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International Journal for

Biotechnology and Molecular


Biology Research
Volume 6 Number 1, January 2015
ISSN 2141-2154
ABOUT IJBMBR
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Prof Atagana, Harrison Dr. Amro Hanora
Institute for Science and Technology Education Suez Canal University, Department of Microbiology
University of South Africa and Immunology,
Faculty of Pharmacy, Suez Canal University,
Prof. UC Banerjee Box 41522 Ismailia, Egypt
Department of Pharmaceutical Technology
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National Institute of Pharmaceutical Education and VIT University
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Punjab, INDIA
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Dr. Y. Omidi Gaziosmanpasa Univerisity
Faculty of Pharmacy, Faculty of Agriculture,
Research Center for Pharmaceutical Nanotechnology, Departmen of Field Crops, Tokat-Turkey
School of Advanced Biomedical Sciences,
Tabriz University of Medical Sciences, Dr. Ahmet Tutus
Tabriz, Iran. KSU (Kahramanmaras Sutcu Imam Universirty)
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University of New Brunswick (UNB-SJ), Kahramanmaras 46100 Turkey
Saint John College, NB,
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Desert Medicine Research Centre,
Dr. Sripada M. Udupa Indian Council of Medical Research
ICARDA-INRA Cooperative Research Project, New Pali Road, Jodhpur, India
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Rabat Instituts, Rabat, Morocco K.M.18 Khazarabad road,
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Sher-e-Kashmir University of Agricultural Sciences & Dr. Shashideep Singhal
Technology 121 Dekalb Ave, Brooklyn,
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grasain Marg, Karnal-132001, Dr. Okonko Iheanyi Omezuruike
Haryana, India Department of Virology,
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University of Karachi,
Karachi-75270, Pakistan
Prof. Reda Helmy Sammour Dr. Chun Chen
Botany Department, College of Life Sciences,
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Tanta University, Xueyuan Street, Xiasha, Hangzhou,
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International Journal for Biotechnology and Molecular Biology Research
International Journal of Medicine and Medical Sciences

Table of Contents: Volume 6 Number 1 January, 2015

ARTICLES

A new method for the detection of oil degrading genes in Pseudomonas


aeruginosa based on transformation and PCR hybridization
Shiju Mathew and Yahya Hasan Hobani
Vol. 6(1) pp. 1-6, January 2015
DOI: 10.5897/IJBMBR2014.0218
Article Number: 927CE3249952
ISSN 2141-2154
International Journal for Biotechnology and
Copyright © 2015 Molecular Biology Research
Author(s) retain the copyright of this article
http:// www.academicjournals.org/IJBMBR

Full Length Research Paper

A new method for the detection of oil degrading genes


in Pseudomonas aeruginosa based on transformation
and PCR hybridization
Shiju Mathew* and Yahya Hasan Hobani
Department of Medical Lab Technology, College of Applied Medical Sciences, Jazan University, P.O. Box No. 114,
Jazan, Kingdom of Saudi Arabia.
Received 14 November, 2014; Accepted 31 December, 2014

Biodegradation is the chemical breakdown of materials by a physiological environment. The term is


often used in relation to ecology, waste management and environmental remediation. A specific gene
from bacteria is identified and sequenced which has the capacity of oil degradation without any obvious
strain specific discrimination using a combination of PCR and hybridization. The parameters of
biodegradation that is culturing and PCR technique provide useful information for an assessment of the
intrinsic biodegradation potential that is present at a site. PCR amplification products in the plasmid
DNA of Pseudomonas aeruginosa was transformed into competent Escherichia coli cells. Thus the E.
coli cells were conferred with oil degrading property and this was confirmed by growing them in
Bushnell Hass medium along with petroleum oil. The E. coli cells were found to be catabolizing the oil.
Results show that the capability for alkane degradation is a common trait in microbial communities. The
method can be a very useful tool for the fast estimation of the biodegradation potential at polluted sites.

Key words: Detection, hydrocarbon biodegradation, PCR-hybridization, transformation.

