Quantitative PCR analysis of diesel degrading genes of Acinetobacter calcoaceticus isolates

Acinetobacter strains LT1 and V2 were grown in Bushnell-Haas medium with 1% diesel and their expression levels of eight diesel-degrading genes were evaluated by quantitative polymerase chain reaction (PCR). LT1 and V2 isolates achieved 86.2 and 89.7% degradation respectively, after 60 days with no significant differences in expression, in comparison with 16S rRNA, rubB and lipB. LT1 showed higher expression levels of rubA, alkM, alkR and xcpR genes during the initial stages of incubation corresponding to higher level of degradation rate. Amplification of alkM, alkR and xcpR genes of V2 indicated more than one enzyme system involved in the process. Low lipB and lipA expression in LT1 and no lipA expression in V2 suggested the absence of lipases in degradation; however, estB gene was predominantly expressed in V2. Thus, isolates LT1 and V2 possessed comparable efficiency in degrading diesel; however, more complex systems have been employed by V2i n degradation process.


INTRODUCTION
A wide array of studies has dealt with biodegradation and bioremediation of petroleum hydrocarbon unraveling the intricacies of degradation and generating a wealth of bacterial, genetic, biochemical, and physiological knowledge (van Beilen and Funhoff, 2007;Rojo, 2009).Characterization of bacterial communities, isolation of prospective degraders, monitoring their response to pollutants, and identifying functional genes and enzymes involved in biodegradation processes have been subject to extensive research (Wentzel et al., 2007;Rojo, 2010).Over the years, a multitude of bacterial species capable of degrading hydrocarbon, have been readily isolated from hydrocarbon-contaminated and non-contaminated sites.Acinetobacter sp. are one of the representative genera capable of utilizing n-alkanes (Tani et al., 2002;Van Hamme et al., 2003;Singh and Lin, 2008;Lee et al., 2012;Mara et al., 2012).
The pathways of alkane degradation and the enzymes involved in it have been reviewed extensively (Coon, 2005;van Beilen and Funhoff, 2007;Wentzel et al., 2007;Rojo 2010).In majority of the cases, aerobic degradation of n-alkanes starts either by the terminal (van Beilen and Funhoff, 2007;Wentzel et al., 2007;Rojo 2010) or subterminal oxidation pathway (Kotani et al., 2006).In this pathway, conversion of methyl group which leaves a primary alcohol, is further oxidized to the corresponding aldehyde, fatty acid and finally processed by β-oxidation pathway to generate acetyl-CoA (Wentzel et al., 2007;Rojo 2010).In some microorganisms, both terminal and sub-terminal oxidation pathway coexists (Watkinson and Morgan, 1990).
In Acinetobacter calcoaceticus, alkane hydroxylases (encoded alkM) require two soluble electron transfer proteins named rubredoxin (rubA) and rubredoxinreductase (rubB) for n-alkane degradation and are obligatory for its function (Tani et al., 2001).The alkM gene expression is regulated by the alkane hydroxylase regulator (alkR) (Ratajczak et al., 1998).The participation of xcpR gene in alkane degradation was proposed in A. calcoaceticus ADP1 after the insertional inactivation of xcpR impeded the secretion of lipase and esterase that lead to the lack of growth on dodecane and slower growth on hexadecane (Parche et al., 1997).In A. calcoaceticus ADP1, the estB gene encodes a functional esterase and is localized in an operon with genes rubA, rubB and oxyR (Geißdörfer et al. 1999).The esterase activity has been reported to be involved in the sub-terminal oxidation pathway (Kotani et al., 2007).The involvement of these enzymes in the degradation of hydrocarbons has been proposed, however their exact function is unknown.Genomes of many Acinetobacter strains have been sequenced recently, including those of Acinetobacter sp.DR1 (Jung et al., 2010), Acinetobacter oleivoran ssp.DR1(T) (Kang et al., 2011), Acinetobacter venetianusRAG-1T (Fondi et al., 2012) and Acinetobacter venetianusVE-C3 (Fondi et al., 2013).Although, momentous gains have been made in understanding the processes in hydrocarbon degradation, the minutiae of individual systems as well as the diversity of systems have yet to be fully articulated (Van Hamme et al., 2003).Therefore, this study investigated the proficiency of A. calcoaceticus isolates in the degradation of diesel and implementing the levels of mRNA expression of diesel degrading genes using quantitative polymerase chain reaction.

