Introduction

Antarctica is one of the last pristine environments on Earth today (Cowan and Tow 2004; Ebinghaus 2008). Human activity over the years has led to hydrocarbon contamination of the soils near active and abandoned research stations (Aislabie et al. 1999; Martin-Laurent et al. 2003; Saul et al. 2005). Fuel storage and refueling of aircrafts and other vehicles has led to localized fuel spills on land. Jet fuels, commonly used in the Antarctic, are complex mixtures of hydrocarbon that partition in the environment according to their physical properties. They may evaporate, be absorbed into the soil, or dissolve in water thereby becoming harmful to the marine ecosystem or be degraded by chemical or biological processes (Chandler and Brockman 1996). Bioremediation of the Antarctic soils has been proposed to clean up the spills (Aislabie et al. 1998, 2004, 2006; Kerry 1993; Mesarch et al. 2000). However, the Antarctic Treaty does not support the introduction of foreign organisms (Eltis and Bolin 1996) on this continent, and the physical removal of contaminated soil is not monetarily feasible. Therefore, use of indigenous biodegradative bacteria seems be the preferred means to best achieve this objective. These bacteria would already be well adapted to the cold, dry, alkaline, low nutrient soil conditions (Campbell et al. 1998).

Jet fuel is mostly composed of alkanes of the which the ~81% is in C8–C17 range, and the remainder of the fuel is aromatics like substituted benzenes and naphthalenes along with low levels of highly volatile chemicals like benzene, toluene and xylene (Zeiger and Smith 1998). Based on the removal of JP4 and JP8 Jet fuel from soil, it was shown that higher molecular weight hydrocarbons were removed significantly faster by the activity of the biodegradative microbial population than in soil treated with chemicals (2% HgCl2) to kill microorganisms (Dean-Ross et al. 1992; Dean-Ross 1993). Historically, research has focused on the catabolic pathways and genetics of hydrocarbon-degrading mesophilic bacteria. But with increasing numbers of investigations of hydrocarbon-contaminated polar soils, degradation of hydrocarbons by psychrotolerant bacteria has been documented and certain biodegradative genes characterized (Baraniecki et al. 2002; Ensminger et al. 1999; Laramee et al. 2000; Panicker et al. 2001). There are more than 1,301 known microbial biocatalytic reactions and 190 biodegradation pathways listed in The University of Minnesota Biocatalysis/Biodegradation Database (http://umbbd.msi.umn.edu/) (Ellis et al. 2006).

The alkane degradation pathway located on the OCT-plasmid, was first genetically characterized in Pseudomonas oleovorans (Kok et al. 1989; van Beilen et al. 1994). Since then the alkane hydroxylase gene (alkB) has been cloned and sequenced in several other bacteria such as Acinetobacter sp., Rhodococcus sp., Mycobacterium sp., Pseudomonas putida P1, P. aureofaciens and P. fluorescens. alkB gene and encoded protein AlkB were found to be diverse in nucleotide as well as amino acid sequence (Ratajczak et al. 1998; Sotsky et al. 1994; van Beilen et al. 2002; Vomberg and Klinner 2000; Wasmund et al. 2009; Whyte et al. 2002a, b, 1998). Aromatic hydrocarbons such as benzene, toluene, xylene and naphthalene that constitute Jet fuel are degraded by microorganisms via catechol-like intermediates to less toxic compounds (Cerniglia 1984; Head et al. 2006). These biodegradative pathways make use of either ortho- or meta-cleavage catechol dioxygenases that have been characterized in several bacteria (Eltis and Bolin 1996; Head et al. 2006), which may possibly be present in many of the Antarctic psychrotolerant bacteria.

Naphthalene degradation has also been well studied in many biodegradative bacteria, in which the first step in the degradation pathway is carried out by naphthalene dioxygenase, which is encoded by the ndoB gene (Kurkela et al. 1988; Ferrero et al. 2002). This gene has been identified in mesophilic as well as in psychrotolerant bacteria from hydrocarbon contaminated soils using polymerase chain reaction (PCR) or Southern blot DNA-DNA hybridization methods (Herrick et al. 1997; Stapleton and Sayler 1998; Whyte et al. 1996; Wilson et al. 2003).

