Application and Construction of Microbial Biosensors in Chemical Forensics

<p>Forensic toxicologists are often required to rapidly determine if a suspicious substance, such as a white powder, contain toxins. Preliminary tests usually include screens for a wide range of 'Potentially Toxic Chemicals' (PTCs) such as cyanide, pesticides, herbicides, medicinal and illicit drugs. Subsequent analyses are generally very time-consuming and costly. Any protocol screening for a range of PTC's, prior to more robust chemical analysis, could therefore save significant analytical time. Microbial biosensors are ideal biological tools that can be utilised for these purposes. In vivo bioassays were developed for a range of PTCs using a suite of microbial biosensors, in a variety of complex matrices including water, white powders, soils and vomit to determine the effect of matrix complexities on the biosensors, as well as the toxins. The lux biosensor, Escherichia coli HB101 pUCD607, showed an EC50, (where EC50 is the effective concentration of toxin causing 50% reduction in bioluminescence), of cyanide in water of 20 mg/L. This biosensor still detected cyanide, in talc and flour, at EC50 values of 589 mg/L and 700 mg/L respectively. Vibrio harveyi showed good sensitivity to cyanide in initial water bioassays with an EC50 of 9.66 mg/L. The V. harveyi biosensor did not detect cyanide spiked in talc or flour when tested up to a maximum concentration of 10,000 mg/L. The Mycena citricolor ATCC 34884 fungal biosensor, showed lower sensitivity levels however it detected the presence of sodium monofluoroacetate (1080) at a concentration 1000 mg/L. Preliminary investigation of a novel, faster, solid-phase sample preparation method was also undertaken and its potential proven, particularly in PTC spiked white powders. Here the biosensor showed sensitivity to arsenate, arsenite, copper, cyanide and PCP at 1000 mg/L.This project highlighted the inability of current biosensors to reliably detect 1080 and the difficulty in constructing a specific biosensor. The utilisation of a reliable vector and inducible promoter are pivotal in biosensor construction.</p>

Application and Construction of Microbial Biosensors in Chemical Forensics

<p>Forensic toxicologists are often required to rapidly determine if a suspicious substance, such as a white powder, contain toxins. Preliminary tests usually include screens for a wide range of 'Potentially Toxic Chemicals' (PTCs) such as cyanide, pesticides, herbicides, medicinal and illicit drugs. Subsequent analyses are generally very time-consuming and costly. Any protocol screening for a range of PTC's, prior to more robust chemical analysis, could therefore save significant analytical time. Microbial biosensors are ideal biological tools that can be utilised for these purposes. In vivo bioassays were developed for a range of PTCs using a suite of microbial biosensors, in a variety of complex matrices including water, white powders, soils and vomit to determine the effect of matrix complexities on the biosensors, as well as the toxins. The lux biosensor, Escherichia coli HB101 pUCD607, showed an EC50, (where EC50 is the effective concentration of toxin causing 50% reduction in bioluminescence), of cyanide in water of 20 mg/L. This biosensor still detected cyanide, in talc and flour, at EC50 values of 589 mg/L and 700 mg/L respectively. Vibrio harveyi showed good sensitivity to cyanide in initial water bioassays with an EC50 of 9.66 mg/L. The V. harveyi biosensor did not detect cyanide spiked in talc or flour when tested up to a maximum concentration of 10,000 mg/L. The Mycena citricolor ATCC 34884 fungal biosensor, showed lower sensitivity levels however it detected the presence of sodium monofluoroacetate (1080) at a concentration 1000 mg/L. Preliminary investigation of a novel, faster, solid-phase sample preparation method was also undertaken and its potential proven, particularly in PTC spiked white powders. Here the biosensor showed sensitivity to arsenate, arsenite, copper, cyanide and PCP at 1000 mg/L.This project highlighted the inability of current biosensors to reliably detect 1080 and the difficulty in constructing a specific biosensor. The utilisation of a reliable vector and inducible promoter are pivotal in biosensor construction.</p>

Constructing of Bacillus subtilis-Based Lux-Biosensors with the Use of Stress-Inducible Promoters

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.

