Agric. Biol. Chem., 41 (10), 1847•`1855, 1977
Isolation of a Strain of Agrobacterium radiobacter and
Its Acidic Polysaccharide
Tomonori NAGAHAMA, Shigeo FUJIMOTO and Matsuo KANIE
Laboratory of Applied Starch Chemistry, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan
Received January 10, 1977
Among the bacteria isolated from polluted water and viscid sludges in the factories
manufacturing sweet potato starch, a group of strains was ascertained to be capable of
producing slimy materials keeping fairly stable viscosity through the alterations in pH.
Representative strain A-1 of the group was assigned to Agrobacterium radiobacter. The
polysaccharide produced by culturing the strain in the medium containing glucose, yeast
extracts and CaCO3 was estimated to be Gal: Glc: succinic acid: pyruvic acid=l: 7.2
•` 7.3: 1: 0.85 in a molar ratio. The IR spectra, basicity and other determinations indicated
that the one of the moieties showing acidic function was succinic acid linking in ester bond,
and another one was pyruvic acid linking to glucose in ketal.
On many sorts of extracellular polysac
charides produced by microbes,1) the physio
logical and biochemical significances have been
reported, and also their applicable uses2) have
been developing.
In the previous paper,3) the authors reported
on a screening examination for slime-producing
bacteria from polluted water and viscid sludges
at the factories manufacturing sweet potato
starch, as well as on the isolation of the strains
of coryneform bacteria producing a series of
viscous polysaccharides composed of mannose,
galactose, glucose, glucuronic acid and pyruvic acid.
This paper is concerned with the isolation
and identification of a certain slime-producing
strain other than the coryneform bacteria.
Strain A-1 isolated here was confirmed to
belong to Agrobacterium radiobacter, and the
chemical constituents of the polysaccharide
produced by it were ascertained to be galactose, glucose, succinic acid and pyruvic acid,
showing more complex acidic constituents than
such known bacterial acidic polysaccharides
made by Alcaligenes4,5) and Rhizobiaceae.6,7)
MATERIALS AND METHODS
Screening for slime producing strains and qualitative tests on the broth. The procedures for isolation and
screening are as described in the previous paper,3)
generally.
Media used for screening tests were: Medium I
(1% potato starch and 1.5% agar in potato extracts),
Medium II (3% commercial mashed potato and 1
CaCO3) and Medium III (3% glucose, 0.25% yeast
extracts and 1% CaCO3).
Microbial sources were picked up from starchy
polluted water and viscid sludges in the factroies
manufacturing sweet potato starch.
For separation of bacterial colonies, Medium I
plates were used. The screening test for slime-produc
ing bacteria was performed by estimating the visual
fluidity of the broth in a test tube containing Medium
II after 4-day cultivation at 34•Ž on a reciprocating
shaker.
Each of the strains screened was incubated in a
300-ml conical flask containing 100ml of Medium II
for 4 days at 34•Ž on a rotary shaker. The broth
was centrifuged, and the viscosity-change in the
supernatant with acid- or alkali-addition was deter
mined for preliminary classification of the respective
slime producers.
Polysaccharide productivity of the strain. As the
strain used in this paper preferred glucose to "mashed
potato" as carbon source for slime production, it was
cultured in 300-m1 conical flasks each containing 100 ml
of Medium III.
The cultured broth was centrifuged at 6000 rpm,
and then pH and absolute viscosity of the supernatant
were determined. The slime polysaccharide was sepa
rated by adding 2 volumes of ethanol to the super
natant. The precipitate was dissolved in water and
centrifuged again at 20,000 rpm to remove the insoluble
1848 T. NAGAHAMA, S. FUJIMOTO and M. KANIE
materials, and the crude polysaccharide was obtained
by being treated with ethanol as noted above, and then,
dried in vacuo.
The polysaccharide-yields were represented as per
centages of crude polysaccharides to initial amounts
of glucose.
Morphological, physiological and chemotaxonomic
examination. General diagnostic examinations were
carried out according to the previous paper,
3) "Laboratory Methods in Microbiology,"8) and other
procedures.9,10)
Incubation temperature was generally maintained
at 32•Ž. For morphological tests, nutrient agar and
nutrient broth were used, unless otherwise noted.
Plant pathogenic experiments were performed by
referring to stock cultures of Agrobacterium radio-
bacter (IAM 1526) and A. tumefaciens (JAM 1037).
