INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 246-251
0020-7713/86/020246-06$02.OOIO
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 2
Rhodococcus chlorophenolicus sp. nov. a
Chlorophenol-Mineralizing Actinomycete
JUHA H. A. APAJALAHTI," PAULIINA KARPANOJA, AND MIRJA S. SALKINOJA-SALONEN
Department of General Microbiology, University of Helsinki, SF-00280, Helsinki, Finland
Strain PCP-I, which was isolated from a pentachlorophenol-mineralizing mixed culture, had the following
characteristics of the actinomycetes assigned to the genus Rhodococcus: DL-diaminopimelicacid, arabinose, and
galactose as cell wall constituents; major menaquinone with nine isoprenoid units and one hydrogenated double
bond (MK-9Hz); mycolic acids containing 33 to 43 carbon atoms; and a marked rod-to-coccus cycle during
growth. None of the previously described species of Rhodococcus contains both MK-9H2 and mycolic acids of
this size, and, unlike other rhodococci, strain PCP-I utilizes rhamnose, inositol, and sorbitol. Based on these
properties, we believe that strain PCP-I represents a new Rhodococcus species. We propose the name
Rhodococcus chlorophenolicus for this new species because of its ability to degrade several chlorophenols. The
type strain is strain PCP-I (= DSM 43826).
pentachlorophenol-mineralizing bacterial mixed culture,
whose properties have been described previously (1,37,41).
Media. For chemical analyses strain PCP-I was grown at
28°C in mineral salts medium (40) supplemented with 2 g of
yeast extract per liter. Mineral salts medium (40) supplemented with (per liter) 1.5 ml of a vitamin solution (40),1.5
ml of a trace element solution (3), and 10 g of rhamnose was
used in temperature and NaCl tolerance tests. After incubation for 1 week growth was recorded as turbidity (absorbance at 600 nm).
Sole carbon sources. The growth of strain PCP-I on 1%
rhamnose, 1% inositol, 1% sorbitol, 1% glycerol, 1%
maltose, 1% trehalose, and 0.1% m-hydroxybenzoic acid
was tested at 28°C in shake flask cultures. Other carbon
sources were tested by using the API 50CH system and API
20NE medium (based on turbidity).
Cell wall diamino acids and sugars. Cell wall preparations
were obtained by alkali treatment of washed cell pellets (25).
Subsequent acid hydrolysis for amino acid determinations
was performed as described by Keddie and Cure (25), and
hydrolysis for sugar determinations was performed in 1 M
HCl for 4 h. Amino acids and sugars were detected by
thin-layer chromatography (TLC). TLC plates (catalog no.
5716; cellulose with no fluorescent indicator; E. Merck AG)
were developed, and the spots were visualized as described
by Staneck and Roberts (39). Strains containing mesodiaminopimelic acid (Brevibacterium linens strain 9), LLdiaminopimelic acid (Nocardioides simplex strain 11), or
lysine (Arthrobacter globiformis strain lo), as well as authentic DL- and DD-diaminopimelic acids (Sigma Chemical
Co.), lysine, and ornithine, were used as references for the
analysis of diamino acids, and galactose, arabinose, glucose,
xylose, rhamnose, maltose, and ribose were used as references for the sugar analysis.
Lipids. Simple fatty acids and mycolic acids were extracted and methylated by whole-cell methanolysis of dried
organisms (30). Methyl esters of the simple fatty acids were
identified by gas chromatography-mass spectrometry. A
Hewlett-Packard model HP5880 gas chromatograph
equipped with a type SE-30 capillary column and parallel
flame ionization and model HP5970A mass selective detectors was used.
The sizes of mycolic acids were estimated by using TLC
(catalog no. 5721 silica gel, without fluorescence indicator),
The classification of actinomycetes containing mycolic
acids was unsatisfactory prior to the use of chemical characteristics as taxonomic markers. Chemotaxonomy has provided a new framework for the classification of these organisms, and simple methods have been developed for routine
analysis (18, 35).