Our aim was to identify biodegradative genes encoding alkane hydroxylase (alks), naphthalene dioxygenase (ndoB), catechol 2,3-dioxygenase (C23DO) and toluene/biphenyl dioxygenase (todC1/bphA1) using PCR in Gram-positive and Gram-negative bacteria isolated from oil-contaminated Antarctic soils and also to assess active transcription of these genes. This would provide a better understanding of the nature and distribution of biodegradative genes in Antarctic soils that are contaminated with Jet fuel, as well as a comparison of gene sequences among mesophiles and psychrotolerant bacteria. We also developed a competitive PCR (cPCR) assay, which utilizes an internal control to quantify the ndoB of a naphthalene-degrading bacterium isolated from this environment.

Materials and methods

Bacterial strains

The hydrocarbon-degrading bacterial strains used in this study were isolated from hydrocarbon-contaminated soils near Scott Base, the former Vanda Station and Marble Point, Antarctica (Table 1) and characterized as described previously (Bej et al. 2000; Panicker et al. 2001). P. putida ATCC 17484 and P. putida mt2 were maintained on Nutrient Agar (Difco, Detroit, MI) at 26°C.

Table 1 Bacterial strains isolated from oil-contaminated Antarctic soils used in this study
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PCR amplification

PCR was used to detect the presence of genes involved in alkane, naphthalene and catechol degradation in the various isolates. Genomic DNA was purified from by the method described by Ausubel et al. (1987). The oligonucleotide primer sets designed from the gene sequence coding for different regions of alkane hydroxylase (alk), an ISPα subunit of naphthalene dioxygenase (ndoB), competitor ndoB (cndoB), catechol 2,3-dioxygenase (C23DO) and toluene/biphenyl dioxygenase (todC1/bphA1) are listed in Table 2. All oligonucleotide primers were custom synthesized based on sequences of respective biodegradative genes from mesophilic bacteria (Table 2). P. putida ATCC 17484 and P. putida mt2 were used as positive controls for ndoB and C23DO amplification, respectively. Each PCR amplification was performed in a 25 μl reaction volume consisting of 1 μg of purified genomic DNA; 200 μM of each of the dNTPs; 1 μM of each of the oligonucleotide primer and 2.0 U AmpliTaq (Perkin Elmer, Norwalk, CT) DNA polymerase; and 1× PCR reaction buffer [10× buffer consisted of 300 mM Tris–Cl (pH 9.0), 75 mM (NH4)2SO4 and 2.0 mM MgCl2]. All PCR amplifications were performed in a GeneAmp PCR system 2400 (Perkin Elmer, Norwalk, CT) thermocycler using the following temperature cycling parameters: initial denaturation at 94°C for 2 min followed by a total of 30 cycles of amplification in which each cycle consisted of denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min and primer extension at 72°C for 2 min. After amplification, final extension of the incompletely synthesized DNA was carried out at 72°C for 7 min. For those isolates that showed a negative result, annealing temperatures of 45, 50 and 55°C were also attempted. For amplification of the C23DO gene an annealing temperature of 55°C was used. The PCR fragments were analyzed by agarose gel electrophoresis (1% wt/vol). The gel was stained with ethidium bromide and visualized under a Photoprep I UV transilluminator (Fotodyne, Inc., Hartland, WI).

Table 2 Oligonucleotide gene-specific primers used for PCR amplification of biodegradative genes
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Cloning and nucleotide sequence analysis

The PCR amplified DNA fragments from the representative groups of different primer sets were cloned in a pCR4-Topo™ plasmid vector using the Topo TA™ cloning kit (Invitrogen, Inc., Carlsbad, CA). After transformation, randomly selected white colonies were grown in (Ausubel et al. 1987) and plasmid DNA extracted using the Qiagen™ mini-prep columns (Qiagen, Valencia, CA). The purified DNA from each putative clone was then treated with EcoRI restriction endonuclease (New England Biolab, Beverly, MA) and the correct molecular weight cloned DNA fragment was determined by agarose gel electrophoresis. The nucleotide sequence of the cloned gene fragments were analyzed by using M13 forward or reverse primers and an ABI Prism automated DNA sequencer (Perkin Elmer, Norwalk, CT). The nucleotide and deduced amino acid sequences were then compared with the respective GenBank (National Institute for Health, Bethesda, MD) sequence database using the BLAST program.

