FEMS Microbiology Letters 130 (1995) 75-80
Phylogenetic analysis of polycyclic aromatic hydrocarbon
degrading mycobacteria by 16s rRNA sequencing
Rong-Fu Wang, Wei-Wen Cao, Carl E. Cerniglia *
Microbiology Dtrmision.National Centerfor To.uicologicalResearch.FDA, Jefferson,AR 72079-950,USA
Received 24 January 1995;revised 8 May 1995;accepted8 May 1995
Abstract
Mycobacterium sp. PYR-1 was previously isolated in our laboratory and was shown to be able to mineralize high
molecular mass polycyclic aromatic hydrocarbons (PAHs) [Heitkamp and Cemiglia, (1988) Appl. Environ. Microbial. 54,
1612-16141. In this research, the 16s rRNA gene (rDNA) of this strain was amplified by polymerase chain reaction (PCR)
and directly sequenced by cycle sequencing method. We compared this sequence with all known mycobacterial 16s rDNA
sequences available from GenBank and found that Mycobacterium sp. PYR-1 16s rDNA differs from the other
mycobacteria, especially in the region of nucleotides 168-200 (in the Escherichia co/i numbering system). Using the 16s
rDNA sequences of the mycobacteria, a phylogenetic tree was constructed. The data from the phylogenetic tree and
similarity values suggest that Mycobacterium sp. PYR-1 is closer to M. aurum and M. uaccue. Using the same approach,
we also determined the 16s rDNA from an another PAH-degrading Mycobacterium sp. PAH135, isolated by Grosser and
colleagues (1991) (Appl. Environ. Microbial. 57, 3462-3469). Mycobacterium sp. PAH135 was found to be closer to M.
aichiense, and different from our MJ>cobacterium sp. PYR-1.
Keywords: PAH-degrading mycobacteria; 16s rRNA; Polymerasechain reaction: Phylogeny
1. Introduction
Bioremediation
of polycyclic
aromatic hydrocarbons (PAHs) contaminated environments
has been
considered as an attractive technology for restoration
of polluted sites to an innocuous state. Generally, the
biodegradation of low molecular mass PAHs such as
naphthalene, phenanthrene and anthracene in pure
culture or in environmental samples were reported to
be performed by Pseudomonas strains [1,2]. Recently, strains from the genus Mycobacterium have
* Corresponding author. Tel: + I (501) 543 7341: Fax: + I
(501) 543 7307; e-mail: [email protected].
been shown to degrade the more recalcitrant and
genotoxic higher molecular mass PAHs to polar
metabolites and carbon dioxide [1,3-S]. Mycobacterium sp. PYR-1 was previously isolated in our
laboratory from an oil-contaminated sediment and is
efficient in the metabolism of high molecular mass
PAHs, such as pyrene, fluoranthene and phenanthrene [5-81. This organism was identified as a
rapidly-growing Mycobacterium sp. [5,6]. Confirmation that strain PYR-1 was a Mycobacterium sp. was
determined by comparing its mycolic acid, cellular
proteins, and nucleic acids with those of known
species [91.
The 16s ribosomal RNA sequences have proved
useful in establishing phylogenetic relationships be-
037%1097/95/$09.50 6 1995 Federationof EuropeanMicrobiological Societies.All rights reserved
SSDI 0378-1097(95)00186-7
76
R.-F. Wang et al. / FEMS Microbiology
cause of their high information content, conservative
nature, and universal distribution [lo,1 11. The 16s
rRNA sequence analysis is a standard method for the
investigation of their phylogenetic relationships [12].
Even though some closely related species may have
only a few bases differences in their 16s rRNA gene
sequences, a phylogenetic tree can be established to
give them an exact taxonomic position [13]. Recently, more reports have been published for the
identification of bacterial species and phylogenetic
relationship analysis by the 16s rRNA sequencing
[12-171.
The taxonomic position of mycobacteria which
degrade PAHs has not been described. Establishing
the phylogenetic placement of Mycobacferium sp.
PYR-1 could be helpful for further application of
this organism, particularly if this strain is identified
as a new species. In addition, determining the 16s
rRNA sequence of Mycobacterium sp. PYR-1 can
result in developing DNA probes or PCR methods to
detect this organism from indigenous soil samples or
inoculated soil samples when this organism is used
as an inoculant in bioremediation of PAHs contaminated sediments.
In this article, we report the amplification and
sequencing of the 16s rRNA gene (rDNA) of Mycobacterium sp. PYR-1. Using this sequence data, a
phylogenetic tree was constructed and its phylogenetic placement was established.
