Vol. 169, No. 4
Lipopolysaccharides of Pseudomonas spp. That Stimulate Plant
Growth: Composition and Use for Strain Identification
LETTY A. DE WEGER,L* B. JANN,2 K. JANN,2 AND BEN LUGTENBERG'
Department ofPlant Molecular Biology, Botanical Laboratory, 2311VJ Leiden, The Netherlands,' and
Max-Planck-Institut fur Immunobiologie, D-7800 Freiburg-Zahringen, Federal Republic of Germany2
Received 14 October 1986/Accepted 31 December 1986
The outer membrane proteins of a series of fluorescent, root-colonizing, plant-growth-stimulating Pseudomo-
nas spp. having been characterized (L. A. de Weger et al., J. Bacteriol. 165:585-594, 1986), the lipopolysac-
charides (LPSs) of these strains were examined. The chemical composition of the LPSs of the three best-studied
plant-growth-stimulating Pseudomonas strains WCS358, WCS361, and WCS374 and ofP. aeruginosa PAO1 as
a reference strain was determined and appeared to differ from strain to strain. The 2,6-dideoxy-2-aminosugar
quinovosamine was the most abundant compound in the LPS of strain WCS358. Analysis by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of purified LPS and of proteinase K-treated cell envelopes revealed
ladderlike patterns for most of these strains. These patterns were not substantially influenced by differences in
culture conditions. Analysis of proteinase K-treated cell envelopes of 24 root-colonizing Pseudomonas spp.
revealed a unique band pattern for each strain, suggesting a great variety in the LPS structures present in these
root colonizers. Therefore, electrophoretic analysis of LPS can be used for characterization and identification
of the fluorescent root-colonizing Pseudomonas strains.
In Dutch fields frequently planted with potatoes, yields are
reduced by the accumulation of deleterious 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
the potato root by the plant-growth-stimulating Pseudomo-
nas strain is thought to be very important 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 of thoroughly washed roots. It is
likely that the bacterial surface plays an important role in the
interaction between plant and bacterium. For this and other
reasons (7), we are interested in the characteristics ofthe 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-
lamide gel electrophoresis (7). Asjudged from their patterns,
including the proteins induced by Fe3"-limited growth, most
strains were mutually distinguishable. Of these 30 strains, 24
were chosen for use in the present study, which is focused
on the lipopolysaccharide (LPS) of these strains.
Research on the bacterial LPS structure in correlation
with the interaction of a bacterium with plant tissue has been
performed preferentially for interactions of plants with
pathogenic bacteria (3, 11, 33, 35). However,
publication on the composition of the LPS of saprophytic
bacteria (4) might reflect increasing interest in this important
group of soil bacteria. Our interest in factors that may be
involved in the colonization of the plant root by the plant-
growth-promoting Pseudomonas spp. prompted us to study
in more detail the LPS structure of the three root-colonizing
strains WCS358, WCS361, and WCS374. Therefore the
compositions of the LPSs of these three strains and of the
well-studied Pseudomonas aeruginosa strain PAO1 were
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-
domonas spp. or varies among the different strains. The LPS
patterns of all these strains appeared to differ from each
other. For this reason analysis of LPS by SDS-polyacry-
lamide gel electrophoresis can be used for characterization
and identification of these root-colonizing Pseudomonas
MATERIALS AND METHODS
Strains and growth conditions. The relevant characteristics
of the 24 Pseudomonas strains used in this study have been
published (7). Ofthe seven strains that are probably identical
(7), only strain WCSS358 was used in this study. After
diluting stationary-phase cultures 100-fold into fresh culture
medium, cells were grown for 64 h under vigorous aeration
at 28°C. The following culture media were used. The com-
position of King B medium, an Fe3+-deficient medium, has
been described previously (17). When required, 100 ,uM
FeCl3 was added. Minimal salts medium (30) was supple-
mented either with 1% glucose as the carbon source or with
root exudate from axenically cultivated potato plants. For
the isolation of LPS, a stationary-phase culture was diluted
100-fold in fresh King B medium and cultivated for 24 h at
Cocultivation of bacteria with sterile potato plantlets. 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 (type GA7; Magenta Corp., Chicago, Ill.) on