Reverse transcriptase PCR amplification

Total RNA was isolated from 1.5 ml of the saturated cultures of Pseudomonas sp. 4/101, 7/156, Ant6 and Rhodococcus sp. 5/1, respectively, using NucleoSpin RNA II kit (Clontech, Mountain View, CA) following manufacturer’s protocol. The purified total RNA was used for One-step RT-PCR using TITANIUM™ One-Step RTPCR kit (Clontech, Mountain View, CA) following manufacturer’s protocol. The amplified product was observed on 1% (w/v) agarose gel.

Competitive PCR

In competitive PCR, the competitor DNA strand (cndoB) was constructed by the amplification of a 501 bp segment of the genomic DNA of Pseudomonas sp. 30/2 (Celi et al. 1993) using L-ndoB and R-cndoB primers (Table 2). The amplified DNA fragment was cloned on pCR4™ plasmid using the Topo TA™ cloning kit (Invitrogen, Carlsbad, CA). The correct DNA insert was confirmed by restriction endonuclease treatment and DNA sequence analyses (Ausubel et al. 1987). The inserted cndoB DNA segment on the pCR4™ plasmid was then used as the template for cPCR amplification. DNA concentration of the competitor DNA was determined by using a spectrophotometer (Lambda 2, Perkin Elmer, Norwalk, CT) at 260 nm wavelength. The PCR conditions and concentration of cndoB, which were necessary to perform competitive PCR were optimized using twofold dilutions of the plasmid ranging from 452 pg to 4.52 fg and a constant amount of target DNA. The ndoB gene copy number was determined using the size and the concentration of the template DNA with the appropriate unit conversion (Mesarch et al. 2000). The annealing temperature was set for 60°C with the PCR conditions as stated above. The PCR products were separated on 1.2% (w/v) agarose gels. The mean intensities of the target and competitor band were visualized and compared by the Kodak 1D software (Scientific Imaging Systems, New Haven, CT). The concentration of the target gene was determined by plotting a standard curve using Microsoft Excel™ software. The experiment was repeated three times.

Results

PCR amplification of alk genes

PCR amplification of Pseudomonas sp. 7/156 and 4/101 exhibited an 870 bp PCR amplified DNA fragment at 55°C annealing temperature with the alkB870G oligonucleotide primers (Table 3). The nucleotide sequence analysis of the amplified DNA confirmed the presence of alkB (Accession number AY034587). The deduced amino acid sequence of the alkB gene from these strains exhibited 94, 93 and 89% homologies with that of P. putida P1 (Smits et al. 1999), P. aureofaciens RWTH 529 (Vomberg and Klinner 2000) and P. oleovorans (Kok et al. 1989), respectively. The four Gram-negative strains i.e. Pseudomonas sp. 19/1, 30/3 and Sphingomonas sp. 35/1, 8/44 isolated from JP8 Jet fuel were PCR-positive for alkB. All other Gram negative strains showed no amplification for alkB genes (Table 3).

Table 3 Results of PCR amplification using different primer sets for biodegradative genes like alkane hydroxylase, naphthalene dioxygenase and catechol 2,3 dioxygenase
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Among Gram positive strains, Coryneform 31/1 isolated from JP8 Jet fuel was PCR-positive for alkB2. Rhodococcus sp. 5/1, 5/103 and 7/1 exhibited amplification of an 870 bp DNA segment with alkB870G primers (Table 3). Similar results were described for biodegradative Pseudomonas sp. and Rhodococcus sp., which were isolated from Canadian high Arctic environment (Whyte et al. 1999a).

The Gram-positive Rhodococcus isolates were PCR positive for alkB1 or alkB2 gene segments when amplified with RH-alkB1, RH-alkB2 or RH-alkB194 primers (Table 3). The RH-alkB194 primer set was designed to amplify a region common to the alkB1 and alkB2 in Rhodococcus isolates. All Rhodococcus isolates were PCR positive for the 194 bp region of RH-alkB primers (6 out of 6 isolates). Also, they were PCR positive for the the 552 bp alkB2 (6 out of 6 isolates), whereas only 2 out of 6 isolates were positive for the 629 bp gene fragment of alkB1. However, Rhodococcus 7/1, which is phylogenetically similar to the R. erythropolis and Rhodococcus strain Q15, exhibited positive PCR amplification of both alkB1 and alkB2 gene segments (Bej et al. 2000). The specificity of the amplified PCR products was confirmed by nucleotide sequence analysis using Sanger di-deoxy chain termination method. Among the Gram negative strains, 13 out of 16 Pseudomonas spp. and 3 out of 5 Sphingomonas spp. were PCR-negative for alkB1, alkB2 and alkB194, which were designed from a Gram positive strain Rhodococcus sp. Q15 (Table 3). These results suggest that these primers sets were specific to alkB sequences of Rhodococcus spp. (Whyte et al. 2002a).

