Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1
Novosphingobium pentaromativorans US6-1. PLoS ONE 9(3): e90812. doi:10.1371/journal.pone.0090812
Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1
Sung Ho Yun 0
Chi-Won Choi 0
Sang-Yeop Lee 0
Yeol Gyun Lee 0
Joseph Kwon 0
Sun Hee Leem 0
Young Ho Chung 0
Hyung-Yeel Kahng 0
Sang Jin Kim 0
Kae Kyoung Kwon 0
Seung Il Kim 0
Alexey Porollo, Cincinnati Childrens Hospital Medical Center, United States of America
0 1 Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea, 2 Department of Biology, Dong-A University , Busan , Republic of Korea, 3 Department of Environmental Education, Sunchon National University , Sunchon , Republic of Korea, 4 Korea Institute of Ocean Science & Technology, Ansan, Republic of Korea, 5 Department of Bio-Analytical Science, University of Science and Technology (UST) , Daejeon , Republic of Korea
Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LCMS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1.
Funding: This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs,
Republic of Korea and the Korea Basic Science Institute Grant (T32414). SHL was partially supported by a National Research Foundation of Korea (NRF) grant
funded by the Korea government (MEST) (2012-0000481). YHC was partially supported by a KBSI grant (T33417). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Environmental contamination by polycyclic aromatic
hydrocarbons (PAHs) is a serious problem due to their toxic,
carcinogenic and recalcitrant properties , and hence their
biodegradation is an important process crucial for bioremediation,
and understanding the breakdown pathways is an important part
of developing clean-up technology. High-resolution analytical
chemistry for metabolomics and high-throughput sequencing for
genomics are essential for resolving PAH biodegradation
pathways. Recently, high-throughput proteomic approaches and
integrated omics technologies have become important tools in
the discovery of related proteins and enzymes [2,3]. For example,
metabolite analysis and proteogenomic methods were recently
used to fully understand the biodegradation of pyrene in
Mycobacterium vanbaalenii PYR-1 . Proteomic studies have also
been conducted on the PAH-degrading Sphingomonas sp. CHY-1
and Mycobacterium sp. KMS [8,9].
Novosphingobium pentaromativorans US6-1 is a Gram negative
halophilic marine bacterium able to utilize several PAHs,
including phenanthrene, pyrene, and benzo[a]pyrene, as sole
carbon and energy sources . Genome sequencing of N.
pentaromativorans US6-1 has been recently completed and the
genome database is accessible from the public NCBI database
. N. pentaromativorans US6-1 contains two plasmids, pLA1 and
pLA2. Large plasmid pLA1 possesses clustered putative aromatic
compound degradation genes. The purpose of this study was to
elucidate the PAH biodegradation pathways active in N.
pentaromativorans US6-1. Proteomic analysis of N. pentaromativorans
US6-1 cultured in the presence of different PAHs was performed
to identify biodegradation-related proteins and revealed the
induction of low-molecular-weight (LMW) aromatic-hydrocarbon
degrading genes located in pLA1. Importantly, this strain uses a
plasmid-born extradiol cleavage pathway (catechol-2,3
dioxygenase pathway) for the degradation of both PAHs and MAHs. In this
study, we report on the role of plasmid pLA1 in PAH
biodegradation and the physiological characterization of N.
pentaromativorans US6-1 using proteomic approach. Genomic
studies on the biodegradation plasmids from several stains have
been conducted previously . However, proteomic
characterization of these extrachromosomal genetic elements has yet to be
performed. This study reports the proteomic analysis of a
Materials and Methods
Bacteria cultivation and sample preparation
N. pentaromativorans US6-1 was cultured according to a method
described previously . A starter culture of bacteria was
prepared by growing cells in marine broth (MB) at 30uCto an
optical density at 600 nm (OD600) of 0.8. Bacteria were harvested
aseptically and equal amounts of bacteria (culture of 500 ml) were
added to fresh Bushnell-Hass broth (incorporating 30 g NaCl/L)
(BD, USA) containing phenanthrene (50 ppm), pyrene (50 ppm),
benzo(a)pyrene (50 ppm), benzoate (50 ppm), and
p-hydroxybenzoate (50 ppm). OD600 values of all cultures were checked at
every six hours until 48 hours using spectrophotometer (Beckman
coulter proteome Lab Du 800, USA). Bacteria were harvested
after 12 or 36 h before suspending in 20 mM Tris-HCl buffer
(pH 8.0) and then disrupting twice in a French pressure cell (SLM
AMINCO, Urbana, IL, USA) at 20,000 lb/in2. Supernatants
(crude cell extracts) were collected by centrifugation (15,0006g,
45 min) and subjected to oxygenase activity assay and proteomic
analysis. Protein concentrations were determined by the Bradford
method . Enzyme activity assay and proteome analysis was
conducted on the basis of same protein amount.
