860

THE

JOURNAL

NANAOMYCINS,

NEW

OF

TAXONOMY,

PRODUCED

BY

1975

A

STREPTOMYCES

ISOLATION,

AND

NOV.

ANTIBIOTICS

ANTIBIOTICS

STRAIN I.

OF

CHARACTERIZATION

BIOLOGICAL

PROPERTIES

HARUO TANAKA, YASUAKI KOYAMA, JUICHI AWAYA, HIROFUTO MARUMO*, RUIKO OIWA, MICHIKO KATAGIRI, TOSHIAKI NAGAI and SATOSHI OMURA** The Kitasato Institute Minato-ku, (Received

culture

silica

filtrate

by extraction

A and

B inhibit

acute toxicities respectively.

(LD50.

Screening in order

with

to discover

the culture

Nanao-shi

antibiotics

by

program

broth

of strain Noto

paper

deals

activities

disease which Japan.

briefly with

against had

Peninsula, 1.

obtained

been isolated

The

has been conducted

successful

KITAME et al.3)

Mycoplasma

against

bacteria,

and

from

gallisepticum

fungi

new

a soil sample

isolation

and

anti-

were obtained collected

properties

of the

report4).

of the producing

strain,

isolation,

characterization

1.

strain

Electron OS-3966

micrograph (JSM-2,

of

JEDL

the

Co.,

spores Ltd.)

culture, isolated

at Nanao-shi

strain from

a

in the Noto

Japan. Morphological

The morphology yeast extract-malt salt-starch agar *

than

The

169 mg!kg,

Strain

is a Streptomyces

soil sample

activities

mycoplasmas

in the preliminary

taxonomy

bacteria.

are 28.2 and

of the antibiotics.

The nanaomycin-producing OS-3966,

The

IKEUCHI et al.2)and

of chickens)

of the Nanaomycin-

producing

Gram-positive

B in mice

more active against

Fig.

Characteristics

and

and

OMURA et al.1),

OS-3966

gel chromatography.

for antimycoplasmal

especially

Peninsula,

and

fungi A

of antibiotics

respiratory

have been reported

and biological

filtrates

A and B, effective

in the

The present

mycoplasmas,

culture

of chronic

nanaomycins

solvent

was desfrom the

that nanaomycins A and B are quinone-related C16H14O6 and C16H16O7, respectively. Nanao-

nanaomycins

new antibiotics

of our screening

(a pathogen

biotics,

of

organic

suggest formulae,

mainly ip)

of Streptomyces

In the course

at

May 26, 1975)

strain OS-3966 which A and B were isolated

mycins

from

for publication

Nanaomycins are new antibiotics produced by the ignated Streptomyces rosa var. notoensis. Nanaomycins physical and chemical properties compounds having the molecular

KP-13

and Kitasato University, Tokyo, Japan

Present

Sunto-gun, ** To

characteristics. of the strain

cultured

on

extract agar and inorganic for 14 days at 27°C was

address:

Pharmaceutical

Shizuoka-ken, Japan. whom all correspondence

Res . Labs., should

Kyowa

be addressed

.

Hakko

Kogyo

Co.

Ltd.,

Nagaizumi-cho,

of

VOL.

XXVIII

NO.

11

THE

JOURNAL

OF

ANTIBIOTICS

861

microscopically observed (Fig. 1).

The aerial mycelium

synthetic or organic agar medium.

It forms no whorls, but extends aerial hyphae forming open

or compact spirals. 2.

of the strain

is abundant

on either

The spores are oval or cylindrical and their surfaces are smooth.

Cultural and physiological characteristics.

The strain

OS-3966 was cultivated

(International

Streptomyces

Projects'

Table Medium

1.

on various

media

at 27°C or 37°C,

Cultural

characteristics

Growth

described by WAKSMAN5)and ISP and the changes

of strain

Reverse

good, light ivory to light melon yellow

light melon to apricot

Glucose-nitrate agar

good, dusty yellow to golden brown

golden brown to chocolate brown apricot

white

white

Glycerolasparagine

agar

good, light melon yellow to orange rust moderate, light melon yellow

yellow

light

light

mycelium

apricot to pearl

Soluble

pink

apricot

light sepia

Tyrosine

agar

good, light wheat to amber topaz

pearl pink to golden brown light melon yellow to nude tan

Nutrient

agar

moderate, colorless to pearl pink

squash yellow bright yellow

to

light melon yellow to pearl pink white, scant

Glucose-peptone agar

moderate, to golden

golden brown sepia brown

to

white

ivy

Yeast extract-mall extract agar

good, colorless to golden brown moderate, colorless to light melon yellow moderate, cream to light wheat

