INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1987, p. 52-59 Vol. 37, No.

1
0020-7713/87/010052-08$02.00/0
Copyright 0 1987, International Union of Microbiological Societies

Reclassification of Pseudomonas acidovorans den Dooren de Jong

1926 and Pseudomonas testosteroni Marcus and Talalay 1956 as
Comamonas acidovorans comb. nov. and Comamonas testostersni
comb. nov., with an Emended Description of the Genus
Comamonas
JIN TAMAOKA, DUK-MO HA,? AND KAZUO KOMAGATA*
Institute of Applied Microbiology, University of Tokyo, Yayoi 1-1- I , Bunkyo-ku, Tokyo 113, Japan

We examined 36 strains of Pseudomonas acidovorans, Pseudomonas testosteroni, and Comamonas terrigena

on the basis of phenotypic characters, chemotaxonomic characters, and deoxyribonucleic acid (DNA)-DNA
homology. Strains of these three species share many phenotypic characteristics; these organisms exhibit higher
levels of DNA-DNA homology and higher levels of electrophoretic enzyme pattern similarity within the three
species than with Pseudomonas aeruginosa. The strains in the three species could be differentiated on the basis
of carbon compound assimilation patterns, cellular fatty acid composition, and DNA base composition. We
propose that Pseudomonas acidovorans and Pseudomonas testosteroni be transferred to the genus Comamonas,
as Comamonas acidovorans comb. nov. and Comamonas testosteroni comb. nav., respectively.

Pseudomonas acidovorans, Pseudomonas testosteroni,
Pseudomonas cruciviae, Pseudomonas dacunhae,
P ~ e u d o m o n a s desmolytica, and Pseudomonas
indoloxidans constitute a related group of species (6, 14,
15,37). Strains of species in this group assimilate a variety of
organic compounds but no sugars (1,37). For example, Gray
and Thornton described P. dacunhae, P. desmolytica,
and P. cruciviae as soil bacteria that decompose certain
aromatic compounds (8). The type strain of P. testosteroni
decomposes testosterone (23), and the strains named P.
desmolytica grow with hydrocarbons as sole carbon
In this paper we describe transfer of P. acidovorans and P.
testosteroni to the genus Cornamonas as Comamonas
acidovorans comb. nov. and Comamonas testosteroni
comb. nov., respectively.
MATERIALS AND METHODS
Microorganisms. We examined 36 strains of P , acido-
vorans, P. testosteroni, C . terrigeua, L P .cruciviae, P.
dacunhae, P. desmolytica, P. indoloxidans, Vibrio
percolans, Vibrio cyclosites, and Vibrio neocistes

sources (8, 16). These strains were isolated by the enrich-
ment culture method, and organic compounds in natural
environments may be degraded by these microorganisms.
Pseudomonas species have been grouped on the basis of
biochemical and physiological characters (14, 15, 30, 34, 37),
ribosomal ribonucleic acid (rRNA)-deoxyribonucleic acid
(DNA) homology (4, 28, 29, 33), 16s rRNA cataloging (41,
42), the allosteric pattern of 3-deoxy-~-arabino-heptul-
osonate 7-phosphate synthetase (40), and quinone system
and cellular fatty acid compositions (26). With all of these
grouping methods, Pseudomonas aeruginosa, the type spe-
cies of the genus Pseudomonas, and P. acidovorans are
clearly separated. According to the International Committee
on Systematic Bacteriology Subcommittee on Pseudomonas
and Related Organisms in 1978 (18), It was unanimously
agreed that both Pseudomonas and Alteromonas were them-
selves heterogeneous groups of organisms, possibly contain-
ing clusters of organisms differentiable at the genus level,
e.g., the P. acidovorans group.
De Vos et al. proposed revival of the genus Comamonas,
with Comamonas terrigena as the type species (5). C.
terrigena, P. acidovorans, and P. testosteroni are in the
same rRNA-DNA homology group (3,the same 16s rRNA
cataloging subdivision (41), and the same guinone system-
cellular fatty acid composition group (26).
* Corresponding author.
t Present address: Department of Food Technology, College of
Engineering, Dongguk University, Seoul 100, Korea.
52
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(Table 1).P . aeruginosa KS 0025T (= ATCC 10145T) (T =
type strain) was used as a reference strain. Species names in
quotation marks are not on the Approved Lists of Bacterial
Names (35). The medium used for maintenance and cultiva-
tion of the strains was nutrient agar that contained 1.0%
(wthol) beef extract (Difco Laboratories, Detroit, Mich.),
1.0% (wtlvol) Polypeptone (Daigo, Osaka, Japan), 0.5%
(wthol) NaCl, 1.5% (wt/vol) Bacto-Agar (Difco), and dis-
tilled water (pH 7.2). The inoculated medium was incubated
at 30C. Stock cultures of the strains were transferred once
every 2 months.
Cell morphology. Gram reactions were determined by
using cells grown on nutrient agar at 30C for 2 days; the
Hucker-Conn method (10) was used. Spore formation was
determined by malachite green staining of cells grown on
nutrient agar. Flagellation was examined by using a model
JEOL 200CX transmission electron microscope at 100 kV
after negative staining with 1% (wthol) phosphotungstic
acid.
Assimilation test of organic compounds. A total of 68
organic compounds were examined. The sodium salts of
organic acids were used unless stated otherwise. The follow-
ing carbohydrates were tested; D-ribose, D-xylose, D-
arabinose, L-arabinose, n-glucose, D-galactose, D-fructose,
sucrose, maltose, cellobiose, lactose, and starch. The
carboxylic acids tested were acetate, caprylate, pelargonate,
oxalate (free acid), malonate, succinate, maleate, fumarate,
glutarate, adipate, pimelate, suberate, azelate, and sebacate.
The hydroxy acids tested were DL-malate, citrate, DL-
tartrate (potassium-sodium salt), DL-P-hydroxybutyrate, DL-
VDL. 37, 1987 GENUS COMAMONAS 53