INTRODUCTION

The use of enormous amounts of petroleum products organic material during the geochemical formation of oil.
contributes highly to environmental pollution. Spills of However, alkanes are also produced by plants and
hydrocarbons occur from several causes, including microorganisms and form part of the biomass (Bird and
blowouts, leakage from tanks, and dumping of waste Lynch, 1974; Taylor and Calvin, 1987; Kunst and
petroleum products. The elevated loading of petroleum Samuels, 2003; Kloos et al., 2006).
hydrocarbons in soil causes a significant decline in soil Geogenic and biogenic alkane fractions differ in the
quality, and these soils have become unusable. Aliphatic complexity of the mixture and the mean chain length
alkanes are a group of hydrocarbons that are present in found (Hellmann, 1991). Introduced in greater amounts
crude and refined oils. They were formed by reduction of into the environment, for example by oil spills, aliphatic

*Corresponding author. E-mail: sjohn@jazanu.edu.sa. Tel: +966-540081303. Fax: +966- 73230028.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License
4.0 International License
2 Int. J. Biotechnol. Mol. Biol. Res.

alkanes may become environmental pollutants. The bio- aeruginosa (ATCC 27853), Staphylococcus aureus and Escherichia
availability and toxicity of n-alkanes depends on their coli. They were grown in NB medium (8 g l−1 nutrient broth No. 4,
Fluka, Buchs, Switzerland).
chain length (Gill and Ratledge, 1972). Only short chain HC medium contained per l : 2g NH4NO3, 4g KH2PO4, 6 g
n-alkanes are directly toxic, acting as solvents for cellular Na2HPO4·2H2O, 200 mg MgSO4, 50mg CaCl2·2H2O, 136.3 μg
fats and membranes (Sikkema et al., 1995). However, ZnCl2, 3.2 mg FeCl3, 2.0 mg MnCl2·4H2O, 170.5 μg CuCl2·2H2O,
long chain n-alkanes can contribute to the formation of oil 475.9 μg CoCl2·6H2O, 61.8μg HBO3, 3.9 μg NaMoO4·2H2O. The Ph
films and slicks (Leahy and Colwell, 1990). These are of the medium was adjusted to 7.0 with KOH before autoclaving.
hazardous to the macro- and microflora of the The appropriate C-source was autoclaved or filter sterilized
separately before addition to the medium
contaminated site blocking the exchange of water, water
soluble nutrients and gases.
A wide range of non-related bacteria and fungi can use Molecular biological methods
n-alkanes as sole carbon and energy source (Rehm and
Reiff, 1981; Watkinson and Morgan, 1990; van Beilen et Preparation of genomic DNA
al., 2003). Bacterial degradation of n-alkanes is possible
For preparation of genomic DNA from pure cultures, cells were
under aerobic and anaerobic conditions (Widdel and
grown over night and harvested by centrifugation. Cells were
Rabus, 2001). resuspended in 320 μl 1×TE (10 mM Tris pH 7.5, 1 mM EDTA) and
However, activation of the otherwise chemically inert incubated for 1 h. After addition of 80 μl 10% SDS, samples were
aliphates with molecular oxygen allows much faster incubated at 65°C for 2 h. The final DNA extraction was done with
degradation rates. Although various mechanisms of the Mini Kit (Qiagen, Germany) starting with the addition of buffer
aerobic activation have been described (Rehm and Reiff, AP2 according to the manufacturer's protocol. The collection site for
soil sample was randomly selected followed by the procedure of
1981; Maeng et al., 1997; van Beilen et al., 2003), only DNA isolation using Genei Kit, Bangalore
the terminal oxidation pathway that involves a key
process of degradation has been investigated in greater
detail so far. Estimation of genomic DNA
Hybridization probes derived from various organisms
have been used for the detection of microbial DNA concentration and purity were determined using ULTROSPEC
2000 spectrophotometer at an absorbance of 260 and 280 nm.
communities after isolation of the bacteria (Sotzsky et al.,
Quality of DNA can be calculated using the formula; Quality of DNA
1994; Whyte et al., 1995; Vomberg and Klinner, 2000). = A260nm / A280nm. If the value ranges from: 1.75 - 1.9 Good quality
Investigations with PCR-based methods were also mostly DNA; ≤ 1.75 Protein contamination; ≥ 1.9 RNA contamination.
performed with cultured isolates (Smits et al., 1999; Quantity of DNA can be calculated using the formula; Quantity of
Vomberg and Klinner, 2000; van Beilen et al., 2002). The DNA = A260nm x 50 x dilution factor / 1000.
high sequence divergence found in Pseudomonas
Aeruginosa bacteria from different taxonomic groups
Detection of oil degrading gene in the genomic DNA
(Smits et al., 1999; van Beilen et al., 2003) caused a
group-specificity in the detection of alkane degraders with PCR primers are responsible for identifying the polymorphism or
the PCR and hybridization methods described in the specific DNA sequence presence for oil degradation. So, along with
literature. The big genetic material of the P. aeruginosa the sample, control strains are also used and analyzed the DNA
support the idea of using this microbes in sample. The primer has been specially designed using the PubMed
software of NCBI. nahC F -5’-AAG GCG CAG GCT TGC AAA TG-
biotechnological applications
3’; nahC R -5’-TCG TCG CTT TCC CAG AAG CC-3’. The PCR mix
The diversity of P. aerginosa different strains and that contained in a final volume of 50 μl: 5 μl buffer (provided with Taq
each strain should be study as a single case were polymerase), 1.5 μl MgCl2 (50 mM), 5 μl dNTP-mix (2.5 mM each
highlighted. Therefore, comprehensive analysis of nucleotide), 5 μl each primer (10 μM), 10 ng purified DNA of
environmental samples required the use of multiple cultured strains or 5 ng soil DNA and 2.5 U Taq-DNA polymerase
primer and probe sets targeting the respective subgroups (Biorad, USA). Cycling was performed with initial denaturation for 3
min at 40°C, 34 cycles with 30 s at 94°C, 45 s at 53°C, 60s 72°C,
(Luz et al., 2004; Heiss-Blanquet et al., 2005; Kloos et al., and final elongation for 5min at 72°C.
2006). PCR products were separated in 1.5% agarose gels. Gels were
Here, an improved PCR based method is described blotted on positively charged nylon membrane by capillary transfer
that in combination with hybridization allowed a specific with 0.4 M NaOH for hybridization. Hybridization was performed
and sensitive detection of the unknown gene in with the oligonucleotide. The oligonucleotide was labeled with
environmental samples without obvious discrimination of DigddUTP using the 3′-end labeling kit (Genei, Bangalore, India)
according to the manufacturer's protocol. Five hundred picomoles
any of the known bacterial groups possessing this gene. of oligonucleotide were used per 50 ml hybridization buffer
containing 5×SSC (20×SSC: 3 M NaCl, 0.3 M Na3-citrate), 0.1%
Na-lauroylsulfate, 0.02% Na-N-lauroylsarcosine, 0.5% blocking
MATERIALS AND METHODS reagent. Hybridization was performed at 42°C over night and was
followed by two washes with 2×SSC/0.1% SDS for 5min at room
Reference strains and media temperature, and two washes with 0.5×SSC/0.1% SDS at 42°C.
The colour detection kit was used according to the manufacturer's
Strains used as references were Bacillus subtilis, Pseudomonas instructions to detect hybrids (Kloos et al., 2006).
Shiju and Yahya 3

