JOURNAL OF BACEIOLOGY, May 1972, p. 758-760 Vol. 110, No.

2
Copyright 1972 American Society for Microbiology Printed in U.SA.

Clostridium barkeri sp. n.

E. R. STADTMAN, THRESSA C. STADTMAN, IRA PASTAN,I AND LOUIS DS. SMITH
Laboratory of Biochemistry, NHLI, Bethesda, Maryland 20014, and Anaerobe Laboratory, College of
Agriculture, Virginia Polytechnic Institute, Blacksburg, Virginia 24061
Received for publication 6 January 1972

Clostridium barkeri sp. n. has been described, and its relationship to other
clostridia is discussed.

A clostridium was isolated from Potomac
River mud by the enrichment culture tech-
nique. [The original strain isolated and studied
by Harary (2) was lost. A similar strain later
isolated by Pastan et al. (11) was employed for
all subsequent biochemical studies; a subcul-
ture of the latter is described here in detail
and has been deposited with the American
Type Culture Collection. The ATCC accession
number is 25849.] This strain ferments nico-
tinic acid to equimolar amounts of propionic
acid, acetic acid, carbon dioxide, and ammonia
according to the equation: C6H502N t 4 H20
- CH3CH2COOH + CH3COOH + CO2 + NH3.
A number of detailed biochemical studies (3,
4, 6-9, 13) with cell suspensions and enzyme
preparations of this microorganism have estab-
lished the following compounds as sequential
intermediates in the anaerobic breakdown of
nicotinic acid (I): 6-oxonicotinic acid (II);
1,4,5, 6-tetrahydro-6-oxonicotinic acid (III); a-
methylene-glutaric acid (IV); methylitaconic
acid (V); dimethylmaleic acid (VI); and py-
ruvic acid (VII) (Fig. 1).
Of particular interest is the finding that the
carbon skeleton of nicotinic acid, after
methylmaleic acid (8, 9). The participation of
B ,2-coenzyme as cofactor in one of the reaction
steps of the overall fermentation process pre-
sumably accounts for the high levels of Bl 2
compounds found in cells cultured on nicotinic
acid as the major carbon, nitrogen, and energy
source (7, 13). On the basis of recent taxonomic
studies by one of us (L. DS. S.) wherein the
nicotinic acid-fermenting organism and a large
number of named species of clostridia were
compared, it is concluded that the nicotinic
acid-fermenting clostridium is sufficiently
different as to be considered a distinct species.
In view of the pioneering work of H. A. Barker
in elucidating the mechanism of many complex
fermentations of the nicotinic acid type and
especially since coenzyme B12, which he dis-
covered, plays an important role in the fermen-
tation, it seems appropriate to suggest the
name Clostridium barkeri for the nicotinic
acid fermenting clostridium.
The characteristics used to distinguish the
new species are included in the general de-
COOH

cleavage of the ring and elimination of the ni- a C_OH COON

trogen as ammonia, undergoes a B,2-coen- NI 0 N

H
II 0
A~ H NH
zyme-dependent rearrangement to form meth- -", --
ylitaconic acid (V). Migration of the double CN2 , COOH
bond next results in the formation of the sym- I2
HOOC
11
CH2
IV + NH3
metrical dicarboxylic acid, dimethylmaleic
acid (VI), which, presumably after hydration,
can yield propionate equally well from either H3C\ CH2

half of the molecule as indicated by the results HOOC , COOH

of the earlier isotope studies (11). Two separ-
able enzymes (7) are required to convert a-
methyleneglutaric acid (IV) to dimethylmaleic
acid (VI). The first, the B,2-coenzyme-de- CH,CH2COOH
pendent a-methyleneglutarate mutase, forms
methylitaconic acid (V) which is then con-
verted by methylitaconic isomerase to di- + Co2
'Present address: National Cancer Institute, Bethesda,
Md. 20014. FIG. 1. Anaerobic breakdown of nicotinic acid.
758
VOL. 110, 1972 NOTES 759
scription below. C. barkeri and a few nonpro- zurin, 0.001 g; and 40 ml of a solution con-
teolytic clostridia (which resemble it in cul- taining (per liter): CaCl2 2H20, 0.2 g; MgSO,
tural characteristics) having terminal oval -7H2O, 0.2 g; K2HPO4, 1 g; KH2PO4, 1 g;
spores are listed in Table 1, together with NaHCO.s, 10 g; and NaCl, 2 g. The other
some properties whereby these can be distin- media and methods were those commonly used
guished. for the identification of anaerobic bacteria (12).
The basal medium for fermentation tests Fermentation products were determined by
contained, per liter: yeast extract, 10 g; pep- gas chromatography and silicic acid chroma-
tone, 20 g; cysteine hydrochloride, 0.5 g; resa- tography (10). Deoxyribonucleic acid (DNA)
TABLE 1. Some characteristics differentiating C. barkeri from similar clostridiaa
Indole Mannose Trehalose Mannitol Sorbitol Fermenationb
Organism Motility frain fermen-
formation tation
fermen-
tation
fermen-
tation
fermen-
tation acids

Clostridium barkeri + + B, L
C. innocuum _ _ + _ + _ A, B
C. ramosum _ _ + + + _ A, F, L, S
C. glycolicum + _ _ _ - + A, IV, P
C. scatologenes + + + A, B, C
a Plus indicates positive reaction, minus indicates no reaction.
bPrincipal acids produced while growing in peptone, yeast extract, glucose medium. Acids are abbreviated
as follows: A, acetic acid; B, butyric acid; C, caproic acid; F, formic acid; IV, isovaleric acid; L, lactic acid;
P, propionic acid; and S, succinic acid.

tm0

MS

wm
FIG. 2. Electron micrograph of Clostridium barkeri. x29,000.
760 NOTES J. BACTERIOL.
isolated (5) from dried cells that had been mycin. Resistant to dihydrostreptomycin, kan-
grown on nicotinate, was hydrolyzed with 70% amycin, and polymixin B.
perchloric acid. The purine and pyrimidine Source: isolated from Potomac River mud.
bases were separated by paper chromatography
is a distinct pleasure for the authors, especially E.R.S.
and estimated spectrophotometrically (1). A andItT.C.S.,
mineral salts-carbonate medium supplemented esteem of a greathave this opportunity to publicly express our
to
scientist and teacher, H. A. Barker.
with 0.5 to 1% nicotinic acid was used for large We are indebted to Blair Bowers for the electronmi-
scale culture of C. barkeri (11, 13). crograph of C. barkeri.
The characteristics of C. barkeri are: LITERATURE CITED
Morphology: slender rods, 0.3 to 0.5 ,m in 1. Bendich, A. 1957. Methods for characterization of nu-
width and 1.6 to 9.7 ym in length (Fig. 2). cleic acids by base composition, p. 715-723. In S. P.
Heat-resistant, oval, terminal spores formed, Colowick and N. 0. Kaplan (ed.), Methods in enzy-
but not abundantly. Gram positive. Not mo- 2. Harary, mology, vol. 3. Academic Press Inc., New York.
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bation are 0.5 to 1.0 mm in diameter with en- II. Anaerobic reversible hydroxylation of nicotinic
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823-831.
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5. Kirby, K. S. 1968. Isolation of nucleic acids with phe-
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Stadtman. 1970. Nicotinic acid metabolism. V. A
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