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A gene expression study of the activities of aromatic ring-cleavage dioxygenases in Mycobacterium gilvum PYR-GCK to changes in salinity and pH during pyrene degradation.

Badejo AC, Badejo AO, Shin KH, Chai YG - PLoS ONE (2013)

Bottom Line: Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants found in the environment which can be removed through the use of physical and biological agents.The pyrene degrading activity of the strain was strongly functional in all the NaCl concentration states, with the least activity found at 1M (∼70% degraded after 48 hours of cultivation).The expression of these genes as proteins has already been studied in our laboratory using proteomics techniques and this validates our current study.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Sciences, Hanyang University, Ansan, Korea.

ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants found in the environment which can be removed through the use of physical and biological agents. The rate of PAH biodegradation is affected by environmental conditions of pH, salinity and temperature. Adaptation of the pyrene degrading bacteria, Mycobacterium gilvum PYR-GCK, to fluctuating environmental conditions during pyrene biodegrading activity was studied using the quantitative real time - Polymerase Chain Reaction (qRT-PCR) technique. Four aromatic ring-cleavage dioxygenase genes: phdF, phdI, pcaG and pcaH; critical to pyrene biodegradation, were studied in pH states of 5.5, 6.5, 7.5 and NaCl concentrations 0 M, 0.17 M, 0.5 M, 0.6 M, 1 M. First, we conducted a residual pyrene study using gas chromatography and flame ionization technologies. Central to a gene expression study is the use of a valid endogenous reference gene, making its determination our next approach, using the geNorm/NormFinder algorithms. Armed with a valid control gene, rpoB, we applied it to a gene expression study, using the comparative critical threshold (2(ΔΔCT)) quantification method. The pyrene degrading activity of the strain was strongly functional in all the NaCl concentration states, with the least activity found at 1M (∼70% degraded after 48 hours of cultivation). The transcripts quantification of three genes backed this observation with high expression levels. The gene expression levels also revealed pH 6.5 as optimal for pyrene degradation and weak degradation activity at pH of 5.5, corroborating the residual pyrene analysis. The expression of these genes as proteins has already been studied in our laboratory using proteomics techniques and this validates our current study.

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Relative expression levels of the ring-cleavage dioxygenase genes after induction.The graphs of quantified transcript levels after various pHs induction (A–D) and different NaCl concentrations induction (E–H). Gene expression is quantified using qRT-PCR and the comparative critical threshold (2ΔΔCT) method. The rpoB gene was used as the endogenous reference, while the expression in the control sample (without pyrene substrate) was used as the calibrator. Two independent experiments were performed. Vertical bars indicate means from standard deviations of triplicate qRT-PCR analyses.
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pone-0058066-g004: Relative expression levels of the ring-cleavage dioxygenase genes after induction.The graphs of quantified transcript levels after various pHs induction (A–D) and different NaCl concentrations induction (E–H). Gene expression is quantified using qRT-PCR and the comparative critical threshold (2ΔΔCT) method. The rpoB gene was used as the endogenous reference, while the expression in the control sample (without pyrene substrate) was used as the calibrator. Two independent experiments were performed. Vertical bars indicate means from standard deviations of triplicate qRT-PCR analyses.

Mentions: The aim of the best endogenous gene search was to analyze the various gene expression levels of the aromatic ring-cleaving dioxygenase genes (phdF, phdI, pcaG and pcaH) in the different states of NaCl concentration and pH levels with accuracy. With the rpoB gene showing the least expression variation in the preliminary experiments, it was therefore used as an internal control to normalize the gene expression for further studies. The control sample, without any carbon source was used as a calibrator to calculate the relative expression levels. All genes were differently expressed in all the samples as expected. In the pH induced cells, a general upregulated expression was observed at pHs 6.5 and 7.5. Three out of the four genes studied, pcaG, pcaH and phdF, had their activities repressed in the pH 5.5 induced cells, with their highest expression values slightly above the calibrator (Fig. 4A, C and D). An exception was made of phdI with an expression activity level of above 2-fold in the pH 5.5 induced cells (Fig. 4B). At pHs 6.5 and 7.5, phdF and pcaH were highly expressed with phdF attaining an expression value of ∼4-fold after 24 hours of cultivation; while pcaH was ∼3-fold expressed at the 24th and 36th hour of induction. pcaG was lowly expressed in all induction conditions and this phenomenon was echoed in an earlier proteomic study [20] and in a similar pyrene degradation systems biology study using Mycobacterium vanbaalenii PYR1 [25].