Dioxygenase activity assay
The activities of catechol 1,2-dioxygenase (CD1,2), catechol
2,3dioxygenase (CD2,3), protocatechuate 3,4-dioxygenase (PCD3,4),
and protocatechuate 4,5-dioxygenase (PCD4,5) were determined
using a UV spectrometer (Beckman Coulter Proteome Lab
DU800, USA), as reported previously . The activities of
CD1,2 and CD2,3 were assayed by monitoring increases in
concentration of cis, cis-muconate at A260 and 2-hydroxymuconic
semialdehyde at A375, respectively. Activities of PCD3,4 was
determined by monitoring the increase in concentration of
bcarboxymuconate at A290 (absorbance decreased as
b-carboxymuconate concentrations increased) and PCD4,5 activity was
measured through the increase in 2-hydroxy-4-carboxy-muconic
semialdehyde at A410, respectively. For each assay, one unit (U) of
enzyme activity is defined as the amount of enzyme producing
1 mmol of product per min. Activity assay of each sample was
conducted at least three times for each sample.
One-dimensional gel electrophoresis (1-DE) and in-gel
SDS-PAGE and in-gel digestion was performed as reported
previously . After electrophoresis and Coomassie blue staining,
1D-gels were divided into 10 fractions according to molecular
weight. Sliced gels containing fractionated protein samples were
digested with trypsin (Promega, Madison, WI, USA) for 16 h at
37uC after reduction with 10 mM dithiothreitol (DTT) and
alkylation of cysteines with 55 mM iodoacetamide. The digested
peptides were recovered with extraction solution (50 mmol/L
ammonium bicarbonate, 50% acetonitrile, and 5% trifluoroacetic
acid). The extracted tryptic peptides were dissolved in 0.5%
trifluoroacetic acid prior to further fractionation by LC-MS/MS
LC MS/MS analysis
LC MS/MS analysis was performed according to a modified
version of a previously published method . Tryptic peptide
samples were loaded onto a 2G-V/V trap column (Waters, USA)
for the enrichment of peptides and the removal of chemical
contaminants. Concentrated tryptic peptides were eluted from the
column and directed onto a 10 cm 675 mm i.d. C18 reverse phase
column (PROXEON, Denmark) at a flow rate of 300 nl/min.
Peptides were eluted using a gradient of 0265% acetonitrile for
80 min. All MS and MS/MS spectra were acquired in a
datadependent mode using a LTQ-Velos ESI Ion Trap mass
spectrometer (Thermo Scientific, Germany). Each full MS (m/z
range of 300 to 2,000) scan was followed by three MS/MS scans of
the most abundant precursor ions in the MS spectra. For protein
identification, MS/MS spectra were analyzed by MASCOT
(Matrix Science, http://www.matrix science.com). The genome
sequence database of N. pentaromativorans US6-1 (GI:359402640)
was downloaded from NCBI and used for protein identification.
Mass tolerance of the parent or fragment ion was 0.8 Da.
Cabamidomethylation of cysteine and oxidation of methionine
were considered to be variable modifications of tryptic peptides in
the MS/MS analysis.