Cell wall analysis is a useful tool in distinguishing members of the genera Corynebacterium, Caseobacter,
Rhodococcus, Nocardia, and Mycobacterium from other
actinomycetes. In representatives of these genera the cell
walls contain meso-diaminopimelic acid, arabinose, and
galactose (12, 25, 26), as well as mycolic acids (long-chain
2-alkyl-branched 3-hydroxy acids). In mycobacteria,
mycolic acids are large and complex and are not extractable
in ethanol-diethyl ether (22). Corynebacterium, Rhodococcus, and Nocardia species contain smaller and easily extractable (free) mycolic acids (22, 29, 33).
In different bacterial species menaquinones (2-methyl-3polyprenyl-1,4-naphthoquinones)vary in the number of isoprenoid units and in the degree of hydrogenation. This can
be used as a classifying marker (8). The menaquinone
compositions of Nocardia species (except Nocardia
amarae) (21) are different from the menaquinone compositions of Corynebacterium, Rhodococcus, and Mycobacterium (4,7, 10, 11, 43).
Rhodococci undergo rod-to-coccus variation during the
growth cycle (23, 27). This kind of morphological variation
does not occur in the true corynebacteria (23, 27). In
contrast, norcardiae form mycelia which fragment, whereas
mycobacteria usually form curved or straight rods which
occasionally branch (18). However, because of dissimilarities in cultural conditions, the morphological studies described in the literature are seldom comparable.
In this paper we describe a novel mycolic acid-containing
actinomycete. The type and only strain, strain PCP-I, mineralizes pentachlorophenol and degrades many other
chlorophenols as well (Apajalahti and Salkinoja-Salonen,
submitted for publication).
MATERIALS AND METHODS
Cultures. The strains which we used are listed in Table 1.
The original isolate, strain PCP-I, was obtained from a
* Corresponding author.
246
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RHODOCOCCUS CHLOROPHENOLICUS SP. NOV.
TABLE 1. Strains used in this study
Laboratory no.
9
10
11
12
Taxon
Strain
Rhodococcus erythropolis
R . rhodochrous
R . coprophilus
R . maris
R . equi
R . fascians
Nocardia brasiliensis
Corynebacterium
glutarnicum
Brevibacterium linens
Arthrobacter globiformis
Nocardioides simplex
R . chlorophenolicus
DSM 43066T
DSM 43241T
DSM 43347T
DSM 20303
DSM 20307
DSM 20131
DSM 43009
ATCC 6872
DSM
DSM
DSM
DSM
DSM
DSM
DSM
ATCC
ATCC 9172T
ATCC 8010T
ATCC 6946T
PCP-I
ATCC
ATCC
ATCC
Lake
sediment
Sourcea
DSM, Deutsche Sammlung von Mikroorganismen, Gottigen, Federal
Republic of Germany; ATCC, American Type Culture Collection, Rockville,
Md.
as described by Minnikin et al. (30). The presence of free
mycolic acids was checked by ethanol-diethyl ether extraction and subsequent TLC as described previously (33). For
mass spectrometric analysis mycolic esters were purified
from methanolysates by preparative TLC (10). A JEOL
model JMS 01-SG2 mass spectrometer (direct inlet; ionization voltage, 24 eV; temperature, 200 to 210°C) was used to
determine the numbers of carbon atoms in the mycolic
esters.
Menaquinones were extracted from dry cells with chloroform-methanol (10) and purified by using a high-pressure
liquid chromatograph (Micromeritics Instrument Corp.). A
Rad-Pak C18 column (5 km; diameter, 8 mm) in a model
RCM-100 compression module (Waters Associates) was
used. The mobile phase was n-butylchloride-methanol
(20:80, vol/vol). The effluent (1.0 ml/min) was monitored
with an ultraviolet light detector at 270 nm. Purified compounds were confirmed as menaquinones by measuring their
absorption spectra (8). The sizes and degrees of hydrogena:
tion of the menaquinones were determined by mass spectrometry. A temperature range of 140 to 180°C and an
ionization voltage of 75 eV were used.