medium as described by Murashige and Skooge (24), final
pH 5.8, supplemented with 2.0% sucrose and solidified with
0.8% agar. The culture vessels were placed in a growth
chamber at 28°C with a day length of 14 h. Prior to cocultiva-
tion of plantlets and bacteria, eight sterile plantlets were
placed on a metal grid and cultivated on 100 ml of liquid
Murashige-Skooge medium. After 1 week the medium was
JOURNAL OF BACTERIOLOGY, Apr. 1987, p. 1441-1446
Copyright © 1987, American Society for Microbiology
1442 DE WEGER ET AL.
TABLE 1. Composition of LPS from strains WCS358, WCS361, WCS374, and P. aeruginosa PAO1
Composition of LPS (% by wt, mean±SD)a
2.4 ± 0.2 3.1 ± 0.7 1.3 ± 0.3 5.0 ± 0.8
3.1 ± 0.3 2.0 ± 0.5 1.3 ± 0.2 9.6 ± 0.8
2.9 ± 0.4 2.1 ± 0.4 1.8 ± 0.3 4.3 ± 0.2 0.6 ± 0.1
1.4 ± 0.1 3.2 ± 0.5 0.8 ± 0.1 9.5 ± 0.2
3.3 ± 0.1
0.9 ± 0.1
50 1.9 ± 0.2 0.6 ± 0.0
aAt least two determinations.
cDetermined by gas-liquid chromatography.
dSingle determination on an amino acid analyzer.
eND, Not determined.
fEstimated from the peak integral.
g Besides alanine, glycine (0.8%) was detected in the LPS of strain WCS374 and was predominantly associated with the lipid A fraction.
replaced by bacterial minimal salts medium (30) without any
carbon source. A 100-fold-diluted stationary-phase culture
ofbacteria was cocultivated with the potato plant roots for 3
days at 28°C under gentle rotation. The optical density ofthe
resulting bacterial suspensions varied from 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 was isolated either
after extraction of the cells with hot phenol-water as de-
scribed by Westphal and Jann (32) or by successive Mg2+
and ethanol precipitations after solubilizing the membranes
with 2% SDS as described by Darveau and Hancock (6).
Contaminating nucleic acids were determined by UV absor-
bance. Cell envelopes were isolated by differential centrifu-
gation after disruption of the cells by ultrasonic treatment
SDS-polyacrylamide gel electrophoresis. Samples were
solubilized by incubation for 15 min at 95°C in the standard
sample mixture described previously (22). Solubilized cell
envelope samples, containing approximately 1 mg of cell
envelope protein per ml, were supplemented with proteinase
K (13) to a final concentration of 50 ,ug/ml and incubated at
56°C for 1 h. Fifteen microliters of the 10-fold-diluted sam-
ples was applied per slot. The gel system described previ-
ously (22) was used, except that gels contained 13% poly-
acrylamide instead of 11%. Fast green (22) was used for
staining proteinase K-resistant protein fragments, while LPS
was stained by the silver-staining procedure described by
Tsai and Frasch (29).
Sugar analysis ofLPS. To liberate the carbohydrate moiety
(core and 0-antigenic sidechain) from lipid A, small amounts
(1 to 3 mg) of LPS were hydrolyzed in 1 M HCl at 100°C for
15 min. Centrifugation at 10,000 x g for 15 min resulted in
separation of lipid A (pellet fraction) from the carbohydrate
moiety (supernatant fraction).
For analysis of neutral sugars by 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 with an integrator (Shimadzu C-R1B).
For thin-layer chromatography, LPS was hydrolyzed in 1
M HCl (neutral sugars) or 6 M HCl (aminosugars) at 100°C
for 4 h. The hydrolysates were lyophilized and dissolved in
demineralized water. Approximately 20 ,ug of hydrolyzed
LPS was spotted on Kieselguhr Silica Gel 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
detected by using an aniine-phthalate spray (31).
For identification of the most abundant aminosugar in the
LPS of strain WCS358, the hydrolyzed LPS was analyzed by
paper electrophoresis on 2043 paper (Schleicher & Schuell)
in pyridine-acetic acid-water (10:4:86, by vol), pH 5.4, at 40
V/cm. The aminosugar was identified by using the Elson-
Morgan reagent (26) and after periodate treatment by using
the Edwards and Waldron reagent (8).
Amino acids and aminosugars were quantitatively ana-
lyzed after hydrolysis in 4 M HCl for 18 h and subjected to
amino 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 was
estimated from the peak integral.
Other analytical procedures. Heptose was determined as
described by Wright and Reber (34), with manno-heptulose
as the reference. 2-Keto-3-deoxyoctanate (KDO) was mea-
sured by the thiobarbituric acid method (16), with commer-
cial KDO (Sigma Chemical Co., St. Louis, Mo.) as a
standard. Phosphate was assayed as described by Ames and
Isolation of LPS. LPS of strains WCS358, WCS361,
WCS374, and P. aeruginosa PAO1 was isolated by the hot
phenol-water method (32) and by the method described by
Darveau and Hancock (6). For the strains under study the
latter method proved to be superior in both yield and purity.
This procedure yielded 5 to 55 mg of LPS per g of cells (dry
weight), depending on the strain (Table 1), with strain
WCS374 always giving the lowest yield. Contaminating
nucleic acids never exceeded 1%. In addition, no protein
could be detected in LPS preparations of strains WCS358,
WCS361, and WCS374 when examined by SDS-polyacryl-
amide gel electrophoresis followed by fast green staining.
