(Becker model 420) with a glass column packed with 3% ECNSS-M on Chromosorb Q (Applied Science Laborato-ries) and equipped withanintegrator (ShimadzuC-R1B).
Forthin-layer chromatography, LPSwas hydrolyzed in 1 M
HCl
(neutral sugars)or 6 MHCl
(aminosugars)at 100°C for4h. Thehydrolysates werelyophilized and dissolvedindemineralized water. Approximately 20 ,ug ofhydrolyzed
LPS wasspotted onKieselguhr SilicaGel G plates (Merck,
Darmstadt). For resolving amino compounds,
chromato-grams were developed in solvent system 1 (pyridine-ethyl acetate-acetic acid-water, 35:35:7:21 by vol) and stained with ninhydrin (31). For resolving neutral sugars, solvent
system 1 or 2 (acetone-chloroform-water, 85:10:5 by vol)
was used to develop the chromatograms, and spots were
detectedbyusingan
aniine-phthalate
spray(31).Foridentification of themostabundantaminosugar inthe
LPSof strainWCS358, the hydrolyzed LPSwasanalyzedby
paperelectrophoresison2043 paper
(Schleicher
& Schuell) inpyridine-acetic acid-water (10:4:86, by vol), pH 5.4, at40V/cm. The aminosugar was identified by using the
Elson-Morgan reagent(26) and afterperiodate treatmentbyusing
theEdwards and Waldronreagent(8).
Amino acids and aminosugars were quantitatively
ana-lyzed after
hydrolysis
in4 M HClfor 18 h andsubjectedtoamino acid analysis in a Chromakon 500 amino acid
ana-lyzer. Since
quinovosamine
was not available as a pure sugar, the value for the amount of quinovosamine wasestimated fromthepeak integral.
Other analytical procedures. Heptose was determined as
describedby Wrightand Reber(34),with manno-heptulose
asthereference. 2-Keto-3-deoxyoctanate (KDO)was
mea-suredby the thiobarbituric acid method (16), with
commer-cial KDO (Sigma Chemical Co., St. Louis, Mo.) as a
standard.Phosphatewasassayedasdescribed
by
Amesand Dubin (2).RESULTS
Isolation of LPS. LPS of strains WCS358, WCS361, WCS374, and P. aeruginosa PAO1 wasisolatedby the hot
phenol-water method (32) and
by
the method describedby
Darveau and Hancock(6). Forthe strains understudy
the latter methodprovedtobesuperior
in bothyield
andpurity.
Thisprocedure yielded5 to55mgofLPSper gofcells(dry weight), depending on the strain (Table 1), with strain WCS374 always giving the lowest yield.Contaminating
nucleic acids never exceeded 1%. In
addition,
noprotein
could be detected in LPS
preparations
ofstrainsWCS358,
WCS361, and WCS374when examined
by
SDS-polyacryl-amide gel
electrophoresis
followedby
fast greenstaining.
Since this
staining
method revealsprotein
bands of 0.5jxg
ormore, we concluded that the percentage of
contaminating
polypeptide
was less than 0.5%by weight.
In the LPSpreparationofstrain
PAQ1
avague elongated band consist-ing ofa(proteinaseK-resistant)polypeptide fragment(s)
wasdetectable in
fast-green-stained
gels,corresponding
to anestimated
polypeptide
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FIG. 1. Band patternsofsilver-stained preparations of
protein-ase K-treated cell envelopes (left lanes) and purified LPS (right lanes) after analysis by SDS-polyacrylamide gel electrophoresis. Arrows indicate bands that canalso be visualized with fastgreen
staining and which therefore presumably represent protein
frag-mentsresistanttoproteinaseK. Forstrain WCS361 the middleand lower part of the LPS profile is not identical to the proffle of proteinase K digests. Similar differences in thispartof theprofile
wereobserved among different proteinase K digests of cell
enve-lopes of this strain(comparetheleft lane for strainWCS361of this figure with lane 18 in Fig. 3).