TABLE 1. List of strains used

Received as: History of strain Other strain designation(s) Reference
P . acidovorans KS 0057 + AJ 3117 + ATCC 15668 + RH 2157 = NCIQ 9681 = NCTC 10683 37
6 P .crkciuiae KS 0005 + AJ 2002 + IAM 1048 t P-8-2 1
P . dacunhae KS 0007 + AJ 2004 + IAM 1123 c A-7-2 1
P adacunhae KS 0065 + AJ 2005 + IAM 1152 + R-10-2 1
P . dacuhhae KS 0066 + AJ 2006 t IAM 1176 t G1-7 1
P . desmolytica KS 0054 + AJ 3111 + ATCC 15006 + RH 1888 + P-13 19
P.desmolytica KS 0059 + AJ 1575 + 1-1(Komagata)b
P.desmolytica KS 0060 + AJ 1577 + 1-3-2 (Komagata)
P.desmolytica KS 0061 + AJ 1578 t 1-4-1 (Komagata)
P . desmolytica KS 0062 + AJ 1582 + 2-3-2 (Komagata)
P . desmolytica KS 0063 + AJ 1583 + 3-1 (Komagata)
P . desmolytica KS 0064 + AJ 1584 + 3-4 (Komagata)
P.desmolytica KS 0072 + AJ 2278 + IAM 1461 t P-3 16
P. desmolytica KS 0073 + AJ 2279 + IAM 1482 t- P-15 16
P.desmolytica KS 0074 + kl 2280 +- IAM 1492 t P-13 16
P . desmolytica KS 0075 + AJ 2304 + 4B 43
P . indoloxidans KS 0080 + AJ 2074 t ATCC 9355 + P. Gray 7
P . indoloxidans KS 0081 + RH 1173 = ATCC 17438 = ATCC 12816 37
P . indoloxidans KS 0089 + NCIB 2760 + NCTC 2760 + P. Gray = ATCC 13751 7
P . testosteroni KS 0043 + AJ 2230 + NRRL B-2611 = ATCC 11996 = NCIB 8955 22
P . testosteroni KS 0048 + AJ 2270 + ATCC 17409 + 27 (Stanier) 37
P . testosteroni KS 0103 + NCIB 8893 + S. Dagley = ATCC 17454 3
P . testosteroni KS 0105 + NCIB 9683 t- T. Wiken = ATCC 15666 37
Pseudomonas sp. KS 0149 + NP 9-2 (Ikemoto)c
Pseudomonas sp. KS 0150 + NP 9-5 (Ikemoto)
Pseudomonas sp. KS 0151 t NP 9-8 (Ikemoto)
Pseudomonas sp. KS 0152 + NP 9-10 (Ikemoto)
Pseudomonas sp. KS 0153 + NP 9-14 (Ikemoto)
C . terrigena KS 0020 + AJ 2083 + RH 247 = ATCC 8461 = NCIB 8193 11
C . terrigena KS 0705 + RH 1174 (Alwine)d
C . terrigena KS 0708 + RH 1177 (Alwine)
C . terrigena KS 0706 t RH 1175 (Alwine)
C.terrigena KS 0702 + RH 608 (Alwine)
V . percolans KS 0079 +- RH 260 = ATCC 17758 20
V . cyclosites KS 0106 + NCIB 2581 + NCTC 2581 + Thornton = ATCC 14635 21
V . neocistes KS 0107 c NCIB 2582 + NCTC 2582 +- Thornton = ATCC 14636 21
P . aeruginosa KS 0025 t AJ 2116 t IAM 1514 t ATCC 10145 = NCIB 8295 = NCTC 10332
a AJ, Central Research Laboratories, Ajinomoto Co., Kawasaki, Japan; ATCC, American Type Culture Collection, Rockville, Md. ; IAM, Institute of Applied
Microbiology, University of Tokyo, Tokyo, Japan; IFO, Institute for Fermentation, Osaka, Japan; NCIB, National Collection of Industrial Bacteria, Torry
Research Station, Aberdeen, Scotland; NCPPB, National Collection of Plant Pathogenic Bacteria, Plant Pathology Laboratory, Harpenden, England; NCTC,
National Collection of Type Cultures, Central Public Health Laboratory, London, England; NRRL, Northern Utilization Research and Development Division,
U.S. Department of Agriculture, Peoria, 111.; RH, R. Hugh, George Washington University, Washington, D.C.
Isolated from chicken feces.
Isolated as aromatic compound-assimilating bacteria.
Alwine, M.S. thesis.