Selection and identification of diesel degrading bacterial isolates
A. calcoaceticus isolates LT1and V2, were obtained from departmental stock cultures.LT1 (accession number JN036553) were originally isolated from diesel-contaminated soil (Singh and Lin, 2008) while V2 (accession number JN036552) was isolated from used engine oil-contaminated soil (Mandri and Lin, 2007) previously from the same laboratory Isolates were streaked on nutrient agar and incubated at 30°C for 24 h to attain pure cultures.

Growth of bacterial isolates on diesel as sole carbon and energy source
To access their ability to degrade diesel, isolates were grown on Bushnell-Haas (BH) minimal medium with diesel as sole carbon source (Singh and Lin, 2008).Isolates were grown in separate 100 ml Erlenmeyer flasks with a total volume of 25 ml of liquid BH medium supplemented with 1% (vol/vol) sterilized diesel using 0.22 μm membrane filters (Whatman) as a carbon and energy source.The isolates were grown overnight at 30°C in Luria broth, washed thrice with normal saline and 1% of the washed cells were inocula-ted in each BH medium (standardized using BH medium to OD600 of 1.0 after centrifugation).The BH medium flasks were incubated aerobically at 30°C in a rotary shaker (160 rpm) for variable time period.An abiotic control flask, with 1% (vol/vol) diesel and devoid of bacterial inoculum, was incubated under the same conditions to ascertain abiotic loss.Bacterial growth was monitored by optical density at 600 nm (OD600) at 5,10,15,20,30,40,50 and 60 days of incubation.The experiments were repeated in triplicate.
Carbon source utilisation patterns were determined by gravimetric analysis (Marquez-Rocha et al., 2001).The remaining diesel in each sample was extracted using the separating funnel with 10 ml dichloromethane three times.The dichloromethane extract was funneled through filter paper containing 5 g anhydrous sodium sulfate (Na2SO4) for the removal of cellular debris and absorption of moisture.The extract was collected in the pre-weighed glass tubes and was left overnight in a fume cupboard to facilitate evaporation of dichloromethane.The combined mass of the pre-weighed tube and diesel was recorded.The mass of the tube was subtracted to determine the mass of the diesel remaining.The mass of the diesel remaining was subtracted from the mass of diesel in the control sample and multiplied by 100 to determine the percentage of diesel remaining.Student t-test (Microsoft Excel 2010) was used to evaluate the differences of diesel degradation between LT1 and V2.Probability (significant level) was set at 0.05.

RNA extraction and cDNA validation
The BH medium containing isolates was centrifuged at 4000 xg for 20 min resulting in a pellet and a cell-free supernatant.Pellet was re-suspended in phosphate-buffered saline (PBS), pH 7.6, and subjected to further centrifugation at 4000 xg for 20 min.Total RNA was extracted from each pellet using Aurum TM Total RNA Mini Kit (BIO-RAD) according to manufacturer's instructions with following modifications :500 µl of lysozyme was added instead of 100 µl of 1000 µg/ml stock dissolved in TE (10 mMTris, 1mM EDTA, pH 7.5) to the bacterial suspension and incubated at room temperature for 30 min.Total RNA purity and yield was determined spectrophotometrically using Nanodrop ND-1000 (BIO-RAD).Purified RNA (1 μg) was used to prepare cDNA by using a first Strand iScript TM cDNA Synthesis Kit (BIO-RAD).The reverse transcriptase PCR was performed (GeneAmp® PCR System 9700, Applied Biosystems) according to manufacturer's instructions.Purified RNA and cDNAwas also assessed electrophoretically by ethidium bromide staining on 2% (wt/vol) agarose in 1 X TAE running buffer at 80V.The OD260/280 ratio obtained ranged from 1.30 to 2.27.Samples LT1 -day 5, with ratio of 1.30, indicated poor RNA quality that resulted in no 16S rRNAPCR product detected.LT1 -day 5 sample was omitted from the real time PCR study.The PCR product of 16S rRNA gene using 63F and 1387R primers (Marchesi et al., 1998) was obtained.DNA concentrations of 16S rRNA PCR products were measured spectrophotometrically using a NanoDrop ND-1000 spectrophotometer and concentrated stock solutions of specific 16S rRNA gene fragments were prepared.These were serially diluted to generate a set of calibration pure standards with known concentrations of target DNA.The cDNA obtained was stored at -20°C.