2. Materials
and methods
2.1. Microorganism
and culture conditions
Mycobacterium sp. PYR-1 was originally isolated
from oil-contaminated sediments [5,6]. Mycobacterium sp. PAH 135 was received from R.J. Grosser,
University of Cincinnati, Cincinnati, OH. Axenic
cultures of Mycobacterium sp. PYR-1 and Mycobacterium sp. PAH 135 were maintained on minimal basal salts medium (MBS) supplemented with
pyrene and nutrients as described previously [5,6].
2.2. Amplification
of the 16s rRNA gene
The amplification and sequencing primers were
located at the highly conserved regions of the 16s
Letters 130 (1995) 75-80
rRNA gene. The 5’-end amplification primer is
GCTGGCGGCGTGCTTAACA,
which is located at
R036-R056 (E. coli numbering system, [IS]). For
the Mycobacterium sp. PYR-1 amplification, the
3’-end amplification primers is AAGGAGGTGATCCARCCGCA, which is located at R1535-R1516. For
the Mycobacterium sp. PAH135 amplification, the
3’-end primers is CGGCTACC’ITGTTACGACTC,
which is located at R1509-R1489 (E. coli numbering
system).
Mycobacterial cells were collected from colonies
or broth culture in MBS containing organic nutrients
as described previously. The cells were washed twice
with PBS (0.05 M NaH,PO,-Na,HPO,,
0.8% NaCl,
pH 7.4) and once with autoclaved distilled water
(dH,O), then resuspended in 0.1 ml dH,O. The cell
suspensions (10 ~1) were diluted 1:lO using 90 ~1
of 1% Triton X-100, heated at 100°C for 5 min,
immediately cooled in ice water, then subjected to
the PCR amplification without isolation of the DNA.
2.3. Sequencing the PCR products by 35S-cycle sequencing method
The DNA bands were visualized with a long wave
UV lamp and excised from agarose gel. Glassmilk
method (The Geneclean II kit, Bio 101 Inc., La Jolla,
CA) was used to recover the DNA following the
manufacturer’s instructions. Direct sequencing of the
PCR products was conducted using the SequiTherm
Cycle Sequencing Kit (Epicentre Technologies,
Madison, WI) with 35S-dATP following the manufacturer’s instructions.
2.4. Phylogenetic analysis
A computer program ‘ALIGN’ (Scientific and
Educational Software, State Line, PA) was used for
alignment and homology analysis of the rDNA sequences. Computer programs PHYLIP version 3.5~
were provided by Dr. Joe Felsenstein (University of
Washington, Seattle, WA) and used for producing
the phylogenetic tree. The sequences were also sent
to Ribosomal Database Project (RDP) at the University of Illinois for phylogenetic analysis [19].
2.5. Nucleotide sequence accession numbers
The sequences of the 16s rRNA gene for Mycobacterium sp. PYR-1 and Mycobacterium sp.
R.-F. Wang et al. / FEMS Microbiology
PAH135 have been deposited in the EMBL GenBank data library with accession numbers X84977
and X84978, respectively.
3. Results and Discussion
The 16s rRNA gene sequence of Mycobacterium
sp. PYR-1 was determined. Phylogenetic analysis
was conducted by comparing the 16s rDNA sequences for this species with other Mycobacterium
species and some closely related High G + C Grampositive bacterial species available from GenBank.
The sequences exhibited very high levels of similarity with other mycobacteria. For example, the similarity values for the 16s rRNA gene sequence of
M.sp.PYRm1
M.sp.PAH135
M.aichiense
M.asiaticum
M.aurum
M.avium
M.bovis
M.celatum
M.chitae
M.chlorophenoll
M.chubuense
M.cookii
M.diernhoferi
M.fallax
M.farcinogenes
M.flavescens
M.fortuitum
M.gadium
cus
M.gastr1
M.gWleVeUSe
M.gilvum
M.gordonae
M.haemophilum
M.interiection
M.intermedium
M.lntracellulare
M.kansasii
M.komossense
M.lepraemurium
M.malmoense
M.marinum
M.obuense
M.paratuberculosis
M.scrofulaceum
M.simiae
M.smeamatis
M.sphagni
M.szulgai
M. terrae
M.thermoresistibile........M.tuberculosis
M.lllCfZrXE
M.VaCCae
Letters
130 11995) 75-80
77
Mycobacterium
sp. PYR-1 is 99% with M. aurum,
98% with M. aaccae, 94-97% with other mycobac-
terial species except for a few species in which
incomplete 16s rDNA sequences available from
GenBank were compared. These high similarity values confirmed that Mycobacterium
sp. PYR-1 is a
mycobacterial species. However, an alignment of
this sequence with the 16s rRNA sequences of 41
other mycobacterial species available from the GenBank demonstrated that this species is different from
all other known mycobacterial species tested, the
most notable variation at the V2 region of 16s rRNA
gene, positions 168-200, (in the E. coli numbering
system, 18). Fig. 1 shows the alignment result of the
variable region (V2) of 16s rDNA for 43 species of
Mycobacterium.
