Real-time reverse transcription PCR analysis of trichloroethylene-regulated toluene dioxygenase expression in Pseudomonas putida F1

Abstract

Toluene dioxygenase (tod) is a multicomponent enzyme system in Pseudomonas putida F1. Tod can mediate the degradation of Trichloroethylene (TCE), a widespread pollutant. In this study, we try to explore the TCE-regulated tod expression by using real-time qRT-PCR. The minimal culture media were supplemented with glucose, toluene, or a mixture of glucose/toluene respectively as carbon and energy sources. The TCE was injected into each medium after a 12-hour incubation period. The TCE injection severely affected bacterial growth when cultured with toluene or toluene/glucose mixtures. The cell density dropped 61 % for bacteria growing in toluene and 36 % for bacteria in the glucose/toluene mixture after TCE injection, but the TCE treatment had little effect on bacteria supplied with glucose alone. The decrease in cell number was caused by the cytotoxicity of the TCE metabolized by tod. The results from the real-time qRT-PCR revealed that TCE was capable of inducing tod expression in a toluene-dependent manner and that the tod expression level increased 50 times in toluene and 3 times in the toluene/glucose mixture after 6 hours of TCE treatment. Furthermore, validation of the rpoD gene as a reference gene for P. putida F1 was performed in this study, providing a valuable foundation for future studies to use real-time qRT-PCR in the analysis of the P. putida F1 strain.

Keywords:

• Toluene dioxygenase
• Pseudomonas putida
• trichloroethylene
• bioremediation
• tod
• rpoD

Acknowledgment

This work was supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology's Global COE Program entitled “Global Eco-risk management from Asian Viewpoints”. We wish to express our sincere gratitude for financial support from of the Ministry of Education, Culture, Sports, Science and Technology.