lactate, glycolate, and gluconate (potassium salt). The mis-
cellaneous acids tested were 2-ketogluconate, a-ketogluta-
rate, laevulinate, citraconate, and itaconate. The alcohols
tested were ethanol, ethylene glycol, propylene glycol, 2,3-
butylene glycol, glycerol, and D-mannitol. The aromatic
compounds tested were benzoate, rn-hydroxybenzoate, p -
hydroxybenzoate, phthalate, phenol, hippurate, and testos-
terone. The amino acids tested were glycine, p-alanine,
L-serine, L-threonine, L-glutamate, L-asparagine, L-arginine,
L-ornithine, DL-methionine, L-phenylalanine, and L-
tryptophan. The miscellaneous compounds tested were
glucono-&lactone, anthranilate, putrescine, betaine,
acetamide, and n-hexadecane.
The basal medium, the standard mineral base of Stanier et
al. (37), was solidified with 1.5% (wthol) purified agar
(Difco). After the basal medium was autoclaved, each com-
pound (sterilized by filtration) was added at a concentration
of 0.2% (wthol); the o d y exception to this was phenol,
which was added at a concentration of 0.02% (wthol).
Growth was examined after 1, 3, 7, and 14 days at 30C.
Biochemical characterization. Requirements for growth
factors were determined in basal medium (37) without agar
containing 1% (wthol) L-glutamate (sodium salt) as the
carbon source. One loopful of fresh cells was suspended in
10 ml of basal medium, 0.1 ml of the suspension was
suspended in another 10 ml of basal medium, and 0.1 ml of
the suspension was inoculated into 10 ml of basal medium
containing 1 yg of each vitamin per ml or 20 yg of an amino
acid per ml. As described by Alwine (H. M. Alwine, M.S.
thesis, George Washington University, Washington, D.C.,
1969),requirements for nicotinamide, sodium folate, calcium
pantothenate, and DL-methionhe were determined by the
omission-addition procedure.
Denitrification was determined by the method of Stanier et
al. (37), using a GasPak anaerobic system (BBL Microbiol-
ogy Systems, Cockeysville, Md.). King A and King B media
were used for production of pigments. Catalase was detected
by formation of bubbles after a 3% H202 solution was
dropped onto a fresh colony. Indophenol oxidase was de-
tected with cytochrome oxidase test paper (Nissui, Tokyo,

