Genomics 105 (2015) 182–190

Contents lists available at ScienceDirect

Genomics
journal homepage: www.elsevier.com/locate/ygeno

Complete genome sequence analysis of Pseudomonas aeruginosa N002

reveals its genetic adaptation for crude oil degradation
Dhrubajyoti Das, Reshita Baruah, Abhijit Sarma Roy, Anil Kumar Singh, Hari Prasanna Deka Boruah ⁎,
Jatin Kalita, Tarun Chandra Bora
Department of Biotechnology, CSIR-North East Institute of Science & Technology, Jorhat 785006, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: The present research work reports the whole genome sequence analysis of Pseudomonas aeruginosa strain N002
Received 2 June 2014 isolated from crude oil contaminated soil of Assam, India having high crude oil degradation ability. The whole ge-
Accepted 15 December 2014 nome of the strain N002 was sequenced by shotgun sequencing using Ion Torrent method and complete genome
Available online 26 December 2014
sequence analysis was done. It was found that the strain N002 revealed versatility for degradation, emulsification
and metabolizing of crude oil. Analysis of cluster of orthologous group (COG) revealed that N002 has significantly
Keywords:
Bioremediation
higher gene abundance for cell motility, lipid transport and metabolism, intracellular trafficking, secretion and
Crude oil vesicular transport, secondary metabolite biosynthesis, transport and catabolism, signal transduction mechanism
Pseudomonas aeruginosa and transcription than average levels found in other genome sequences of the same bacterial species. However,
Genome lower gene abundance for carbohydrate transport and metabolism, replication, recombination and repair, trans-
lation, ribosomal structure, biogenesis was observed in N002 than average levels of other bacterial species.
© 2014 Elsevier Inc. All rights reserved.

1. Introduction However, bioremediation efficiency is limited due to complex and

Crude oil contamination in soil and water bodies is a major threat to
the pristine ecological condition and is reported at regular intervals
across the globe. Contamination occurs during exploration, abandon-
ment of drill sites, accidental spillage from production units, refining
processes and transportation [1–3]. Due to the non degradability of
crude oil, the contaminants remain in the environment for long time
and affect the physical, chemical and biochemical behavior of the
contaminated soil/water [4]. Further, because of xenobiotc nature of
crude oil and oil products, their difficult degradation results continued
environmental threats which require utmost attention for remediation
[5]. Remediation of crude oil contaminated environment employs
different processes like physical, chemical and biological. Physical and
chemical processes are costly, not eco-friendly and requires extensive
site restoration. In contrast to these, bioremediation processes are
being preferred presently due to their cost effectiveness, less stress to
environment and complete recovery of contaminated sites [6,7]. In bio-
remediation, free living or immobilized bacteria having high crude oil
degradation ability are used individually or in consortium. Addition of
crude oil degrading fungi or algae can also be done. In many cases bio-
remediation is coupled with phytoremediation employing plant species
highly tolerant to crude oil and other hydrocarbon contaminants.
⁎ Corresponding author.
E-mail addresses: 1010anil@gmail.com (A.K. Singh), dekaboruah@yahoo.com,
dekabaruahhp@rrljorhat.res.in (H.P. Deka Boruah).
toxic nature of crude oil and intricacies associated with adaptation
and survival of microorganisms under adverse crude oil contaminated
environment [6,8,9]. However, in the recent past, screening of different
crude oil utilizing bacteria from different ecological niche and their
utilization in clean-up of oil contaminated environment have been
reported [2–9].
Several research work have reported the crude oil degradation po-
tential of different bacterial strains belonging to the genus Lysinibacillus,
Brevibacillus, Bacillus, Paenibacillus, Stenotrophomonas, Alcaligenes,
Delftia, Achromobacter and Pseudomonas [10–16]. Experimental findings
from different research reports revealed that action of crude oil utilizing
bacteria on degradation and mineralization depends on the nature of
crude oil composition, genes and traits responsible for their adaptation,
edaphic and related ecological factors [13–18]. The search for critical
factors regulating crude oil mineralization leads different catabolic
genes that encode enzymes involved in a variety of known bacterial
hydrocarbon degradation pathways and expression [17–19]. The identi-
fied genes includes alkane monooxygenase (alkB) from Pseudomonas
putida, alkane monooxygenase (alkm) from Acinetobacter sp. strain
ADP-1; alkB1; alkB2, alkane monooxygenase from Rhodococcus sp.;
xylE, catechol-2, 3-dioxygenase from P. Putida; naphthalene dioxygenase
(ndoB) from P. putida; and nidA, pyrene dioxygenase large subunit from
Mycobacterium sp. strain PYR-1 [17,19]. However, adaptation of mi-
crobes in oil polluted environment is not limited to the different gene
regulating degradation and mineralization of crude oil and its various
components. Adaptation in general is ensured by factors that governs

