STREPTOMYCES NODOSUS SP. N.

, THE AMPHOTERICIN-PRODUCING
ORGANISM
WILLIAM H. TREJO AND R. E. BENNETT
Squibb Institute for Medical Research, New Brunswick, New Jersey
Received for publication 10 September 1962

ABSTRACT MATERIALS AND METHODS

TREJO, WILLIAM (Squibb Institute for Medi- Cultural studies were carried out using the
cal Research, New Brunswick, N.J.) AND RALPH standard diagnostic media recommended by the
E. BENNETT. Streptomyces nodosus sp. n., the Subcommittee on Taxonomy of Actinomycetes
amphotericin-producing organism. J. Bacteriol. (Gottlieb, 1958). Inocula from aqueous spore
85:436-439. 1963.-Streptomyces nodosus, the suspensions of 10-day-old slants were used to
amphotericin-producing organism, is described streak plates of the diagnostic media in uniform
as a new species in conformity with the rules of cross-hatch design. The plates were then incu-
nomenclature as applied to streptomycetes. The bated at 28 to 30 C and examined periodically
relationship between S. nodosus and S. rutgersen- over a 14-day interval. Descriptive colors were
sis is discussed, and the basis for separation of matched to Ridgway's (1912) color standards.
the species is presented. The morphology of the sporophores was
determined by direct microscopic examination
of 14-day-old petri dish cultures. Spore color
The biosynthesis and biological properties of groups and morphology sections refer to the
the amphotericins were first described by Gold designations of Pridham, Hesseltine, and Bene-
et al. (1956). The organism producing these dict (1958). Spore morphology was determined
materials was a streptomycete isolated from a by examination of impression mounts in an
soil sample taken from the Orinoco River region electron microscope.
of Venezuela and was designated in their paper The following techniques were used in physi-
as Streptomyces sp. M-4575. In more recent ological studies.
publications (Dutcher et al., 1959; Waksman, Proteolysis. Proteolytic activity was determined
1961), this streptomycete has been referred to as by the casein-plate method (Gordon and Smith,
S. nodosus. 1955) and by the gelatin-plate method (Frazier,
It is common knowledge to those engaged in 1926). Plates were examined during a 14-day
streptomycete taxonomy that a wealth of taxo- incubation period for evidence of proteolytic
nomic information lies buried in the patent activity.
literature, little of which finds its way into the Chromogenicity. Chromogenicity was deter-
scientific journals. The explanation for this may mined after growth of the culture on a modifica-
lie in differences in interpretation of Rule 11 of tion of Matsumoto's tyrosine agar (Shinobu,
the International Code of Nomenclature of Bacteria 1958). The production of brown, dark brown, or
and Viruses (1958) which deals with the pre- black soluble pigment was interpreted as a posi-
requisites for effective publication of names. The tive reaction. Other media used to check chro-
question of whether a patent constitutes effective mogenicity included tyrosine agar (Gordon and
publication is one which must be decided by the Mihm, 1957) and other proteinaceous media,
Judicial Commission of the International Com- i.e., nitrate broth (Difco), Bennett's agar, and
mittee on Bacteriological Nomenclature. The 12 % gelatin.
purpose of this paper is to describe validly, in Production of H2S. Hydrogen sulfide production
