Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster.

Satamita Deb,Soumik Basu,Achintya Singha,Tapan K Dutta

doi:10.3389/fmicb.2018.00254

Abstract

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and para-isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.

Highlights

Various anthropogenic impacts based on agricultural and industrial practices, often introduce a variety of organic pollutants including nitroaromatic compounds into the environment at a concentration toxic to existing life forms (Kovacic and Somanathan, 2014)
We report the construction of a highly specific and sensitive 2NBA-sensing bioreporter by strategically integrating a reporter gene into 2NBA-inducible gene cluster of strain ST-14 based on the structural gene information, even without the need for identifying the exact location of the associated promoter-operator sequence or the role of the encoded regulatory protein
The BRprox and BRdistmutants of strain ST-14 were constructed by disrupting the chromosomal onbFCA and onbIGDB gene segments with the incorporation of the fluorescent reporter gene egfp by homologous recombination using the protocols as described in the ‘Materials and Methods’ section

Summary

Introduction

Various anthropogenic impacts based on agricultural and industrial practices, often introduce a variety of organic pollutants including nitroaromatic compounds into the environment at a concentration toxic to existing life forms (Kovacic and Somanathan, 2014). Among the wide array of nitroaromatic compounds, nitrobenzoates are widely used in the production of dyes, plastics, explosives, pharmaceuticals, polyurethane foams, elastomers, and pesticides (Peres and Agathos, 2000; Ju and Parales, 2010). These compounds are lethal to living beings because of their genotoxicity, mutagenicity, and hematologic toxicity (Grummt et al, 2006; Arora and Sharma, 2015). In spite of being toxic and lethal to higher organisms, bacteria, the most ubiquitous organism in the ecosystem, encounter such pollutants soon after they get released into environment, and often evolve in order to survive, by expanding their capability to utilize these pollutants as carbon sources. The degradative ability of bacteria is often exploited to clean up

Methods

Results

Conclusion