Gram stain. Besides usual staining, nutrient broth
containing crystal violet8) (1/500,000 in a final con
centration) and another containing sodium azide8)
(1/4000 in a final concentration) were used as selective
media for Gram-negative and Gram-positive bacteria,
respectively.
Ability to form colonies on sucrose-salts medium.
The medium11) was composed of Nitsch's macro
elements and microelements plus 0.9mg per liter of
FeCl3 and 0.5% sucrose, adjusted to pH 7.0 with
NaOH, and the cultures were incubated for 7 days
at 27•Ž.
Litmus milk reaction. The cultures in litmus milk
(Difco Co.) were incubated at 27•Ž without further
mixing or shaking, and were scored after 3 weeks.
Production of 3-ketolactose. The method described
by Bernaerts and DeLey12) and DeLey et al.13) was
used. Cultures on lactose-yeast extracts agar plates
were maintained at 27•Ž for 2 days, and the produc
tivity was estimated by Benedict's reagent.
Growth on calcium glycerophosphate mannitol nitrate
agar. The medium was prepared according to
Hofer,14) and the strains were incubated on plates at
27•Ž.
Plant pathogenic tests. Discs of carrot roots were
used according to Lippincott and Lippincott.15)
Carrot roots were peeled and sterilized with 0.2
HgCl2 and sliced about 5mm thick. Four slices were
placed in a petri dish containing 20ml of 1% agar in
distilled water. Each strain in water suspension was
inoculated on 16 slices, incubated at 27•Ž for 3 weeks,
and tumor and/or root formation on discs were
estimated.
Infectivity to leguminous plants. The bacterial
suspension was inoculated to 16 seedlings of Trifolium
repens L., and the seedlings were cultured on slides at
25•Ž for a week under 3000 Lux to observe infective
symptoms, according to Fahraeus16) and Higashi.17)
On the other hand, the seedlings of Trifolium pra
tense L., Astragalus sinicus L., Medicago sativa L. and
Pisum sativum L. var. arvense Poir. were subjected to
test by Gibson's tube method.18)
GC ratio of DNA. The organisms were grown on
medium consisting of 1% glucose, 1% yeast extracts,
0.1% (NH4)2SO4, 0.025% KH2PO4, and 2.5% agar for
about 2 days at 30•Ž. The bacterial cells were har
vested with cold saline-EDTA (0.15M NaCl and 0.1M
ethylene diamine tetraacetate, pH 8.0), centrifuged
and washed twice with the above reagent.
Cell-DNA was extracted and purified according
to the procedures of Marmur.19) The GC ratio of
DNA was determined spectrophotometrically according
to the previous paper8) and Skidmore and Duggan.20)
Preparation of polysaccharide in large scale. The
seed culture was shaken in 300-ml conical flasks con
taining 100ml each of Medium III without addition of
CaCO3 for 36 hr at 32•Ž on a rotary shaker. The
seed culture was inoculated to 10 liters of Medium
III in a 15-liter jar fermenter. The fermenter was
maintained at 31•‹•`33•Ž, with stirring at 100 rpm and
passed through 15-16 liter/min of air.
Purification of the polysaccharide. The crude poly
saccharide was disssolved in water and passed
through Amberlite IR 120 (H+) column to remove
cation. Addition of 10% cetylpyridinium chloride
(CPC) solution into the above clear eluate resulted in
precipitation of the acidic polysaccharide-CPC complex.
The complex was dissolved in warm 10% NaCl solu
tion. The saline solution was centrifuged and dialysed,
and then the purified polysaccharide was recovered
by precipitation with addition of ethanol as Na-form.
H-form was prepared by treating Na-form with
Amberlite IR 120 (H+).
Methanolysis and gas liquid chromatography. The
polysaccharide (10mg) was treated with 0.7 N HCl in
methanol (4ml) in a sealed tube at 80•Ž for 24 hr.
After treatment with AgCO3, the methanolysate was
converted into trimethylsilyl (TMS) derivatives in the
usual way21) and analysed by a gas liquid chromato
graph (Shimazu GC 4BM-PF) equipped with 5%
SE-30 column (ƒÓ 3mm •~ 2m) at 170•Ž and FID at
210•Ž under N2 carrier gas.
Deacylation and hydrolysis of the polysaccharide.