Using the degenerate primers TS2S/deg1RE, only two of the isolates, Rhodococcus 5/1 and 5/103, exhibited a 550 bp DNA fragment, while the others amplified multiple non-specific fragments in the range of 600–900 bp fragments. The deduced amino acid sequence from the 550 bp fragment from a representative strain (Rhodococcus 5/1) exhibited 87% nucleotide sequence identity to the alkB gene. Previous studies by Smits et al. (2002) and van Beilen et al. (2002) showed that although majority of the isolates amplified a 550 bp fragment, only a few of them had sequences similar to the alkB gene. All other primer sets targeting the alkane-degradation genes such as alkM (496 bp) and alkB (546 bp) tested in this study exhibited PCR-negative results (Table 3).

PCR amplification of ARHDs

Amplification of the ndoB gene

The L-ndoB and R-ndoB oligonucleotide primers amplified a 648 bp DNA fragment from all isolates that utilized naphthalene as a sole carbon source (Table 3). Three Pseudomonas spp. isolated on different sources i.e. JP8 jet fuel, m-toluene and sodium salicylate also were PCR-positive for ndoB. The Sphingomonas strains Ant 18 and Ant 20 utilizing 1-methyl naphthalene were negative for the ndoB. One Sphingomonas strain 35/1 utilizing JP8 jet fuel was positive for the ndoB (Table 3). A 648 bp amplified region of the ndoB from a representative strain Pseudomonas sp. 30/2 was sequenced (Accession no. AY034588). The Blast N and Blast P (Genbank) analysis revealed 82% nucleotide and 97% amino acid sequence identity with the known ndoB sequences from P. putida. Among Gram positive strains, all Rhodococcus sp. were PCR negative except Coryneform strain 31/1 utilizing JP8 jet fuel which showed positive amplification for the ndoB gene (Table 3).

Amplification of the catechol 2,3 dioxygenase gene

The cat238 primers developed by Mesarch et al. (2000) (Table 2) were designed to amplify the I.2.A subfamily of C23DO genes, which are involved in biodegradation of wide variety of aromatic compounds. The primers were tested on all isolates that were able to use toluene, xylene or naphthalene as sole carbon source. Three isolates, which were m-toluene, m-xylene or JP8 jet fuel degraders: Ant 10, Ant 11, 7/22, 30/3 and 8/51 amplified a 238 bp band under the PCR conditions used (Table 3). Nucleotide and amino acid sequences derived (Accession number AY034589) were 95 and 100% similar to the known sequences of xylE, which codes for C23DO in the mesophilic P. putida strain and only one isolate, Pseudomonas sp. 30/2, out of the eight naphthalene degraders showed a positive amplification with C23DO primers (Table 3). We designed and tested two other primers xylE and cat2, 3 (Laramee et al. 2000; Nakai et al. 1983) (Table 2). The naphthalene degrader Pseudomonas spp. as well as others utilizing JP8 jet fuel and one out of five Sphingomonas sp. i.e. Ant 29 showed positive amplification for both the xylE and cat2,3 genes (Table 3), indicating the presence of naphthalene degradation pathway. Interestingly, 5 out of 6 Rhodococcus spp. also were PCR-positive for either xylE or cat2,3 or both because the primers for both the genes were designed from Pseudomonas putida ATCC 33015 (Tables 2, 3).

Amplification of the toluene/biphenyl dioxygenase gene

None of the isolates tested in this study showed amplification for the todC1 (Table 3). And only two strains i.e. Sphingomonas 35/1 and Coryneform 31/1 amplified an 830 bp band for the bphA1 gene.