Figure 1. Clear genomic DNA bands obtained for each of the sample in the agarose gel
when viewed under UV transilluminator. Lane 1 controlled Genomic DNA and Lane 2
collected Genomic DNA samples of each microorganism DNA that is Bacillus, Stapyllococcus
and Pseudomonas.

Transformation and cloning allowed to distinguish specific and non-specific products


(Figure 2). Hybridization signals were obtained only for
The amplified specific DNA fragments were cloned to competent E.
coli using the Novagen Kit, Germany. Plasmids from transformants
the expected products of the degraders. The detection
were prepared using the method of Birnboim and Doly (1979) and limit of the combined PCR/hybridization assay was
checked for the presence of an insert of the expected size. A fast estimated with chromosomal DNA of the reference
initial screening of the transformants was performed by colony strains P. aeruginosa to be 0.1ng DNA. Assuming a
hybridization according to the method of Grunstein and Hogness mean bacterial genome size of 3.6 Mb (Fogel et al.,
(1975). Plasmids used for sequencing were further purified with the 1999; Kloos et al., 2006) this detection limit corresponded
Plasmid Mini Kit (Qiagen, Germany). Both strands were read from 4
double stranded plasmid with the opposing vector primers.
to 2.6 X 10 gene copies.
Sequences were compared to Genbank with BLASTN (Altschul et
al., 1997). Trees were calculated with NTSYS software (Dubey et
al., 2002; Boyer, 2006; Walker, 2007). Evaluation of the detection method with the reference
isolates