golden orange

to

light melon yellow to light apricot light melon yellow to light apricot

ivy

scant, white to colonial yellow

none

Oatmeal

agar

Peptone-yeast extract iron agar Tryptone-yeast extract broth

light melon yellow to nude tan colonial

yellow

wheat brown

melon apricot

saltsagar

brown rust

pigment

pearl pink to light melon yellow

Inorganic starch

colorless brown

to flesh pink

to

yellow

none to dark

light

laurel

tan

light

ivory

white

none

surface growth. moderate

pearl

pink

none

light

apricot pink

Gelatin

surface

Nitrate

good surface growth, moderate

pearl pink chartreuse

pearl laurel

broth

Cellulose Table 2. 3966

growth,

light

none Physiological

properties

none of

strain

formation

Tyrosinase

OS-

none

none Table

3.

Response

reaction

Positive

H2S production reduction

Hydrolysis Liquefaction Peptonization

of gelatin of milk of milk

Cellulolytic

activity

range

Negative

of starch

Coagulation

Temp.

ivory

to celadon

to

none

Utilization

of carbon

sources

by

OS-3966

Melanin

Nitrate

white gray white

to tint

to

dark luggage tan to sepia brown light wheat to melon yellow

surface growth, moderate, light ivory

Milk

aerial

OS-3966

Aerial

Sucrose-nitrate agar

of growth,

for growth

15-45°C

Carbon

source

D-glucose, D-fructose, D-xylose, L-arabinose, glycerol, mannose, rhamnose, maltose, D-mannitol sucrose, raffinose, i-inositol

strain

THE

862

mycelium

and soluble

Table

4.

between

chain

was tested

Spore

surface

by growth and

Inorganic agar

GOTTLIEB'S mediums6) carbon

R*

sources.

SP*

names and hue

Tyrosine

numbers indicated were

AM

those of the Color Har-

R

Carbon

tion) published by ConCooperation

OS-3966

and

Streptomyces

ISP 5533

Spirals Smooth

Spirals Smooth

Flesh pink (4 ca) Beige camel (3 ea) Slate (15 ih)

Lt. melon yellow (3 ea) Bamboo chamois (3 gc) Chocolate (4 nl)

Flesh pink (4 ca) Lt. melon yellow (4 ea) Dusty yellow (1/2gc)

Flesh pink (4 ca) Lt. melon yellow (4 ea) Flesh pink (4 ca)

agar

SP

mony Manual (4th editainer

strain

salt-starch

AM*

containing 1 % of various

the

1975

Morphology Spore

Color

NOV.

OS-3966

Utiliza-

PRIDHAM

ANTIBIOTICS

ISP 5533

tion of carbon sources on

OF

Comparison

rosa

pigment were observed after a period of 7, 14 and 21 days.

JOURNAL

utilization

Sucrose

of

Inositol

America. The

cultural

Nanaomycin production

and

physiological characteristics of the strain OS3966 are listed is shown

in Tables

in Table

follows:

*

growth

3.

AM:

Aerial

mycelium

1 and 2, respectively. The

is yellow

cultural

and

to brown

The utilization

physiological

on either

yellowish orange to pink on various media; media; it produces no melanoid pigment. From

the above

Among

known

ology",

8th

comparison

morphological former

"The

of strain

OS-3966

characteristics

of the

tion of sucrose

former

and

and

with those

i-inositol

was designated

has been deposited Technology

with accession

different

The stock agar

slant

culture

of strain

(KRAINSKY's

agar

rosa var.

OS-3966

from

notoensis

Research

FERM-P

medium).

pigment those

related

No.

the

to the

on some

various series. Bacteri-

ISP

reports

strain

by

OS-3966.

characteristics in Table media

of the latter.

Agency

on

is

rosa ISP 5533, all of the

as shown

AWAYA var.

Institute,

brown

Determinative

but the latter did not. be assigned to a new variety

the Fermentation

Production

of

as

mass color

to the red color

physiological

However,

of soluble

by the strain summarized

aerial

is yellowish

Streptomyces and

be

media;

and belongs

closely

pigment.

sources

can

WAKSMANS' and

was

of the latter.

were slightly

number

by

of the cultural

formation

as Streptomyces with

II

rosalr'

produced the new antibiotics, nanaomycins, Therefore, the strain OS-3966 should and

pigment

in "BERGEY'S Manual Vol.

