Ted to 1 ml with nitrogen gas aftercDNA synthesis and gene expression quantificationReverse transcription of RNA samples were performed as described by Kim et al. [30] using 2 mg of total RNA sample, 1 ml random hexamers (per reaction) and PrimeScript 1st strand cDNA synthesis Kit (Takara, Japan). Briefly, random hexamers and RNA templates were mixed and 
denatured at 65uC for 5 min followed by cooling for 2 min on ice. Primescript buffer (56), RTase and RNAse inhibitor were added to the cooled template mix and incubated for 1 hour at 50uC before enzyme inactivation at 70uC for 15 min. Negative controls for reverse transcription were performed to test for the presence of genomic DNA contamination in the RNA samples. Complementary DNA samples were diluted 1.5-fold and a quantitative real-time PCR run was employed on AB-7500 Real-time thermal cycler (Applied Biosystems, California, USA) using SYBR Premix Ex-Taq II (Takara Bio, Shiga, Japan) according to manufacturer’s directions. PCR volume was 20 ml consisting of 0.4 mM of each primer (Table 1). Each PCR run included a no-template control with water instead of cDNA as well as a RT negative control for each gene. Triplicate measurements were performed for all reactions. pH and NaCl concentration samples were carried on separate Hexokinase II Inhibitor II, 3-BP web 96-well plates for the gene expression experiments while all samples were analyzed on a single plate for the endogenous control determinationFigure 2. A chart indicating the value M of candidate reference genes. The M value refers to the average expression stability of reference genes TA-01 site during a stepwise exclusion of the least stable expressed reference gene. Starting from the least stable gene at the left, the genes are ranked according to increasing expression stability, ending with the two most stable genes on the right (dnaG and rpoB). doi:10.1371/journal.pone.0058066.gRing-Cleavage Dioxygenase Genes in MycobacteriaFigure 3. Histogram of cycle thresholds (CT) of the four presumed house-keeping genes. The transcript quantifications were determined in nine different conditions: pH 5.5, pH 6.5, pH 7.5, NaCl concentrations of 0 M, 0.17 M, 0.5 M, 0.6 M, 1 M and a control state of no pyrene induction. For each condition, CT was measured from two independent cDNA; the means are represented in this histogram. doi:10.1371/journal.pone.0058066.gexperiment. Results were analyzed using the critical threshold (DCT) and the comparative critical threshold (DDCT) method in the AB-7500 software, the Normfinder and the geNorm-plus algorithms. (Primer Design, Southampton, UK).Results Pyrene degradation in the various pH and NaCl concentrationsAt various times during bacterial growth/induction, 10 ml samples were extracted for residual pyrene determination. Fig. 1A of the residual pyrene profile shows reduced pyrene degradation activity at pH 5.5 with 30 of total pyrene degraded after 48 hours of cultivation. Extreme degradation activities were observed at the 48th hour of cultivation in the pH 6.5 and 7.5 cultures. The highest degradation activity was recorded in the pH 6.5 culture with zero residual pyrene after 58 hours. This makes a slightly acidic condition, probably, the best condition for pyrene degradation in our experimental strain. The salinity conditions conferred to the cultures showed results highlighting the halotolerant nature of the strain at our experimental conditions (Fig. 1B). High degree of pyrene degrading activities were observed in the 0 M and 0.17 M induced.Ted to 1 ml with nitrogen gas aftercDNA synthesis and gene expression quantificationReverse transcription of RNA samples were performed as described by Kim et al. [30] using 2 mg of total RNA sample, 1 ml random hexamers (per reaction) and PrimeScript 1st strand cDNA synthesis Kit (Takara, Japan). Briefly, random hexamers and RNA templates were mixed and denatured at 65uC for 5 min followed by cooling for 2 min on ice. Primescript buffer (56), RTase and RNAse inhibitor were added to the cooled template mix and incubated for 1 hour at 50uC before enzyme inactivation at 
70uC for 15 min. Negative controls for reverse transcription were performed to test for the presence of genomic DNA contamination in the RNA samples. Complementary DNA samples were diluted 1.5-fold and a quantitative real-time PCR run was employed on AB-7500 Real-time thermal cycler (Applied Biosystems, California, USA) using SYBR Premix Ex-Taq II (Takara Bio, Shiga, Japan) according to manufacturer’s directions. PCR volume was 20 ml consisting of 0.4 mM of each primer (Table 1). Each PCR run included a no-template control with water instead of cDNA as well as a RT negative control for each gene. Triplicate measurements were performed for all reactions. pH and NaCl concentration samples were carried on separate 96-well plates for the gene expression experiments while all samples were analyzed on a single plate for the endogenous control determinationFigure 2. A chart indicating the value M of candidate reference genes. The M value refers to the average expression stability of reference genes during a stepwise exclusion of the least stable expressed reference gene. Starting from the least stable gene at the left, the genes are ranked according to increasing expression stability, ending with the two most stable genes on the right (dnaG and rpoB). doi:10.1371/journal.pone.0058066.gRing-Cleavage Dioxygenase Genes in MycobacteriaFigure 3. Histogram of cycle thresholds (CT) of the four presumed house-keeping genes. The transcript quantifications were determined in nine different conditions: pH 5.5, pH 6.5, pH 7.5, NaCl concentrations of 0 M, 0.17 M, 0.5 M, 0.6 M, 1 M and a control state of no pyrene induction. For each condition, CT was measured from two independent cDNA; the means are represented in this histogram. doi:10.1371/journal.pone.0058066.gexperiment. Results were analyzed using the critical threshold (DCT) and the comparative critical threshold (DDCT) method in the AB-7500 software, the Normfinder and the geNorm-plus algorithms. (Primer Design, Southampton, UK).Results Pyrene degradation in the various pH and NaCl concentrationsAt various times during bacterial growth/induction, 10 ml samples were extracted for residual pyrene determination. Fig. 1A of the residual pyrene profile shows reduced pyrene degradation activity at pH 5.5 with 30 of total pyrene degraded after 48 hours of cultivation. Extreme degradation activities were observed at the 48th hour of cultivation in the pH 6.5 and 7.5 cultures. The highest degradation activity was recorded in the pH 6.5 culture with zero residual pyrene after 58 hours. This makes a slightly acidic condition, probably, the best condition for pyrene degradation in our experimental strain. The salinity conditions conferred to the cultures showed results highlighting the halotolerant nature of the strain at our experimental conditions (Fig. 1B). High degree of pyrene degrading activities were observed in the 0 M and 0.17 M induced.