c /ryM B B .056 .060 119 .092 .070 .065
) P M M - 0 -0 .0
94 25 23 82 .02 26 51 89 95 20 .02 63 78 74
.1 .2 .0 .2 0 .5 .2 .3 .7 .3 0 .1 .0 .2 B
0 2 0 - 0 0 0 0 2 - 0 - 0 0 0 0 2 0 0 0 M
ryohd llceuub reeMm ltsaopm rteeuMm ltsyaopm rteeuMm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm lirsaepm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm ltsyaopm
c S Inn yC O C O C C C C C C C C C P C C C C C C C C C C C C
liifrtssaaaeeeoonnogddgpm iitrsceaenopNDm ilifrrrrt//cccyaFeBonpAAADm IfrtsssaaSe6100onop lrrtaeoug ilrsyyaaSeeeeeehhonddddgm -rrtttceeeeeBnonnoddpp i---rrttcxyyvaeee22uohhondpm lirrtsscxyaaaeeeoobm ilifrtttssxyvaaaeeeeuuunoonggpb illiftttsssxyvaaaeeeuuunoongpbm ,,iilil---ttyvaee2312unhodppb isxyaeeongd ililfrtttt/svaaeeeeuuunooungpb itszxyaaeeeeonongdb illilftttt/ssvaaeeeuunuoounpbm itszxyaaeeeeonongdb i---rrttsxyyaeee44ohonhnhohdpp rsyaeeeohnddg lilll-rtssscxyyyaaaaaee1hohuundpb lilli-rttssscxyyyaaaaeee1houundbb lif-rrttssaaaSTeeehunnog ii-rttccxyyvae2uohuondpm lilrssyyaaeeeehhodddm ,il-tsccxyaaeee32hoondg iil-rsccxyyyaeee2ohonuhdddmm rsyaeeeohnddg ,ii---rttttxyyvaaeeee224uohnnoddpp rtsyaaehd lrtscyyaaaeeeeeehhonddddg lll---rrtsxxyyavaaaeee42ohooodd ll-rrttsccxxyaaaeee4ooonooodb llilrsscyyyaaaeeeeehhonddddg ilirrsyyaeeeohhoondddddg liirtttcyLaSee_1136hohonpppAUN iffrrrttsccxaeeeeeounoonnoddpm isxyaeeongd iil()-ttttszcyvaaeeeeuhnopbm ilirrsyyaeeeohhoondddddg
/M 8 6 0 8 9
c) ryP .260 - .160 .420 .520 .500 ryP ryP - - ryP ryP ryP
io n 4 n 4 9 9 4 n n n n n n
t e 4 e 8 3 3 0 e e e e e e
tra hP .70 Ph .70 .90 .80 .01 Ph Ph Ph Ph - Ph Ph
liteav /aPM .154 .039 .0033 .812 .112 aP aP aP
R B 0 - 0 2 0 0 B B - - - - B
4 1 en en 6 en 1 en 7 en
.13 .37 .31 Ph .36 Ph .23 Ph .52 Ph .71 Ph
0 1 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1
LA LA LA LA LA LA LA LA LA LA LA LA LA
p p p p p p p p p p p p p
1 2 7 1 2 3 7 4 0 5 7 9 0
LS_111pAU LS_111pAU LS_111pAU LS_112pAU LS_112pAU LS_112pAU LS_112pAU LS_113pAU LS_114pAU LS_115pAU LS_115pAU LS_115pAU LS_116pAU
N N N N N N N N N N N N N
lxyQ F88RO hbpC 1hdbpA 2hdbpA Khpb lxyG aFhn aEhn a1hnpA a2nhpA -
3 4 9 3 4 5 9 6 2 7 9 1 2
i|95304246g i|39520446g i|93520446g i|59342047g i|95320447g i|59324047g i|59304247g i|53940248g i|53904249g i|59342050g i|95340250g i|95324051g i|93502451g
Nano-UPLC-MSE Tandem Mass Spectrometry and
An alternative MS/MS analysis was conducted using a
nanoACQUITY Ultra Performance LC ChromatographyTM
equipped SynaptTM HDMS System (Waters Corporation, MA,
USA) as described previously . The flow-through peptides
were applied to a nano-ACQUITY UPLC BEH300 C18 RP
column (180 mm 6250 mm, particle size, 1.7 mm).
Trypsindigested peptide mixtures were loaded onto the enrichment
column (180 mm 620 mm, particle size, 5 mm), which was
equilibrated with mobile phase A (3% acetonitrile in water with
0.1% formic acid) to remove salts and concentrate the peptides.
Flow-through peptides were directly applied to a
nanoACQUITY UPLC BEH300 C18 RP column (180 mm
6250 mm, particle size, 1.7 mm) at a flow rate of 300 nl/min.
The step gradient was as follows: 3240% mobile phase B (97%
acetonitrile in water with 0.1% formic acid) for 95 min, 40270%
mobile phase B for 20 min, and then a sharp increase to 80%
mobile phase B for 10 min. MS data analysis was carried out
with the continuum LC-MSE data using ProteinLynx
GlobalServer (PLGS) version 2.3.3 (Waters Corporation). The criteria
for protein identification used in the PLGS search engine were
applied with a peptide tolerance of 100 ppm, a fragment
tolerance of 0.2 Da, and a missed cleavage allowance of 1.
Analysis of quantitative changes in protein abundance (.95%
confidence based on peptide ion peak intensities observed in low
collision energy mode (MS) in a triplicate set) was conducted
using ExpressionTM Software version 2(Waters Corporation).
