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(P347) Quantitative Estimation of Expression and Substrate Specificity of Naphthalene Dioxygense of Pseudomonas putida by Real-Time PCR.
Narasimmalu, Rajendran*,1, Urushigawa, Yoshikuni2, Kanazawa, Nobuhiro2, 1 Yokohama National University, Yokohama, Kanazawa Pref., Japan2 Akita Prefectural University, Honjo, Akita Pref., Japan
ABSTRACT- Quantitative estimation of catabolic gene expression can be useful to efficiently monitor microbial activity in polluted environments. The objectives of the present study are to quantify the naphthalene degrading gene (nahAc) of Pseudomonas putida grown in the presence of different substrates by Real-time PCR which is a precise and sensitive method for quantification. Bacterial culture was carried out using mineral medium supplemented with different substrates (naphthalene, succinate, salicylate and glucose) and their growth was monitored regularly. Samples collected at a regular interval of 5h were analyzed for total organic carbon (TOC), and RNA and nahAc gene. Growth, as measured at 610nm, showed distinct variations among the substrates. Marked variations in TOC, which is an indicator of available organic carbon were observed for the different substrates. RNA extracted from cells showed a gradual increase during the experiment for all the substrates indicating that cells have high RNA levels at increased growth rates. Gene copy numbers, as quantified by Real-time PCR, varied from 103 to 106/ng RNA, indicating that the nahAc gene of P. putida was expressed in the presence of different substrates. High number of nahAc gene copies in naphthalene grown culture during the first 20h indicated the successful induction of naphthalene dioxygenase, whereas, low gene copy numbers in glucose added medium suggested that glucose might not be able to induce the gene expression. Gene copies in salicylate grown culture reached the maximum number at 25h and then sustainable gene expression was observed till 40h, indicating that salicylate is a potential inducer. Succinate was also found to induce the gene expression that can be related to the growth. These variations in gene copy numbers revealed substrate specificity of nahAc gene expression. The proportions of gene copies in total number of bacteria (16SrRNA) were also determined. From these results, it can be suggested that quantification of gene expression is essential to better monitoring of degradation of the pollutants and also to evaluating the effectiveness of bacteria in bioremediation processes.
Key words: Real-time PCR, Catabolic gene, Naphthalene, Substrate
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