http://dx.doi.org/10.1016/j.ygeno.2014.12.006
0888-7543/© 2014 Elsevier Inc. All rights reserved.
 D. Das et al. / Genomics 105 (2015) 182–190
two-component systems (TCS) [19]. The TCS like EnvZ/OmpR for osmo-
larity sensing [20], CheA/CheY for chemotaxis [21] and DesR/DesK for
thermo sensing [22] are found in bacteria which can sense and respond
to the corresponding environmental situation.
Several studies have reported the bacterial complete genome
sequence information based on their biological activities [23–27]. Simi-
larly, bacteria capable of degrading crude oil isolated from different oil
contaminated site have been sequenced and the genetic basis of their
crude oil mineralization potential has been studied [24,27]. Complete
genome analysis of hydrocarbon degrading bacterial strains provide us
the useful insights into the hydrocarbon degradation/ uptake mecha-
nism and also the genetic adaptations of the strains for proliferating in
the crude oil stressed environment. Research on the complete pathway
elucidation of crude oil degradation in different bacterial strains is still
in its nascent stage and only few research reports have highlighted
the degradation pathway [17–19]. Detailed investigation of crude oil
degradation and uptake mechanism by the strain Pseudomonas
aeruginosa is very important because it is one of the most frequently
reported bacterial strains from crude oil condition across the globe
[11,16,27]. Several researchers have highlighted the importance and
suitability of the strain for crude oil bioremediation of different contam-
inated ecological regions [2,5–7]. This research work is the first report of
whole genome analysis of P. aeruginosa highlighting to its crude oil deg-
radation ability from the north east region of India, one of the leading
biodiversity hot spot under Indo Burma Mega Biodiversity hot zone of
the world [28]. The reported strain N002 is a potent hydrocarbon
degrader [29] and has been isolated from a crude oil contaminated
site of Geleky (Sibsagar district), Assam, India. The focus of the research
reported in this report is the whole genome analysis of the strain
P. aeruginosa N002. Emphasis was given in understanding the spectrum
of intricacies and factors associated with crude oil degradation, uptake
and general adaptation in crude oil contaminated environment by the
strain N002 at genetic level.
2. Results and discussion
2.1. General features of P. aeruginosaN002 genome
2.1.1. COG analysis
P. aeruginosa N002 has single circular chromosome having total
sequence length 65,37,648 contains 5764 annotated genes with 100%
total genome coverage (Table 1). The assembled genome consists of
235 de novo based contigs with an average contig size of 2574 bp and
G + C content of 62.36% [29]. The genome contains 11,038 open reading
frames (ORFs) and encodes 5629 protein coding genes (CDS), 135 RNA
genes, 63 tRNA genes and 12 rRNA genes. Comparison of the N002
genome with other available genomes revealed highest synteny be-
tween N002 and P. aeruginosa PA01 genomes and rest genomes were
in following order (Pseudomonas putida F1 N Pseudomonas fluorescence
PF5 N Burkholderia cepacia 363 N Polymorphum gilvum SL003B-26A1 N
Table 1
Key features of Pseudomonas aeruginosa N002 genome.
Features Genome
DNA, total number of bases 65,37,648
DNA coding number of bases 54,91,038
DNA G + C content (%) 62.36
Genes total number 5,764
Protein coding genes 5,629
Pseudo genes 0 to adapt to changing environment and also with different selective pres-
RNA genes 135
rRNA genes 12
5S rRNA 4 genes often regarded with horizontal origins [32] and N002 witnessed a
16S rRNA 4 number of horizontal gene transfer events assisted by transposons and
23S rRNA 4 other origins. There are 40 GIs predicted in the genome of the strain
tRNA genes 63
ORF number 11,038
183
Paracoccus denitrificans PD1222) compared to N002 genome. Among
5629 CDS 4668 are the cluster of orthologous groups (COGs). In addition
all CDS could be assigned to 21 different categories (Fig. 1), including