Cluster analysis of proteomic data and prediction of
The emPAI values were used in the cluster analysis of all
analyzed proteins, and the proteome dataset was z-transformed
and median-centered normalized. Analysis of the proteome
dataset was performed by Pearson correlation and average linkage
hierarchical clustering by Cluster and TreeView . All proteins
identified by proteomic analysis were classified according to the
cluster of orthologous groups (COG) functions, and their
subcellular localization of the identified proteins was predicted
by Cello (v. 2.0; http://cello.life.nctu.edu.tw/) . Prediction of
trans-membrane topology was performed using Phobius (http://
Purification and characterization of plasmid pLA1
N. pentaromativorans US6-1 was grown in LB broth containing
10 g/L NaCl. Plasmid DNA was isolated using a standard alkaline
lysis procedure, and purified on NucleoBond columns
(NucleoBond Plasmid BAC Maxi kit, Clontech, USA). Genomic DNA was
shared by careful pipetting of the DNA solution up and down
several times with a 200 ml Pipetman tip. Purified plasmid DNA
was analyzed in a 1% (w/v) agarose gel by clamped homogeneous
electrical field (CHEF) gel electrophoresis (Bio-Rad, USA). CHEF
parameters were set according to the manufacturers protocol,
including a circulating temperature of 14uC, electric current of
350 mA, and a pulse time of 35 s for 28 h. After electrophoresis,
the gel was stained with ethidium bromide solution for 30 min,
washed with TBE for 30 min, and DNA bands were detected
using the Gel-Doc System (Bio-Rad, USA).
iv s 25 27 28 30 31 32
t u 1 1 1 1 1 1
a c 1 1 1 1 1 1 3 4 5 7 2 4 5 6 7
m lo LA LA LA LA LA LA 62 62 62 62 63 63 63 63 67
ro e _p _p _p _p _p _p _3 _3 _3 _3 _3 _3 _3 _3 _3
a n U U U U U U U U U U U U U U U
t e S S S S S S S S S S S S S S S
J K id
en lE lG lJ n n m
y y y h h
G x x x p p - - - - - - - - - - s
thw yehd ian iahn ian iahn m
iilirrtttxaaaaeeonooogdddbpw iitrsceaeonnpDm ,li-tsccxyaaeee23noohgd lii-rsccxyyyaeee2hnohuodddmm ,ii---rtttxyyvaeee422nonouhddpp lll---rrtsxxyyaaaaveee24ooohodd ll-rrttsccxxyaaaeee4oooooondb iilrtttcLyaS_ee1132nohohpppAUN il-----rrtsccxxyyyaaaeee624nouoohddbmm rsyaeeeohngdd ,,i-rttttscccxyaaaaeeeee54nooohuhgdbp ,,il-rtttscccxyaaaaaeeee54nooohuhgdpp l-rttsscxyaaaaaeee4hoondm ,lli---rrrtscxyyyaaaaeee462honoddbp ,,i-rttttscccxyaaaaeeeee54nooohuhgdbp ,,il-rtttscccxyaaaaaeeee54nooohuhgdpp --rtszxxyyyaaeeee3oonooohnngdbpm ll(-rtttscxyaaaaaaeeeeeeoonohgddm +li))(rtcxyaaePongdbAND ,;;,-rrrtszcxyyaeepPBhhonoooodbCHHm .t030128
Comparative analysis of dioxygenase enzymes in
Novosphingobium pentaromativorans US6-1 in response
to polycyclic and monocyclic aromatic hydrocarbons
N. pentaromativorans US6-1 were pre-cultured in MB to obtain
sufficient biomass to determine dioxygenase activity and
proteomic analysis. Approximately equal quantities of cells were
transferred into PAH and MAH culture media. Although all
culture has same cell mass, each has different OD600 values
because of different absorbance of PAHs and MAHs. OD values
are as follow; benzo(a)pyren (0.5146 at OD600), pyren (0.5612 at
OD600), phenanthren (0.604 at OD600), benzoate (0.4242 at
OD600), and p- hydroxybenzoate (0.4366 at OD600). After 12 h
incubation, delta OD values of benzo(a)pyren, pyren,
phenanthren, benzoate, and p-hydroxybenzoate were +0.0101, +0.1461, +
0.0628, 20.0347, 20.0199, respectively. After 36 h incubation,
delta OD values of benzo(a)pyren, pyren, phenanthren, benzoate,
and p-hydroxybenzoate were +0.0013, +0.0254, 20.008, 2
0.0492, +0.5695, respectively. The bacteria were harvested after
12 h and 36 h incubation for enzyme activity assay and proteome
analysis. Highest delta OD values of benzo(a)pyren and pyren
were +0.0101 and +0.1461 at 12 hours incubation, respectively.