A gene expression study of the activities of aromatic ring-cleavage dioxygenases in Mycobacterium gilvum PYR-GCK to changes in salinity and pH during pyrene degradation.

Badejo AC, Badejo AO, Shin KH, Chai YG - PLoS ONE (2013)

Relative expression levels of the ring-cleavage dioxygenase genes after induction.The graphs of quantified transcript levels after various pHs induction (A–D) and different NaCl concentrations induction (E–H). Gene expression is quantified using qRT-PCR and the comparative critical threshold (2ΔΔCT) method. The rpoB gene was used as the endogenous reference, while the expression in the control sample (without pyrene substrate) was used as the calibrator. Two independent experiments were performed. Vertical bars indicate means from standard deviations of triplicate qRT-PCR analyses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585252&req=5

pone-0058066-g004: Relative expression levels of the ring-cleavage dioxygenase genes after induction.The graphs of quantified transcript levels after various pHs induction (A–D) and different NaCl concentrations induction (E–H). Gene expression is quantified using qRT-PCR and the comparative critical threshold (2ΔΔCT) method. The rpoB gene was used as the endogenous reference, while the expression in the control sample (without pyrene substrate) was used as the calibrator. Two independent experiments were performed. Vertical bars indicate means from standard deviations of triplicate qRT-PCR analyses.
Mentions: The aim of the best endogenous gene search was to analyze the various gene expression levels of the aromatic ring-cleaving dioxygenase genes (phdF, phdI, pcaG and pcaH) in the different states of NaCl concentration and pH levels with accuracy. With the rpoB gene showing the least expression variation in the preliminary experiments, it was therefore used as an internal control to normalize the gene expression for further studies. The control sample, without any carbon source was used as a calibrator to calculate the relative expression levels. All genes were differently expressed in all the samples as expected. In the pH induced cells, a general upregulated expression was observed at pHs 6.5 and 7.5. Three out of the four genes studied, pcaG, pcaH and phdF, had their activities repressed in the pH 5.5 induced cells, with their highest expression values slightly above the calibrator (Fig. 4A, C and D). An exception was made of phdI with an expression activity level of above 2-fold in the pH 5.5 induced cells (Fig. 4B). At pHs 6.5 and 7.5, phdF and pcaH were highly expressed with phdF attaining an expression value of ∼4-fold after 24 hours of cultivation; while pcaH was ∼3-fold expressed at the 24th and 36th hour of induction. pcaG was lowly expressed in all induction conditions and this phenomenon was echoed in an earlier proteomic study [20] and in a similar pyrene degradation systems biology study using Mycobacterium vanbaalenii PYR1 [25].

Bottom Line: Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants found in the environment which can be removed through the use of physical and biological agents.The pyrene degrading activity of the strain was strongly functional in all the NaCl concentration states, with the least activity found at 1M (∼70% degraded after 48 hours of cultivation).The expression of these genes as proteins has already been studied in our laboratory using proteomics techniques and this validates our current study.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Sciences, Hanyang University, Ansan, Korea.

ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants found in the environment which can be removed through the use of physical and biological agents. The rate of PAH biodegradation is affected by environmental conditions of pH, salinity and temperature. Adaptation of the pyrene degrading bacteria, Mycobacterium gilvum PYR-GCK, to fluctuating environmental conditions during pyrene biodegrading activity was studied using the quantitative real time - Polymerase Chain Reaction (qRT-PCR) technique. Four aromatic ring-cleavage dioxygenase genes: phdF, phdI, pcaG and pcaH; critical to pyrene biodegradation, were studied in pH states of 5.5, 6.5, 7.5 and NaCl concentrations 0 M, 0.17 M, 0.5 M, 0.6 M, 1 M. First, we conducted a residual pyrene study using gas chromatography and flame ionization technologies. Central to a gene expression study is the use of a valid endogenous reference gene, making its determination our next approach, using the geNorm/NormFinder algorithms. Armed with a valid control gene, rpoB, we applied it to a gene expression study, using the comparative critical threshold (2(ΔΔCT)) quantification method. The pyrene degrading activity of the strain was strongly functional in all the NaCl concentration states, with the least activity found at 1M (∼70% degraded after 48 hours of cultivation). The transcripts quantification of three genes backed this observation with high expression levels. The gene expression levels also revealed pH 6.5 as optimal for pyrene degradation and weak degradation activity at pH of 5.5, corroborating the residual pyrene analysis. The expression of these genes as proteins has already been studied in our laboratory using proteomics techniques and this validates our current study.

Show MeSH
Related in: MedlinePlus