Isolation of a Strain of Agrobacterium radiobacter and Its

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.


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


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


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-


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.


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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