Phenanthren was continually increased until 48 hours (+0.0628).
Unexpectedly, cell mass under benzoate culture condition was not
increased, whereas degrading enzyme activities were significantly
increased. The activities of four major dioxygenases (CD1,2,
CD2,3, PCD3,4 and PCD4,5) were assayed using protein extracts
from N. pentaromativorans US6-1 cultured in the presence of three
PAHs and two MAHs to determine which biodegradation
pathways were induced. Analysis of the N. pentaromativorans
US61 genome indicated the presence of only extradiol oxygenase
genes; however, many unspecified oxygenase genes were
identified. In attempting to determine if N. pentaromativorans US6-1 also
expressed intradiol oxygenase activities, we selected the four
dioxygenase enzymes (CD1,2, CD2,3, PCD3,4 and PCD4,5) that
we considered would cover most degradation pathways in aerobic
cultures. No activity of the intradiol dioxygenases CD1,2 and
PCD3,4 was detected. Activity of CD2,3 was high in cells cultured
in media containing phenanthrene (1.59 U/mg at 12 h
cultivation), benzoate (0.53 U/mg at 36 h cultivation), and
p-hydroxybenzoate (0.31 U/mg at 36 hr cultivation), whereas, CD2,3
activity was detected to some degree in cells cultured under all
conditions (0.0220.04 U/mg), including MB media (Fig. 1).
Activity of PCD4,5 was only detected in cells cultured in
phydroxybenzoate (0.20 U/mg at 36 hr cultivation) media (Fig. 1).
This suggests that the CD2,3 pathway could be a primary pathway
for the degradation of PAHs and benzoate, while
p-hydroxybenzoate is broken-down via the PCD4,5 or CD2,3 pathway in N.
Proteomic analysis of Novosphingobium
pentaromativorans US6-1 cultured with polycyclic
Cytosolic proteins were prepared for shotgun proteomics using
1-DE/LC MS/MS from cells cultured in different PAHs.
Approximately 6502718 proteins were identified in cells grown
in the presence of phenanthrene, pyrene, and benzo(a)pyrene.
Comparative analysis between the three PAHs was made with cells
grown in MB as a control (Table S1 and Fig. S1). Analysis
revealed that 494 proteins were commonly induced by all culture
media, with 26232 unique to the different aromatic hydrocarbons
used as sole carbon sources. The identified proteins were divided
into six groups (C12C6) according to cluster analysis with each
protein group arranged according to COG functions (Fig 2).
Enzymes of the PAH and MAH catabolic pathways were clustered
in group C2 and group C6, and were expressed at higher levels in
the presence of phenanthrene. This was particularly noticeable for
the degradation enzymes associated with secondary metabolite
biosynthesis, transport and catabolism [Q] (Table S2). Proteins in
group C6 were strongly induced in those cells grown in MB and
the primary up-regulated COG protein group was Translation,
ribosomal structure and biogenesis [J]. The ribosomal proteins
induced during growth in MB increased by more than 1.69-fold
compared to PAHs. Proteins uncharacterized or unknown [R or
S] were relatively higher in C3 group proteins, which were
abundant under benzo[a]pyrene culture conditions.
A notable outcome of the proteomic analysis was the strong
induction of genes originating from pLA1. This large plasmid
encodes 199 genes, and approximately 27 are clustered,
considered to be a putative coding region for LMW
aromatichydrocarbon degradation. These genes could regulate
biodegradation of bicyclic aromatic ring compounds through to
tricarboxylic acid cycle metabolites, such as acetaldehyde. Approximately
27236 proteins coded by genes located on pLA1 were induced by
three PAHs. Among the putative biodegradation genes, 24, 19,
and 12 proteins were up-regulated in the presence of
phenanthrene, pyrene, and benzo(a)pyrene, respectively, compared to MB
(Table 1). Comparative results showed that the greatest amount of
protein induction occurred in the presence of phenanthrene,
which is consistent with the results of the dioxygenase activity assay
(Fig. 1). Enzymes catalyzing the breakdown of bicyclic aromatic
compounds to acetyl CoA were strongly induced.