Reverse transcriptase PCR amplification

The reverse transcriptase PCR for three genes i.e. alkB2, C23DO and ndoB from three different strains i.e. Rhodococcus sp. 5/1, Pseudomonas sp.7/156 and Pseudomonas sp. Ant 6, which showed positive amplification for these genes in conventional PCR, also exhibited amplification of the cDNA of the transcript of these biodegradative genes (Fig 1). This suggests that these biodegradative genes are in fact are functional and contribute to the biodegradation of the targeted compounds.

Fig. 1
figure 1

A photopositive of a representative agarose gel of the RT-PCR amplified gene fragments. Lane 1: 100-bp size standard (Clone-Sizer, Norgen Biotek); Lane 2: 0.552 kb alkB2 from Rhodococcus sp. 5/1; Lane 3: negative control (no DNA).; Lane 4: PCR with RNA from 5/1; Lane 5: 100-bp size standard; Lane 6: 0.642 kb ndoB from Pseudomonas sp.7/156; Lane 7: negative control (no DNA).; Lane 8: PCR with RNA from Pseudomonas sp.7/156; Lane 9: 100-bp size standard; Lane 10: 0.408 kb C23DO from Pseudomonas sp. Ant 6; Lane 11: negative control (no DNA); Lane 12: PCR with RNA from Pseudomonas sp. Ant 6

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Competitive PCR

Due to the conserved nature of the ndoB gene in psychrotolerant as well as in mesophilic bacteria, a quantitative analysis of this gene by cPCR was developed in this study. First, the cPCR reaction was optimized with equal concentrations of the 648 bp target and the 501 bp competitor DNA fragments of the ndoB gene to ensure equal levels of amplified products results from the cPCR. Presence of DNA bands of equal intensity suggested that the PCR conditions used to amplify both the target and competitor gene were adequate. Next, twofold dilutions of the competitor template were co-amplified with constant amounts of genomic DNA from Pseudomonas sp. 30/2 to determine the target gene concentration (Fig. 2). The mean intensities of the competitor and the target DNA exhibited a good correlation (R 2 = 0.96) with the concentration of the targeted DNA being 48.8 pg/μl (SD ± 0.06) (Fig. 3), which corresponds to 1 × 107 copies of the template DNA. These results were consistent among three individual experiments conducted at different times. The results suggest that cPCR using the ndoB gene can potentially be applied in the Antarctic soils to quantify relatively low concentrations of targeted ndoB gene.

Fig. 2
figure 2

A photonegative of a representative agarose gel showing the PCR amplified cndoB and ndoB gene fragments from Pseudomonas sp. 30/2. Lane 1: 100-bp size standard (Clone-Sizer, Norgen Biotek); Lane 2: 452 pg cndoB; Lane 3: 226 pg cndoB; Lane 4: 113 pg cndoB; Lane 5: 56.5 pg cndoB; Lane 6: 45.2 pg cndoB; Lane 7: 28.2 pg cndoB; Lane 8: 14.52 pg cndoB; Lane 9: ndoB (positive control); Lane 10: cnodB; Lane 11: negative control (no DNA). The amount of target DNA used for cPCR was the same in all the reactions

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Fig. 3
figure 3

Standard curve to calculate the gene copy number of the target ndoB gene using competitive PCR. The points plotted correspond to the concentrations of cndoB listed in lanes 4, 5, 6, 7 and 8 of Fig. 1. Regression analysis performed on the results exhibited an R 2 = 0.96. The experiment was repeated three times with consistent results

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Discussion

Oil-spills in pristine Antarctic continent are a threat to the indigenous flora and fauna. In order to implement bioremediation using indigenous hydrocarbon-degrading microorganisms, it is necessary to understand the physiology, community structure as well as the genetics behind the catabolic pathways. In this study we have used conventional PCR to identify the occurrence and distribution and expression of the biodegradative genes in microorganisms isolated from Antarctic oil-contaminated soils. With regard to naphthalene degradation, ndoB has been found to be highly conserved in Pseudomonas B17, B18 and other psychrotolerant and mesophilic bacteria (Whyte et al. 1996, 1997). It has been reported that, although the biodegradative pseudomonads are not the only species that degrade naphthalene in soil, they are prevalent in oil contaminated environments (Aislabie et al. 2000; Eriksson et al. 2003; Whyte et al. 2002a). Similarly in our study, Pseudomonas spp. with ndoB was commonly isolated from hydrocarbon-contaminated Antarctic soils. Psychrotolerant bacteria with ndoB gene sequence homology to that in mesophiles have been documented (Whyte et al. 1996). Our results also suggest that there is a considerable similarity in the catabolic pathway involved in naphthalene degradation between mesophilic and psychrotolerant bacteria.

