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Applied and Environmental Microbiology, February 1999, p. 549-552, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isolation and Characterization of a
Mycobacterium Species Capable of Degrading Three- and
Four-Ring Aromatic and Aliphatic Hydrocarbons
Sharon A.
Churchill,1,*
Jennifer P.
Harper,2 and
Perry F.
Churchill2
Department of Civil and Environmental
Engineering, Washington State University, Pullman, Washington
99164-2910,1 and
Department of
Biological Sciences, University of Alabama, Tuscaloosa, Alabama
35487-03442
Received 8 May 1998/Accepted 20 November 1998
 |
ABSTRACT |
Mycobacterium sp. strain CH1 was isolated from
polycyclic aromatic hydrocarbon (PAH)-contaminated freshwater sediments
and identified by analysis of 16S rDNA sequences. Strain CH1 was
capable of mineralizing three- and four-ring PAHs including
phenanthrene, pyrene, and fluoranthene. In addition, strain CH1 could
utilize phenanthrene or pyrene as a sole carbon and energy source. A
lag phase of at least 3 days was observed during pyrene mineralization. This lag phase decreased to less than 1 day when strain CH1 was grown
in the presence of phenanthrene or fluoranthene. Strain CH1 also was
capable of using a wide range of alkanes as sole carbon and energy
sources. No DNA hybridization was detected with the nahAc
gene probe, indicating that enzymes involved in PAH metabolism are not
related to the well-characterized naphthalene dioxygenase gene. DNA
hybridization was not detected when the alkB gene from
Pseudomonas oleovorans was used under high-stringency conditions. However, there was slight but detectable hybridization under low-stringency conditions. This suggests a distant relationship between genes involved in alkane oxidation.
| |
INTRODUCTION |
Polycyclic aromatic hydrocarbons
(PAHs) are fused-ring aromatic compounds whose presence in contaminated
soils and sediments poses a significant risk to the environment, and
they have cytotoxic, mutagenic, and in some cases carcinogenic effects
on human tissue (13, 18, 25). PAHs are hydrophobic
compounds, whose persistence within ecosystems is due chiefly to their
low aqueous solubility. Microbial biotransformation is a major
environmental process affecting the fate of PAHs in both terrestrial
and aquatic ecosystems. The microbial degradation of PAHs having two or
three rings is well documented (5-7, 11). Only within the
last decade have a number of bacteria that metabolize larger PAH
molecules been isolated. These include Alcaligenes
denitrificans (33), Rhodococcus sp. strain
UW1 (32), several Pseudomonas species (20,
31), and various Mycobacterium species (1, 3, 12,
23).
Different degradative genes are involved in the metabolism of aromatic
and aliphatic hydrocarbons (26). The genes coding for the
enzymes involved in the degradation of alkanes (alk) and naphthalene (nah) have been extensively characterized
(16, 17, 24). Assessment of hydrocarbon utilization capacity
for a large number of environmental isolates indicated that strains
were capable of mineralizing either aromatic or aliphatic hydrocarbon
compounds. Although Foght et al. postulated that bacteria having
multidegradative capacity might exist, no strains were found with the
capability to degrade both classes of compounds (10). A more
recent report has established that the abilities to degrade aliphatic
and aromatic hydrocarbons are not necessarily mutually exclusive
(27). Two Pseudomonas strains that can degrade
both naphthalene and alkanes have been isolated. These pseudomonads
possess both the alk and nah catabolic pathways
for hydrocarbon biodegradation (34).
Recent reports (27, 34) that both naphthalene and alkanes
can be degraded by a single bacterial species have raised the question
whether bacteria capable of degrading larger (three- or four-ring) PAHs
could also metabolize alkanes and, if so, whether they would possess
well-characterized hydrocarbon degradation genes, such as
nahAc and alkB. We report here the isolation and characterization of Mycobacterium sp. strain CH1. This
strain is capable of mineralizing phenanthrene, pyrene, and
fluoranthene. In addition to PAHs, strain CH1 could use branched
alkanes and n-alkanes (dodecane, hexadecane, and pristane)
that are liquids at 25°C and also solid-phase alkanes (octadecane,
docosane, and octacosane) as the sole carbon and energy source. These
degradative capabilities were found to be unrelated to the
nahAc gene and only distantly related to the alkB gene.
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MATERIALS AND METHODS |
Chemicals.