Ring-hydroxylating dioxygenase, dihydrodiol dehydrogenase, putative
biphenyl-2,3-diol-1,2-dioxygenase, and catechol 2,3-dixoygenase were
included. Although the activity of catechol 2,3-dixoygenase in
those cultures containing pyrene was low, proteomic results
showed a strong induction of both this enzyme and others
involved in related biodegradation steps (Table 1). The signal
intensity for those enzymes produced by plasmid pLA1 in the
presence of benzo(a)pyrene was lower than that for the other
PAHs phenanthrene and pyrene.
These results were verified by proteomic analysis using
NanoUPLC MS, confirming the up-regulation of N. pentaromativorans
US6-1 biodegradation genes by phenanthrene, pyrene, and
benzo(a)pyrene (Table 2). These results showed that the
biodegradation genes located on pLA1 play a major role in
the utilization of PAHs as sole carbon sources in N.
Proteomic analysis of Novosphingobium
pentaromativorans US6-1 cultured with monocyclic
In response to the differential induction of extradiol
dioxygenases of N. pentaromativorans US6-1 in the presence of
benzoate and p-hydroxybenzoate, a comparative proteome
analysis was conducted. Approximately 1,475 proteins were
identified. 126 were induced by benzoate, and 85 by
phydroxybenzoate (Table S1 and Fig. S1.). Genomic analysis
revealed the presence of a CD2,3 gene and a ring-hydroxylating
gene on pLA1, which are responsible for the initial step of the
lower catabolic pathway of aromatic hydrocarbons. Notably,
four PCD4,5 genes (small and large subunits) were found to be
localized on the chromosome. Proteome analysis showed that
when N. pentaromativorans US6-1 was cultured in
p-hydroxybenzoate, two PCD4,5 and p-hydroxybenzoate degradation
enzymes were induced (Table 3 and Fig. 3). These genes (gene
number NSU_36232NSU_3811) were clustered on contig 58 of
the chromosome (Table S3). However, none of the three PAHs
or benzoate induced the expression of PCD4,5 and
phydroxybenzoate degradation enzymes, suggesting that the
chromosomal-born protocatechuate pathway plays a role in
the breakdown of p-hydroxybenzoate, but not in the
degradation of PAHs or benzoate. Unexpectedly, the biodegradation
genes on pLA1 were expressed in response to
p-hydroxybenzoate, despite having no direct role in the breakdown of this
The genomic sequence of the bacterium N. pentaromativorans
US6-1, which is able to utilize PAHs as its sole carbon source, was
reported recently [10,11]. However, the genes coding proteins that
are important for the biodegradation of PAH have not been
completely annotated, and until now, their function has remained
largely speculative. Two putative clusters containing the genes
necessary for the breakdown of aromatic compounds were
detected in the biodegradation gene region of plasmid pLA1 and
contig 58 of the chromosome (Table S3). In this study, a
proteomic analysis of N. pentaromativorans US6-1 cultured in the
presence of three different PAHs was conducted to examine the
differential expression of biodegradative genes. The results
indicate that PAHs strongly induced the expression of
biodegradation genes located on plasmid pLA1 (Fig 2 and Table 1), but
that other biodegradation gene clusters on contig 58 were not
induced. These results suggest that the biodegradation genes on
plasmid pLA1 are essential for the biodegradation of PAHs.
Semiquantitative proteomic analysis using emPAI revealed that
tri(phenanthrene), tetra- (pyrene), and penta-aromatic compounds
(benzo(a)pyrene) induce a differential biodegradation capacity
(Table 1 and 2). The total amount of biodegrading enzymes
induced in the presence of phenanthrene was estimated to be nine
times greater than that of benzo(a)pyrene (Table 1). However, we
found a discrepancy between enzyme activity and the amount of
CD2,3 expression induced in cells cultured in the presence of
pyrene, and the reason for the instability of CD2,3 induced by
pyrene-containing culture media remains unclear. Because genes
that regulate the biodegradation of bicyclic aromatic compounds
were identified on pLA1, genes for high-molecular-weight PAHs
should be found on the chromosome. An analysis using the
program pFam revealed that several putative aromatic
compounddegrading genes are scattered throughout the chromosome
(contigs 58, 54, and 55), although they were not induced
significantly under our culture conditions. We also considered
the possibility that the cytochrome P450 monooxygenases (CYPs)
may provide alternative biodegradation pathways. Proteomic
analysis showed that four cytochrome P450 proteins
(NSU_2269, 2261, 2259, and 2257) were present, with the
hypothetical protein NSU_2261 assigned as a cytochrome P450
monooxygenases in uniprot Blast analysis. Induction level was very
low, suggesting that CYP has no direct involvement with the
biodegradation of PAHs (Table S1), and this was further
supported by the genomic analysis of N. pentaromativorans US6-1.