LPS preparations stained after SDS-polyacrylamide gel electrophoresis showed different ladderlikepatternsforeach strain (Fig. 1). Cell envelopes treated with proteinase K revealed thesamepatternsaspurified LPS,exceptforsome
extra bands (indicated by arrows in Fig. 1). These extra
bandswerealsoobserved in fast-green-stained gels,
indicat-ing that these bands areproteinase K-resistantpolypeptide
fragments. Proteinase K-resistant bandswerealsoobserved
inproteinase K digests of Coxiella burnetii cells (12). Composition of LPS.Colorimetric determinations showed the presence of various amounts of KDO, heptose, and phosphate in the LPS of strains WCS358, WCS361,
WCS374,and P. aeruginosaPAO1 (Table 1). Analysis of the LPS by gas-liquid chromatography revealed differences in neutralsugarcompositionamongthe various strains (Table
1). Our results confirmed previousones(19) whichindicated
that the LPS of P. aeruginosa contains glucose and rhamnose. Glucose was present in all three plant-root-colonizing Pseudomonas strains (Table 1). Besides glucose,
noneutral sugars weredetected in strainWCS358, while in
strain WCS361 low levels ofmannose and rhamnose were
detected(Table 1). In strainWCS374glucoseaswellasthe
two 6-deoxysugars, rhamnose and fucose, were present (Table 1).
Analysis of the amino compounds indicated thepresence
ofalanine,glucosamine, and itsphosphorylatedderivative in
the LPS of all three root-colonizing strains and of galactosamine in strains WCS358 and WCS361 (Table 1). Furthermore, the LPS of WCS358 contained another aminosugar as the most abundant constituent. In paper
electrophoresis this aminosugar had a mobility relative to glucosamine
(MGICN)
of 1.06. It could be stained with the Elson-Morgan reagent, which is indicative ofa2-deoxy-2-aminosugar, and afterperiodatetreatmentwith the Edwards andWaldronreagent,which is indicative ofa6-deoxygroup. Thus, the aminosugarwas probably a
2,6-dideoxy-2-amino
sugar. Itelutedfrom the amino acidanalyzer withanelutiontime relative to glucosamine (tGICN) of 1.126. This was
identical with the elution time of
2,6-dideoxy-2-amino-glucose (quinovosamine;tGlcN,
1.123) and different fromthoseofrhamnosamine
(tGIcN,
1.088) andfucosamine(tGlCN,
1.178). These results indicate that the LPS from WCS358 contains, in addition to
glucosamine,
2,6-dideoxy-2-amino-glucose(quinovosamine).
The presenceofglucosamine phosphate inhydrolysates of LPS indicates incomplete
hydrolysis
of thisconstituent,
which ischaracteristic of lipidA. Toassessthedistribution of the aminosugars between
lipid
A and thecarbohydrate
moiety (core and
0-antigenic
side chain), both of these fractions were analyzed for aminosugars. Alllipid
A frac-tions containedglucosamine
andglucosamine phosphate
and practicallynoother aminosugars.Thecarbohydrate fraction from strain WCS374 contained onlyglucosamine,
thatfrom strain WCS361containedglucosamine
andgalactosamine
in the molar ratio of 1:1, and that from strain WCS358con-tained
galactosamine
andquinovosamine
in the molar ratio of1:10. Alanine waspredominantly
found in thecarbohy-drate
moiety
of these strains.Influence of culture conditions on the LPS patterns in silver-stained gels. Cell envelopes of strains
WCS358,
WCS361, and WCS374 were treated with
proteinase
K.Growth in minimal medium with either glucose or root
exudate from sterile potato plants as the carbon source
resulted in LPS
patterns indistinguishable
from those ob-served aftergrowth
inKing
B medium. Neitheraddition of 100 ,uMFeCl3
to the medium nor variation in growthtemperaturefrom4to 44°C alteredthe LPS ladder
patterns
significantly (data not shown). Also, the growth phase at
28°C had no effect on the LPS
patterns
ofstrains WCS361 and WCS374. An extension of the ladder pattern in thehigh-molecular-weight
region of the gel was observed for strain WCS358 when cells arrived at the stationary phase(Fig.
