International Journal of
Biotechnology and Molecular Biology Research

  • Abbreviation: Int. J. Biotechnol. Mol. Biol. Res.
  • Language: English
  • ISSN: 2141-2154
  • DOI: 10.5897/IJBMBR
  • Start Year: 2010
  • Published Articles: 100

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*
  • Shiju Mathew*
  • Department of Medical Lab Technology, College of Applied Medical Sciences, Jazan University, P.O. Box No. 114, Jazan, Kingdom of Saudi Arabia.
  • Google Scholar
Yahya Hasan Hobani
  • 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.
  • Google Scholar


  •  Received: 14 November 2014
  •  Accepted: 31 December 2014
  •  Published: 31 January 2015

 ABSTRACT

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 contributes highly to environmental pollution. Spills of hydrocarbons occur from several causes, including blowouts, leakage from tanks, and dumping of waste petroleum products. The elevated loading of petroleum hydrocarbons in soil causes a significant decline in soil quality, and these soils have become unusable. Aliphatic alkanes are a group of hydrocarbons that are present in crude and refined oils. They were formed by reduction of organic material during the geochemical formation of oil. However, alkanes are also produced by plants and microorganisms and form part of the biomass (Bird and Lynch, 1974; Taylor and Calvin, 1987; Kunst and Samuels, 2003; Kloos et al., 2006).

Geogenic and biogenic alkane fractions differ in the complexity of the mixture and the mean chain length found (Hellmann, 1991). Introduced in greater amounts into the environment, for example by oil spills, aliphatic alkanes may become environmental pollutants. The bio-availability and toxicity of n-alkanes depends on their chain length (Gill and Ratledge, 1972). Only short chain n-alkanes are directly toxic, acting as solvents for cellular fats and membranes (Sikkema et al., 1995). However, long chain n-alkanes can contribute to the formation of oil films and slicks (Leahy and Colwell, 1990). These are hazardous to the macro- and micro flora of the contaminated site blocking the exchange of water, water soluble nutrients and gases.

A wide range of non-related bacteria and fungi can use n-alkanes as sole carbon and energy source (Rehm and Reiff, 1981; Watkinson and Morgan, 1990; van Beilen et al., 2003). Bacterial degradation of n-alkanes is possible under aerobic and anaerobic conditions (Widdel and Rabus, 2001).

However, activation of the otherwise chemically inert aliphates with molecular oxygen allows much faster degradation rates. Although various mechanisms of aerobic activation have been described (Rehm and Reiff, 1981; Maeng et al., 1997; van Beilen et al., 2003), only the terminal oxidation pathway that involves a key process of degradation has been investigated in greater detail so far.

Hybridization probes derived from various organisms have been used for the detection of microbial communities after isolation of the bacteria (Sotzsky et al., 1994; Whyte et al., 1995; Vomberg and Klinner, 2000). Investigations with PCR-based methods were also mostly performed with cultured isolates (Smits et al., 1999; Vomberg and Klinner, 2000; van Beilen et al., 2002). The high sequence divergence found in Pseudomonas Aeruginosa bacteria from different taxonomic groups (Smits et al., 1999; van Beilen et al., 2003) caused a group-specificity in the detection of alkane degraders with the PCR and hybridization methods described in the literature. The big genetic material of the P. aeruginosa support the idea of using this microbes in biotechnological applications

The diversity of P. aerginosa different strains and that each strain should be study as a single case were highlighted. Therefore, comprehensive analysis of environmental samples required the use of multiple primer and probe sets targeting the respective subgroups (Luz et al., 2004; Heiss-Blanquet et al., 2005; Kloos et al., 2006).

Here, an improved PCR based method is described that in combination with hybridization allowed a specific and sensitive detection of the unknown gene in environmental samples without obvious discrimination of any of the known bacterial groups possessing this gene.


 MATERIALS AND METHODS

Reference strains and media

Strains  used  as  references  were  Bacillus subtilis,  Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus and Escherichia coli. They were grown in NB medium (8 g l1 nutrient broth No. 4, Fluka, Buchs, Switzerland).