Since this staining method reveals protein bands of0.5 jxg or
more, we concluded that the percentage of contaminating
polypeptide was less than 0.5% by weight. In the LPS
preparation of strain PAQ1 a vague elongated band consist-
ing of a (proteinase K-resistant) polypeptide fragment(s) was
detectable in fast-green-stained gels, corresponding to an
estimated polypeptide contamination of 1 to 2%.
LPS OF PSEUDOMONAS SPP.
FIG. 1. Band patterns of silver-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 can also be visualized with fast green
staining and which therefore presumably represent protein frag-
ments resistant to proteinase K. For strain WCS361 the middle and
lower part of the LPS profile is not identical to the proffle of
proteinase K digests. Similar differences in this part of the profile
were observed among different proteinase K digests of cell enve-
lopes of this strain (compare the left lane for strain WCS361 of this
figure with lane 18 in Fig. 3).
LPS preparations stained after SDS-polyacrylamide gel
electrophoresis showed different ladderlike patterns for each
strain (Fig. 1). Cell envelopes treated with proteinase K
revealed the same patterns as purified LPS, except for some
extra bands (indicated by arrows in Fig. 1). These extra
bands were also observed in fast-green-stained gels, indicat-
ing that these bands are proteinase K-resistant polypeptide
fragments. Proteinase K-resistant bands were also observed
in proteinase 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. aeruginosa PAO1 (Table 1). Analysis ofthe
LPS by gas-liquid chromatography revealed differences in
neutral sugar composition among the various strains (Table
1). Our results confirmed previous ones (19) which indicated
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,
no neutral sugars were detected in strain WCS358, while in
strain WCS361 low levels of mannose and rhamnose were
detected (Table 1). In strain WCS374 glucose as well as the
two 6-deoxysugars, rhamnose and fucose, were present
Analysis of the amino compounds indicated the presence
ofalanine, glucosamine, and its phosphorylated derivative 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 of a 2-deoxy-2-
aminosugar, and after periodate treatment with the Edwards
and Waldron reagent, which is indicative ofa 6-deoxy group.
Thus, the aminosugar was probably a 2,6-dideoxy-2-amino
sugar. It eluted from the amino acid analyzer with an elution
time 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 from
those of rhamnosamine(tGIcN, 1.088) and fucosamine (tGlCN,
1.178). These results indicate that the LPS from WCS358
contains, in addition to glucosamine, 2,6-dideoxy-2-amino-
The presence ofglucosamine phosphate in hydrolysates of
LPS indicates incomplete hydrolysis of this constituent,
which is characteristic of lipid A. To assess the distribution
of the aminosugars between lipid A and the carbohydrate
moiety (core and 0-antigenic side chain), both of these
fractions were analyzed for aminosugars. All lipid A frac-
tions contained glucosamine and glucosamine phosphate and
practically no other aminosugars. The carbohydrate fraction
from strain WCS374 contained only glucosamine, that from
strain WCS361 contained glucosamine and galactosamine in
the molar ratio of 1:1, and that from strain WCS358 con-
tained galactosamine and quinovosamine in the molar ratio
of 1:10. Alanine was predominantly found in the carbohy-
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 after growth in King B medium. Neither addition of
100 ,uM FeCl3 to the medium nor variation in growth
temperature from 4 to 44°C altered the LPS ladder patterns
significantly (data not shown). Also, the growth phase at
28°C had no effect on the LPS patterns of strains WCS361
and WCS374. An extension of the ladder pattern in the
high-molecular-weight region of the gel was observed for
strain WCS358 when cells arrived at the stationary phase
LPS patterns of a collection of antagonistic Pseudomonas
root isolates. Cell envelopes of24 Pseudomonas root isolates
(7) were treated with proteinase K, and the LPS species
were electrophoretically separated and stained with the
silver reagent. The majority of the root isolates showed
ladderlike LPS patterns (Fig. 3). Instead of discrete bands,
elongated spots were observed in the LPS patterns of strains
WCS312, WCS317, WCS324, and E6. Only one strain,
WCS429, showed a pattern consistent with LPS lacking the
0-antigenic side chain.
Each of these 24 strains showed a unique LPS pattern,
except for strains WCS374 and WCS375, which are colony
variants (7). Strain WCS358 and the six strains (WCS345,
WCS348, WCS357, WCS359, WCS360, WCS364) mentioned
in a previous paper (7) as most likely being descendants of
one ancestor had indistinguishable LPS patterns.
Composition of LPS. Our results on the composition of the
LPS of P. aeruginosa PAO1 (Table 1) were very similar to
those reported by Kropinski et al. (19), except for the
phosphorus content, which was lower in our assays. The
relative amounts of KDO and heptose (Table 1) were very
similar in the three root-colonizing Pseudomonas strains and
P. aeruginosa PAO1 (2.5 to 3% and 2 to 3%, respectively),
VOL. 169, 1987
1444DE WEGER ET AL.