2).
LPS
patterns
of a collection of antagonistic Pseudomonas root isolates.Cellenvelopes of24Pseudomonasrootisolates (7) were treated with proteinase K, and the LPS specieswere electrophoretically separated and stained with the silver reagent. The
majority
of the root isolates showed ladderlike LPSpatterns
(Fig. 3). Instead of discrete bands,elongated
spotswereobserved in the LPSpatterns of strains WCS312, WCS317, WCS324, and E6. Only onestrain,
WCS429, showedapattern consistent with LPSlacking the
0-antigenic
sidechain.Each ofthese 24 strains showed a unique LPS pattern, exceptfor strains WCS374 and WCS375, which are
colony
variants (7). Strain WCS358 and the six strains (WCS345, WCS348,WCS357,
WCS359,
WCS360,WCS364) mentioned ina previouspaper(7)as mostlikely being descendants ofoneancestor had
indistinguishable
LPSpatterns.
DISCUSSION
Compositionof LPS. Ourresults on the
composition
ofthe LPS ofP. aeruginosaPAO1
(Table 1) were very similartothose reported by Kropinski et al. (19), except for the
phosphorus content, which was lower in our assays. The
relative amounts of KDO andheptose
(Table
1) wereverysimilarinthethree
root-colonizing
Pseudomonasstrains andP. aeruginosaPAO1 (2.5to3% and 2 to
3%, respectively),
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12
FIG. 2. Silver-stained LPS patterns of cell envelopes of strain WCS358 treated with proteinase K after growth for 64 h (lane 1) or 8 h (lane 2) in King B medium. The additional weak band in the middle of the ladderpattern in lane 2 was not observed in other proteinase K digests of these cell envelopes.
exceptthat theKDOcontent instrain WCS374wasslightly lower (1.4%). Glucose was present as the major neutral sugarin each of these three strains.Lowlevels ofmannose
and rhamnose weredetected in the LPS of strainWCS361, whereas fucose and rhamnose were found in the
LPS
of strainWCS374
(Table1). TheLPS of strainWCS358 didnotcontain other neutral sugars.
Analysis
of the aminocompounds revealed that the lipidAfrom strains WCS358, WCS361, and WCS374 contained glucosamine and its phosphorylated derivative butnoother amninosugars. The aminosugar composition of the carbohy-drate moiety (core and
0-antigenic
side chain) differedamongthe strains(Table 1).InstrainWCS358,the verylow
relative
contentof galactosamine makes it difficulttoenvis-age both galactosamine and quinovosamine as part of the repeating unit. Since the carbohydrate fraction consists of
0-antigenic
sidechain
linked to the core, the possibility exists that in contrast to quinovosamine, galactosamine is not aconstituent of the0-antigenic side chain but of thecoreof the LPS of strainWCS358.
Quinovosamine
has been reported to be present in theLPS
ofmany other bacterialspecies (e.g.,
someP. aerugi-nosa strains [20], Salmonella spp., Proteus vulgaris [21], Vibrio cholerae [14], and Rhizobium legu'minosarum [27]).In the LPS ofP. aeruginosa PAO, another
2,6-dideoxy-2-aminohexose,
fucosamine (2,6-dideoxy-2-aminogalactose),wasfound (17). Likethe quinovosamine in strainWCS358,
the
2,6-dideoxy-2-aminosugars
in the LPS of several P. aeruginosa strains were shown to be constituents ofthe0-antigenic
sidechain
(19, 20).Anderson(4) detected both rhamnose and glucose in both
pathogenic(Pseudomon,assyringae strains) and saprophytic
Pseudomonas
spp.(P.
fluorescens
and P.aeruginosa).
SaprophyticP.
putida
strains, whicharedistinguished
from other Pseudomonas spp. by their agglutination by aplantarabinogalactan
protein complex (15), showedaunique LPS composition as (i) they contained glucose as the major neutralsugar,(ii) they hadahigh
ratio of aminoover neutralsugars, and (iii) they lacked rhamnose and fucose (4). We found a similar result for strain WCS358.