The pruified polysaccharide (native polysaccharide)
(4.005g) was treated with N/20 NaOH (400ml) at
60•Ž, for 3 hr under N2 atmosphere, and was neutraliz
ed. After dialysis of the reaction mixture, the
A Strain of Agrobacterium radiobacter and Its Acidic Polysaccharide 1849
deacylated polysaccharide (3.2g) in the inner solution
was recovered by ethanol precipitation. The outer
solution was slightly acidified with diluted H2SO4 and
extracted with ether by Soxhlet's extractor. The ether
extracts were carefully evaporated, and the crystalline
residues (216mg) were subjected to qualitative tests
for organic acids.
The polysaccharide (250mg) was hydrolysed with
1 N H2SO4 (50ml) for 6 hr at 100•Ž. The hydroly
sates were neutralized with diluted NaOH and sub
jected to sugar and organic acid determinations.
Enzymatic analysis of sugars. The hydrolysates
were analysed for glucose by a system of glucose
oxidase-peroxidase (Blood sugar test, Boehringer Co.)
and for galactose by a system of galactose oxidase
peroxidase (Galactostat, Worthington Biochemical
Co.).
Analysis for pyruvic acid. Pyruvic acid in the
hydrolysate was estimated spectrophotometrically as
2,4-dinitrophenyl (2,4-DNP-) derivatives.22)
Determination of neutral equivalent. About 0.1%
solution of the polysaccharide was manually titrated
with N/20 NaOH to pH 7.0 with a pH meter (Hitachi
Horiba M-5) on a magnetic stirrer.
Paper and thin-layer chromatography. On paper
chromatography, the upper layer of a mixture of
n-BuOH-formic acid-H2O (4:1.5: 1)23) (Solvent A)
and a mixture of n-PrOH-c.NH4OH (6: 4)24) (Solvent
B) were used as the ascending system for organic acids.
2,4-DNP-derivatives of keto acids extracted with
ethylacetate from the hydrolysates were applied on
silica gel plates and chromatographed with isoAmOH-
0.25 N NH4OH (20: 1)25) (Solvent C) or benzene-
tetrahydrofuran-acetic acid (20: 9: 1)26) (Solvent D).
Sugar components were chromatographed as methyl
glycosides on silica gel plate by two solvent systems:
n-BuOH-acetic acid-ethyl ether-H2O (9: 6: 3: 1) (Sol-
vent E) or benzene-EtOH (100: 15) (Solvent F). For
freed sugars in the hydrolysate, formic acid-butanone-
tert-BuOH-H2O (15: 30: 40: 15) (Sovlent G) and
cellulose powder plate were used.
Gel chromatography. Gradient chromatography
was carried out, using a column of DEAE-Sephadex
A-25 (Cl•L form, ƒÓ 14mm •~ 470mm), and eluting with
0.25M to 1M NaCl. Fractions of 5ml each were
analysed for carbohydrate content by the phenol-
H2SO4 method.
Infrared spectroscopy. Spectra were recorded with
an infrared spectrophotometer (Hitachi EPI-G2) in
form of KBr tablet.
Viscosity. Absolute viscosity (cp) was determined
by a B-type viscosimeter (Tokyo Keiki Co.) at 25•Ž.
RESULTS AND DISCUSSION
Isolation of strain A-1
As reported in the previous paper,3) 20
strains were found to be slime producers on
shaking culture in Medium II.
For preliminary grouping of these strains,
the mode of viscosity change was examined
on addition of acid or alkali. The broths of
4 strains showed 2000 to 3000 cp and fairly
stable viscosity between pH 2 and 11, differing
from the broths of the group of coryneform
bacteria3) showing 14,000 cp and remarkably
decreased viscosity at both alkaline and acidic
sides.
Strain A-1 was the representative of these
4 strains.
Identification of strain A-1
Identification studies were performed by
referring to the 8th edition of "Bergey's
Manual.""10)
Strain A-1 is rod-shaped (0.3•`0.5 •~ 1.2
•` 2.0ƒÊ), motile with several sparse peritrichous
flagella, Gram-negative, aerobic and does
not form endospores. Electronmicrograph of
Strain A-1 is shown in Fig. 1.
The strain grows well on potato extract-agar,
nutrient-agar and nutrient-broth without any
special additives, forming non-colored and
FIG. 1. Electronmicrograph of Strain A-1.
Strain A-1 cultured for 24 hr at 26•Ž on peptone
glucose agar. Electron microscopy, by JEOL 100•Ž
at 80 kV, shadowed with Cr.