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 ladder pattern in lane 2 was not observed in other
proteinase K digests of these cell envelopes.
except that the KDO content in strain WCS374 was slightly
lower (1.4%). Glucose was present as the major neutral
sugar in each of these three strains. Low levels of mannose
and rhamnose were detected in the LPS of strain WCS361,
whereas fucose and rhamnose were found in the LPS of
strain WCS374 (Table 1). The LPS of strain WCS358 did not
contain other neutral sugars.
Analysis ofthe amino compounds revealed that the lipid A
from strains WCS358, WCS361, and WCS374 contained
glucosamine and its phosphorylated derivative but no other
amninosugars. The aminosugar composition of the carbohy-
drate moiety (core and 0-antigenic side chain) differed
among the strains (Table 1). In strain WCS358, the very low
relative content of galactosamine makes it difficult to envis-
age both galactosamine and quinovosamine as part of the
repeating unit. Since the carbohydrate fraction consists of
0-antigenic side chain linked to the core, the possibility
exists that in contrast to quinovosamine, galactosamine is
not a constituent ofthe 0-antigenic side chain but ofthe core
of the LPS of strain WCS358.
Quinovosamine has been reported to be present in the
LPS of many other bacterial species (e.g., some P. aerugi-
nosa strains , Salmonella spp., Proteus vulgaris ,
Vibrio cholerae , and Rhizobium legu'minosarum ).
In the LPS of P. aeruginosa PAO, another 2,6-dideoxy-2-
aminohexose, fucosamine (2,6-dideoxy-2-aminogalactose),
was found (17). Like the quinovosamine in strain WCS358,
the 2,6-dideoxy-2-aminosugars in the LPS of several P.
aeruginosa strains were shown to be constituents of the
0-antigenic side chain (19, 20).
Anderson (4) detected both rhamnose and glucose in both
pathogenic (Pseudomon,as syringae strains) and saprophytic
Pseudomonas spp. (P. fluorescens and P. aeruginosa).
Saprophytic P. putida strains, which are distinguished from
other Pseudomonas spp. by their agglutination by a plant
arabinogalactan protein complex (15), showed a unique LPS
composition as (i) they contained glucose as the major
neutral sugar, (ii) they had a high ratio ofamino over neutral
sugars, 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 of other gram-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 not share specific characteristics.
LPS patterns of plant-growth-promoting Pseudomonas spp.
Analysis by SDS-polyacrylamide gel electrophoresis re-
vealed the same ladderlike patterns for purified LPS and for
cell envelopes treated with proteinase K. Since the latter are
faster and easier to obtain than purified LPS, we used these
preparations to study the influence of various growth condi-
tions on the LPS patterns and to study the LPS patterns of24
fluorescent root-colonizing Pseudomonas strains. No influ-
ence of varying the growth conditions was observed, except
that for strain WCS358 a slight increase in the ladder pattern
in the high-molecular-weight part of the gel was detected
when the cells entered the stationary phase (Fig. 2). Appar-
ently a slight change in the size distribution of the LPS
molecules in favor of LPS molecules with increasing length
of the 0-antigenic side chains took place. Analysis of cell
envelopes treated with proteinase K resulted for most of the
24 strains in ladderlike patterns (Fig. 3). This multitude of
bands observed in the LPS patterns is supposed to be due to
LPS molecules having varying lengths of 0-antigenic side
chains (10, 25). For each strain a different pattern was
observed, except WCS374 and WCS375 (Fig. 3), which are
colony variants (7). This result showed that the LPSs of
3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
FIG. 3. Silver-stained patterns ofproteinase K-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
resistant to proteinase K. For details see the legend to Fig. 1.
LPS OF PSEUDOMONAS SPP.
root-colonizing Pseudomonas strains are not well-preserved
Previously we reported the analysis of the membrane
proteins of the Pseudomonas strains used in this study (7).
Most of these strains were mutually distinguishable by their
membrane protein pattern. However, a few strains were
hard to discriminate by their protein patterns (e.g., the pairs
WCS358 and Al and WCS141 and WCS312 ). Analysis of
the LPSs of these strains revealed that they were actually
distinct. Since we showed that the LPS patterns of the
Pseudomonas strains tested are not substantially influenced
by culture conditions and that the ladder patterns are unique
for each of these strains, they can be used to identify each
individual strain. Therefore LPS patterns, in combination
with the membrane protein patterns (7), provide a powerful
tool to accurately identify these fluorescent root-colonizing
Pseudomonas spp., e.g., reisolates from field experiments.
We thank Lia van der Vlugt and Harold Klaassen for technical
These investigations were supported by the Netherlands Technol-
ogy Foundation (STW).
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