Real-time PCR data analysis and quantification of gene expression
The nucleotide sequences of the genes of interest were acquired from the National Center for Biotechnology Information (NCBI) nucleotide sequence databases with the following accession numbers: AF047691 (lipA and lipB), AJ233398 (alkM and alkR), EF524340 (16S rRNA), Y09102 (xcpR) and Z46863 (rubA, rubB,  and estB).Primers for real-time PCR were designed with the aid of primer design software Primer3 (Kubista et al., 2006) and were synthesized by Inqaba Biotech.The real-time PCR primer sequences are presented in Table 1.PCR amplifications of alkM, alkR, rubA, rubB, estB, lipA, lipB, xcpR and 16S rRNA genes were performed using LightCycler® Instrument Version 3.5real-time PCR system (RocheDiagnostics).LightCycler® reactions were performed in 20 µl glass capillary tubes using LightCycler® FaststartDNA Master SYBR Green I kit (Roche Diagnostics) according to the manufacturer's instructions.The evaluating parameters selected for data analysis were fluorescence (d[F1]/dT), melting temperature (Tm) and crossing point (Cp) (Pfaffl et al., 2002;Rasmussen, 2001).Conditions of real-time PCR for the respective target genes under the optimized conditions are presented in Table 2. Specificity of real-time PCR primers was determined using LightCycler Software®, Version 3.5 (Roche Diagnostics).Due to the multiple products in some samples, the cDNA of LT1was selected as the calibrator for 16S rRNA, alkM, alkR, rubA, lipA, lipB and xcpR genes and that of V2for estB gene.Each amplification product for the target genes demonstrated a specific and characteristic melting curve (supplementary Figure 1S).Specificity of real-time PCR products were documented by agarose gel electrophoresis and resulted in a single product of anticipated length.No PCR amplification product was observed for gene rubB, despite rigorous optimization strategies and redesigning of primers.
Equal aliquots of cDNA obtained were standardized to 1000 ng/μl.
To generate a standard curve, the serially diluted cDNA standard (1,000 to 0.001 ng) was quantified in each real-time PCR runin duplicates.The efficiency of each standard curve was determined using the equation: Efficiency (E) = 10 -1/slope -1 (Rasmussen, 2001).Descriptive statistics (minimal (Min) and maximal (Max) mean values, standard deviation (SD), and coefficient of variance (CV %), of the derived Cp values were computed for each investigated gene to determine intra-sample variation.
Gene expression was quantified using the Pfaffl model which combines gene quantification and normalizationand was calculated with the aid of Microsoft Excel® based application, Relative Expression Software Tool -XL (REST-XL©) -Version 2 (Pfaffl et al., 2002).The Cp values of both, control and the samples of interest were normalized based on the PCR product of 16S rRNA gene in each sample.The copy number of each gene was calculated based on the 16S rRNA standard curve.