accordance with the rules of the International was observed by growing cultures for 24 hr on
Code of Nomenclature of Bacteria and Viruses peptone iron agar (Difco) supplemented with
(1958), the organism producing the amphoteri- 0.1% yeast extract (Tresner and Danga, 1958).
cins, and to discuss its relationship to Streptomyces The presence of bluish-black pigment was taken
rutgersensis, a relationship mentioned by Waks- as evidence of H2S production.
man (1961). Carbon utilization. The pattern of utilization of
436
VOL. 85, 1963 STREPTOMYCES NODOSUS SP. N.
carbon compounds was tested by the method
of Pridham and Gottlieb (1948) with the following
modifications: the tests were run on plates
instead of slants, and the number of carbon
sources was reduced to include only glucose,
adonitol, mannitol, inositol, sorbitol, xylose,
melibiose, trehalose, arabinose, rhamnose, fruc-
tose, raffinose, cellulose, sucrose, and lactose.
Growth response was determined after 10 days
of incubation at 28 to 30 C.
Nitrate reduction. The procedure outlined by
the Society of American Bacteriologists Com-
mittee on Bacteriological Technic (1946) was
used to study nitrate reduction by the cultures
grown for 14 days in nitrate broth (Difco).
Starch hydrolysis. Hydrolysis of starch was
determined by flooding a 14-day-old culture
grown on inorganic salts-starch agar (Pridham
et al., 1957) with Lugol's iodine.
DESCRIPTION
Streptomyces nodosus Trejo, sp. nov.
Nodosus. L. adj. knotty.
Morphology. Aerial mycelium: forms open
and closed spirals, the latter predominating as
tightly knotted coils (Fig. 1). Spores are spherical
to oval (0.5 to 1.0 by 1.0 ,t) as shown in Fig. 2.
Spore-color series. S. nodosus is assigned to the
gray spore-color series. On media which support
sporulation, the culture produces spores which
L.
FIG. 1. Sporophore morphology of Streptomyces
nodosus. Magnification 640 X.
437
FIG. 2. Electron micrograph of Streptomyces
nodosus spores. Magnification 14,000 X.
range from light gray through light olive-gray to
grayish olive: ISCC-NBS numbers 264, 112,
and 110, respectively (Kelly and Judd, 1955). The
variations of spore color and mycelial color with
growth in different media are summarized in
Table 1.
Physiological characteristics. S. nodosus is
nonchromogenic since it does not produce
melanoid pigment on any of the tyrosine-
containing media tested. It is H2S-negative,
strongly proteolytic, hydrolyzes starch readily,
and reduces nitrate to nitrite. The following
carbon sources are utilized on basal medium of
Pridham and Gottlieb (1948): glucose, mannitol,
inositol, xylose, rhamnose, fructose, trehalose,
and melibiose. No growth occurs on adonitol,
sorbitol, arabinose, cellulose, sucrose, lactose,
and raffinose.
Antagonistic properties. Produces the antifungal
antibiotics, amphotericin A and amphotericin B.
Source. Soil isolate from sample collected in
Orinoco River region of Venezuela.
Temperature optima for growth. Good growth
is obtained when cultures are incubated over
the range of 25 to 37 C. No growth occurs at 45 C.
Type culture. American Type Culture Col-
lection no. 14899.
DISCUSSION
Waksman (1961) commented that S. nodosus
"appears to be closely related to S. rutgersensis."
However, he did not indicate the basis for such a
438 TREJO AND BENNETT J. BACTERIOL.