1850 T. NAGAHAMA, S. FUJIMOTO and M. KANIE
TABLE 1. PHYSIOLOGICAL CHARACTERISTICS OF STRAIN A-I
usually viscid colonies. Abundunt slime was
produced on shaking culture in potato-glucose broth containing calcium carbonate.
The physiological properties of the strain
are described in Table I.
The strain utilizes oxidatively glucose, lactose
and most of the other common carbohydrates,
forming acid without any visible gas produc
tion, but does not hydrolyse cellulose and
starch. The strain does not grow in mannitol
mineral salts medium devoid of nitrogen com
pounds, though it utilizes ammonium salts, nitrates and amino acids as nitrogen sources.
According to the key to genera described in "Bergey's Manual
,"10) the above findings designate Strain A-1 to the family of Rhizo
biaceae. Furthermore, no visible symptoms
were found on roots of leguminous plants infected with the strain. Accordingly, it was considered that Strain A-1 belongs to the
genus Agrobacterium.As shown in Table I, Strain A-1 is positive
for calcium glycerophosphate mannitol nitrate agar,14,28) litmus milk, 3-keto-lactose production, Congo red-mannitol agar and sucrosemineral agar, and 62.2 moles% in GC ratio of DNA.13,27) Moreover, Strain A-1 induces no tumor to carrot-root discs,15) or no nodules and tumor-like entities29) to leguminous plants tested.
Although it has been controvertible to differentiate any soil habitates of rhizobia and agrobacteria on the basis of their infectivity to
plants, these diagnostic features of Strain A-1
A Strain of Agrobacterium radiobacter and Its Acidic Polysaccharide 1851
agree clearly with the descriptions on Agro
bacterium radiobacter in "Bergey's Manual"10)
and the improved proposals to confirm its
taxonomic status by DeLey et a1.13) and
Lippincott and Lippincott.15)
Production of the polysaccharide
Since Medium II used for the screening tests
was disadvantageous for the production and
purification of the polysaccharide, glucose and
yeast extracts were used as carbon source and
nitrogen source, respectively. When 0.25
yeast extracts was added to 3% glucose and 1
calcium carbonate in medium, as shown in
Table II, the broth showed maximum values in
broth-viscosity and in polysaccharide-yield,
while additional amounts caused lower poly-
saccharide production.
TABLE II. EFFECT OF DIFFERENT AMOUNTS OF
YEAST EXTRACTS ON POLYSACCHARIDE
PRODUCTION BY STRAIN A-1
Basal medium, consisting of glucose 3g, CaCO3
1g in 100ml. Cultured in 300-m1 conical flask at
34•Ž, on a rotary shaker.
a) Yield was represented as a percentage of crude
polysaccharide to initial amount of glucose.
Accordingly, the medium consisting of
glucose 3g, yeast extracts 0.25g and calcium carbonate 1g in 100ml was adopted for poly
saccharide production. A time course of poly
saccharide production in conical flasks is re
presented in Fig. 2. Accumulation of the
polysaccharide appeared to be at peak after 6-day cultivation and, after that, it held to
almost a constant level.
On the other hand, polysaccharide produc-
FIG. 2. Time Course of Polysaccharide Production
in Flasks by Strain A-1.•›•\•›
, viscosity; ƒ¢•\ƒ¢, yield;_??_, pH.
Cultured on 100ml of medium consisting of glucose
3g, yeast extracts 0.25g and CaCO3 1g in a 300-m1
conical flask, at 34•Ž on a rotary shaker. See the
text in details on determinations.
tion in a jar fermenter proceeded in a time course similar to that in the flask culture, and crude polysaccharide was obtained from 136-hr cultured broth at 27% yield to the initial amount of glucose.
In qualitative comparison with the polysaccharide obtained from 6-day cultural broths in a flask and preparation in large scale, the IR spectra and the gas liquid chromatograms of the TMS derivatives from methanolysates of the both preparations showed the same characteristics as described later.
General properties and chemical composition of the polysaccharideThe precipitate with ethanol from the broth,
which seemed to be partly in Ca-form, showed 5100 cp in 1.0% solution, but the purified polysaccharide in Ca-form was highly viscous, showing 23,400 cp in 0.5% solution.
The polysaccharide precipitated in a complex with CPC, and there remained little residue to be precipitated with ethanol in the filtrate. It was assumed that a large part of the product was acidic polysaccharide.