Growth of bacterial isolates on diesel and their carbon utilization pattern
The growth behavioral patterns of the bacterial isolates on diesel were monitored over a period of 60 days and determined by optical density (OD 600 ).The results obtained obtained are presented in Figure 1.After 60 days of incubation, a 9.40% mass reduction in the diesel of the abiotic control flask was detected (data not shown).Both isolates displayed comparable proficiency in the overall utilization of diesel as a sole carbon and energy source.Rapid degradation of alkanes occurred during the first 5 days of incubation with attributes to the exponential growth phase of the bacterial cells.The OD 60 0 increased progressively up to 15 days, and remained relatively constant until the end of the culture time.Both isolates LT 1 and V 2 showed diesel degrading capability.LT 1 and V 2 achieved 58.6 and 48.3% degradation after 5 days of incubation and 86.2and 89.7% degradation after 60 days of incubation, respectively (Figure 1).During the initial days of incubation, V 2 degraded less diesel as compared to LT 1 .However, after a rapid increase in diesel degradation at day 15, V 2 emerged as the most proficient diesel degrader by the end of the incubation period (Figure 1), though the diesel degradation ability between these two strains did not differ significantly (p=0.506) at the end of the experiment.
The presence of an opaque substance was observed in the hydrocarbon and hydrocarbon -culture medium fraction of V 2 samples throughout the incubation process.
After 10 days of incubation, however, the diesel of the V 2 samples no longer appeared as a confluent layer over the culture medium phase, but rather as minuscule droplets.This occurrence was not observed for LT 1 samples for the duration of the culture time.

Optimizing the components of the real-time PCR master mix
LightCycler analyses of alkM, alkR, rubA, rubB, xcpR, estB, lipA, lipB and16S rRNA were performed and optimized.Melting temperature (T m ) of target gene amplification products ranged from 79.3°C for alkR to 91.2°C for 16 rRNA (Figure 1S).A high degree of efficiency ranging from 1.76 to 2.24 was achieved indicating a stable and reliable assay.All generated standard curves illustrated high linearity with r 2 values of 1.00 over three orders of magnitude.An expression profile of lipA could not be created for LT 1 , and V 2 samples due to very low expression levels of lipA and therefore, E lipA could not be deter- The 16S rRNA gene was stably expressed in LT 1 samples throughout the time course and showed the lowest degree of intra-sample variation based on SD and CV% of C p .The 16S rRNA gene of V 2 was not signi-ficantly different from 16S rRNA of LT 1 with p values of 0.072 demonstrating an accepted level of gene expres-sion variability.The findings also show the similar growth and diesel degradation patterns of both isolates as shown in Figure 1.Hence, the expressions of 16S rRNA of both isolates were used to normalize all other target gene expressions respectively for comparison.