[4,5,9,10-14C]pyrene (specific
activity, 32.2 mCi/mmol; purity, >95% by high-pressure liquid
chromatography [HPLC]), [9-14C]phenanthrene (specific
activity, 8.3 mCi/mmol; purity, >98% by HPLC),
[3-14C]fluoranthene (specific activity, 55 mCi/mmol;
purity, >95% by HPLC),
[ring-U-14C]anthracene (specific activity,
15.5 mCi/mmol; purity, >97% by HPLC), [9-14C]fluorene
(specific activity, 11 mCi/mmol; purity, >98% by HPLC), [1-14C]naphthalene (specific activity, 10.1 mCi/mmol;
purity, >98% by gas chromatography [GC] and HPLC),
[1-14C]octadecane (specific activity, 3.6 mCi/mmol;
purity, >98% by HPLC and GC), and [1-14C]dodecane
(specific activity, 3.7 mCi/mmol; purity, >98% by GC) were purchased
from Sigma Chemical Co. The radiochemical purity of each
14C-labeled PAH or linear alkane was assayed by either GC
or HPLC, using an in-line radiometric flow cell detector. Unlabeled
pyrene, phenanthrene, fluoranthene, fluorene, anthracene, naphthalene, octadecane, dodecane, docosane, hexadecane, octacosane (purity, >95%
by HPLC or GC), and chloramphenicol also were obtained from Sigma
Chemical Co. The branched alkane pristane
(2,6,10,14-tetramethylpentadecane) was obtained from Fluka. Amplitaq
DNA polymerase was purchased from Perkin-Elmer. [32P]dATP
was purchased from New England Nuclear. Bacteriological media and
reagents were purchased from Life Technologies. All solvents and
chemicals used were analytical grade reagents.
Culture conditions.
The minimal mineral medium used for
enrichment of PAH-degrading bacteria contained (per liter) 1.0 g
of (NH4)2SO4, 5.0 g of KH2PO4, 0.1 g of MgSO4
· 7H2O, 5 mg of
Fe(NH4)2(SO4)2, and 1.0 ml of sterile trace metal solution (3). The pH of the
culture solution was adjusted to 7.0 with sodium hydroxide. Riverine
sediments known to be heavily saturated with large PAHs were obtained
from the Great Lakes Environmental Research Laboratory (Ann Arbor, Mich.). Pyrene-degrading bacteria were isolated from an enrichment culture obtained by adding riverine sediment samples to minimal medium
containing 400 mg of pyrene per ml and were detected on pyrene-coated
mineral medium agar plates (14). Initial identification of
the bacterial strain as a Mycobacterium species was made by Gram staining and fatty acid analysis conducted by Microbial ID Inc.
(Newark, Del.).
Hydrocarbon biodegradation.
Hydrocarbon mineralization by
Mycobacterium sp. strain CH1 was measured as
14CO2 in triplicate bioreactors
(15). Cells were pregrown on 5.0 mM acetate to stationary
phase in either the presence or absence of 300 mg of PAHs per liter.
The cells were collected by low-speed centrifugation (7,700 × g) at 5°C, washed, and resuspended in mineral medium before
use. The growth of strain CH1 was monitored spectrophotometrically at
600 nm.
DNA amplification, sequence analysis, and probe
hybridization.
DNA was extracted by a method developed for
gram-positive bacteria (35). The 16S rDNA was amplified by
PCR (Rapid-Cycler, Idaho Falls, Idaho). The forward-primer (50F)
sequence was 5'-ACCACATGCAAGTGCAACG-3', and the
reverse-primer (1392R) sequence was 5'-ACGGGCGGTGTGTAC-3'. PCR was conducted by using a touchdown protocol from 65 to 55°C as previously described (21). The amplification product was purified by agarose gel electrophoresis and cloned into the pGEM-T vector (Promega Scientific, Santa Barbara, Calif.), and both strands were sequenced (Iowa State University Sequencing Facility). The sequences were compared to those in GenBank by using the Blast alignment tool (2).
Pseudomonas putida NCIB 9816 was used as the sequence source
for the naphthalene dioxygenase gene probe. Total DNA was used, and
amplification of the nahAc gene (1,245 bp) was carried out with 5'-AAGCACCTGATTCATGGCGATGA-3' as the forward primer and
5'-GAACTCAGCCCAGTTGGAGCTGCTG-3' as the reverse primer.
P. oleovorans ATCC 29347 was used as the sequence source for
the alkane hydroxylase gene probe. Amplification was carried out with
5'-GCAAATGAAACTGGTTGGG-3' as the forward primer and
5'-GGGTAACCCGTCGGAAGAGC-3' as the reverse primer to a 906-bp
fragment. The gene probes were labeled with [32P]dATP in
a PCR. P. putida NCIB 9816 DNA was used as a positive control for nahAc, and P. oleovorans DNA was used
for alkB. Escherichia coli DNA was used as a negative
control in the dot blot hybridization assays. A 10-µg portion of
total DNA was denatured and applied to nylon membranes under low vacuum
(Micron Separations, Inc.). The DNA was hybridized at 42°C in the
presence of 50% formamide by standard procedures (19). The
filters were washed under high-stringency (0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] plus 0.5% sodium dodecyl sulfate
[SDS] at 68°C), intermediate-stringency (0.1× SSC plus 0.5% SDS
at 37°C), or low-stringency (2.0× SSC plus 0.5% SDS at 23°C) conditions.