those for amino acid transport and metabolism (category E, 9.35%),
transcription (K, 9.27%), energy production and conversion (C, 6.3%),
inorganic ion transport and metabolism (P, 5.69%), and signal transduc-
tion mechanisms (T, 6.56%) (Table S1).
A one-sample t-test was used to evaluate possible significant differ-
ences of the gene abundances of each COG categories between
P. aeruginosa N002 and other genomes in the IMG genome database
(Table S1). The results showed that the abundances of amino acid
transport and metabolism (E, 9.35%), carbohydrate transport and
metabolism (G, 4.31%), cell motility (N, 2.92%), coenzyme transport
and metabolism (H, 4.03%), energy production and conversion (C, 6.3%),
functions unknown (S, 10.04%), general function prediction (11.62%)
lipid transport and metabolism (I, 4.43%), nucleotide transport and
metabolism (F, 2.04%) post translation modification, protein turnover,
chaperons (O, 3.8%), RNA processing and modification (A, 0.06%), second-
ary metabolites biosynthesis, transport and catabolism (Q, 3.09%), signal
transduction mechanisms (T, 6.56%) were appreciably higher than the
average values. However, the genes responsible for cell cycle regulation,
cell division, chromosome partitioning (D, 0.67%), defense mechanisms
(V, 1.41), inorganic ion transport and metabolism (P, 5.69%), intracellular
trafficking, secretion, and vesicular transport (U, 3.24%), replication,
recombination and repair (L, 2.42%), transcription (K, 9.27), translation,
ribosomal structure and biogenesis (J, 3.8%) were found comparatively
(P b 0.001) lower than the average levels.
One-sample t-test was also performed to evaluate the difference in
gene abundance of different COG categories between P. aeruginosa
N002 and other hydrocarbon degrading bacteria of IMG database
(Table S2). The strain N002 showed significant (P b 0.001) higher
abundance of genes related to aminoacid transport and metabolism
(E, 9.335%), cell motility (N, 2.92%), inorganic ion transport and metab-
olism (P, 5.69), intracellular trafficking, secretion and vesicular trans-
port (U, 3.24%), posttranslational modification, protein turnover,
chaperones (O, 3.8%), signal transduction mechanisms (T, 6.56%), and
also the genes with unknown functions (S, 10.04%) compared to that
of hydrocarbon degrading bacteria [24,28,30,32]. Moreover, abun-
dances of genes belonging to chromatin structure and dynamics (B),
RNA processing and modification (A) categories in N002 were similar
compared to other hydrocarbon degrading strains. In contrary to
these, abundance of genes related to carbohydrate transport and metab-
olism (G, 4.31%), cell cycle control, cell division, chromosome
partitioning (D, 0.67%), chromatin structure and dynamics, coenzyme
transport and metabolisms (H), defense mechanisms (V), energy pro-
duction and conversion (C), general functions predictions (R), lipid
transport and metabolism (I), nucleotide transport metabolism (F), rep-
lication, recombination and repair (L), secondary metabolites biosyn-
thesis, transport and catabolism (Q), translation, ribosomal structure
and biogenesis (J), are significantly lower (P b 0.001) (Table S2).
Based on COG analysis, 347 genes in the genome were assigned to the
signal transduction category (Table S3) and 74 genes are involved in
the two component system (TCS) category (Table S4). The genes in-
volved in signal transduction and TCS signifies the strain's ability to reg-
ulate cell metabolism to a wide variety of environment [19–22].
2.1.2. Horizontal gene transfer and genomic islands
Gene transfer, especially horizontal gene transfer (HGT), is a univer-
sal process for microorganisms to acquire functions that enables them
sures [31]. Genomic islands (GIs) in prokaryotic genomes are clusters of
N002 by SeqWord Sniffer and Island Viewer methods [25,27] and local-
ization of predicted GIs is shown in Fig. 2. The 40 GIs identified in
184 D. Das et al. / Genomics 105 (2015) 182–190

