Abstract
Here, we present a new lux-biosensor based on Bacillus subtilis for detecting of DNA-tropic and oxidative stress-causing agents. Hybrid plasmids pNK-DinC, pNK-AlkA, and pNK-MrgA have been constructed, in which the Photorhabdus luminescens reporter genes luxABCDE are transcribed from the stress-inducible promoters of B. subtilis: the SOS promoter PdinC, the methylation-specific response promoter PalkA, and the oxidative stress promoter PmrgA. The luminescence of B. subtilis-based biosensors specifically increases in response to the appearance in the environment of such common toxicants as mitomycin C, methyl methanesulfonate, and H2O2. Comparison with Escherichia coli-based lux-biosensors, where the promoters PdinI, PalkA, and Pdps were used, showed generally similar characteristics. However, for B. subtilis PdinC, a higher response amplitude was observed, and for B. subtilis PalkA, on the contrary, both the amplitude and the range of detectable toxicant concentrations were decreased. B. subtilis PdinC and B. subtilis PmrgA showed increased sensitivity to the genotoxic effects of the 2,2′-bis(bicyclo [2.2.1] heptane) compound, which is a promising propellant, compared to E. coli-based lux-biosensors. The obtained biosensors are applicable for detection of toxicants introduced into soil. Such bacillary biosensors can be used to study the differences in the mechanisms of toxicity against Gram-positive and Gram-negative bacteria.
Highlights
For testing chemical impurities and other biologically active substances, primarily medications, luminescent biosensor cells are currently used in two ways: (1) based on bioluminescence quenching [1,2,3], and (2) based on bioluminescence induction [4,5,6,7,8,9,10,11,12,13]
The characteristics of B. subtilis 168 pNK-DinC were investigated using the antibiotic mitomycin C (MitC), a well-known drug inducing the SOS-response in E. coli cells [27]
After MitC was added to final concentrations of 10 μM, 1 μM, 100 nM, and 10 nM, cells were incubated at room temperature for 3 h with periodic luminescence measurements
Summary
For testing chemical impurities (toxins) and other biologically active substances, primarily medications, luminescent biosensor cells (lux-biosensors) are currently used in two ways: (1) based on bioluminescence quenching [1,2,3], and (2) based on bioluminescence induction [4,5,6,7,8,9,10,11,12,13]. The construction of a lux operon that is efficiently expressed in Gram-positive bacteria is complicated due to the need to replace the Shine–Dalgarno sequence upstream of each reading frame. Such works were carried out and luminescent bacteria of Streptococcus, Staphylococcus, Bacillus, and a number of other genera were obtained, which were used mainly for medical applications [2,15,16]. The luminescence of these bacteria was constitutive, and it was used to determine the integral effect of biologically active substances on bacterial cells
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