The target gene expressions and comparison between LT 1 and V 2 isolates
The expression levels of rubA, alkM, alkR, and xcpR genes in LT 1 samples exhibited their maximums during the initial stages of incubation (Figures 2 and 3a) and with high degrees of intra-sample variation in C p with SD and CV% (Table 3).The higher expression (8.71 x 10 12 gene copies) of alkM gene was observed at day 10 as compared to other genes involved and its expression declined as incubation time progressed.The above results revealed the involvement of these gene products of LT 1 in the diesel degradation.Previous studies showed that Acinetobacter sp.strain ADP1 requires at least five essential genes, namely rubA, rubB, alkM, alkR, and xcpR, for n-alkane utilisation (Ratajczak et al., 1998) confirming rubA, rubB, and alkM constitute a three component alkane hydroxyase system in A. calcoaceticus (Ratajczak et al., 1998;Geißdörfer et al., 1999;van Beilen et al., 2002).The overall alkR gene expression of LT 1 , samples was comparatively low in relation to the alkane hydroxylase complex genes, alkM and rubA.This inference is in accordance with preceding studies that found alkR to be expressed at lower levels during alkane degradation despite its indispensability in the degradation process (Ratajczak et al., 1998).The xcpR gene encodes a type IV pilus-related system that may not only be required for the secretion of proteins but may represent a more universal transport system for a variety of macromolecules.It may also facilitate the alleviation of membrane stress incurred by bacterial cells during alkane degradation (Parche et al., 1997).All genes except lipB gene demonstrated a similar pattern.The lipB gene was expressed in LT 1 at all sampling times, however, low expression levels was observed as depicted by the high C p values (Table 3).Unlike other target genes that were expressed in the early stage of diesel degradation, expression levels of lipB gene increased with incubation time (from 5.62x10 8 gene copies at day 10 to 2.82 x 10 9 gene copies at day 20) for LT 1 and declined thereafter showing greater intra-sample variation (SD and CV% values of 10.27 and 25.10 respectively) (Table 3).Positive peak formations of desired estB and lipA gene products were observed for many samples.However, such peaks are a consequence of amplification occurring very late in the PCR often with C p values > 51.00 in a 55 cycle amplification segment, therefore are considered invalid and are indicative of false-positive results.Lack of target-specific amplifications of estB and lipA in LT 1 samples, suggest either the absence of gene expressions or low levels of expression.
In comparison with the diesel-degrading target gene expression levels of LT 1 , the expression profile of the rubA gene of V 2 had a similar trend as LT 1 (Figure 2), exhibiting the maximum during the initial stage of incubation and declining as incubation time progressed.The rubA expression level of V 2 was down-regulated by the factor 10.115 as compared to that of LT 1 .Despite the down-regulation, expression levels for rubA of V 2 were not significantly different from rubA of LT 1 with p values of 0.066.
During the amplification of alkM of V 2 samples, the alkM primer pair concurrently amplified the anticipated alkM gene (designated as alkM1 as shown in Figure 3A with a T m of 80.36°C) as well an additional product; presumed to be another alkane hydroxlase that was tenta-tively designated alkM2 exhibiting a much higher T m exceeding 92°C (Figure 3B) that was evident between 20 and 50 days of incubation.Amplification of alkM 1 and alkM 2 genes of V 2 samples was repeated and results were highly reproducible.Agarose gel electrophoresis of alkM 1 amplicons from LT1 confirmed the presence of the anticipated product of 189 bp from day 5 to 60 (Figure 3Ca).An additional product of 336 bp was evident (Figure 3Cb) from day 20 to 50 of V 2 samples which coincided with the melting curve analysis of alkM gene.The alkM gene amplified products of the V 2 at day 15 and LT 1 were sequenced and aligned.Partial sequence alignment (Figure 3D) shows 100% homogeneity of these two sequences.The alkM gene of LT 1 samples is therefore analogous to alkM 1 of V 2 isolate.Additionally, the obtained sequences showed 99 and 95% homogeneities with the partial sequences of alkane monoxygenase (alkB) from Acinetobacter sp.49A and A. calcoaceticus PHEA-2, respectively.Sequencing of the day 20 sample of alkM 2 gene from isolate V 2 proved to be unsuccessful even after numerous attempts.
Formation of additional products could also be attributed to co-amplification of two or more genes as a result of sequence similarity.The presence of multiple alkane hydroxylases in a single bacterial strain is not an atypical occurrence (Tani et al., 2001;Marín et al., 2003).Multiple alkane hydroxylases enable alkane degraders to grow on a broad range of alkane substrates, where each alkane hydroxylase may exhibit unique properties and have different induction patterns (van Beilen et al., 2003).The alkMa and alkMb expressions of Acinetobacter sp.M1 were differentially induced in response to n-alkane chain length (van Beilenet al., 2003).Differential expressions of alkB1 and alkB2 of P. aeruginosa were not based on alkane chain length but rather on the levels of oxygen (Marín et al., 2003).In addition to two genes encoding AlkB-type alkane hydroxylase homologues in Acinetobacter sp.strain DSM 17874, almA, which encodes a putative flavin-binding monooxygenase, was also involved in the metabolism of long-chain n-alkanes (Throne-Holst et al., 2007).The A. venetianus VE-C3 genome possesses different alkane degrading genes which encode AlkBlike, soluble cytochrome P450 monooxygenases, AlmA and LadA (Fondi et al., 2013).V 2 might have been evolved to possess at least two alkane hydroxlases/monooxygenases to cope with the stress of the longer carbon chain length and/or under oxygen levels as alkM1 was expressed at the late exponential/early stationary phase and alkM2 throughout the late stationary phase.