Fig. 1. Circular chromosome map of Pseudomonas aeruginosa N002.

combination can cover 19,81,504 bp or 30.3% of the whole chromosome
and would comprise 537 genes in total, starkly contrasting with other
group of well-known hydrocarbon utilizing bacteria, Alcanivorax
borkumensis [24], Oleispira antarctica [33], and Polymorphum gilvum
SL003B-26A1 [34]. Identification and analysis of horizontally transferred
genomic islands revealed identical genes available in other organisms.
The GIs having probable horizontal origins might have a significant
role in adapting the bacteria to different abiotic stress, besides confer-
ring antimicrobial resistance and secondary metabolite production
[35,36]. This might have been acquired from other organisms or evolved
after transfer in response to the changed environmental condition
(crude oil contamination) in its vicinity. GIs associated with adaptation
and environmental interest has had substantial impact on bacterial
evolution [31,32]. In these 40 GIs, all identified genes were summarized
in Table S5, including genes encoding for major facilitator transporter,
type II secretion protein, hydrogen cyanide synthase HcnB and
adenylatecyclase ExoY. As per SeqWord Sniffer GI23 (52,43,628 bp to
53,93,030 bp) found to be the prominent island representing forty
eight different gene clusters while GI24 (61,91,799 bp to 62,14,676 bp)
is the lowest having twenty gene clusters.
Plenty of putative 19 CDS for transposase coding genes or their frag-
ments were scattered in the chromosome that could support potential
active HGT in the strain (Table S6A). IS3 family transposases are pre-
dominantly present in N002 genome and the most transposon affected
region in the genome is situated inside GI-19 where three IS3 trans-
poses (or their fragments) were inserted between genetic loci coding
for predicted ADP-ribose pyro phosphatase (related to pentose phos-
phate pathways), thio esterase, tRNA_Lys_TTT (related to aminoacyl
t-RNA biosynthesis) and PreQ (0) biosynthesis protein QueC (related
to purine metabolism). Further, two IS3 transposes are situated on GI-1
between genetic loci for Rhs element Vgr protein, Type VI secretion sys-
tem effectors Hcp1 family (role in mediating host interactions) [19] and
 D. Das et al. / Genomics 105 (2015) 182–190 185

Fig. 2. Genomic island prediction of Pseudomonas aeruginosa N002.

tRNA_Arg_CCG, Succinate-semialdehyde dehydrogenase. Multiple
genes for chaperones, alkane degrading gene, organic solvent trans-
porters, phenol degrading gene, outer membrane efflux proteins,
heavy metal and drug resistance proteins and various transcriptional
regulators were found to be associated with predicted GIs (Table S5).
These genes are possibly being used to adapt to the hydrocarbon degra-
dation, biosurfactant synthesis and oil-contaminated soil with changing
temperatures. All the forty GIs had the DNA composition very similar to
the P. aeruginosa chromosome, possibly due to the acquired composi-
tion similarity over a long time after insertion, however, the next choice
of homology also been mentioned in Table S5. It can be concluded that
P. aeruginosa N002 might have evolved and received GIs which make
them genetically strong to utilize/degrade or mineralize crude oil [35].
The P. aeruginosa N002 genome contains small number of mobile ge-
netic elements such as transposons, insertion (IS) elements (Table S6A
and S6B). These less mobile elements may help the strain N002 in selec-
tion of the bacteria under variant environment.
containing a CheY-like receiver domain and an HTH DNA-binding
domain (COG2197). A total of 19 proteins were found to belong to
DNA-directed RNA polymerase specialized sigma subunit, sigma 24
homolog (COG1595). A further 50 proteins belong to AraC-type DNA-
binding domain-containing proteins (COG2207); 4 proteins belong
to transcriptional regulator containing PAS, AAA-type ATPase and
DNA-binding domains (COG3829); 11 belong to transcriptional regula-
tor containing an amidase domain and an AraC-type DNA-binding HTH
domain (COG4977) (Table S7).
2.1.5. Detoxification and nutrient uptake
Crude oil contaminated environment causes rapid lowering of ele-
mental nutrients such as N, P, S, and Fe [11]; instead growth and survival
of microorganisms were affected by the toxicity of crude oil hydrocar-
bons, various antimicrobial compounds and heavy metals. Efficient
transporter systems for uptake of nutrient elements and detoxification
therefore, play an important role for bacteria survival under such

2.1.3. Genomic comparisons with closely related bacteria

The results revealed that major proteins of P. aeruginosa N002 were
closely related to P. aeruginosa DK2, an isolate from airways of cystic fi-
brosis patients [37], when seven P. aeruginosa strains were taken from
IMG database (Fig. 3, Table 2). Moreover, in the comparison of the ge-
nome of N002 strain with related bacterial genome from NCBI database,
two P. aeruginosa strains DK2 [37] and LESB58 [38] were seen to be very
close which were isolated from cystic fibrosis patient and from infant
with community-acquired diarrhea disease respectively (Fig. 3).

2.1.4. Regulation pattern

A total of 486 CDS in the N002 genome were assigned to the tran-
scription category (K) based on the COG analysis, among which 100
proteins are LysR type transcriptional regulators (LTTRs) (COG0583)
(Table S7). This is the most abundant type of transcriptional regulator
in the prokaryotic kingdom, especially throughout the different subdivi-
sions of proteobacteria, which could play a regulatory role over genes
involved in catabolism of aromatic compounds, cell motility and quo-
rum sensing [24]. Another 26 proteins were found to belong to response
regulators consisting of a CheY-like receiver domain and a winged-helix Fig. 3. Phylogenetic tree of Pseudomonas aeruginosa N002 with other seven reference
DNA-binding domain (COG0745) and 22 belonged to response regulator strains of Pseudomonas available in IMG database.
186 D. Das et al. / Genomics 105 (2015) 182–190

Table 2
Comparison genome statistics of reference strains of Pseudomonas aeruginosa available retrieved from http://img.jgi.doe.gov and genomic comparisons with Pseudomonas aeruginosa
N002.

