• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1125-1131
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• 2006

A kan'::luxCDABE fusion strain that was both highly bioluminescent and responsive to benzoic acid was constructed by transforming E. coli strain W3110 with the plasmid pUCDK, which was constructed by digesting and removing the 7-kb KpnI fragment from the promoterless luxCDABE plasmid pUCD615. Experiments using buffered media showed that this induction was dependent on the pH of the media, which influences the degree of benzoic acid protonation, and the expression levels seen are likely due to acidification of the cytoplasm by uncoupling of benzoic acid. Consequently, the sensitivity of this strain for benzoic acid was increased by nearly 20-fold when the pH was shifted from 8.0 to 6.5. Benzoic acid derivatives and several phenolics also resulted in significantly increased bioluminescent signals. Although these compounds are known to damage membranes and induce the heat-shock response within E. coli, bacterial strains harboring mutations in the fadR and rpoH genes, which are responsible for fatty acid biosynthesis during membrane stress and induction of the heat-shock response, respectively, showed that these mutations had no effect on the responses observed.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1254-1261
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• 2007

In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate ($R^2=0.98$), 1 to 6 ppm for nitrite ($R^2=0.99$), and 1 to 7 ppm for ammonium ($R^2=0.99$). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.59-62
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• 1999

The recombinant bacteria strain DPD2540, containing a fabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150 min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.103-106
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• 2001

Bioluminescent bacteria fusing the stress promoter and lux gene have been developed as a toxicity biosensor. The light emitting bioluminescent bacteria have been used to measure the toxicity of many different chemicals. In this study, specially, DPD2540 (fabA::luxCDABE) was used to detect and classify phenolic toxicity to the cells membrane fatty acids, and then the relationship between phenolic toxicity and the distribution of various phenols in the cell was determined, with a model and equations provided. In addition, to show the possibility of detecting and classifying the toxicity of a chemical mixture, which may be present in wastewater, various bioluminescent bacteria having different stress promoters were used and their distinct response to the sample mixture was measured. To extend the applicable area of these bioluminescent bacteria to field, the portable biosensor using freeze-drying methods was developed and confirmed successfully.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.212-215
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• 2000

Recombinant bioluminescent bacterial strains that use specific promoters fused to the bioluminescence genes (lux genes) have been applied in environmental monitoring. Advantages of using recombinant bioluminescent bacteria as blosensing cells include rapid responses, low costs, and improved reproducibility. In this study, a recombinant Escherichia coli, GC2, containing a lac::luxCDABE fusion immobilized with solid agar media and glass beads was used to estimate the effect of this soil flushing technique. This bacterium constitutively emits light under normal conditions (no toxic chemicals). When growth and metabolism of these bioluminescent bacteria is inhibited by their exposure to toxic chemicals, the bioluminescence (BL) is reduced. A biosurfactant, rhamnolipids, was used to extract phenanthrene from the soil after flushing.

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.135-135
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• 2008

Various biosensors were evaluated for identifying and detecting foodborne pathogens in a rapid and effective manner. First, five strains of Escherichia coli and six strains of Salmonella were identified using Fourier transform infrared spectroscopy and a statistical program. For doing this, lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) were extracted from a cell wall of each bacterial strain. As a result, each strain was identifed at the level of 97% for E. coli and 100% for Salmonella. Second, E. coli O157:H7, S. Enteritidis, and Listeria monocytogenes were identified by multiplex PCR products from four specific genes of each bacteria using a capillary electrophoresis (CE). Also, ground beef for E. coli O157:H7, lettuce for S. Enteritidis, and hot dog for L. monocytogenes were used to determine the possibility of detecting pathogens in foods. Foods inoculated with respective pathogen were cultivated for six hours and multiplex PCR products were obtained and assessed. The minimum detection levels of tested bacteria were <10 cells/g, <10 cells/g, and $10^4$ cells/g for E. coli O157:H7, S. Enteritidis, and L. monocytogenes, respectively. Third, it was possible to detect S. Typhimurium in a pure culture and lettuce by a bioluminescence-based detection assay using both recombinant bacteriophage P22::luxI and a bioluminescent bioreporter. In addition, bacteriophage T4 was quantitatively monitored using E. coli including luxCDABE genes.