Nucleotide sequence accession number.
The 16S rDNA sequence
has been deposited in GenBank (accession no. AF054278).
| |
RESULTS AND DISCUSSION |
Isolation and characterization of Mycobacterium sp.
strain CH1.
The PAH-degrading strain was obtained from
contaminated riverine sediment by standard culture enrichment
techniques with pyrene as the sole source of carbon and energy. When
colonies were grown on pyrene-coated agar plates, zones of clearing
appeared, indicating pyrene degradation. Upon isolation, the bacterium
was found to be gram positive with yellow pigmentation. Fatty acid
analysis was conducted, and the isolate was found to contain large
amounts of tuberculosteric acid (10Me-18:0), a defining characteristic of Mycobacterium species. DNA sequence analysis of
PCR-amplified 16S rDNA was also performed and confirmed the genus
identification of the pyrene-degrading microorganism as
Mycobacterium. DNA sequence analysis also differentiates
strain CH1 from the other PAH-utilizing mycobacteria,
Mycobacterium sp. strains RJG11-135, PYR-1, and PAH 135.
Degradation of PAHs and polycyclic aliphatic hydrocarbons.
Biodegradation of PAHs was monitored by measuring the conversion of
14C-radiolabeled substrate to
14CO2. Previous studies have demonstrated that
Mycobacterium sp. strain PYR-1 is subject to enzyme
induction upon exposure to pyrene (12). The mineralization
of pyrene and fluoranthene by Mycobacterium sp. strain CH1
when pregrown on acetate is shown in Fig.
1. Cells pregrown in the presence of
pyrene or fluoranthene had a lag phase of less than 1 day prior to
initiation of mineralization of either PAH. When cells are pregrown in
the absence of PAHs, there is a significant lag phase prior to the
onset of mineralization for both fluoranthene and pyrene degradation.
The metabolism of other PAHs was examined by growing strain CH1 on
acetate in the presence of phenanthrene, anthracene, fluorene, or
naphthalene and measuring the mineralization of these PAHs.
Phenanthrene was mineralized with a lag phase of less than 1 day,
whereas anthracene, fluorene, and naphthalene were not mineralized
(data not shown). To further investigate the expression of degradative
enzymes, the effect of the protein synthesis inhibitor chloramphenicol
on PAH mineralization was examined (data not shown). Chloramphenicol
eliminated mineralization by cells grown on acetate only; however, CH1
cells grown in the presence of phenanthrene were still capable of
converting this PAH to carbon dioxide in the presence of the inhibitor.
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FIG. 1.
Effect of pregrowth conditions on the mineralization of
pyrene and fluoranthene by Mycobacterium sp. strain CH1.
Cells were resuspended in minimal salts medium at a concentration
equivalent to 0.10 absorbance unit at 600 nm. Bioreactors were
incubated in the presence of 25 mg of [14C]pyrene or
[14C]fluoranthene per liter. The production of
14CO2 was measured as described in Materials
and Methods. Pregrowth on acetate in the presence of pyrene is
represented by  and  , and pregrowth on acetate in the presence of
fluoranthene is represented by  and  . Cells pregrown on acetate
in the absence of PAHs are represented by  and  . Mineralization
of pyrene is represented by , , and . Mineralization of
fluoranthene is represented by , , and .
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|
The ability of strain CH1 to metabolize liquid- and solid-phase
long-chain aliphatic compounds was also investigated. The
rate of
mineralization of radiolabeled alkane is not directly
related to the
aqueous solubility of these sparingly soluble hydrocarbons
(Fig.
2). Octadecane is mineralized at
approximately the same
rate as dodecane, although the aqueous
solubility in distilled
water of dodecane (0.0037 mg/liter) is greater
than that of octadecane
(0.0021 mg/liter) (
28). The ability
of both aromatic and aliphatic
hydrocarbons to serve as the sole carbon
and energy source for
aerobic growth of
Mycobacterium sp.
strain CH1 was examined. Either
pyrene or phenanthrene could be used as
the sole carbon and energy
source for growth. Although fluoranthene
could be mineralized,
it was unable to support cell growth. The liquid
linear alkanes
(dodecane and hexadecane) and the liquid branched-chain
alkane
(pristane), as well as the solid-phase long-chain alkanes
(octadecane,
docosane, and octacosane), were all able to serve as the
sole
carbon and energy source for growth. Cyclohexane was unable to
support the growth of
Mycobacterium sp. strain CH1 cells.