HC medium contained per l : 2g NH4NO3, 4g KH2PO4, 6 g Na2HPO4·2H2O, 200 mg MgSO4, 50mg CaCl2·2H2O, 136.3 μg ZnCl2, 3.2 mg FeCl3, 2.0 mg MnCl2·4H2O, 170.5 μg CuCl2·2H2O, 475.9 μg CoCl2·6H2O, 61.8μg HBO3, 3.9 μg NaMoO4·2H2O. The Ph of the medium was adjusted to 7.0 with KOH before autoclaving. The appropriate C-source was autoclaved or filter sterilized separately before addition to the medium.

 

Molecular biological methods

Preparation of genomic DNA

For preparation of genomic DNA from pure cultures, cells were grown over night and harvested by centrifugation. Cells were resuspended in 320 μl 1×TE (10 mM Tris pH 7.5, 1 mM EDTA) and incubated for 1 h. After addition of 80 μl 10% SDS, samples were incubated at 65°C for 2 h. The final DNA extraction was done with the Mini Kit (Qiagen, Germany) starting with the addition of buffer AP2 according to the manufacturer's protocol. The collection site for soil sample was randomly selected followed by the procedure of DNA isolation using Genei Kit, Bangalore

 

Estimation of genomic DNA

DNA concentration and purity were determined using ULTROSPEC 2000 spectrophotometer at an absorbance of 260 and 280 nm. Quality of DNA can be calculated using the formula; Quality of DNA = A260nm / A280nm. If the value ranges from: 1.75 - 1.9 Good quality DNA; ≤ 1.75 Protein contamination; ≥ 1.9 RNA contamination. Quantity of DNA can be calculated using the formula; Quantity of DNA = A260nm x 50 x dilution factor / 1000.

 

Detection of oil degrading gene in the genomic DNA

PCR primers are responsible for identifying the polymorphism or specific DNA sequence presence for oil degradation. So, along with the sample, control strains are also used and analyzed the DNA sample. The primer has been specially designed using the PubMed software of NCBI. nahC F -5’-AAG GCG CAG GCT TGC AAA TG-3’; nahC R -5’-TCG TCG CTT TCC CAG AAG CC-3’.  The PCR mix contained in a final volume of 50 μl: 5 μl buffer (provided with Taq polymerase), 1.5 μl MgCl2 (50 mM), 5 μl dNTP-mix (2.5 mM each nucleotide), 5 μl each primer (10 μM), 10 ng purified DNA of cultured strains or 5 ng soil DNA and 2.5 U Taq-DNA polymerase (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, and final elongation for 5min at 72°C.

PCR products were separated in 1.5% agarose gels. Gels were blotted on positively charged nylon membrane by capillary transfer with 0.4 M NaOH for hybridization. Hybridization was performed with the oligonucleotide. The oligonucleotide was labeled with DigddUTP using the 3′-end labeling kit (Genei, Bangalore, India) according to the manufacturer's protocol. Five hundred picomoles 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 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 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 instructions to detect hybrids (Kloos et al., 2006).

 

Transformation and cloning

The amplified specific DNA fragments were cloned to competent E. coli using the Novagen Kit, Germany. Plasmids from transformants were prepared using the method of Birnboim and Doly (1979) and checked for the presence of an insert of the expected size. A fast initial screening of the transformants was performed by colony hybridization according to the method of Grunstein and Hogness (1975). Plasmids used for sequencing were further purified with the Plasmid Mini Kit (Qiagen, Germany). Both strands were read from double stranded plasmid with the opposing vector primers. 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).