Electron microscopy. Samples were prefixed with 3%
(voVvo1) glutaraldehyde (Leiras, Turku, Finland) in 0.1 M
sodium phosphate buffer (pH 7.2) for 2 h at room temperature and washed three times in the same buffer. The specimens were postfixed for 2 h in buffered 1% (wt/vol) osmium
tetroxide, dehydrated in a graded series of ethanol and
propylene oxide, and embedded in Epon LX-112 (Ladd).
Thin sections were cut with a diamond knife on an LKB
Ultrotome 111 ultramicrotome and double stained with
uranyl acetate and lead citrate. The grids were examined
with a JEOL model JEM-100CX electron microscope at an
operating voltage of 60 kV.
RESULTS AND DISCUSSION
Strain PCP-I was isolated from a pentachlorophenol enrichment culture inoculated from lake sediment (1, 37, 41);
FIG. 1. Morphological cycle of R . chlorophenolicus grown on
yeast extract-glucose agar (13) at 28°C for 0 h (A), 12 h (B), 2 days
(C), 4 days (D), and 7 days (E). Bars = 5 pm. Phase-contrast
micrographs of live cells.
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247
248
INT. J . SYST.BACTERIOL.
APAJALAHTI ET AL.
FIG. 2. Electron micrographs of thin sections of R . chlorophenolicus cells grown on yeast extract-glucose agar (13) at 28°C. (A) Cells
grown for 4 days. Bar = 1 pm. (B) Cells grown for 14 days. Bar = 200 nm.
this strain has been shown to degrade pentachlorophenol and
many other chlorophenols (Apajalahti and SalkinojaSalonen, submitted).
Strain PCP-I had the major characteristics of the genus
Rhodococcus and was unlike any previously described spe-
cies of the genus. We propose the species description given
below.
Rhodococcus chlorophenolicus sp. nov. Rhodococcus
chlorophenolicus (chlor. 0.phen. 6 li. cus. N.L. adj. chloro
containing chlorine, fr. Gr. adj. chloros pale green; N . L . n.
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VOL. 36, 1986
RHODOCOCCUS CHLOROPHENOLICUS SP. NOV.
TABLE 2. Differentiating characteristics of Corynebacterium,
Caseobacter, Rhodococcus, Nocardia, and Mycobacterium"
Genus
Major
menaquinone
No. of C
atomsacids
in
mycolic
Mycelial
fragmentation
Corynebacterium
Caseobacter
Rhodococcus
Nocardia
Mycobacterium
8H2 or 9H2
9H2
8H2 or 9H2
8H4 (9H2Ib
9H2
20-38
30-36
30-66
46-60
60-90
+
+
+
+
a
45.
-
Data from references 2, 4, 6, 7, 10, 20, 21, 23, 24, 27, 32, 34,42, 44, and
The data in parentheses are for Nocardia amarae.
pheno phenol, hydroxybenzene, fr. Gr. phaino shine, to be
shining, appear; N.L. adj. chlorophenolicus relating to
chlorophenols) cells are gram positive, cytochrome oxidase
negative, catalase positive, strictly aerobic, and rod shaped
(1.0 to 1.5 by 3 to 4 pm) and become coccoid upon prolonged
incubation.
Figure 1 shows the morphological cycle on yeast extractglucose agar (13). The inoculum form is coccoid (Fig. 1A).
After 12 h of incubation, unbranched rods predominate (Fig.
1B). After 7 days of incubation the rods fragment into
coccoids (Fig. 1E). Figure 2A shows an electron micrograph
of a culture that was grown for 4 days on yeast extractglucose agar. A thick capsulelike material surrounds the
cells after 2 weeks of incubation (Fig. 2B).
On yeast extract agar and rhamnose agar, colonies are
orange pigmented and slightly mucoid. Visible colonies are
formed after 1 to 2 weeks of incubation at 28°C; after 1
month of incubation the colonies are 2 to 4 mm in diameter.
Growth occurs at 18, 28, (optimum), and 37"C, but not at
10 or 45°C.