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54 TAMAOKA ET AL.
Japan). Extracellular deoxyribonuclease was detected with
DNase test agar (Difco). Aerobic and anaerobic production
of acid (OF reaction) from D-glucose, D-fructose, and D-
galactose (13) was determined in OF basal medium (Difco).
A sugar solution sterilized by filtration was added at a
concentration of 1% (wt/vol), and acid production was
recorded after 7 days of incubation. The accumulation of
poly-P-hydroxybutyrate was determined by the method de-
scribed by Stanier et al. (37).
Isolation of DNA. DNA was isolated by the phenol method
of Saito and Miura (32), with some modifications. A mixture
of phenol and chloroform (l:l, vol/vol) was used instead of
phenol to remove protein, and a solution containing 50 pg of
ribonuclease A (Sigma Chemical Co., St. Louis, Mo.) per ml
and 20 U of ribonuclease TI (Sankyo, Tokyo, Japan) per ml
was used for hydrolysis of the ribonucleic acid. Isopropanol
precipitation was also used to remove polysaccharides and
ribonucleic acid (24).
Determination of DNA base composition, DNA base com-
position was determined by reverse-phase high-performance
liquid chromatography (HPLC) after enzymatic hydrolysis
of DNA into nucleosides (39). DNA was treated with nucle-
ase PI (EC 3.1.30.1; Yamasa, Chiba, Japan) at 50C for 1h,
and this was followed by treatement with bacterial alkaline
phosphatase (EC 3.1.3.1; Sigma) at 37C for 1h. The mixture
of nucleosides was analyzed with an HPLC system consist-
ing of a model LC4A pump (Shimadzu, Kyoto, Japan), a
Cosmosil 5Cls column (150 mm by 4.6 mm [inside diameter];
Nakarai, Kyoto, Japan), a model 481 Lambda-Max detector
(Waters Associates, Inc., Milford, Mass.) set at 270 nm, and
a Chromatopac C-R2AX data analyzer (Shimadzu). The
eluent was a mixture Containing 0.2 M NH4H2P04 and
acetonitrile (20:1, voVvol); the flow rate was 1 ml/min.
DNA-DNA hybridization. DNAs were hybridized by the
membrane filter method (25). DNAs of P . acidovorans KS
0057T,P . testosteroni KS 0043T,and C. terrigena KS 0020T
were labeled by nick translation (31), using a type TRK.700
kit (Radiochemical Centre, Amersham, United Kingdom).
[3HlDNAwas hybridized on a membrane filter bearing 30 pg
of sample DNA in 1 ml of 2x SSC buffer ( l x SSC is 0.15 M
NaCl plus 0.015 M sodium citrate) containing 0.1% (wthol)
sodium dodecyl sulfate at 65C for 45 h. The filter was
washed with 2 ml of 2X SSC buffer twice and then with 1and
5 ml of 5 mM tris(hydroxymethy1)aminomethane buffer (pH
9.5). The amount of hybridized [3H]DNAwas determined by
using a Tri-Carb 300 liquid scintillation counter, (Packard
Instrument Co., Inc., Rockville, Md.). A DNA filter con-
taining Escherichia coli K-12 strain IAM 1268 DNA was
used as a negative control.
Determination of cellular fatty acid composition. Methyl
esters of cellular fatty acids were prepared from lyophilized
cells by using a 5% methanolic hydrochloride acid solution
(Kokusan Chemical Works, Tokyo, Japan) according to the
method of Ikemoto et al. (17). After extraction with hexane,
methyl esters of fatty acids were analyzed by gas-liquid
chromatography. We used a model GC-6A gas-liquid chro-
matography system (Shimandzu) equipped with a DIACOT
Silar 1OC capillary column (30 m by 0.28 mm [inside diam-
eter]) at 190C and a flame ionized detector. The carrier gas
was nitrogen (flow rate, 0.4 mumin). Gas-liquid chromatog-
raphy chromatograms were analyzed with a Chromatopac
C-R1A analyzer (Shimadzu).
Determination of quinone system. Quinones were extracted
from lyophilized cells with a mixture of chloroform and
methanol (2:1, vol/vol) and were purified by thin-layer
chromatography, using the method of Collins et al. (2).
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INT. J. SYST. BACTERIOL.
Quinone compositions were determined by reverse-phase
HPLC (38). The HPLC system used consisted of a model
LC-SA pump (Shimadzu), a Zorbax ODS column (250 mm
by 4.6 mm [inside diameter]; Du Pont Co., Wilmington,
Del.), a model SPD-2A spectrophotometric detector
(Shimadzu) set at 270 nm, and a Chromatopac C-R1B data
analyzer (Shimadzu). The eluent was a mixture of methanol
and isopropyl ether (3:1, vol/vol) at a flow rate 1ml/min. The
results of the HPLC analysis were confirmed by direct-inlet
mass spectrometry with a model LKB-9000 instrument
(Shimadzu).
Electrophoretic pattern of enzymes. Zymograms were pre-
pared by a previously described method (9). The enzymes
examined were glutamate dehydrogenase (nicotinamide ad-
enine dinucleotide phosphate, oxidized) (EC 1.4.1.4), malate
dehydrogenase (EC 1.1.1.37), isocitrate dehydrogenase
(nicotinamide adenine dinucleotide phosphate, oxidized)
(EC 1.1.1.41), aspartate aminotransferase (EC 2.6.1.1),
fumarate hydratase (EC 4.2.1.2), and catalase (EC 1.11.1.6).
Similarity values for the electrophoretic mobilities of the
enzymes were calculated by using the simple matching
method (9), and a dendrogram was made by using the
unweighted average linkage method (36).
RESULTS
Phenotypic characteristics. All 36 strains studied were
gram-negative, nonsporeforming rods. They were motile and
possessed one to five polar flagella. Catalase and indophenol
oxidase were produced. All strains accumulated poly-p-
hydroxybutyrate in their cells, and did not exhibit
denitrification, hydrolysis of DNA, production of water-
soluble pigments or fluorescent pigments on King A and
King B media, or aerobic and anaerobic production of acid
from D-glucose, D-frUCtOSe, or D-galactose (OF media).
These phenotypic characteristics were the same for all 36
strains studied. The 36 strains cannot be differentiated on the
basis of these phenotypic characters.
Assimilation of organic compounds. The 36 strains were
separated into three clusters designated C. terrigena, P .
acidovorans, and P.testosteroni on the basis of assimilation
patterns. Strains in the cluster designated C. terrigena
(which included C.terrigena KS 0020*, V . percolans KS
0079, V . cyclosites KS 0106, and V . neocistes KS
0107) did not grow on the defined media because methionine
and nicotinamide were required for growth. The remaining
strains, which did not require growth factors, were separated
into two clusters based on their patterns of assimilation of 14
compounds. The cluster of strains designated P . acido-
vorans (which included P . acidovorans KS 0057T, nine
strains of P. desmolytica, three strains of P. indo-
loxidans, and two strains of C. terrigenu) assimilated
D-fructose, maleate, DL-tartrate, ethylene glycol, propylene
glycol, 2,3-butylene glycol, glycerol (except strains KS 0059
and KS 0062), D-mannitol, p-alanine (except KS 0059, KS
0062, and KS 0068), L-threonine, L-tryptophan (except KS
0073, and acetamide (except KS 0089) and did not assimilate
DL-P-hydroxybutyrateor testosterone. The cluster of strains
designated P . testosteroni (which included the type strain
and three other strains of P . testosteroni, one strain of P.
cruciviae, three strains of P.ducunhae, two strains of
P . desmolytica, five strains of Pseudomonas sp., and two
strains of C. terrigena) assimilated DL-P-hydroxybutyrate
and testosterone and did not assimilate the 12 compounds
which were assimilated by P . acidovorans.
VOL.37, 1987 GENUS COMAMONAS 55