Genome features Pseudomonas aeruginosa strains available in IMG database P

value
P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa
N002 NCGM2.S1 M18 DK2 LESB58 PA7 PAO1 UCBPP-PA14

DNA, total number of bases 6537648 6764661 6327754 6402658 6601757 6588339 6264404 6537648 1.1234
DNA coding number of bases 5491038 5999144 5645326 5688215 5863385 5916354 5617954 5864198 4.3166
DNA G + C number of bases 4076629 4474404 4208227 4243057 4376792 4377914 4169320 4333951 4.5068
Genes total number 5764 6358 5769 5960 6026 6396 5671 5994 6.5532
Protein coding genes 5629 6269 5690 5884 5925 6294 5568 5892 8.1890
Pseudo Genes 0 0 6 0 0 8 2 0 0.1210
RNA genes 135 89 79 76 101 102 103 102 4.3105
rRNA genes 12 12 13 12 13 12 13 13 6.7445
5S rRNA 4 4 5 4 4 4 4 4 1.3105
16S rRNA 4 4 4 4 4 4 4 4 –
23S rRNA 4 4 4 4 4 4 4 4 –
tRNA genes 63 77 61 64 67 65 63 63 2.7958

harsh conditions. Transport system analysis was performed by compar-
ing each predicted protein against Transporter classification database
(http://www.tcdb.org/). A total of 905 genes (15.70% of total CDS)
were found to be involved in the transport systems for aromatic com-
pounds, amino acids, carbohydrates, lipid, nucleotides and inorganic
ions (Tables S8 and S9). It was found that transporter found in the strain
N002 is 0.05% higher to that of other seven strains of IMG data base.
Phosphorus is considered to be the major rate-limiting element
for biological activity in oil reservoirs [11]. N002 contains a typical
ABC-type phosphate/phosphonate transport system (A222_01647,
A222_01650), which is under control of the phosphate regulon tran-
scriptional regulatory protein PhoB-PhoR (A222_05556-A222_05557)
[26,27]. Phosphate may also be taken up by a Na_/phosphate symporter
(A222_05001, A222_05678) and three members of the PiT (inorganic
phosphate transporter) family (A222_00459, A222_04418, A222_
05400). Typical sulfate ABC-type transporter (A222_00283, A222_
00285) and sulfate permease (SulP) (A222_00029, A222_00108,
A222_02471, A222_03397) were found. N002 utilizes both primary
and secondary transporters for export of various drugs and heavy
metals. Iron uptake seems to be very important for N002, as 3 sets
of iron chelate ABC-type systems (A222_00807-A222_00809,
A222_02061-A222_02063, A222_04875- A222_04877). FeoB protein
(A222_04486) for Fe2 + uptake and low affinity Fe2 +/transporter
(FeT) family (A222_02843) proteins were found.
No specific uptake transporters for Zn2+was observed in the strain
N002, however two sets of Mn2 +/Zn2 + transport systems (A222_
02631-A222_02632; A222_05706-A222_05707) and one set of Zn2+-
Fe2 +Permease (ZIP) family (A222_04602) were found. Other metal
transporters including, Co2 + (CbtAB, A222_02023-A222_02024),
Mg2 +/Co2 +(CorA, A222_03152, A222_03263, A222_05464), Mn2 +/
Fe2 +(Nramp, A222_04246, A222_04462), Mg2+(MgtC, A222_00044,
A222_02478, A222_02879, A222_04792 and MgtEA222_04135) Cu2+
(CopD, A222_04001, A222_04765), Fe/Pb2 + (ILT, A222_00544) and
arsenate-antimony (ArsAB, A222_02755) were observed. The putative
benzoate organic solvent uptake gene (A222_03393) were found,
which frequently detected at crude oil contaminated site [28]. In
addition, two more putative organic solvent transporter system
(A222_01761, A222_04590) were also identified which are obtained
by HGT (Table S5). We also found 9 putative transporter genes
(A222_0773, A222_01041, A222_01105, A222_01666, A222_01667,
A222_02084, A222_02479, A222_02807, A222_03827) for fatty acids
(FAT family), the common degradation products of n-alkanes along
with one putative gene (A222_03023) of short chain fatty acid uptake
(A to E) family. The N002 genome encoded various systems for the
uptake and biosynthesis of osmoprotectant such as glycin-betain,
carnitine, choline, and betaine. Three betaine/carnitine/choline trans-
porter (BCCT) family (A222_01032, A222_05488, A222_05577), four
other ABC superfamily transporters (A222_01076-A222_01079),
(A222_01737) (A222_05282-A222_05284), and (A222_05291). Pre-
sumably, these osmoprotectants accumulate in the cytoplasm in
response to osmotic stress [21,38].
N002 genome contains at least 63 transporters (Table S10) belong-
ing to five families plausibly associated with drug resistance, including
the ATP binding cassette (ABC), drug/metabolite transporters (DMT),
membrane fusion protein (MFP), major facilitator super family (MFS),
and resistance-nodulation-cell division (RND). The arrays of transporter
systems noted in N002 are probably of importance for persistence in
crude oil contaminated environment where nutrients are scarce and
xenobiotics pollutants are diverse.
2.2. Genetic mechanisms of crude oil degradation
2.2.1. Biosurfactant synthesis and crude oil emulsification
Biosurfactant is a surface active compound produced by many mi-
croorganisms that reduce the surface tension, critical micelle concentra-
tion and interfacial tension in both aqueous solutions and hydrocarbon
mixtures. Crude oil components are hydrophobic and of low availability
to the environment microbes and emulsification is a crucial step en-
abling bacteria to easily assimilate and degrade crude oil. Association