Mycobacterium
sp. PYR-1 has a
CCGAATAC-ACCCTTCTGGCTGCATGGTCTGGKTGGGGAAAGC--TTTT.......
T-GAT.A.-G.C........mG.T.TG.T.......-.. .........
G-GAT.A./G.C..T.....mG.T.TG.T.......-.. ..... G...G-GA..-A.G..A.....-...CT.TG.T.......-.. ..m
--GGA..A.GC.A.....-...GT.TG.T.......-.......
.. ..... G...G-GA..-..AA.AC....-....TCTG.T.......-.. ..... G...G-GA.:A.G..A.....-....T.TG.TN......GC...A
... G...G-GA..A.~G..A.....-....T.TG.T.......-.. .........
T-GAT.A.~G.C..T.....-G.T.TG.T.......-... .
.......
G-GA..-A.G."........~A.T.TG.T.......-... .
... G...G-GA..~A.G.CA......TG..-.TG.T.......-... .
.G...--GGA..A.G.A..N...N....-.TG.T.......-.....
.......
T-GA..-G.GCT..T.....GG.-.TG.T.......-.. ..m
... G...T-GAT.A.GC.C../......G.-.TG.T.......-.. ....G...--GGA..A.GC..........G.-.TA.T.......-.
.. .........
T-T....NN...TC......C....TA.........-.. ........
T-GA../G.GCA..T.C....G./.TG.T.......-.. ... ..G...--GGA..A.GC.A.....~...GT.TG.T.......-.. ... ..G...G-GA..ACT....~....-.C..T.TG.T.......-.. ....G...T-GA..~A.G.AAC....-..T.T.TG.T.......-.
.....
.......
G-GA..-G.AT...T...A..G.-.TG.T.......-.....
G-GA.:A.A..ACA.
-...CT.TG.T.
:-___
G...G-GA...CAA.../..
CCT.T.TG.T.
.--...
G...G-GA...CGA...m..
:.C..T.TG.T.
.--___
G...G-GA.:...C.GC..
CCTA..AG.T.
_--...
. . ..TTAG.T.
r G...G-GA....TA...-..
_--...
G...G-GA..ACT...:..
.-.C..T.TG.T.
.--.__
. ..G...T-GA..A.-G.A..T.
C..:.T.TG.T.
.--.__
G..:-GGA....AA.GC..
. . ..T.TG.T.
.T
G-GA..CCGA.../..
.-.C..T.GG.T.
_--_., .G...G-GA.:A.G..A.".
-...CT.TG.T.
_--...
G...G-GA.:A.G.CA.T.
TG..-.TG.T.
_--...
. ..G...G-GA..-..AA.AC....-....TCTG.T.......--....G...G-GA..ACT..../....-.C..T.TG.T.......--....... G...G-GA..ACT....-....-.C..T.TG.T.......-.. ........
G....TC......C....TA.........-..- .....
.. ..m
.......
TG...A.G.:-..T.....GG./.TG.T.......-... .
... G...G-GA..CCGA.../....-.C..T.GG.T.......-.. ..m
... G...G-GA..A.-G..A......T...~.TG.T.......-... .
.... .G..A.TC......C..A.TG.........--G
. ..... G...GmGA..-A.G..A.....-....T.TG.T.......GC...A
... G...G-GA..-A.G..A.T.../...CT.TG.T.......-.. ..m
......
..................
T......GA.........-.. ..-
(
(
(
(
i
(
(
(
(
(
(
(
(
(
(
(
(
c
(
c
(
(
c
c
1
1
t
(
t
(
(
(
(
(
(
(
(
(
(
(
(
(
(
173)
171)
172)
172)
172)
1741
176)
161)
171)
172)
172)
173)
172)
170)
172)
174)
171)
173)
174)
174)
172)
172)
175)
1741
1751
163)
174)
174)
188)
1741
1681
172)
1741
173)
173)
1761
1711
1731
174)
175)
176)
168)
174)
Fig. 1. Alignment result of one variable region (V2) of 16s rDNA for 43 species of Mycobacterium.
The sequences of Mycobacterium
sp.
PYR-1 and Mycobacterium
sp. PAH135 were determind by us. The other sequences were available from the GenBank. ” shows the same
bases with the first line bases; ” shows lacking bases the letters showed different bases, K represents T or G. The base numbers shown on
the right should be added 38 each to give the numbers in the E. co/i numbering system [18].
R.-F. W’ang ef ul. / FEMS MI<
78
different sequence in the V2 region from all other
Mycobacterium species examined.