RESULTS AND DISCUSSION The PCR/hybridization method was used to screen soil
isolates for the occurrence of alkB by colony-PCR.
Detection of oil degrading gene by PCR and Isolates were obtained under non-selective conditions
hybridization from a reference strains. Fifty randomly picked isolates
were screened for each of the two independent samples.
Specific detection of gene was achieved combining PCR A single PCR product of the expected size was obtained
amplification with oligonucleotide hybridization. To for 8 and 10% of the isolates. None or unspecific
confirm the presence of DNA and to separate it from products were observed for all other isolates. Again, only
protein and RNA, agarose gel electrophoresis (AGE) was PCR products of the expected size hybridized with the
done (Figure 1). The PCR primers target essentially the internal probe (Figures 3 and 4). Sequencing of these
same regions as the primers described by Smits et al. PCR products after cloning confirmed their homology to
(1999). However, the reverse primer was shifted 6 bases, alkB. Derived protein sequences showed highest
and both primers used here were more degenerate. The homologies to AlkB from other bacterial gene (Smits et
expected band of 500bp was amplified from DNA of the al., 1999; Khalameyzer et al., 1999; Andreoni et al., 2000;
reference strains P. aeruginosa. Sequences of these Kloos et al., 2006)
PCR products differed only in the primer sequences from
those published. Unspecific PCR products of various Transformationtion of oil degrading gene by PCR and
sizes were obtained with the nondegrading strains hybridization
Bacillus subtilis and Escherichia coli strain. Hybridization
with an oligonucleotide probe binding within the amplicon The results have confirmed that an oil degrading gene is
4 Int. J. Biotechnol. Mol. Biol. Res.

Figure 2. The confirmation of amplified DNA


product is only present in Pseudomonas
whereas in Bacillus and Stapyllococcus it is
absent as shown in agarose gel.

Figure 4. Figure showing the confirmation


of plasmid DNA of Pseudomonas
aeruginosa has oil degrading genes. The
band appeared has a size of about 200bp.
All other strains show a negative result and
had no amplification in response to the oil
degrading primer.

present in the P. aeruginosa. An annex to this work was


concentrated on the transfer this particular oil degrading
gene into a non oil degrading bacteria (Figure 5). The
non-oil degrading bacteria were selected. E. coli cells
were selected which were made competent using calcium
chloride and were transformed using Bushnell Hass (BH)
medium along with petroleum oil layered on top of the
medium and further showed that a normally non-oil
degrading bacteria has been successfully transformed to
oil degrading E. coli (Figure 6).

Figure 3. Figure shows that there was no Conclusion


amplification of DNA in any of the
bacterial samples. This infers that the
genomic DNA of these bacteria lacks any Biodegradation is the chemical breakdown of materials
oil degrading genes used along with a by a physiological environment. Oily wastewater,
100 bp marker. especially from oil field, has posed a great hazard for
Shiju and Yahya 5

Figure 6. The transformed E. coli cells have degraded the


petroleum oil present in the medium which could be seen as small
bubbles on the surface of the medium.

Conflict of Interests

The author(s) have not declared any conflict of interests.


Figure 5. The transformed E. coli cell which can degrade
the oil that is E. coli cells were selected which were made
competent using calcium chloride and were transformed
using Bushnell Hass (BH) medium. ACKNOWLEDGEMENT

I thank our lab colleagues for their assistance with this


project and valuable comments on this manuscript. I also
thank Dr. Gowher Nabi for their continuous support in
terrestrial and marine ecosystems. During biodegra- research. The work was supported by the university
dation, crude oil is used as an organic carbon source by research funding.
a microbial process, resulting in the breakdown of crude
oil components to low molecular weight compounds. The
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