Abstract
Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given environmental stress substance. In this study, we utilized Escherichia coli (E. coli) as a model system for the preparation of both cell lysates and genomic DNA fragments. Through enriching protein-bound DNA fragments to construct luciferase reporter libraries, we found that, of 280 clones collected and sequenced, 131 clones contained either the promoter-35 and -10 conservative sequences and/or an operator transcription factor binding sites (TFBS) region. To demonstrate the functionality of the identified clones, five of 131 clones containing LexA binding sequence have been demonstrated to be induced in response to mitomycin C treatment. To evaluate our libraries as a functional screening library, 80 randomly picked up clones were cultured and treated with and without MMC, where two clones were shown to have greater than twofold induction. In addition, two arsenite-responsive clones were identified from 90 clones, one having the well-known ArsR and another having the osmotically inducible lipoprotein (OsmE1). The newly discovered osmE1 has been quantitatively validated to be induced by arsenite treatment with real-time PCR in a dose response and time course manner. This enriching protein-bound DNA luciferase reporter libraries and functional screening facilitate the identification of stress-responsive transcriptional factors in microbes. We developed functional libraries containing E. coli genomic-wide protein-bound DNA as enhancers/operators to regulate downstream luciferase in response to stress.
Highlights
Microbes are highly adaptable to environmental toxic stress such as heavy metals, pesticides, and polychlorinated biphenyls (PCBs) (Chowdhury et al 2018; Caine 2012)
Bacteria biosensors act as a new class of detectors to produce a detectable signal upon activation of a promoter reporter gene induced by specific stimuli, which have been used for monitoring environmental pollutants such as heavy metals or pesticides (Gutiérrez et al 2015)
This study presents a novel approach to enriching protein-bound genomic DNA fragments for the construction of luciferase libraries conducting directly functional screening to identify substance-responsive transcription factor binding sites (TFBS) elements
Summary
Microbes are highly adaptable to environmental toxic stress such as heavy metals, pesticides, and polychlorinated biphenyls (PCBs) (Chowdhury et al 2018; Caine 2012). The adaptation to changes in their environment is controlled by the induction or repression of gene expression (Balleza et al 2009; Cases et al 2003). Environmental genomic toxic stresses such as certain types of chemical reagents and UV irradiation can cause changes in gene expression and cellular metabolism of microbe (Foster 2007). The distinguishing feature of these genes is the presence within the promoter region of a binding sequence for transcriptional repressors, such as LexA (Butala et al 2009) and ArsR (Chen et al. Li et al AMB Expr (2020) 10:199. In response to any DNA damage, the LexA repressor undergoes dissociation from its binding sequences and activate DNA repair genes (Butala et al 2009). The protein dissociates from the promoter, subsequently activating relevant gene expression (Shi et al 1994). Many toxic substances and their corresponding genes are not well characterized due to lack of simpler and more efficient methods
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