 RESULTS AND DISCUSSION

Detection of oil degrading gene by PCR and hybridization

Specific detection of gene was achieved combining PCR amplification with oligonucleotide hybridization. To confirm the presence of DNA and to separate it from protein and RNA, agarose gel electrophoresis (AGE) was done (Figure 1). The PCR primers target essentially the same regions as the primers described by Smits et al. (1999). However, the reverse primer was shifted 6 bases, and both primers used here were more degenerate. The expected band of 500bp was amplified from DNA of the reference strains P. aeruginosa. Sequences of these PCR products differed only in the primer sequences from those published. Unspecific PCR products of various sizes were obtained with the nondegrading strains Bacillus subtilis and Escherichia coli strain. Hybridization with an oligonucleotide probe binding within the amplicon allowed to distinguish specific and non-specific products (Figure 2). Hybridization signals were obtained only for the expected products of the degraders. The detection limit of the combined PCR/hybridization assay was estimated with chromosomal DNA of the reference strains P. aeruginosa to be 0.1ng DNA. Assuming a mean bacterial genome size of 3.6 Mb (Fogel et al., 1999; Kloos et al., 2006) this detection limit corresponded to 2.6 X 104 gene copies.

 

 

 

Evaluation of the detection method with the reference isolates

The PCR/hybridization method was used to screen soil isolates for the occurrence of alkB by colony-PCR. Isolates were obtained under non-selective conditions from a reference strains. Fifty randomly picked isolates were screened for each of the two independent samples. A single PCR product of the expected size was obtained for 8 and 10% of the isolates. None or unspecific products were observed for all other isolates. Again, only PCR products of the expected size hybridized with the internal probe (Figures 3 and 4). Sequencing of these PCR products after cloning confirmed their homology to alkB. Derived protein sequences showed highest homologies to AlkB from other bacterial gene (Smits et al., 1999; Khalameyzer et al., 1999; Andreoni et al., 2000; Kloos et al., 2006).

 

 

 

Transformationtion of oil degrading gene by PCR and hybridization

The results have confirmed that an oil degrading gene is 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).

 

 


 CONCLUSION

Biodegradation is the chemical breakdown of materials by a physiological environment. Oily wastewater, especially from oil field, has posed a great hazard for terrestrial and marine ecosystems. During biodegra-dation, crude oil is used as an organic carbon source by a microbial process, resulting in the breakdown of crude oil components to low molecular weight compounds. The focus of the research is identifying novel oil degrading gene in the cosmopolitan available Pseudomonas and also to evaluate the highly sophisticated PCR technique to detect the oil degrading gene in Pseudomonas species. Though the target of the work is to concentrate on Pseudomonas the other two strains are used as controls. It is observed after PCR amplification (Agarose gel) that Pseudomonas DNA contain oil degrading gene. Resulting into the plasmid DNA of Pseudomonas was transformed into competent E. 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. Molecular data were in accordance with physiological experiments for detection and enumeration of alkane degrading bacteria. Results show that the capability for alkane degradation is a common trait in soil microbial communities. Thus above results can be concluded that the PCR mediated detection and identification of oil degrading gene in Pseudomonas aeruginosa.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

I thank our labouratory colleagues for their assistance with this project and valuable comments on this manuscript. I also thank Dr. Gowher Nabi for their continuous support in research. The work was supported by the university research funding.



 REFERENCES

 

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
Crossref
 
Andreoni V, Bernasconi S, Colombo M, Van Beilen JB, Cavalca L (2000). Detection of genes for alkane and naphthalene catabolism in Rhodococcus sp. strain 1BN. Environ. Microbiol. 2:572-577.
Crossref
 
Bird CW, Lynch JM (1974). Formation of hydrocarbons by microorganisms. Chem. Soc. Rev. 3:309-328.
Crossref
 
Birnboim HC, Doly JA (1979). Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.
Crossref
 
Boyer R (2006). Biochemistry Laboratory: Modern Theory and Techniques. Mod. Exp. Biochem. 2:140-144.
 