The degradation of PAHs was catalyzed by enzymes with broad
substrate specificities. Consequently, further biochemical assay
and overexpression are required to determine related
biodegrading genes , coupled with a more accurate proteomic study
and optimization of culture conditions to understand PAH
metabolism in N. pentaromativorans US6-1. Plasmids similar to
pLA1 have been identified in Sphingomonas aromaticivorans F199 and
Sphingomonas sp. strain KA1 [24,25]. S. aromaticivorans F199 contains
a large plasmid, pNL1, which possesses 186 ORFs and 79 genes
that mediate catabolism or transport of aromatic compounds, such
as mono-aromatic compounds (m-xylene and p-cresol) and bicyclic
aromatic compounds (biphenyl and naphthalene). Plasmid
pCAR3 from Sphingomonas sp. strain KA1 contains a number of
carbazole degradation genes. A comparative analysis of DNA
sequence of pLA1 and pNL1 reveals that they are significantly
homologous (more than 36%) and each of the ORFs in pLA1 has
more than 61% amino acid sequence homology with its
corresponding ORFs (Fig S2). These plasmids are categorized
into four groups according to the repA proteins, with pLA1 from
N. pentaromativorans US6-1 belonging to the Rep_3 superfamily.
The proteomic characterization in this study revealed that N.
pentaromativorans US6-1 utilizes different pathways for the
breakdown of the two MAHs. Benzoate was degraded via the CD2,3
pathway encoded by genes located on plasmid pLA1, whereas
phydroxybenzoate was broken down by the PCD4,5 route. The
lower induction of biodegradation enzymes coded by pLA1 in the
presence of MAHs such as benzoate and p-hydroxybenzote
suggests that their regulation was optimized by PAHs or their
metabolites. This assumption should be confirmed by further
studies. The genes on plasmid pLA1 have been confirmed only by
genomic sequencing and gene assembly. In a previous study, the
presence of plasmid pLA1 in N. pentaromativorans US6-1 was not
confirmed due to difficulties in the purification of large plasmid (.
200 kb). Here, we confirmed the presence of pLA1 in N.
pentaromativorans US6-1 by CHEF gel electrophoresis, and
identified an open-circled plasmid of approximately .250 kb (Fig. 4).
The presence of biodegradation genes on the plasmid was
confirmed by PCR using the purified DNA as the template (data
In conclusion, two extradiol degradation pathways mediated by
genes located in the plasmid and chromosome were separately
induced by PAHs and MAHs. The large plasmid pLA1 plays a
pivotal role in the degradation of bicylic aromatic compounds to
acetyl CoA in N. pentaromativorans US6-1.
Figure S1 Total number of identified proteins of N.
pentaromativorans US6-1 by LC-MS/MS analysis. A & B;
The proteomes induced in PAHs and MAHs were identified using
N. pentaromativorans US6-1 genome database. Benzoate (Ben),
phydroxybenzoate (PHB), phenanthrene (Phen), pyrene (Pyr),
banzo(a)pyrene (BaP). C & D; the proteomes induced in PAHs
and MAHs were identified using plasmid (pLA1) database of N.
Figure S2 Comparative analysis of pLA1 of N.
pentaromativorans US6-1 and pNL1 of N. aromaticivorans
pentaromativorans F199. Biodegradation genes were
indicated with red boxes.
proteins by LC-MS/MS
Table S3 Putative biodegradation gene clusters of N.
pentaromativorans US6-1 and their proteomic result
according to PAHs and MAHs.
Conceived and designed the experiments: SIK SJK KKK. Performed the
experiments: SHY CWC SYL JK SHL YHC. Analyzed the data: SIK
YGL. Wrote the paper: SIK HYK KKK.
1. Sarma PM , Duraja P , Deshpande S , Lal B ( 2010 ) Degradation of pyrene by an enteric bacterium, Leclercia adecarboxylata PS4040 . Biodegradation 21 : 59 - 69 .
2. Kim SJ , Kweon O , Cerniglia CE ( 2009 ) Proteomic applications to elucidate bacterial aromatic hydrocarbon metabolic pathways . Curr Opin Microbiol 12 : 301 - 309 .
3. Kim SI , Choi JS , Kahng HY ( 2007 ) A proteomics strategy for the analysis of bacterial biodegradation pathways . OMICS 11 : 280 - 294 .