between hydrocarbon biodegradation and biosurfactant production
are of great interest which lead to applications of bacteria in microbial
enhanced oil recovery [39,40], bioremediation of hydrocarbon-
contaminated sites [16]. Further, biosurfactant production is very im-
portant as it increases the emulsification and solubilization of the hy-
drocarbons by lowering the interfacial surface tension which in turn
increases their microbial uptake.
N002 genome encodes 25 genes involve in biosurfactant
synthesis and regulation (Table S11). The synthesis genes include
L -rhamnosyl transferase (RhlC), glycosyl transferases (RhlB) and
phosphomannomutase (AlgC). At the regulation level, MvfR and PtxR
are the transcriptional regulators, AlgR the response regulator of the
LytR/AlgR family and RsmA acts as carbon storage regulator (csrA)
(Table S11). In N002 eleven different bisurfactant producing genes are
recognized which are similar to strain P. aeruginosa UCBPP-PA14.
The genes responsible for type IV pili and flagella assembly are well
equipped in the strain, which can function to emulsifying the hydrocar-
bon for crude oil degradation. Flagella assembly plays an important role
in cell motility and chemotaxis, which could also help bacteria move to
relatively close niches and attach to the oil-water interface where the
utilization of hydrocarbons as carbon source takes place (Table S12,
Table S13). Out of 43 different cells motility and chemotaxis gene
identified in N002, only 18–65% genes were present in other crude oil
degrading reference strain (NCGM2.S1, DK2, M18 and UCBPP-PA14 of
IMG data base).
 D. Das et al. / Genomics 105 (2015) 182–190
2.2.2. Crude oil components degradation
Several genes were identified in P. aeruginosa N002 which are
actively involved in the degradation of oil components, including
alkane, alkene and aromatic compounds (Fig. 4 and Table S14) and
putative aromatic, alkane and alkene degradation pathways is described
in Fig. 5a and b. For aromatic compounds e.g., benzoate and xylene
degrading genes include, benzoate 1,2-dioxygenase (A222_02521-
02522), 2-polyprenylphenol hydroxylase and related flavodoxin
oxidoreductases (A222_02523) and dehydrogenases (A222_02524).
Aminobenzoate degrading genes include 2-polyprenylphenol hy-
droxylase and related flavodoxin oxidoreductases (A222_02525),
acylphosphatases (A222_04091) and alkaline phosphatase (A222_
01670). 2-polyprenylphenol hydroxylase (A222_02523), 3-hydroxyacyl-
CoA dehydrogenase (A222_03417), acetyl-CoA acetyl transferases
(A222_02033), benzoate 1,2-dioxygenase (A222_02521) for benzoate
degradation. 2-haloalkanoic acid dehalogenase (A222_04245) and alco-
hol dehydrogenase (A222_03895) for chloroalkane and chloroalkene
degradation. 2-haloalkanoic acid dehalogenase (A222_04245) and
catechol 1, 2-dioxygenase (A222_02532) for chlorocyclohexane and chlo-
robenzene degradation. S-(hydroxymethyl) glutathione dehydrogenase/
class III alcohol dehydrogenase (A222_01345) is responsible for naphtha-
lene degradation. Toluene degradation (Table S14) is traced back to
catechol 1,2-dioxygenase (A222_02532), dienelactone hydrolase and
related enzymes (A222_02328) and succinate dehydrogenase locus
(A222_03463-A222_03466) whereas 2-polyprenylphenol hydroxylase
(A222_02523) and benzoate 1,2-dioxygenase (A222_02521-A222_
02522) for xylene degradation. Caprolactam degradation is mainly carried
out by the gene enoyl-CoA hydratase/carnithineracemase (A222_01532,
A222_03208, A222_03288). The genes acyl CoA: acetate/3-ketoacid CoA
transferase, alpha subunit (A222_00224-A222_00225) and Asp-
tRNAAsn/Glu-tRNAGln amido transferase A subunit and related amidases
(A222_04347, A222_04470, A222_00803) along with homogentisate
1,2-dioxygenase locus enzymes (A222_03016- A222_03018) are
Fig. 4. Alkane–alkene and other aromatic hydrocarbon degrading gene locus Pseudomonas aeruginosa N002.
187
involved in styrene degradation. Benzoate 1,2-dioxygenase (A222_
02521-A222_02522) is involved in fluorobenzoate degradation. Even
though, alkane 1-monooxygenase (EC 1.14.15.3) (alkB, A222_02440)
is a part of the core genome, it is located in the region of GI-10.
Several putative regulatory genes were found in the genome of
N002, related to catabolism of aromatic compounds (Fig. 5, Table S14).
PcaR transcription regulator (A222_00160) was found with β-
ketoadipate pathway, which is required for chemotactic response to
aromatic compounds (e.g., benzoate degrading pathways) [34]. Several
LysR-type transcriptional regulators (LTRRs) related to various
compounds were identified, such aschloroalkane (A222_01344),
chloroalkene (A222_05631), for benzoate degradation (A222_00231),
for catechol degradation (A222_02529). GntR family transcriptional
regulators found in the strains are associated with aromatic compound
degradation, including putative GntR regulator, vanR (A222_05088)
related to the regulation of vanilate catabolism. Several AraC-type
transcriptional regulators related to various compounds were identi-
fied, for benzoate and fluorobenzoate (A222_02520), aminobenzoate