In APISO CH tests performed with API 20NE medium,
growth is produced on erythritol, L-arabinose, ribose
(weak), D-xylose, adonitol, glucose (weak), fructose,
rhamnose, inositol, mannitol, sorbitol, N-acetylglucosamine
(weak), arbutin (weak), sucrose, trehalose, xylitol, Darabitol, L-arabitol, and gluconate. No growth is produced
on glycerol, D-arabinose, L-xylose, P-methyl-D-xyloside,
galactose, mannose, sorbose, dulcitol, a-methyl-Dmannoside, a-methyl-D-glucoside, amygdalin, esculin,
salicin, cellobiose, maltose, lactose, melibiose, inulin,
melezitose, raffinose, starch, glycogen, gentiobiose, Dturanose, D-lyxose, D-tagatose, D-fucose, L-fucose, 2-ketogluconate, or 5-keto-gluconate. After 7 days of incubation in
a mineral salts medium supplemented with vitamins and
trace elements, growth is produced with 1% (wthol) inositol,
rhamnose, sorbitol, and trehalose, but not with 1%glycerol,
1%maltose, or 0.1% m-hydroxybenzoic acid.
Growth occurs in the presence of 0.003 and 3.0% NaCl,
but not in the presence of 7% NaCl after 7 days of incubation
at 28°C.
The cell wall contains meso-diaminopimelic acid,
arabinose, galactose, and glucose.
The following simple fatty acids are released by acid
methanolysis (relative abundance values are indicated in
parentheses): C14:O (23.61, C15:0 (12.21, C16:O (100.0), c16:1
(20.3), C18:o(11.6), C18:1(87.5),10CH3C18(tuberculostearic
acid) (67.4), C20:0(3.3, C22:o (4.3), and c24:O (4.7).
Mycolic acids are present and contain 33 to 43 carbon
atoms.
Menaquinones with nine isoprenoid units and one hydrogenated double bond (MK-9H2)predominate.
249
The type strain, strain PCP-I (= DSM 43826), was isolated
from a lake sediment. This strain degrades pentachlorophenol, 2,3,4,5-tetrachlorophenol, 2,3,4,6- tetrachlorophenol, 2,3,5,6-tetrachlorophenol, 2,3,5-trichlorophenol,
and 2,3,6-trichlorophenol (Apajalahti and Salkinoja-Salonen,
submitted).
Table 2 shows the chemical and morphological characteristics that are essential for differentiating the genera Corynebacterium, Caseobacter, Rhodococcus, Nocardia, and
Mycobacterium. The question of whether Caseobacter
should be included in the genus Rhodococcus or considered
a distinct genus is still unanswered. However, when the
properties shown in Table 2 are taken as classification
criteria, strain PCP-I is clearly a member of the genus
Rhodococcus.
Menaquinones. The menaquinone composition of most
nocardiae is different from the menaquinone compositions of
related taxa (Table 2). The major menaquinone of most
nocardiae has a side chain of eight isoprenoid units and two
hydrogenated double bonds (MK-8H4) (10). Most animalpathogenic corynebacteria (Corynebacterium sensu stricto)
contain MK-8H2, whereas Corynebacterium glutamicum,
Corynebacterium bovis, and the mycobacteria contain
MK-9H2 as the predominant menaquinone ( 8 ) . In
Rhodococcus either MK-8H2 or MK-9H2 predominates (8,
10, 16, 23, 24, 34). High-pressure liquid chromatography
followed by mass spectroscopy showed that strain PCP-I
contains MK-9H2 almost exclusively. In reverse-phase TLC
(11) the menaquinones of strain PCP-I cochromatographed
with those of C. gfutamicum (strain 8), which contains
MK-9H2 as a major menaquinone (4).