The
maximum growth attained, as measured by absorbance at 600 nm,
for
each substrate was as follows: phenanthrene, 0.82 ± 0.06;
pyrene,
0.41 ± 0.08; dodecane, 0.80 ± 0.04; hexadecane, 0.80 ±
0.06; octadecane, 0.84 ± 0.11; docosane, 0.60 ± 0.04;
octacosane,
0.48 ± 0.07; and acetate, 0.88 ± 0.03.
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FIG. 2.
Mineralization of [14C]octadecane and
[14C]dodecane by Mycobacterium sp. strain CH1.
Cells were pregrown on acetate in the presence (, ) or absence
(, ) of octadecane. Cells were added to triplicate bioreactors at
an initial concentration of 0.10 absorbance unit at 600 nm.
Mineralization was measured by the evolution of
14CO2, as described in the text. , ,
mineralization of octadecane; , , mineralization of dodecane.
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|
The
nahAc gene, which codes for the large subunit of
naphthalene dioxygenase from
P. putida NCIB 9816, was used
as a hybridization
probe to assess the homology between naphthalene
dioxygenase and
PAH-degradative enzymes used by
Mycobacterium sp. strain CH1 for
PAH mineralization.
Even under low-stringency conditions, there
was no detectable
hybridization to indicate that homology exists
between the
nahAc gene and genes encoding PAH-degradative enzymes
in
strain CH1. The
alkB gene was used to assess homology
between
the aliphatic degradative pathway in strain CH1 and alkane
hydroxylase
from
P. oleovorans. Weak hybridization under
low-stringency conditions
between CH1 DNA and the
alkB gene
was detected. This suggests
slight homology between the genes involved
in alkane oxidation
(data not
shown).
In the last decade, several bacteria species capable of degrading PAHs
containing more than three rings have been isolated
(
1,
4,
12,
20,
23,
31-33). Of these microorganisms,
few have been shown to
utilize four-ring PAHs for growth in the
absence of cofactors or
surfactants (
1,
4,
31-33). This capacity
differentiates
Mycobacterium sp. strain CH1 from both
Mycobacterium sp. strain PYR-1 (
12) and
Mycobacterium sp. strain RJG II-135
(
23), which
require additional cofactors for cell growth on
pyrene.
Mycobacterium sp. strain BB1 can grow on fluoranthene,
phenanthrene, and pyrene (
4), differentiating it from
Mycobacterium sp. strain
CH1.
It has been demonstrated that genes coding for both aromatic and
aliphatic hydrocarbon degradation exist in bacteria isolated
from
Alaskan shoreline sediments affected by the
Exxon Valdez oil
spill of 1989 (
27). Additionally, two hydrocarbon-degrading
psychrotolerant bacteria strains possess both alkane (
alk)
and
naphthalene (
nah) catabolic pathways (
34).
Recently, a PAH-degrading
Mycobacterium species isolated
from a PAH-contaminated site in
New Zealand was shown to grow on
dodecane and hexadecane (
1).
Mycobacterium sp.
strain CH1 is capable of using liquid- and solid-phase,
high-molecular-weight
n-alkanes, as well as a branched-chain
alkane
(pristane), as the sole source of carbon and energy. These
results
suggest that the occurrence of both aromatic and aliphatic
hydrocarbon
degradative capacity within an individual strain may be
more common
than was previously thought. The lack of hybridization of
DNA
isolated from strain CH1 with the
nahAc gene under
low-stringency
conditions indicates that the enzyme system involved in
PAH degradation
is unrelated to the well-characterized naphthalene
dioxygenase
pathway. The weak hybridization of the
alkB
gene, even under low-stringency
conditions, suggests that there is only
limited homology between
the genes involved in alkane oxidation in
P. oleovorans and
Mycobacterium sp. strain CH1.
The lack of hybridization of the
nahAc and
alkB gene probes by bacteria capable of degrading naphthalene and alkanes
has been reported previously (
22,
34). Our results reinforce
the need for isolation of additional clones encoding enzymes involved
in the initial oxidation step of three- and four-ring PAHs and
alkanes.
The isolation of these genes will provide an improved
method for
assessing the potential of microorganisms to degrade
these sparingly
soluble compounds in pristine and hydrocarbon-contaminated
environments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Civil and Environmental Engineering, Washington State University,
Pullman, WA 99164-2910. Phone: (509) 335-3227. Fax: (509) 335-7632. E-mail: trouble{at}wsu.edu.
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Applied and Environmental Microbiology, February 1999, p. 549-552, Vol. 65, No. 2
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