However,
the features mentioned do not seem to be a general property of the LPS of saprophytic P. putida strains, since the LPS of the other P. putida strain in our study, WCS361, neither contained high levels of aminosugars nor lacked rhamnose. Fucose, reported to be present in P. fluorescens and P. syringae strains (4), was also found in the plant-growth-promoting P. fluorescens strain WCS374.In conclusion, the composition of the LPS of the
plant-growth-promoting Pseudomonas strains is comparable to that ofothergram-negative bacteria. No common features were found in their LPSs, suggesting that the LPSs of Pseudomonas spp. isolated from the roots of potato plants
do
notsharespecific
characteristics.LPS patterns of plant-growth-promoting Pseudomonas spp. Analysis by SDS-polyacrylamide gel electrophoresis
re-vealed thesameladderlike patterns for purified LPS and for
cellenvelopes treated with proteinase K. Since the latter are
faster and easiertoobtain than purified LPS,weusedthese
preparationstostudytheinfluence of various growth
condi-tionsontheLPSpatternsandtostudy the LPS patterns of24
fluorescent root-colonizingPseudomonas strains. No influ-enceofvarying the growth conditionswasobserved, except
thatfor strain WCS358aslight increaseinthe ladderpattern
in the high-molecular-weight part of the gel was detected whenthecellsenteredthe stationaryphase (Fig. 2).
Appar-ently a slight change in the size distribution of the LPS
moleculesinfavor ofLPSmolecules with increasinglength
of the
0-antigenic
side chains took place. Analysis of cell envelopes treatedwithproteinaseKresulted formostof the 24 strains inladderlike patterns (Fig. 3). This multitude of bandsobserved in the LPS patterns issupposedtobe duetoLPS molecules having varying lengths of 0-antigenic side
chains (10, 25). For each strain a different pattern was
observed, except WCS374 andWCS375 (Fig. 3),which are
colony variants (7). This result showed that the LPSs of
1 2 3 4 5 6 7 8 9 10 11 1213 141516 17 18 19 20 21 22 23
FIG. 3. Silver-stainedpatternsofproteinaseK-treated cell enve-lopes obtained after SDS-polyacrylamide gel electrophoresis. Lanes:1, WCS358; 2,Al;3,WCS141; 4, WCS312; 5,WCS317;6,
WCS321; 7, WCS327; 8, WCS374; 9, WCS375; 10, WCS007; 11, WCS085;12,WCS134;13,WCS307;14,WCS314; 15,WCS315; 16,
WCS324; 17,WCS326;18,WCS361;19,WCS365;20,WCS366;21,
WCS379; 22, WCS429;23, E6. Arrows indicate protein fragments
resistanttoproteinase K. Fordetailssee thelegendtoFig. 1.
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root-colonizingPseudomonas strainsarenotwell-preserved structures.
Previously we reported the analysis of the membrane
proteins of the Pseudomonas strains used in this study (7). Mostof thesestrainsweremutuallydistinguishable by their
membrane protein pattern. However, a few strains were
hardtodiscriminatebytheir proteinpatterns(e.g.,thepairs WCS358 and Al and WCS141 and WCS312 [7]). Analysis of the LPSs of these strains revealed that theywere actually
distinct. Since we showed that the LPS patterns of the Pseudomonas strains testedarenotsubstantiallyinfluenced
byculture conditions and that the ladderpatternsare unique
foreach of these strains, they canbe used to identifyeach individual strain. Therefore LPS patterns, in combination with the membrane proteinpatterns(7), provideapowerful
toolto accurately identify these fluorescent root-colonizing
Pseudomonas spp., e.g., reisolates from fieldexperiments.
ACKNOWLEDGMENTS
Wethank Liavander Vlugt and Harold Klaassen for technical
assistance.
Theseinvestigationsweresupported by the Netherlands
Technol-ogyFoundation (STW).
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