Mycolic acids. The large 60- to 90-carbon mycolic acids
found in mycobacteria are practically insoluble in ethanoldiethyl ether, whereas the mycolic acids of other bacteria
can readily be extracted with this solvent (22, 32, 33). The
mycolic acids of strain PCP-I were not of the Mycobacterium type, since they were extractable in ethanol-diethyl
ether (33). On TLC plates the mycolate esters from strain
PCP-I moved slightly faster (Rf, 0.17) than the mycolate
esters from Rhodococcus equi (strain 5 ; Rf, 0.16), but slower
than the mycolate esters from Rhodococcus erythropolis
(strain 1;Rf,0.18). The mycolic acids of R . equi have been
reported to contain 30 to 38 (6) carbon atoms, and the
mycolic acids of R . erythropolis contain 36 to 48 carbon
atoms (16, 21). The mycolic esters of strain PCP-I were
purified by preparative TLC and analyzed by mass spectrometry. The obvious anhydromycolate peaks (6, 31) in the
mass spectrum ranged from mle 504 to 646, corresponding to
mycolic acids with 33 to 43 carbon atoms.
Rhodococcus is a relatively heterogeneous genus which
contains species with different types of mycolic acids and
menaquinones. Three members of the genus, Rhodococcus
bronchialis, Rhodococcus rubropertinctus, and Rhodococcus terrae, have been reported to contain MK-9H2 as a
major isoprenoid quinone (16, 20, 21, 23). However, the
mycolic acids containing 46 to 66 carbon atoms in these
species (16, 20, 21) are larger than those of R .
chlorophenolicus. Rhodococcus maris, Rhodococcus luteus,
R . erythropolis, Rhodococcus coprophilus, R . equi,
Rhodococcus rhodochrous, and Rhodococcus ruber strains
contain mycolic acids that are approximately the same size
as those in strain PCP-I (6, 16, 20, 32, 34). However, all of
these species contain MK-8H2 as a predominant menaquinone (10, 16, 21, 23, 24, 34). Table 3 lists differential
properties for R . chlorophenolicus and 13 other Rhodococcus species.
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APAJALAHTI ET AL.
INT. J . SYST.BACTERIOL.
TABLE 3. Diagnostic characteristics of Rhodococcus species
~~
~~
Species
R . chlorophenolicus
R . bronchialis’
R . rubropertinctusb
R . terrae’
R . coprophilusb
R . equib
R . erythropolisb
R . rhodniib
R . rhodochrousb
R . rubeP
R . fasciansh
R . maris‘
R . luteus
R . rnarinonascensf
Mycolic acids
Major
menaquinone
GroUpa
No. of
C atoms
B
D
D
D
B
A-B
B
C
B
B
B
B
B
C
33-43
56-66
46-62
52-64
38-48
30-38
36-48
34-52
38-48
38-50
38-52
NR
NR
NR
Utilization of
Inositol
(l%, wt/vol)
Rhamnose
Sorbitol
Trehalose
+
+
+
+
+
+
+
-
-
+
+
+
+
+
v
+
+
+
-
-
-
+
-
-
+
+
+
+
+
+
-
-
N
N
R
-
R
Growth at:
Glycerol
Maltose
+
+
+
+
+
v
+
+
+
+
+
v
-
+
+
-
m-H ydroxybenzoic
acid (0.1%,
wt/vol)
-
-
-
-
-
‘OoC
450c
Growth
in the
presence
Of
%
’
NaCl
-
+VC
+
+
+
NR
NR
NR
+
+
+
V
NR~
NR
NR
+
+
-
-
+
+
NR
+
+
+
Groups based on motility (Rfvalues) on TLC plates (A < B < C < D) (30).
Data from references 6, 10, 15, 16, 19, 20, 21, 23, 32, and 36.
‘v , Variable.
NR, Not reported.
Data from reference 34.
Data from reference 24.
’
ACKNOWLEDGMENTS
We are indebted to M. Goodfellow for valuable advice and for
critically reading the manuscript. We thank S. Kaltia for mass
spectroscopy, E.-L. Nurmiaho-Lassila for electron microscopy, T.
Koro for preparing the thin sections, and V. Sundman and M.
Hameranta for helpful comments. The Department of Electron
Microscopy at the University of Helsinki kindly provided the
equipment used for electron microscopy.
This work was supported by the Academy of Finland and the Maj
and Tor Nessling Foundation.
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