In compliance with multiple alkM gene expressions, multiple alkR and xcpR genes of V 2 samples were also expressed to enhance the regulation of alkM expression (Figure 4).The expected amplification product of alkR gene in LT 1 samples, was only observed at late stage of degradation (days 30, 50, and 60) and in the V 2 samples, and there was shift in T m of approximately 2°C for day-30 and 60 samples (Figure 4A).The presence of a second product (alkR2) with T m exceeding 84°C was also observed at the early stage of diesel degradation (days 5, 10 and 15 samples).Despite the disparity in T m , the amplification product size was identical to that of day 50.No alkR amplification occurred for days 20 and 40 samples.The alkR 1 gene of V 2 might be analogous to alkR of LT 1 on the basis of T m and amplification product size.A. calcoaceticus sp.M1 that contained the two alkane hydroxylases alkMa and alkMb was also found to possess two transcription regulators alkRa and alkRb (Tani et al., 2001).Each alkane hydroxylase was regulated by either alkRa or alkRb responded to the alkane chain lengths.The alkR 1 gene of V 2 is comparable to alkR of LT 1 on the basis of T m and amplification product size.
The xcpR gene, that encodes a subunit of the general secretory pathway, is required for alkane degradation in Acinetobacter sp.ADP1 and DSM 17874 strains (Parche et al., 1997;Throne-Holst et al., 2007).The expression of xcpR in conjunction with rubA and rubB in A. calcoaceticus is known to be constitutive (Ratajczak et al., 1998).Figure 4B shows that the presence of additional amplification products for xcpR of V 2 samples, illustrated by three defined peaks, exhibited higher T m s.The results obtained for V 2 samples were highly reproducible yielding multiple products with identical melting temperatures.Real-time PCR amplification products of xcpR of V 2 samples were verified by gel electrophoresis (Figure 5).The presence of two distinct products was observed.The expected 192 bp product (xcpR1) was observed in all samples and the presence of a larger fragment of approximately 500 bp (xcpR2) was also observed in all samples except on days 30 and 40.The metabolism of nalkanes in the V 2 isolate appears to be more complex.Its broad substrate range, diversity and functions of the enzymes involved in alkane degradation, and its contrasting regulation of both constitutive and differential gene expression may be reminiscent of the fact that two and perhaps more xcpR genes are required to provide an adequate transport system during alkane degradation.
Rubredoxin (rubA) activity was detected in all investigated samples of LT 1 and V 2 isolates.LT 1 and V 2 sam-ples exhibited similar rubA expression trends and the differences in expression levels were not significant.However, rubredoxinreductase (rubB) activity was not detected in any of the isolates despite rigorous optimising strategies and redesigning of primers.Plausible reasons for the inability to detect rubB activity could be attributed to very low expression levels, or poor primer design.
Theoretically, rubB would have exhibited a similar  expression trend to that of rubA due to their synergistic association (Geißdörfer et al., 1999).Interestingly, novel genes encoding AlkB-Rubredoxin fusion proteins were used in the hydroxylation of long-chain alkanes by Gram positive bacteria (Nie et al., 2011).In addition, rubredoxin-rubredoxin reductase systems are present in many other organisms that are unable to degrade alkanes, where they serve other functions such as rapid transport of reducing equivalents to the final receptor (Hagelueken et al., 2007).Although, rubA, rubB and estB constitute an operon in Acinetobacter sp.ADP1, it was established that no association could be ascertained between the expression of rubA and estB in accordance with the findings by Geißdörfer et al. (1999).The expression of estB was predominantly observed in V 2 samples and either nonexpression or extremely low expression of the estB gene in LT 1 samples (Figure 3).During the diesel degradation phase of V 2 inocula, an opaque, waxy material was observed, probably due to bioemulsifiers in the hydrocarbon and hydrocarbon-culture medium fraction, but not in flasks containing LT 1 inocula.These findings are suggestive that the involvement of estB in the release of the bioemulsifier may be plausible (Shabtai and Gutnick, 1985).
Low expression of lipA in LT 1 and lack of expression in V 2 (Table 3) is an indication of its non-involvement in the degradation of diesel.The lipB gene was expressed in all samples in this study.Expression of lipB amongst V 2 samples was moderately constant exhibiting lower SD and CV% values of 1.41 and 3.55, respectively (Table 3).The expression profile of lipB gene in V 2 (Figure 4C) was found in low level, depicted by the high C p values exhibiting maximum expression at day 15.Thereafter, expression levels declined fairly constantly.In comparison to LT 1 , lipB of V 2 was up-regulated by the factor 1.976 with no significant difference (p= 0.436).In this study, the expression trends of lipA and lipB were incongruent, and expression of lipB was still evident in the absence of lipA expression amongst V2 samples.The lipB gene encodes a lipase chaperone that prevents the complete folding of the lipase before it traverses the outer cell membrane (Kim et al., 2008).Alternatively, steric chaperones may have other functions such as the autotransporter protein that can be used to direct a variety of proteins to the cell surface in addition to LipA folding (Wilhelm et al., 2007).Kim et al. (2008) also reported that there are 3 different lipaA genes present in Acinetobacter sp.DYL 129.The involvement of lipA and lipB in alkane degradation is still unknown.
The findings of this study confirm the importance of rubA, alkM, alkR, and xcpR genes in the process of alkane degradation.The results are also suggestive that different members of the alkane degraders, such as Acinetobacter LT 1 and V 2 strains in this study employ a variety of alkane degrading pathways for alkane oxidation, albeit both isolates achieved comparable levels of diesel degradation.It is apparent that alkane oxidation in V 2 appears to be complex due to its diverse regulations of multiple alkane hydroxylases, alkane hydroxylase transcription regulators and the secretory pathways.However, involvement of other alkane degrading proteins such as cytochrome P450 alkane hydroxylases, Lad and AlmA cannot be ruled out.