Soluble

of carbon

or organic

with the type culture, most

as the

synthetic soluble

species described Actinomycetes"

SP:

characteristics

is non-chromogenic

GOTTLIEB8-11), Streptomyces

were in agreement

acteristics

the strain

Streptomyces ed.7)

SHIRLING and In

results,

, R: Reverse,

nov.

of

4, such

the char-

and the utilizaAlso,

the former

of Streptomyces The

of Industrial

strain

rosa,

OS-3966

Science

and

2209.

of Nanaomycin (Streptom)ces

A and B rosa var. notoensis)

A 7-day culture

of the agar

was maintained

slant was inoculated

as an into

VOL.

XXVIII

NO.

11

THE

JOURNAL

OF

ANTIBIOTICS

a medium (100 ml) in a SAKAGUCHI'S flask, incubated for two days at 27°C, and then used as a seed culture for production of nanaomycin. Fermentation was carried out using a 30-liter jar or a 400-liter tank fermentor containing 20 or 200 liters, respectively, of medium for 4 days at 27°C. The composition of the seed or fermentation medium was 2 % glycerol, 2 % soybean meal and 0.3 % sodium chloride (the pH value was adjusted to 7.0 with 6-ON sodium hydroxide before sterilization). The time course of a typical fermentation is shown in Fig. 2. The nanaomycin produced was assayed by the methed reported by ITOH et al.16) using Mycoplasma

863

Fig. 2. Time course of nanaomycin production by Streptomyces rosa var. notoensis. Cultivation was performed using a 30-liter jar fermentor containing 20 liters of the medi um described in the text. Culture conditions were as follows: agitation, 300 rpm; temp., 27°C; aeration, 10 liters/minute. Antimycoplasma activity was assayed by the method reported by ITOH et al.6) pH

Inhibition

Mycelial

Time

gallisepticum KP-13. The nanaomycin production started The total

I day after the inoculation amount

of nanaomycins

and the maximum

A and

zone

B accumulated

concentration

growth,

(days)

was reached

at the 4th day was about

at 3-4

days.

100 ,ug/ml.

Isolation and Characterization of Nanaomycins Culture broth (200 liters) of Streptomyces rosa var. notoensis, obtained by incubation in a 400-liter tank, was used as a starting material for the isolation of nanaomycins. Nanaomycins were detected by antimicrobial activity16). After the culture supernatant was adjusted to pH 2.0 with 6 N hydrochloric acid, nanaomycins were extracted with butyl acetate and then transferred into 1 % sodium bicarbonate solution. From the aqueous solution, nanaomycins were extracted with ethyl acetate after adjusting to pH 2.0 with 6 N hydrochloric acid. A crude powder (10.9g) of nanaomycins was obtained by evaporating the solvent

layer

dried

sulfate

(anhydrous).

with

sodium

The

Chemical

benzene -ethyl active

Co.)

acetate.

component

nanaomycin

(designated

acetate

as

Nonoomycin

B

(A)

(B)

B) with benzene - ethyl

(3: 1).

The fractions first

(B)

as

the second

(designated

active

containing

component

A and B

A

(A)

with

the column,

nanaomycin

Nonoomycin

with

(4 : 1) from

component

of nanaomycins

90% MeOH or in 0.1 N HCl-90 in 0.1 N NaOH-90 %1 MeOH.

on 923

benzene - ethyl acetate

active

UV-Spectra

The first

A) was eluted

and then

3. (A) (B)

crude

powder was chromatographed a column of silica gel No. (Davison

Fig.

the were

Wavelength

(nm)

' MeOH.

864

THE

combined and concentrated A were obtained

JOURNAL

OF

NOV.

ANTIBIOTICS

under reduced pressure to dryness.

from an ethanol

1975

Orange needles of nanaomycin

solution of the powder.

The crystals were recrystallized

from an ethanol solution: yield 317 mg; mp 178 -180'C. Anal. Found:

C, 63.35; H, 4.47; N, 0.

Calcd. for C16H14O6:C, 63.57; H, 4.66; N, 0 % UV 2MeOHmax: 250(9,850), 274(12,200), 423(4,0450). [a]26D:-27.5° 31:1
100

was

were

more

active than

at

a

little

low

concentrations

serum;

the

results

Toxicity

of

mycin

in mice using

0.8

12.5

one

1.6

12.5

was calculated

P

1.6

12.5

KARBER'S

P

1.6

12.5

P

0.8

25

P

0.2

P

3.1

25

and

P

0.4

12.5

toneally,

P

25

25 >25

At pH values at

con-

of nanaomycin

H E

* Abbreviations used: N , nutrient agar (pH 7.0, 2 days, 37'C), P, potato agar (pH 6.4, 4 days, 27°C), H, Hokken PPLO agar (pH 7.8, 8 days, 37'C); E, Eiken PPLO agar (pH 7.8, 8 days, 37°C).

nanao-

5 mice

group,

toxicities

3.12

3.12

is con-

mycin.

P