TABLE 2. DNA base compositions and cellular fatty acid compositions of strains of C. terrigena, P. acidovorans, and P . testosteroni
G+C Cellular fatty acid composition (%)
Received as: Strain content
(mol%) 12:Ob 14:O 15:O 16:l 16:O 17:O 18:l 18:O A-17 A-19 2-oH 2-oH 3-0H
14:O 16:O 10:0
P . acidovorans KS 0057T 67.1 3 1 t 29 37 t 18 t 7 t 3
P . desmolytica KS 0054 66.2 2 1 t 31 39 t 17 t 6 t 3
P . desmolytica KS 0059 66.2 4 1 t 25 37 t 18 1 5 1 3
P.desmolytica KS 0062 66.7 2 1 t 32 36 t 20 1 3 1 t 3
P . desmolytica KS 0063 67.0 3 1 t 30 38 t 18 t 3 1 4
P . desmolytica KS 0064 67.1 6 1 1 37 30 1 16 t 1 t t 5
P . desmolytica KS 0072 66.8 1 1 1 40 36 1 15 t 1 t 3
P . desmolytica KS 0073 66.5 3 1 2 24 37 1 17 1 9 t t 3
P . desmolytica KS 0074 66.9 3 1 1 37 38 t 13 1 t t 6
P . desmolytica KS 0075 66.6 3 1 t 29 34 t 21 t 1 1 4
L Pindoloxidans
. KS 0080 66.6 2 1 1 36 35 t 18 t 1 t 3
P . indoloxidans KS 0081 66.9 3 1 2 22 58 1 18 t 7 2 t 4
P . indoloxidans KS 0089 66.3 2 1 t 39 37 t 16 t 1 t 2
C . terrigena KS 0702 66.6 1 1 t 32 41 t 17 t t t 3
C.terrigena KS 0706 66.8 1 1 2 22 44 1 15 t 7 2 3
P . testosteroni KS 0043T 61.5 4 t t 24 28 2 22 1 3 1 t 4 3
P . testosteroni KS 0048 61.4 2 1 t 34 30 1 24 1 1 t t 2 3
P . testosteroni KS 0103 61.7 2 t t 36 31 1 29 t 1 t t 2 3
P . testosteroni KS 0105 62.0 2 1 t 34 30 1 23 t 2 t t 2 3
P . cruciviae KS 0005 61.9 2 t t 35 30 1 24 t 1 t t 2 3
P . dacunhae KS 0007 62.0 2 t t 32 31 1 22 1 2 t t 3 3
P.desmolytica KS 0060 61.7 3 t t 32 28 1 24 1 1 t t 3 4
P . desmolytica KS 0061 61.5 2 t t 34 30 1 26 1 t t t 2 3
P . dacunhae KS 0065 61.8 3 t t 29 24 2 25 1 t t t 4 4
P . dacunhae KS 0066 62.1 3 t t 33 30 1 24 t 1 t t 3 3
Pseudomonas sp. KS 0149 61.2 2 t t 29 26 1 31 1 2 t t 4 3
Pseudomonas sp. KS 0150 61.5 3 t t 33 28 1 24 1 2 t t 3 4
Pseudomonas sp. KS 0151 61.5 4 t t 29 26 2 25 1 2 1 t 4 4
Pseudomonas sp. KS 0152 61.0 2 t t 33 30 1 23 1 2 t t 3 3
Pseudomonas sp. KS 0153 61.2 3 t t 34 29 1 25 1 1 t t 3 3
C. terrigena KS 0705 61.9 2 1 1 29 40 1 16 t 2 t t 3 4
C . terrigena KS 0708 61.5 4 1 1 31 32 2 20 t t t t 4 5
C . terrigena KS 0020T 65.1 2 3 1 42 31 1 15 t t t t 4
V . percolans KS 0079 65.1 3 4 1 42 30 t 14 t t t t 4
V. cyclosites KS 0106 65.2 2 3 2 30 29 2 26 t 1 t t 3
V. neocistes KS 0107 65.3 2 3 2 34 36 1 14 1 2 t t 3
t, Trace (less than 0.5%).
The number before the colon indicates the number of carbon atoms of the fatty acid, and the number after the colon indicates the number of double bonds. A
indicates cyclopropane acid. 2-OH and 3-OH indicate 2-hydroxy and 3-hydroxy acids, respectively.

