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Using Molecular Methods to Identify and Monitor Xenobiotic-Degrading Genes for Bioremediation
Published in Ederio Dino Bidoia, Renato Nallin Montagnolli, Biodegradation, Pollutants and Bioremediation Principles, 2021
Edward Fuller, Victor Castro-Gutiérrez, Juan Carlos Cambronero-Heinrichs, Carlos E. Rodríguez-Rodríguez
Transposons are mobile elements capable of random insertion within a host’s genome. These mobile elements can therefore be used as powerful molecular tools to disrupt genes throughout the genome, whilst simultaneously inserting selectable markers into the host genome to identify mutated genes. The advantage of this insertional mutagenesis approach, relative to random mutagenesis, is a reduced lethality rate and a potentially higher mutation rate (Seifert et al. 1986). Transposon mutagenesis has been used successfully to identify numerous catabolic genes capable of degrading dicholprop, long chain alkanes, and rubber, amongst other compounds (Schleinitz et al. 2004, Throne-Holst et al. 2007, Kasai et al. 2017, Qiu et al. 2018). As transposons are typically transformed into the host cell through a suicide vector, the main limitation regarding this approach is that it is largely restricted to naturally competent microorganisms (Rabausch et al. 2013).
Pesticide induced up-regulation of esterase and aldehyde dehydrogenase in indigenous Bacillus spp.
Published in Bioremediation Journal, 2019
Pankaj Bhatt, Saurabh Gangola, Parul Chaudhary, Priyanka Khati, Govind Kumar, Anita Sharma, Anjana Srivastava
Upregulation of pesticide induced genes occurs due to the synthesis of specific transcription factors during stress conditions. Expression of the est gene was observed in this study but their expression levels were different in different bacteria. Bacterial strains SG4 and Sulfo3 showed higher level of expression as compared to other and were approximately 30- and 60-fold, respectively, than the control. Similarly Inoue et al. (2016) predicted that monooxygenase (α-subunit) plays a role in the biodegradation of 1,4-dioxane and tetrahydrofuran. Genes encoding periplasmic binding proteins are over-expressed which indicate that bacterial cells can take up all types of carbon sources during glucose limited conditions in E. coli. It means that during glucose scarcity bacterial cells can easily uptake the pesticides through membrane and degrade it using cellular metabolism. Unregulated gene expression during glucose limited condition was reported by Alessandro and Thomas 2006). The results were confirmed by RT-PCR. According to literature till date more than 300 functional genes playing important role in catabolism of toxic aromatic compounds have been characterized from culturable bacteria using genetic engineering. Different strategies like shotgun cloning using indigo formation (Ensley et al. 1983; Goyal and Zylstra 1996; Sato et al. 1997), meta-cleavage activity based technique, cloning, proteomics (2-D electrophoresis) of xenobiotic-inducible proteins to get genetic information (Khan et al. 2001; Pankaj et al. 2016a, 2016b), transposon mutagenesis to attain defective mutants (Fought et al. 1996), use of degenerate primers (Saito et al. 2000), and application of a short probe from a similar gene (Moser and Stahl 2001), have been approached to locate catabolic genes to degrade aromatic xenobiotic compounds in different bacteria. Application of RT-PCR with different primer set to identify useful catabolic genes directly from environmental samples has been documented by Okuta, Ohnishi, and Harayama (1998). Fragments of catechol 2,3-dioxygenase (C23O) genes were identified from environmental samples by PCR using degenerate primers.