4. Kim SJ , Jones RC , Cha CJ , Kweon O , Edmondson RD , et al. ( 2004 ) Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods . Proteomics 4 : 3899 - 3908 .
5. Kim YH , Freeman JP , Moody JD , Engesser KH , Cerniglia CE ( 2005 ) Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1 . Appl Microbiol Biotechnol 67 : 275 - 285 .
6. Kim SJ , Kweon O , Jones RC , Freeman JP , Edmondson RD , et al. ( 2007 ) Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology . J Bacteriol 189 : 464 - 472 .
7. Kweon O , Kim SJ , Holland RD , Chen H , Kim DW , et al. ( 2011 ) Polycyclic aromatic hydrocarbon metabolic network in Mycobacterium vanbaalenii PYR1 . J Bacteriol 193 : 4326 - 4337 .
8. Liang Y , Gardner DR , Miller CD , Chen D , Anderson AJ , et al. ( 2006 ) Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp . strain KMS. Appl Environ Microbiol 72 : 7821 - 7828 .
9. Demaneche S , Meyer C , Micoud J , Louwagie M , Willison JC , et al. ( 2004 ) Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a sphingomonas strain that degrades various polycyclic aromatic hydrocarbons . Appl Environ Microbiol 70 : 6714 - 6725 .
10. Sohn JH , Kwon KK , Kang JH , Jung HB , Kim SJ ( 2004 ) Novosphingobium pentaromativorans sp. nov., a high-molecular-mass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment . Int J Syst Evol Microbiol 54 : 1483 - 1487 .
11. Luo YR , Kang SG , Kim SJ , Kim MR , Li N , et al. ( 2012 ) Genome sequence of benzo(a)pyrene-degrading bacterium Novosphingobium pentaromativorans US6-1 . J Bacteriol 194 : 907 .
12. Aylward FO , McDonald BR , Adams SM , Valenzuela A , Schmidt RA , et al. ( 2013 ) Comparison of 26 sphingomonad genomes reveals diverse environmental adaptations and biodegradative capabilities . Appl Environ Microbiol 79 : 3724 - 3733 .
13. Bradford MM ( 1976 ) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem 72 : 248 - 254 .
14. Kim YH , Cho K , Yun SH , Kim JY , Kwon KH , et al. ( 2006 ) Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis . Proteomics 6 : 1301 - 1318 .
15. Yun SH , Park GW , Kim JY , Kwon SO , Choi CW , et al. ( 2011 ) Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT . J Proteomics 74 : 620 - 628 .
16. Choi CW , Lee YG , Kwon SO , Kim HY , Lee JC , et al. ( 2012 ) Analysis of Streptococcus pneumoniae secreted antigens by immuno-proteomic approach . Diagn Microbiol Infect Dis 72 : 318 - 327 .
17. Moon YJ , Kwon J , Yun SH , Lim HL , Kim MS , et al. ( 2012 ) Proteome analyses of hydrogen-producing hyperthermophilic archaeon Thermococcus onnurineus NA1 in different one-carbon substrate culture conditions . Mol Cell Proteomics 11 : M111 015420.
18. Eisen MB , Spellman PT , Brown PO , Botstein D ( 1998 ) Cluster analysis and display of genome-wide expression patterns . Proc Natl Acad Sci U S A 95 : 14863 - 14868 .
19. Yu CS , Chen YC , Lu CH , Hwang JK ( 2006 ) Prediction of protein subcellular localization . Proteins 64 : 643 - 651 .
20. Kall L , Krogh A , Sonnhammer EL ( 2004 ) A combined transmembrane topology and signal peptide prediction method . J Mol Biol 338 : 1027 - 1036 .
21. Jouanneau Y , Meyer C ( 2006 ) Purification and characterization of an arene cisdihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols . Appl Environ Microbiol 72 : 4726 - 4734 .
22. Singleton DR , Hu J , Aitken MD ( 2012 ) Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium . Appl Environ Microbiol 78 : 3552 - 3559 .
23. Kasai Y , Shindo K , Harayama S , Misawa N ( 2003 ) Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp . strain A5. Appl Environ Microbiol 69 : 6688 - 6697 .
24. Romine MF , Stillwell LC , Wong KK , Thurston SJ , Sisk EC , et al. ( 1999 ) Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199 . J Bacteriol 181 : 1585 - 1602 .
25. Shintani M , Urata M , Inoue K , Eto K , Habe H , et al. ( 2007 ) The Sphingomonas plasmid pCAR3 is involved in complete mineralization of carbazole . J Bacteriol 189 : 2007 - 2020 .