Dubey RC, Gupta CP, Maheshwari DK (2002). Plant growth enhancement and suppression of Macrophomina phaseolina causing charcoal rot of peanut by fluorescent Pseudomonas. Biol. Fertil. Soils. 35:399-405.
Crossref
 
Fogel GB, Collins CR, Li J, Brunk CF (1999). Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb. Ecol. 38:93-113.
Crossref
 
Gill CO, Ratledge C (1972). Toxicity of n-alkanes, n-alk-1-enes, nalkan- 1-ols and n-alkyl-1-bromides towards yeasts. J. Gen. Microbiol. 72:165-172.
Crossref
 
Grunstein M, Hogness DS (1975). Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. 72:3961-3965.
Crossref
 
Heiss-Blanquet S, Benoit Y, Maréchaux C, Monot F (2005). Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR. J. Appl. Microbiol. 99:1392-1403.
Crossref
 
Hellmann H (1991). IR-spectroscopic analysis of alkanes in soil, water and sediment—differenciation of mineral oil and biogene fractions (German). Z. Wasser- und Abwasser-Forsch. Appl. Environ. Microbiol. 24:226-232.
 
Khalameyzer V, Fischer I, Bornscheuer UT, Altenbuchner J (1999). Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones. Appl. Environ. Microbiol. 65:477-482.
Pubmed
 
Kloos K, Munch JC, Schloter M (2006). new method for the detection of alkane- onooxygenase homologous genes (alkB) in soils based on PCR-hybridization. J. Microbiol. Meth. 66:486-496.
Crossref
 
Kunst L, Samuels AL (2003). Biosynthesis and secretion of plant cuticular wax. Prog. Lipid Res. 42:51-80.
Crossref
 
Leahy JG, Colwell RR (1990). Microbial degradation of hydrocarbons in the environment. Microbiol. Rev. 54:305-315.
Pubmed
 
Luz AP, Pellizari VH, Whyte LG, Greer CW (2004). A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil. Can. J. Microbiol. 50:323-333.
Crossref
 
Maeng JH, Sakai Y, Tani Y, Kato N (1997). Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1. J. Bacteriol. 178:3695-3700.
 
Rehm HJ, Reiff I (1981). Mechanisms and occurrence of microbial oxidation of long-chain alkanes. Adv. Biochem. Eng. 19:175-215.
 
Sikkema J, De Bont JA, Poolman B (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev. 59:201-222.
Pubmed
 
Smits TH, Röthlisberger M, Witholt B, van Beilen JB (1999). Molecular screening for alkane hydroxylase genes in Gramnegative and Gram-positive strains. Environ. Microbiol. 1:307-317.
Crossref
 
Sotzsky JB, Greer CW, Atlas RM (1994). Frequency of genes in aromatic and aliphatic hydrocarbon biodegradation pathways within bacterial populations from Alaskan sediments. Can. J. Microbiol. 40:981-985.
Crossref
 
Taylor SE, Calvin M (1987). Hydrocarbons from plants: biosynthesis and utilization. Commun. Agric. Food Chem. 1:1-26.
Crossref
 
Van Beilen JB, Li Z, DuetzWA, Smits THM, Witholt B (2003). Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci. Technol. 4:427-440.
Crossref
 
Van Beilen JB, Smits THM, Whyte LG, Schorcht S, Röthlisberger M, Plaggenmeier T, Engesser KH, Witholt B (2002). Alkane hydroxylase homologues in Gram-positive strains. Environ. Microbiol. 4:676-682.
Crossref
 
Vomberg A, Klinner U (2000). Distribution of alkB genes within nalkane- degrading bacteria. J. Appl. Microbiol. 89:339-348.
Crossref
 
Walker J, Wilson K (2007). The Molecular Evolution of species in the Presence of Slightly Deleterious Mutations and Population Size Change. Princ. Tech. Biochem. Mol. Biol. 5:454-455.
 
Watkinson RJ, Morgan P (1990). Physiology of aliphatic hydrocarbon- degrading microorganisms. Biodegrad. 1:79-92.
Crossref
 
Whyte LG, Greer CW, Inniss WE (1995). Assessment of the biodegradation potential of psychrotrophic microorganisms. Can. J. Microbiol. 42:99-106.
Crossref
 
Widdel F, Rabus R (2001). Anaerobic biodegradation of saturated and aromatic hydrocarbons. Curr. Opin. Biotechnol. 12:259-276.
Crossref

 

 




          */?>