Deuterium oxide enhances the SOS response of Escherichia coli induced by bactericidal agents

Изучали генотоксическое действие бактерицидных средств диоксидина, фурацилина и налидиксовой кислоты на клетки дейтерированной культуры lux-биосенсора E.coli MG1655 (pColD::lux), люминесцирующего в результате активации промотора гена колицина colD в ответ на повреждение ДНК. Впервые показано, что оксид дейтерия (D2O) в концентрации 9% усиливает SOS-ответ, индуцированный исследуемыми лекарственными препаратами, в 1,6-2,8 раза в клетках E. coli. We studied the genotoxic effect of bactericidal agents: dioxine, furaciline and nalidixic acid on cells of the deuterated culture lux-biosensor E. coli MG1655 (pColD::lux), which luminesces as a result of activation of the colicin gene promoter colD in response to DNA damage. For the first time, it was shown that deuterium oxide (D2O) at a concentration of 9% increases the SOS response by 1.6-2.8 times in E. coli cells induced by the studied drugs.

ASSESSING ANTIOXIDANT AND PROOXIDANT PROPERTIES OF LACTOBACILLI AND ENTEROCOCCI STRAINS BY USING ORAC AND LUX-BIOSENSOR METHODS

Lactobacilli are widely used in medicine as probiotic bacteria. Lactobacilli are considered one of the most important types of intestinal microbes. These bacteria have an antioxidant, gene protective effect on the immune and nervous systems of the host. But some strains of enterococci can be pathogenic microorganisms and cause diseases such as urinary tract infections, bacteremia, infections at the surgical sites, bloodstream infections, diarrhea. The need to study the beneficial and harmful properties of bacteria for humans determines the relevance of the study. The antioxidant and prooxidant properties of 11 Enterococcus strains and 7 metabolites of Lactobacillus strains were analyzed using an oxygen radical absorption capacity (ORAC) analysis and a lux biosensor test. Bacteria were incubated in cow's milk. Unfermented milk was used as a control. A study showed that milk fermented with enterococci did not have significant differences in antioxidant ability compared to control. In contrast, almost all lactobacilli increased the antioxidant ability of sour milk compared to unfermented milk. That is, the metabolites of Lactobacillus strains have demonstrated strong antioxidant properties even at low concentrations. The metabolites of Enterococcus strains possessed prooxidant properties. They enhanced the action of other prooxidants, such as paraquat, hydrogen peroxide, dioxidine, and showed a synergistic effect. The Lux biosensor test, used to evaluate the effect of substances on living cells with a complex metabolism, was more informative than the ORAC analysis, which allowed to evaluate the antioxidant and prooxidant properties of probiotic bacteria metabolites. The study revealed not only the influence of the direct effect of the test substance on the target molecule, but also the effect of indirect action by interfering with other biochemical processes of a living cell, which confirmed the need to use the biosensor lux test for further work when choosing strains of probiotic bacteria.

Toxic effect of 2,2’-bis(bicyclo[2.2.1] heptane) on bacterial cells

ABSTRACTHere we present the study of the genotoxic effect of a 2,2’-bis(bicyclo[2.2.1]heptane) (BBH), which is promising as a fuel component for liquid rocket engines. The use of Escherichia coli lux-biosensors showed that in addition to DNA damage causing SOS-response, there is also an oxidative effect on cells. The greatest toxicity is determined by the mechanism of formation of superoxide anion radical and is detected by the lux biosensor E. coli pSoxS-lux, in which the genes of bacterial luciferases are transcriptionally crosslinked with the promoter of the soxS gene. It is assumed that the oxidation of BBH leads to the formation of reactive oxygen species, which should give the main contribution to the toxicity of this substance.

Extracts of medical plants suppress the SOS response and reduce mutagenesis in E. coli

One of the promising directions in the fight against the emergence and spread of farm animal microbiota resistance factors is the development and search for feed additives that can inhibit the SOSresponse. SOS-response is one of the main mechanisms of the occurrence of mutations in bacteria. Plants used in traditional medicine can be a promising source of safe substances that reduce the SOS-response of bacteria. A screening of plants potentially containing substances with antiSOS activity was performed. During the initial screening, the E. coli MG 1655 pRecA-lux biosensor strain with ciprofloxacin as RecA inducer was used. Seven plants were identified whose extracts reduced the expression of the RecA operon. In further experiments on bacteria exposed to antibiotics, we identified four plants whose exstracts significantly reduced the mutagenesis rate of clinical E. coli strains: Austrian broom (Cytisus austriacus), greater celandine (Chelidonium majus), walnut (Juglans regia) and smooth sumac (Rhus glabra).