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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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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.
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pone-0058066-g003: 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.

Mentions: GeNorm calculates expression stability value (M) for each candidate gene based on pairwise comparisons of variability. Each gene is compared to every other gene to determine two genes with the least variation and the stability value is used to rank genes from the most stable to the least stable. The authors of the method give an M-value of 1.5 as a cut-off for suitability of an endogenous control gene [32] and all the genes used for this study were well below the cut-off value (Fig. 2). Gene expression levels of candidate endogenous control genes are displayed in Fig. 3. The most stably expressed gene identified by the geNorm software was rpoB.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0058066-g003: 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.
Mentions: GeNorm calculates expression stability value (M) for each candidate gene based on pairwise comparisons of variability. Each gene is compared to every other gene to determine two genes with the least variation and the stability value is used to rank genes from the most stable to the least stable. The authors of the method give an M-value of 1.5 as a cut-off for suitability of an endogenous control gene [32] and all the genes used for this study were well below the cut-off value (Fig. 2). Gene expression levels of candidate endogenous control genes are displayed in Fig. 3. The most stably expressed gene identified by the geNorm software was rpoB.

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