Strains in the clusters designated P . acidovorans and P .
testosteroni assimilated the following 24 compounds: acetate
(except KS 0005 and KS 0007), succinate (except KS 0005
and KS 0007), fumarate, glutarate, adipate (except KS 0055),
pimelate (except KS 0059, suberate, azelate (except KS
0054), sebacate (except KS 0056, KS 0057, KS 0062, KS
0063, and KS 0064), DL-malate (except KS 0005, KS 0007,
KS 0061, and KS 0066), citrate, DL-lactate, glycolate,
gluconate (except KS 0066),laevulinate, citraconate, itacon-
ate (except KS 0071), m-hydroxybenzoate, p-hydroxybenz-
oate, glycine (except KS 0061), L-gtutamate, L-asparagine
anthranilate, putrescine, betaine, and n-hexadecane (except
KS 0064).
Phenol was assimilated by strains KS 0043, KS 0060, KS
0061, KS 0064, KS 0066, KS 0149, KS 0150, KS 0151, KS
0152, and KS 0153.
DNA base compositions. Strains in the three clusters des-
ignated C. terrigena, P . acidovorans, and P . testosteroni
showed clear differences in DNA base composition (Table
2). The guanine-plus-cytosine ( G + C ) contents of the DNAs
of the 15 strains in the P . acidovorans cluster ranged from
66.2 to 67.1 mot% (mean standard deviation, 66.6 2 0.4
+_

(except KS 0079, L-phenylalanine (except KS 0005 and KS
0105), and glucono-S-lactone.
The following 29 compounds were not assimilated by
strains in the clusters designated P . acidovorans and P.
testosteroni: D-ribose, D-xylose, D-arabinose, L-arabinose,
D-glucose, D-galactose, sucrose, maltose, cellobiose, lac-
tose, starch, malonate (except KS 0057 and KS 0089),
caprylate, pelargonate, oxalate, 2-ketogluconate, a-
ketoglutarate, ethanol (except KS 0057 and KS 0068), benzo-
ate (except KS 0061), phthalate (except KS 0060), hippurate
(except KS 0060 and KS 0061), L-serine, L-arginine (except
KS 0054 and KS 0059), L-ornithine, DL-methionhe,
mol%). The G + C contents of the DNAs of four strains in the
C. terrigena cluster were between 65.1 and 65.3 mol% (65.2
2 0.1 mol%), and the G+C contents of the DNAs of 17
strains in the P . testosteroni cluster were between 61.0 and
62.1 mol% (61.5 k 0.4 mol%).
Cellular fatty acid composition. The major fatty acids in the
36 strains were hexadecanoic acid (16:0), hexadecenoic acid
(16:1), and octadecenoic acid (18:l) (Table 2). Each of the 36
strains also contained more than 2% 3-hydroxydecanoic acid
(3-OH 1O:O). Differences in the fatty acid compositions of the
three clusters of strains were as follows: the levels of
tetradecanoic acid (14:O) were more than 3% in the cluster of

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56 TAMAOKA ET AL. INT.J. SYST.BACTERIOL.

TABLE 3. DNA-DNA hybridization of strains of C. terrigena, P . DISCUSSION

acidovorans, and P . testosteroni
% Hybridization with [3H]DNA from: The genera Pseudomonas and Comamonas both consist of
~