The polysaccharide was recovered from CPC complex by treatment with saline solution, and
1852 T. NAGAHAMA, S. FUJIMOTO and M. KANIE
finally the purified polysaccharide was prepared in H-form and Na-form.
These preparations were, generally, designated here as native polysaccharide.
A portion of native polysaccharide was deacylated with diluted NaOH solution, and the deacylated polysaccharide was prepared in H-form and Na-form.
Generally, these polysaccharides were sub
jected to analyses in H-form unless otherwise noted.
The IR spectra of the native Na-form and the deacylated Na-form are shown in Fig. 3. While the spectrum of the former was characterized at a peak due to ester at 1720cm-1 and
FIG. 3. Infrared Spectra of the Polysaccharide.
•\, native polysaccharide (Na-form); ---, deacylated
polysaccharide (Na-form) in KBr tablet.
FIG. 4. DEAE-Sephadex Column Chromatograms
of the Polysaccharide.
•›•\•›, native polysaccharide; _??_ deacylated
polysaccharide.
Column: DEAE-Sephadex A-25 (Cl-form), ƒÓ 14 •~
470mm. OD50: Phenol-H2SO4 method.
another peak due to carboxylate at 1610cm-1,
the spectrum of the latter showed no peak at
1720cm-1 and the peak retained at 1620cm-1.
On DEAE-Sephadex column chromato
graphy, deacylated and native polysaccharides
were eluted with 0.34M and 0.45M solutions
of NaCl, respectively, showing the respective
single peaks as shown in Fig. 4.
These facts indicate that native polysac
charide eluted later contains ester linkage and
also acts as a compound more acidic: de
acylated polysaccharide eluted earlier is brou
ght out owing to cleavage of the ester bond
and also to release of a part of carboxylates
in native polysaccharide, and acts as a
compound less acidic. Accordingly, it was
considered that native polysaccharide is in
possession of two kinds of acidic components
acting as carboxylate, that is, one linked in
ester form and the other linked in alkali
stable form.
Neutral equivalent values of native and
deacylated polysaccharide (H-forms) estimated
by alkali titration were 861 and 1739, re
spectively.
One of the two acidic components of the
native polysaccharide was isolated by extrac
tion with ether from the deacylated solution.
The ether extracts were examined for organic
acids by paper chromatography, and only one
spot corresponding with authentic succinic
acid was recognized, showing Rf 0.81 in
Solvent A and Rf 0.38 in Solvent B. The
residues after removal of ether amounted to
5.4% of the weight of native polysaccharide.
The residues (100mg) were derived into p
bromophenacyl ester (195mg) in the usual
manner, 30) and were identified to be succinic
acid, mp 212•‹•`214•‹, and 213•‹•`214•Ž on
admixture with authentic specimens, on a
micro hot plate.
Another acidic component retained in de
acylated polysaccharide was liberated by acid
hydrolysis. The hydrolysate was treated with
2,4-dinitrophenylhydrazine reagent, and esti
mated colorimetrically to be containing 5.2%
pyruvic acid in deacylated polysaccharide.
The resulting 2,4-DNP derivatives were ex-
A Strain of Agrobacterium radiobacter and Its Acidic Polysaccharide 1853
TABLE 111. MOLAR RATIO OF SUGAR AND ACIDIC MOIETIES OF THE POLYSACCHARIDE
a) Wt% was calculated on dehydrated moiety .b) H+/100g was calculated on the neutral equivalent value .c) The value was calculated on a presumptive molar ratio of pyruvic acid: succinic acid =1: 1.
tracted with ethyl acetate and applied on silica
gel plates. The main yellow spot having Rf
0.12 in Solvent C and two spots having Rf 0.26
and 0.47 in Solvent D coincided with those of
authentic specimens derived from pyruvic acid.
It has been known that these two spots on TLC
have resulted from stereoisomers occurring
in derivation.25,26)
On the other hand, the sugar components of
both polysaccharides were enzymatically deter-
mined on acid hydrolysates to be Gal: Glc=
1: 7.2•`7.3 in molar ratio.
Molar ratios of sugar and acidic moieties of
native and deacylated polysaccharides are
summarized in Table III. Although succinic
acid was not directly determined, the molar
ratio of pyruvic acid and succinic acid in
native polysaccharide was calculated to be
1: 1.2 on the basis of the difference between
neutral equivalents of native and deacylated
polysaccharides.