The conserved nature of ndoB in all these isolates proved helpful in the design and application of the cPCR method. The assay conditions tested in this study can potentially be used as the first step for the enumeration of the ndoB gene-copy number in oil-contaminated Antarctic soils. Quantification of the ndoB from total genomic DNA extracted from contaminated soils could provide an assessment of the population dynamics of culturable as well as non-culturable naphthalene degrading microorganisms. cPCR has been widely used in the medical diagnostics but has also found its application in environmental biotechnology. The use of cPCR with 16s rRNA gene or the biodegradative genes such as ammonia monooxygenase (amoA), atrazine (atzC), cadmium (cadA) as targets have been used in quantification of non-culturable bacteria as well as to assess population density in both pristine and contaminated soil environments (Ka et al. 2001; Kurkela et al. 1988; Martin-Laurent et al. 2003; Oger et al. 2001). Mesarch et al. (2000) has demonstrated the usefulness of the cPCR for C23DO gene present in contaminated soil samples. Therefore, the development of the cPCR would help in situ analysis of the distribution and quantitation of naphthalene and C23DO degrading microorganisms in pristine oil-contaminated soils in Antarctica. Due to the diverse nucleotide sequences of alkB, selection of a single set of primer to establish a comprehensive detection of this gene in total microbial biomass by using cPCR was not possible. However, the cPCR protocol targeting alkane degradation genes present in Rhodococcus spp. can be applied in polar soils (Whyte et al. 2002a).

Catechol 2,3-dioxygenase was chosen as a target for PCR amplification because of its broad-specificity for a number of hydrocarbons like benzene, xylene, toluene and naphthalene, which are present in Jet fuel. The amino acid residues deduced from the 238 bp conserved region of the C23DO gene exhibited 100% sequence identity to that found in mesophilic bacteria suggesting that most likely they have similar degradative pathways. However, a number of bacterial isolates listed in Table 3 were PCR negative for this gene. But, the PCR amplification using the xylEb-F and xylEB-R; and cat2, 3 1a-F and cat2, 3a-R, targeting different segments of C23DO gene successfully amplified in most of the bacteria. In addition, 4 out of 5 Sphingomonas spp. did not amplify for any of the three primer sets. The results suggest the presence of a different pathway for the degradation of catechol, such as the ortho-cleavage dioxygenase or a different gene altogether is present in these isolates. Alternatively, there is need for designing of new primers from genes specific for the Sphingomonas isolates. Therefore, the presence of the ndoB gene but absence of the C23DO gene in the same organism may suggests that the psychrotolerant bacteria possess a modified or an alternative naphthalene degradation pathway.

The alkB gene has been reported to have diverse sequences in different genera, especially among Gram-negative and Gram-positive isolates (Smits et al. 1999). The alkB sequence is also thought to be dependent on the chain-length of the alkane and/or types of pathways utilized by the alkane-degrading bacterium (Heider et al. 1999; Rehm and Reiff 1981; Vomberg and Klinner 2000). Therefore, not all microorganisms in our study isolated from JP5 Jet fuel contaminated soils exhibited amplification of the alkB gene using a single set of primers. However, the majority of the rhodococcal isolates exhibited positive amplification for alkB2. This could be due to the bacteria being isolated from Antarctic soils typically contaminated with C8–C20 n-alkanes that include C10–C16 n-alkanes, which is the proposed substrate range for alkB2 (Bej et al. 2000; Chandler and Brockman 1996; van Beilen and Funhoff 2007). This in contrast to the study by Whyte et al. (2002a) in which the alkB1 was predominantly identified in rhodococcal population in diesel fuels contaminated soils in Antarctic and Arctic environments. Thus, it is apparent that the distribution of different alkB genes in rhodococcal population correlates with the types of alkanes present in the soil.