(A222_02528). One Xenobiotic response element (XRE) transcriptional
regulator (A222_00223) and one IclR-type regulator (A222_03015)
both were identified in link with styrene degradation pathways.
Furthermore, genes for transferring the intermediary metabolites
from aromatic compounds metabolism into the central metabolism
were also predicted in the genome, indicating the presence of complete
pathways for aromatic compounds degradation and metabolism. These
genes found in the genome also support the genetic basis of
P. aeruginosa N002 for using crude oil component as C-sources.
2.2.3. Comparisons among oil degrading bacteria
Analysis of the abundance of the COG categories of Polymorphum
gilvum SL003B-26A1T, Acinetobacter baylyi ADP1, Acinetobacter lwoffii
SH145, Alcanivorax borkumensis SK2, Bacillus thuringiensis Bt407,
Burkholderia cepacia 383, Geobacillus thermodenitrificans NG80-2,
188 D. Das et al. / Genomics 105 (2015) 182–190
Fig. 5. Alkane–alkene and other aromatic hydrocarbon degrading gene locus of Pseudomonas aeruginosa N002. a. Putative chloroalkane and chloroalkene degradation pathways of
Pseudomonas aeruginosa N002. b. Putative aromatic compound degradation pathways of Pseudomonas aeruginosa N002.
Gordonia bronchialis DSM 43247, P. aeruginosa PAO1, Pseudomonas
fluorescens Pf-5, Rhodococcus jostii RHA1, Desulfococcus oleovorans
Hxd3, Desulfatibacillum alkenivorans AK-01, Marino bacteralgicola
DG893, Mycobacterium bovis AF2122/97, Nocardia –farcinica IFM
10152, Paracoccus denitrificans PD1222 and Xylella fastidiosa 9a5c com-
pared with N002 revealed varied similarities among these hydrocarbon-
degrading strains (Table S2).
The toxicity and scarce availability of oil components as carbon
sources could be the driving forces for these bacteria to evolve sensing
and response systems to avoid damage by hydrocarbons and pursue
co-metabolic pathways. In the genomes of N002, SL003B-26A1T, SK2,
NG80-2 and AK-01, the abundances of protein categories responsible
for carbohydrate transport and metabolism are lower than the average
value of other hydrocarbon degrading bacteria. This is in accord with the
low carbohydrate availability in the environments where these strains
were isolated. Fatty acids are important intermediate products in the al-
kane degradation pathway and lipid transport and metabolism are
therefore important for the further degradation of alkanes.
3. Conclusion
Crude oil contaminated soil generally is an extremely hostile habitat
where various xenobiotic hydrocarbon compounds and heavy metals
are persistent. These compounds have toxic effect on microbial activity
of the soil. The N002 genome analysis revealed the underlying mecha-
nism and degradation pathways for crude oil and some of its major
components, besides throwing light upon different bacterial adaptive
responses in such harsh environment. Several genes involved in envi-
ronment stress sensing and response, signal transduction, cell defenses
etc. have also been reported. The N002 genome carries several laterally
acquired regions for survival in hostile environments. Presence of
stress-response genes and transporter equipped genome gives N002
an advantage for survival in crude oil polluted soil. These findings
might have significant potential for bioremediation of crude oil-
polluted environments and restoration of such sites.
4. Materials and methods
4.1. Isolation and characterization
P. aeruginosa N002 was isolated from crude oil contaminated soil of
Geleky, Assam, India, using enrichment culture method in M1 mineral
media (g L−1) (4.0, NaNO3; 3.61 Na2HPO4; 1.75 g, KH2PO4; 0.2,
MgSO4.7H2O; 0.01, FeSO4; 0.05, CaCl2, trace element solution 1 ml/L)
with 2% crude oil as the sole carbon source. Details of isolation, identifi-
cation, degradation ability of crude oil components i.e., aliphatic frac-
tion, aromatic fraction, asphaltene fractions of N002 is described in
our earlier reports [29]. The accession number for 16S rRNA gene PCR
and sequencing is JX035794.1.
4.2. Whole genome sequencing
The whole genome sequencing of P. aeruginosa N002 was obtained
by whole-genome shotgun sequencing using Ion Torrent method [41,
42]. Sequencing was carried out as per the Ion 316 chip sequencing
protocol. The genome sequence was assembled by using GS de novo
Assembler version 2.6. The total number of reads generated using refer-
ence based approach was 1,074,106, with a mean length of 123 bp. The
mapping of Ion Torrent 2.0 high-quality reads on the reference genome
was performed using TMAP v0.0.28, to get consensus sequence.
4.3. Genome analysis
The genome of N002 was submitted to Integrated Microbial
Genomes (IMG) server (http://img.jgi.doe.gov) of Joint Genome
Institute (JGI) for deep analysis and genome comparisons [43]. The
rRNA genes, tRNA genes, total ORFs, GC and circular map of
P. aureginosa N002 was retrieved from IMG server (http://img.jgi.
doe.gov). For phylogenetic relationship the sequence of N002 was
compared with reported genome by NCBI-BLAST tool (http://blast.
ncbi.nlm.nih.gov/blast/treeview.cgi) [44]. The similar sequence was
 D. Das et al. / Genomics 105 (2015) 182–190 189