The gas chromatographic patterns of the
methanolysates prepared after acid hydrolysis
of native and deacylated polysaccharides were
almost identical with the chromatographic
pattern of the methanolysate of an authentic
mixture of galactose and glucose (1: 7.2).
However, on chromatograms of methanoly
sates of both polysaccharides, peaks corres
ponding to methyl ƒ¿- and ƒÀ-D-glucosides were
broader and shouldered, and it seemed that
there were unknown peaks overlapping the
peaks of methyl glucosides. Figure 5A shows
a chromatogram of the methanolysate of de
acylated polysaccharide.
Thus, deacylated polysaccharide (75mg) was
treated with methanolic hydrogen chloride
(20ml), and the products were detected to be
methyl D-galactoside (Rf 0.39), methyl D-
glucoside (Rf 0.46), methyl ƒÁ-D-galactoside
(Rf 0.55) and unknown "spot 1" (Rf 0.75) on
silica gel plate developed in Solvent E. The
above methanolysate was preparatively chro
matographed on silica gel plate in Solvent F,
and "spot 1" (Rf 0.29) was isolated from the
plates.
A portion of "spot 1" was hydrolysed (2 N
H2SO4, 100•Ž, 6 hr) and the products were
identified to be glucose (Rf 0.34 in Solvent G)
and pyruvic acid (as 2,4-DNP-derivative; Rf
0.12 in Solvent C, Rf 0.47 in Solvent D).
Another portion of "spot 1" was chromato
graphed as TMS derivatives on GLC and it was
ascertained that two peaks (No. 6 and 7)
appeared a little later than methyl ƒ¿- and ƒÀ-D-
glucosides, respectively, as shown in Fig. 5B.
The above two peaks of "spot 1" were assumed
to be the peaks of the anomers.
These facts suggest that the polysaccharide
includes glucose moiety linked with pyruvic
acid in ketal which is cleaved by aqueous
hydrolysis but resistant to methanolysis.
Existence of glucose moiety combined with
pyruvic acid in polysaccharide has been re
ported on Xanthomonas campestris NRRL
B-1459.31)
Harada4) and Misaki et al.5) reported on
succinoglucan, polysaccharide containing
succinic acid produced by Alcaligenes faecalis
var. myxogenes 10C3. However, they did not
detect the presence of pyruvic acid as a com
ponent of succinoglucan.
1854 T. NAGAHAMA, S. FUJIMOTO and M. KANIE
FIG. 5. Gas Liquid Chromatograms of TMSDeri
vatives of Sugar Components of the Polysaccharide.
A, Methanolysate with 0.7 N HCl in MeOH, for
24 hr at 80•Ž in a sealed tube.
B, The pyruvic acid containing moiety (spot 1)
separated from A by TLC.
Peak No. 1, methyl ƒÁ-D-galactoside; 2, methyl ƒ¿-D-
galactoside; 3, methyl ƒÀ-D-galactoside; 4, methyl
ƒ¿-D-glucoside and peak 6; 5, methyl ƒÀ-D-glucoside
and peak 7.
Chromatography: Column 5% SE-30, 170•Ž; de
tector FID, 210•Ž; injector 210•Ž; carrier gas N2.
On exopolysaccharides of rhizobia and agro
bacteria, Zevenhuizene6) reported the presence
of galactose, glucose, and/or glucuronic acid
and pyruvic acid and acetic acid as chemical
components. Recently, Nakanishi et a1.32)
reported on the production of ƒÀ-1,3-glucans by
some stock cultures of Agrobacterium radio
bacter. However, it has not been described
on such polysaccharides as those containing
pyruvic acid, together with succinic acid.
Thus, it is confirmed that Agrobacterium
radiobacter Strain A-1 produces a poly
saccharide containing complex acidic moieties.
Further structural studies on the polysaccharide
are now proceeding.
Acknowledgement. The authors thank Messrs.
Mitsuo Moriuchi, Koji Inuzuka and Masahiro Kuwata
for their assistance in screening and cultural work.
Sincere acknowledgement is also made to Dr. Kei
Arai, Faculty of Agriculture and Dr. Shiro Higashi,
Faculty of Science, Kagoshima University, for their
cooperation on electron microscopy and plant patholo
gical work.
A part of this work was presented at the 135th
meeting of the Nishinihon branch of Japanese Agricul
tural Chemical Society held in Miyazaki, on October
21, 1972.
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A Strain of Agrobacterium radiobacter and Its Acidic Polysaccharide 1855
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