chemoorganotrophic, nonsporeforming, strictly aerobic,

Received as: Strain P. P. C.
acidovorans testosteroni terrigena gram-negative rods with polar flagella. The type species of
KS 0057T KS 0043= KS 0020T these two genera are P . aeruginosa and C . terrigena, respec-
tively. When Comamonas was revived, it was mentioned
P . acidovorans KS 0057= 100 18 26
90 26 23 that some of the named taxa belonging to the P . acidovorans
P . desmolytica KS 0054
P . desmolytica KS 0059 78 22 24 rRNA branch should be assigned to the genus Comamonas
P . desmolytica KS 0063 65 6 3 (5).
P . desrnolytica KS 0075 69 19 24 Various procedures have been used to group the species of
P . indoloxidans KS 0080 77 19 24 Pseudomonas. P . aeruginosa and P . acidovorans always
P . indoloxidans KS 0089 91 19 23 occur in different groups, and often P . acidovorans, P .
C. terrigena KS 0702 65 6 6 testosteroni, and C . terrigena are assigned to one group (5,
C . terrigena KS 0706 63 7 3 26, 41). Palleroni et al. divided Pseudomonas species into
P . testosteroni KS 0043= 26 100 21 five groups based on rRNA homology (29). P . aeruginosa is
L P cruciviae
. KS 0005 26 55 23
P . dacunhae KS 0007 25 77 22
in group I, and P . acidovorans and P . testosteroni are in
P . testosteroni KS 0048 31 92 24 group I11 (29). Pseudomonas species groupings based on the
P . desmolytica KS 0060 30 87 28 allosteric patterns of 3-deoxy-~-arabino-heptulosonate 7-
P . desmolytica KS 0061 27 73 18 phosphate synthetase (40) and on quinone system and cellu-
P . testosteroni KS 0103 45 73 14 lar fatty acid compositions (26) are similar to rRNA group-
P . testosteroni KS 0105 36 81 33 ings. Groupings based on rRNA-DNA homology data by De
Pseudomonas sp. KS 0149 28 74 31 Vos and De Ley produced similar results (4). P . aeruginosa
Pseudomonas sp. KS 0150 40 79 34 was assigned to superfamily 11, whereas P . acidovorans,
Pseudomonas sp. KS 0151 32 76 28 P . testosteroni, and C . terrigena were assigned to superfam-
Pseudomonas sp. KS 0152 31 70 24
Pseudomonas sp. KS 0153 25 65 24 ily I11 (5). Groupings based on oligonucleotide cataloging
C. terrigena KS 0705 42 62 12 also showed separation of rRNA group I and rRNA group I11
C . terrigena KS 0708 34 54 9 at the subdivision level (41,42). Based on our results, the
C.terrigena KS 0020* 18 8 100 three clusters of strains designated P . acidovorans, P .
V . percolans KS 0079 38 17 92 testosteroni, and C . terrigena shared phenotypic character-
V . neocistes KS 0107 29 22 77 istics that distinguish these three clusters from P . aerugi-
V . cyclosites KS 0106 17 10 48 nosa. P . aeruginosa produces fluorescent pigments; C .
terrigena does not produce fluorescent pigments. P . aerugi-
nosa uses glucose; C . terrigena does not use glucose. P .
strains designated C . terrigena and less than 1%in the other aeruginosa does not accumulate poly-P-hydroxybutyrate in
two clusters of strains; the levels of 2-hydroxy-hexadecanoic its cells; C. terrigena accumulates poly-P-hydroxybutyrate.
acid (2-OH 16:O) were more than 2% in the cluster of strains These characters which distinguish P . aeruginosa from C .
designated P . testosteroni and less than 1% in the other terrigena also distinguish P . aeruginosa from P . acidovorans
clusters. and P . testosteroni. On the basis of DNA-DNA homology
Quinone system. All strains had ubiquinone-8 (Q-8) as the and electrophoretic comparison of enzymes, the three clus-
major quinone (more than 90%) and Q-7 and Q-9 as minor ters of strains designated P . acidovorans, P . testosteroni,
quinones. Some strains also contained Q-6 or Q-lo. Mena- and C . terrigena show closer relationships to one another
quinone was not detected. than to the type strain of P . aeruginosa. Although these
DNA-DNA hybridization. Table 3 shows the levels of groups of Pseudomonas species may have different taxo-
DNA-DNA homology among the three clusters of strains. nomic implications for the genus Pseudomonas, all suggest
Strains in each of the three clusters showed greater hybrid- that P . aeruginosa and P . acidovorans should be classified in
ization to the type strain of their cluster than to the type separate genera and that P . acidovorans, P . testosteroni,
strains of the other two clusters. P . aeruginosa K S 0025T and C . terrigena should be classified in one genus.
showed a low level of hybridization (less than 2%) to the A small number of characteristics differentiate P .
type strains of C. terrigena, P . acidovorans, and P . acidovorans, P . testosteroni, and C . terrigena. It had been
tes tosteroni. proposed that P . testosteroni and C . terrigena should be
De Vos et al. reported 100% DNA homology between V . classified in one species (12). P . acidovorans and P .
cyclosites NCIB 2581 (= KS 0106) and the type strain of C. testosteroni have been described as synonyms of C. ter-
terrigena (3, while our result was 48%. De Vos et al. rigena (6). In addition, strains named P . acidovorans or P .
determined homology levels from the renaturation rate, testosteroni have been renamed as C. terrigena (Alwine,
whereas we used the membrane filer method. The data from M . S . thesis). However, De Vos et al. believed that these
different hybridization methods may be not adequate for three species are independent and prepared a differential
comparison. table (5). We confirmed the conclusion of these authors by
Electrophoretic pattern of enzymes. A dendrogram based determining assimilation patterns of carbon compounds,
on the electrophoretic patterns of enzymes is shown in Fig. DNA base compositions, cellular fatty acid compositions,
1. We identified the following four clusters of strains: P . electrophoretic patterns of enzymes, and levels of DNA-
aeruginosa, C . terrigena, P . acidovorans, and P . DNA homology. The type strains of P . acidovorans, P .
testosteroni. The C. terrigena, P . acidovarans, and P . testosteroni, and C . terrigena are included among the clus-
testosteroni clusters are linked at a similarity level of 0.4, but ters of strains designated P . acidovorans, P . testosteroni,
P . aeruginosa is linked to C. terrigena, P . acidovorans, and and C . terrigena, respectively. Therefore, strains in each
P . testosteroni at a similarity level of 0.2. cluster are identified as follows: P . desmolytica K S 0054,

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VOL.37, 1987 GENUS COMAMONAS 57

::%%
0025~- P. aeruginosa
F l u s t e r of strains of
0 0 2 0 ~ C. terrigena

, of
0059

I I
0089

[
0061
0149

I
iiii:fzi 1
Cluster of strains of
testosteroni

0
1 1
0.2
I
0.4
I
0.6
1
0.8
- I
1.o
0105
0007
0043=
KS 0048

SM s
FIG. 1. Dendrogram of the electrophoretic patterns of enzymes calculated by the unweighted average linkage method. See reference 36.
S S M , Simple matching coefficient.