Among other Gram negative isolates, 13 out of 16 Pseudomonas spp. and 3 out of 5 Sphingomonas spp. were PCR-negative for alkB1, alkB2 and alkB194 which were designed from a Gram positive strain Rhodococcus sp. Q15 (Table 3) suggesting that these primers sets are more successful in amplifying alkB sequences of Rhodococcus spp. (Whyte et al. 2002a). This is probably due to the presence of a gene with low homology to known alkB sequences or a novel gene altogether. In fact, genes with low homology to known alkB have been detected in several microorganisms that were isolated from alkane-contaminated soils (Smits et al. 2002; van Beilen et al. 2002). The functionality of a few of these genes has been investigated to prove that they play a role in alkane degradation, but still others remain ambiguous (Smits et al. 2002). Another reason we were unable to detect short-chain alkane degrading microorganisms from Antarctic soils could be due to decreased volatilization of the short chain alkanes (<C10) at cold temperatures. This results in increased solubility of alkanes in the aqueous phase, which becomes toxic to the microorganisms (Whyte et al. 1999b). Thus, further investigation is necessary to understand if the alkane degrading genes in psychrotolerant bacteria evolved independent of alkB gene found in mesophiles. Recently, Kuhn et al. (2009) described the predominant presence of a novel alkM gene in the sediments of a pristine site near contaminated sediments in the Admiralty Bay, King George Island, Peninsula Antarctica. However, this novel alkM gene was described only in Acinetobacter sp. In our study, none of the contaminated soil samples exhibited the presence of Acinetobacter sp. therefore the test for this gene was irrelevant. In another study by Lo Giudice et al. (2009), 253 biodegradative indigenous bacterial isolates from the diesel fuel-enriched marine surface water from the Victoria Land coast of Antarctica showed the presence of Rhodococcus, Sphingomonas, Pseudomonas and Corynebacterium similar to the isolates in our study from Antarctic Dry Valleys soils. This suggests that they are found in both continental spill sites and coastal waters of the Antarctic continent (Atlas 1981; Lo Giudice et al. 2009; Michaud et al. 2004a, b).

Almost all isolates did not show positive amplification for the todC1 and bphA1 gene. The todC1 gene, located on the chromosome of P. putida F1, encodes for the α-subunit of terminal dioxygenase, one of the three components of toluene dioxygenase, the first enzyme involved in the toluene degradation pathway (Baraniecki et al. 2002). The xylE and cat2, 3 genes, located on the TOL plasmid of P.putida ATCC 33015, encode for catechol 2,3 dioxygenase, a key enzyme involved in the lower degradation pathway of aromatic compounds, such as toluene and xylene (Laramee et al. 2000; Nakai et al. 1983). Though these organisms did not show amplification for the first gene of the pathway i.e. todC1 but presence of the enzyme C23DO from the lower degradation pathway of toluene or xylene suggests the occurrence of this pathway in these organisms. The bphA gene, located on the chromosome of the polychlorinated biphenyl-degrading strain P. pseudoalcaligenes KF707, encodes for biphenyl dioxygenase, a multi-component enzyme responsible for the catalysis of the initial oxidation of biphenyl and chlorobiphenyls (Furukawa and Arimura 1987). Appropriate primers targeting the specific genes for the complete pathway are yet to be determined. In our study, we also tested the functionality of three genes i.e. alkB, ndoB and C23DO by RT-PCR. Positive amplification of the cDNA from the transcripts of these genes suggests that in fact these genes are transcriptionally active thereby the microbial isolates bearing these genes are capable of biodegradation of the targeted PAH compounds.

It is apparent from studies by our laboratory as well as others that degradation of hydrocarbon compounds can be achieved by microorganisms that have been isolated from hydrocarbon-contaminated Antarctic soils. In this study, we further confirmed the presence, nature and functionality of the degradative genetic elements. Therefore, bioremediation of the Jet fuel contaminated Antarctic soils may be possible because of these hydrocarbon-degrading pathways present in the indigenous microbial populations. Moreover, the growth, survival and adaptation of some of these biodegradative psychrotolerant microorganisms have already been elucidated (Bej et al. 2000; Aislabie et al. 2000; Panicker et al. 2001; Whyte et al. 1999b). Thus, as our understanding of the workings of the indigenous psychrotolerant bacteria increases, we can harness the biodegradative capabilities of the hardy indigenous microbial populations to degrade a wide range of pollutants.