TABLE 1. Color* of Streptomyces nodosus on various media

Medium Color of aerial mycelium Color of reverse

Tomato paste Deep olive-gray to dark olive-gray Olivaceous to black with a peripheral
Oatmeal agar (plate LI) ring cream-buff to chamois (plate
XXX)

Bennett's agar Deep olive-gray (plate LI) Fuscous to black

Glucose aspara- Deep olive-gray (plate LI) Chamois (plate XXX)

gine agar
Salts-starch agar Pearl-gray to dawn-gray (plate LII) Colorless to gnaphalium green (plate
XLVII)
* Color designations from Ridgway's Color Standards.

by the above procedures a number of gray-spored,

spiral, nonchromogenic streptomycetes, including
representatives of the following known species:
S. halstedii, S. aureus, S. hygroscopicus, and S.
vinaceus-drappus. As a result of these studies. we
have concluded that S. nodosus ATCC 14899
is distinctly different. The fact that S. nodosus
is readily separable from S. rutgersensis and
cannot be identified with any of the described
species in Bergey's M1anual of Determinative
Bacteriology (7th ed.) or by Waksman (1961),
leaves no alternative but to consider this a new
species.
ACKNOWLEDGMENTS
We are indebted to R. E. Gordon for the
culture of S. rutgersensis and to J. Q. Ochs for
FIG. 3. Sporophore morphology of Streptomyces assistance with the electron micrographs.
rutgersensis. Magnification 640 X.
LITERATURE CITED
conclusion. We have compared S. rutgersensis DUTCHER, J., W. GOLD, J. F. PAGANO, AND J.
strain IMRU 3350 with S. nodosus and find that VANDEPUTTE. 1959. Amphotericin B, its
they differ markedly in morphology of the production and its salts. U.S. Pat. 2,908,611.
sporophores. S. nodosus clearly belongs to the Oct. 13.
spira group, whereas S. rutgersensis is in the FRAZIER, W. C. 1926. A method for the detection
rectus flexibilis group. Furthermore, examination of changes in gelatin due to bacteria. J.
of the type culture S. rutgersensis IMRU 3350 Infect. Diseases 39:302-309.
shows that a disparity exists between the organ- GOLD, W., H. A. STOUT, J. F. PAGANO, AND R.
ism we studied and its published description DONOVICK. 1956. Amphotericins A and B,
antifungal antibiotics produced by strepto-
(Waksman and Curtis, 1916; Waksman, 1961). mycete. I. In vitro studies. Antibiotics Ann.
The description refers to the culture as having 1955-56, p. 579-586.
abundant production of closed and open spirals, GORDON, R. E., AND J. M. MIHM. 1957. A compara-
and our studies show that the aerial mycelium is tive study of some strains received as nocar-
straight with a slight tendency toward flexuous- diae. J. Bacteriol. 73:15-27.
ness in old cultures (Fig. 3). We have examined GORDON, R. E., AND M. M. SMITH. 1955. Proposed
VOl,. 85, 1963 STREPTOMYCES NODOSUS SP. N.
group of characters for the separation of
Streptomyces and Nocardia. J. Bacteriol.
69:147-150.
GOTTLIEB, D., Chairman, Subcommittee on Tax-
onomy of the Actinomycetes. 1958. Methods
for use in cooperative studies on criteria for
description of the streptomycetes. Multilith,
17 p.
INTERNATIONAL COMMITTEE ON BACTERIOLOGICAL
NOMENCLATURE, EDITORIAL BOARD. 1958.
International Code of Nomenclature of Bac-
teria and Viruses, p. 61. State College Press,
Ames, Iowa.
KELLY, K. L., AND D. B. JUDD. 1955. The ISCC-
NBS method of designating colors and a
dictionary of color names. U.S. Dept. Com-
merce Circ. 553. Washington, D.C.
PRIDHAM, T. G., P. ANDERSON, C. FOLEY, L. A.
LINDENFELSER, C. W. HESSELTINE, AND R.
G. BENEDICT. 1957. A selection of media for
maintenance and taxonomic study of Strepto-
myces. Antibiotics Ann. 1956-57, p. 947-953.
PRIDHAM, T. G., AND D. GOTTLIEB. 1948. The
utilization of carbon compounds by some
actinomycetales as an aid for species de-
termination. J. Bacteriol. 56:107-114.
439
PRIDHAM, T. G., C. W. HESSELTINE, AND R. G.
BENEDICT. 1958. A guide for the classifica-
tion of Streptomycetes according to selected
groups. Placement of strains in morphological
sections. Appl. Microbiol. 6:52-79.
RIDGWAY, R. 1912. Color standards and color
nomenclature. Published by the author,
Washington, D.C.
SHINOBU, R. 1958. Physiological and cultural
study for the identification of soil actinomy-
cetes species. Mem. Osaka Univ. B Nat.
Sci. 7:1-76.
SOCIETY OF AMERICAN BACTERIOLOGISTS, COM-
MITTEE ON BACTERIOLOGICAL TECHNIC. 1946.
A manual of methods for pure culture study
of bacteria. Biotech. Publ., Geneva, N.Y.
TRESNER, H., AND F. DANGA. 1958. Hydrogen
sulfide production by Streptomyces as a
criterion for species differentiation. J. Bac-
teriol. 76:239-244.
WAKSMAN, S. A., AND R. E. CURTIS. 1916. The
actinomyces of the soil. Soil Sci. 1:99-134.
WAKSMAN, S. A. 1961. The actinomycetes, vol.
II. Classification, identification, and de-
scriptions of genera and species. The Williams
& Wilkins Co., Baltimore.