A phylogenetic tree was constructed based on a
comparison of the 16s rDNA sequences of mycobacteria from positions 39 to 1460 (E. coli numbering
system) because of incompleteness of the 16s rDNA
sequences of a number of the reference strains in the
GenBank. The tree is shown in Fig. 2.
Placement in the tree and similarity values both
suggest that the Mycobacterium sp. PYR-1 is most
similar to Mycobacterium aurum and Mycobacterium uaccae.
The sequences were also sent to Ribosomal
Database Project (RDP) at the University of Illinois
for phylogenetic analysis. A large unrooted tree including many other common bacterial species was
M.
M.
M.
M.
ulcerans
marinum
tuberculosis
bovis
M. paratuberculosis
M. intracellulare
M. gaslri
M. kansasii
M. malmoense
M. haemophilum
TM.
smegmatis
FM.
aeneveuse
M. flavescens
A -M.
terrae
\ -M.
komossense
r
M. aichiense
f-M. diernhoferi
M. thermoresistibile
M. chlorophenolicus
--M.
sphagni
M. obuense
pl.
vaccae
Corynebacterium
Fig. 2. Phylogenetic tree for the two mycobacterial species with other 41 mycobacterial species and 5 high G + C Gram-positive bacterial
species. The tree is based on a comparison of 16s rDNA sequence on positions 38 to 1460 in the E. coli numbering system [18].
R.-F. Wang et al. / FEMS Microbiology
produced by maximum likelihood method, which
also indicated that the strain PYR-1 is in the genus
Mycobacterium (data not shown). A small unrooted
tree including only mycobacterial species was also
generated by RDP with maximum likelihood method
(data not shown). The phylogenetic position of Mycobacterium PYR-1 in these trees is identical to the
results in the parsimony tree, i.e., Mycobacterium
sp. PYR-1 is closest to M. aurum and M. rlaccae.
Previously we found that the polypeptide pattern
of Mycobacterium sp. PYR-1 most closely resembled that of M. austroufiicanum. However, restriction enzyme analysis and Southern blot hybridization
revealed differences between the chromosomal DNA
of our isolate and that of M. austroufiicanum [9].
We did not include M. austroafiicanum in the phylogenetic tree since to our knowledge the 16s rRNA
sequence has not been published or available from
GenBank.
Grosser et al. [4] reported the isolation of a Mycobacterium sp. PAH135 which was also PAH degrading, rapid-growing Mycobacterium similar to
Mycobacterium sp. PYR-1. Genetic differences between the two PAH-degrading mycobacteria were
studied and reported in an abstract [20]. They reported that ‘the Mycobacterium sp. PAH135 (Grosser
strain) is closest to that of M. fullax by 16s rDNA
sequence comparison; The same approach on the
PYR-1 (H-C strain) has yielded a sequence which
verifies that organism as a Mycobacterium; Completion of this approach will allow construction of a
phylogenetic tree...‘. However, sequence data for the
16s rRNA gene for either species was not presented
in the abstract or any subsequent publication, and
these sequences have not been deposited in GenBank. Therefore, we also determined the 16s rDNA
sequence and did the phylogenetic analysis for Mycobacterium sp. PAH135. The similarity values for
the 16s rRNA gene sequence of Mycobacterium sp.
PAH135 is 99% with M. aichiense, 98% with 12 of
other mycobacterial species, 97% with Mycobacterium sp. PYR-1. Placement in phylogenetic tree
and similarity values for this species indicated that
Mycobacterium sp. PAH135 is most similar to M.
aichiense, and distinct from Mycobacterium sp.
PYR-1.
In conclusion, the 16s rDNA of Mycobacterium
sp. PYR-1 and PAH135 were sequenced and the
Letters 130 (1995) 75-80
79
phylogenetic placements for these two species were
established. The sequences will be used to develop
DNA probes or PCR primers to determine the genetic diversity of PAH degrading strains. The research results will also be helpful for further applications of these organisms as inoculants in bioremediation of PAH contaminated sediments.
Acknowledgements
This research was supported in part by appointments (for W.W.C.) to the Postgraduate Research
Program at the National Center for Toxicological
Research administered by the Oak Ridge Institute for
Science and Education through an interagency agreement between the U.S. Department of Energy and
the U.S. Food and Drug Administration. We thank
Robert J. Grosser and David Warshawsky for kindly
providing the Mycobacterium sp. PAH135. We appreciate Dr. Joe Felsenstein at University of Washington for providing the computer programs PHYLIP
version 3.5~ and helping us use it for producing the
phylogenetic tree. We also appreciate Mike McCaughey at RDP for the helping in constructing an
alternative phylogenetic tree using our sequence data.
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