Conclusions
We concluded that both Acinetobacter strains LT 1 and V 2 in this study showed comparable diesel degrading ability, however real-time PCR experiments revealed more than one enzyme system involved by V 2 indicating its diverse regulations of multiple alkane hydroxylases, its transcription regulators and the secretory pathways.Low or no expression of lipB and lipA genes suggested the absence of lipases in degradation.This study also confirms the importance of rubA, alkM, alkR and xcpR genes in the process of alkane degradation, although, future experiments would be necessary to elucidate the involvement of other proteins such as cytochrome P450 alkane hydroxylases, Lad and AlmA in alkane degradation.

Figure 1S . 0 Figure 1 .
Figure 1S.Specificity of LightCycler® PCR.Amplification of target genes (A) 16S rRNA, (B) alkM, (C) alkR, (D) rubA, (E) lipA, (F) lipB, (G) xcpR, and (H) estB, as determined by melting curve analysis.Melting peaks were determined by plotting the continuous negative derivative of fluorescence emitted by each sample as PCR products were melted.The cDNA template control sample for each reaction does n ot show fluorescence, confirming the absence of primer-dimers.

Table 1 .
Primers sequences for real-time PCR amplification of target genes.

Table 2 .
Composition of reaction Mastermix and amplification parameters for LightCycler®real-time PCR assays.

Table 3 .
Descriptive statistics and variation data output for target genes of LT1, and V2 samples.
-Invalid Cp values due to nonspecific binding; * invalid Cp values due to multiple product formation.Analysis of 16S rRNA gene expression.