KS 0059, KS 0062, KS 0063, KS 0064, KS 0072, KS 0073, with the data obtained with conventional methods. Table 4
KS 0074, and KS 0075, b P .indoloxidans KS 0080, KS differentiates the three species by conventional and che-
0081, and KS 0090, and C . terrigena KS 0702 and KS 0706 motaxonomic methods. Alwine reported that C. terrigena
are strains of P . acidovorans; b b Pcruciviae
. KS 0005, b P . requires methionine, nicotinamode, and folate for growth
dacunhae K S 0007, KS 0065, and KS 0066, P . (Alwine, M.Sc. thesis); however, we found that this orga-
desmolytica KS 0060 and KS 0061, Pseudomonas sp. nism requires methionine and nicotinamide but not folate.
strains KS 0149, KS 0150, KS 0151, KS 0152, and KS 0153, The DNA base compositions of a lot of Pseudomonas
and C. terrigena KS 0705 and KS 0708 are strains of P . species were first determined by Mandel (22). Means and
testosteroni; and V . percolans KS 0079, V . cyclosites standard deviations for the G+C contents were 66.8 k 1.0
KS 0106, and V . neocistes KS 0107 are strains of C. mol% for 15 strains of P . acidovorans and 61.8 k 1.1 mol%
terrigena. The differential table of De Vos et al. (5) based on for 9 strains of P. testosteroni (22); these values were
data from the API System seems to be difficult to compare determined from the buoyant densities of DNAs. G+C
contents determined by the HPLC method were 66.6 k 0.4
mol% for 15 strains of P . acidovorans, 65.2 2 0.1 mol% for
TABLE 4. Differential characteristics of C. terrigena, 4 strains of C. terrigena, and 61.5 k 0.4 mol% for 17 strains
C. acidovorans, and C . testosteroni of P . testosteroni. The ranges of G+C content were about
C. C. C. two times of standard deviation. We believe that the G+C
Characteristic terrigena acidovorans testosteroni content range of a well-defined species must be less than 2
(4 strains) (15 strains) (17 strains) mol% (maybe less than 1 mol%).
Growth factor Methionine, The data presented above are interpreted to indicate that
requirement nicotinamide P . acidovorans and P. testosteroni should be transferred to
Assimilation o f a the genus Comamonas. Therefore, Comamonas acid-
D-Fructose ovorans comb. nov. (synonym, Pseudomonas acidovorans)
D-Mannitol and Comamonas testosteroni comb. nov. (synonym, Pseu-
DL-Tartrate domonas testosteroni) are proposed.
Ethylene glycol Comamonas De Vos et al. 1985. The description of the
Propylene glycol
DL-P-Hydroxybutyrate genus Comamonas (5) should be expanded. Poly-p-
Testosterone hydroxybutyrate is accumulated in cells. The major cellular
Fatty acid compositon fatty acids are hexadecanoic acid (16:0), hexadecenoic acid
(%I (16:1), and octadecenoic acid (18:l). Cells always contain
14:0 >3 <1 <1 3-hydroxydecanoic acid (3-OH 1O:O). The major quinone is
2-OH 16:O <1 (1 >2 Q-8. Menaquinone is not produced. The DNA G+C contents
G + C content (mol%)b 65.1-65.3 66.2-67.1 61.O-62.1 range from 61.0 to 67.1 mol% (as determined by the HPLC
a Determined on the basal medium of Stainer et al. (37). method).
Determined by the HPLC method (39). Comamonas terrigena De Vos et al. 1985. The description of

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58 TAMAOKA ET AL.
Comamonas terrigena as reported by De Vos et al. (5) and
Hugh (11,12) was verified. Methionine and nicotinamide are
required as growth factors. The DNA G+C content ranges
from 65.1 to 65.3 mol% (as determined by the HPLC
method).
Comamonas acidovorans (den Dooren de Jong 1926)
Tamaoka, Ha, and Komagata comb. nov. The characteristics
which we investigated are in good agreement with those of
Stanier et al. (36) and those of Palleroni (27) for Pseudomo-
nus acidovorans. The DNA G+C content ranges from 66.2
to 67.1 mol% (as determined by the HPLC method). The
type strain of Comamonas acidovorans is strain KS 0057 (=
IAM 12409 = JCM 5833 = ATCC 15668 = NCIB 9681).
Comamonas testosteroni (Marcus and Talalay 1956)
Tamaoka, Ha, and Komagata comb. nov. The characteristics
which we investigated are in good agreement with those of
Stanier et al. (37) and those of Palleroni (27) for Pseudomo-
nus testosteroni. The DNA G +C content ranges from 61.O to
62.1 mol% (as determined by the HPLC method). The type
strain of Comamonas testosteroni is strain KS 0043 (= IAM
12419 = JCM 5832 = ATCC 11996 = NCIB 8955).
Characteristics which differentiate C. terrigena, C.
acidovorans, and C . testosteroni are shown in Table 4.
ACKNOWLEDGMENTS
We thank R. Hugh, Department of Microbiology, George Wash-
ington University, Washington D.C., for supplying the strains of C .
terrigena and T. Kaneko, Japan Collection of Microorganisms,
RIKEN, Saitama, Japan, for calculating the similarity values and
the dendrogram of the zymograms.
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