Fig. 5 (continued).

downlodadd and phylogenetic tree was prepared using Mega6 soft-
ware. Frameshifts identified by the NCBI submission check tool,
were manually checked. Insertion sequence (IS) elements and trans-
posons were identified using the IS finder database (http://www-is.
biotoul.fr/). Transporter and Genomic Islands were analyzed using
SeqWord Sniffer [44,45] and Island Viewer [43,46,47], respectively.
A one-sample t-test was used to evaluate the statistically significant
differences of gene abundance in each COG category between
P. aeruginosa N002 and other referred genomes deposited in the
IMG bacteria genome database. The taxonomic position of the strain
P. aeruginosa N002 was performed one hand by comparing seven al-
ready sequenced genomes of other P. aruginosa strains in IMG data-
base (P. aruginosa NCGM2.S1 [48], M18 [49], DK2 [26], LESB58 [50],
PA7 [51], PAO1 [52], UCBPP-PA14 [53]).
190 D. Das et al. / Genomics 105 (2015) 182–190
4.4. Statistics
All the statistical analysis was performed by using statistical analysis
tools OriginPro8, USA.
4.5. Nucleotide sequence accession number
The whole genome shotgun project has been deposited in DDBJ/
EMBL/GenBank under accession number ALBV00000000.2.
Acknowledgments
Authors are gratefully acknowledged the support received from
Director, CSIR-NEIST, Jorhat. Authors are also duly acknowledge the
financial support received from CSIR New Delhi and to DST for
INSPIRE fellowship. Authors are thankful to three unknown reviewer
for their critical comment in revising the Ms.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ygeno.2014.12.006.
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