• Journal of Korean Society of Environmental Engineers
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• v.27 no.8
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• pp.900-906
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• 2005

The responses of two luminescence-based biosensors were studied on various heavy metals in aqueous solutions. One was recombinant E. coli ($DH5{\alpha}$/pSB311), genetically modified luminescence-based bacteria, and the other was Vibrio fisheri used for the LumisTox system. The recombinant E. coli was marked with the lux CDABE gene from multicopy plasmid, pACYC184, originally isolated from Photorhabdus luminescens. The $DH5{\alpha}$/pSB311 had a characteristic of no organic substrate for its luminescence reaction. Among the tested heavy metals Zinc and cadmium were less toxic than copper and mercury. The recombinant E. coli was more sensitive to toxicity of heavy metals than the LumisTox. The order of toxicity of the heavy metals to the recombinant E. coli was $Hg^{2+}>Cu^{2+}>Zn^{2+}>Cd^{2+}$. In case of the LumisTox, the order of the toxicity of heavy metals was $Hg^{2+}>Cu^{2+}>Cd^{2+}>Zn^{2+}$. The genetically modified luminescence-based biosensor offers a range of sensitive, rapid, and easy to use methods for assessing the potential toxicity of heavy metals in aqueous samples.

• KSBB Journal
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• v.17 no.6
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• pp.571-576
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• 2002

Recombinant E. coli DH5 ${\alpha}$/pSB311 was made by cloning the genes encoding bacterial luciferase and aldehyde substrate proteins from Photohabdus luminescense, to complement defects of Lumistox, which is normally used in bioassays to monitor toxic substances in water environmental systems. The conditions for stable light production by the recombinant strains were investigated with respect to cell growth stage, cell number, and buffer conditions. The optimum growth stage was a middle-exponential stage with an OD$_{660nm}$ value of 0.6-0.7. ADout 10$^{6}$-10$^{7}$ cells per test tube was optimum for stable light emission. The effect of buffer was not significant if an optimum viable cell number was maintained. The bioluminescence of the recombinant E. coli harboring the lux operon of Photohabdus luminescense was not affected by temperature, while the bioluminescence of Lumistox was temperature sensitive. The recombinant E. coli was more sensitive to heavy metals (Cd, Cu, Hg, Zn) than Lumistox, because it does not require high concentrations of NaCl in the buffer.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.466-469
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• 2004

The recombinant bioluminescent Escherichia coli strains, DPD2511 and TV 1061 containing the katG and grpE promoters, respectively, from Vibrio fischeri fused to luxCDABE, were used to detect the adaptive and repair responses to oxidative damage caused by hydrogen peroxide $(H_2O_2)$, and protein damage due to phenol. The response ratio, represented as the bioluminescence induced in subsequent inductions of DPD2511 and TV1061 with the mother cells previously induced by each chemical, i.e., $H_2O_2$ and phenol during the previous induction stage, decreased suddenly compared with the ratio of the control culture of each strain, meaning there is a possible adaptive response to stress caused by chemicals. Protein damage due to phenol was completely repaired by the second culturing after the initial induction, as was oxidative damage caused by $H_2O_2$ which was also rapidly repaired, as detected by the recovery of bioluminescence level. This result suggests that E. coli promptly adapt and repair oxidative and protein damage by $H_2O_2$ and phenol completely.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1706-1712
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• 2006

Because of the need for new antimicrobial substances with novel mechanisms of action, we report here the use of an Acinetobacter reporter system for high-throughput screening of active antimicrobial agents. The bioreporter Acinetobacter strain DF4/PUTK2 carrying luciferase genes luxCDABE was chosen because of its ecological importance and it is widespread in nature. This bioreporter is genetically engineered to emit light constitutively that can be measured in real time by luminometry. Hence, this reporter system was employed to determine the bacteriostatic actions of spent-culture supernatants derived from twelve bacterial isolates. Out of the results, the strongest bioluminescence inhibitory effect of the supernatants was recorded with Bacillus cereus strain BAC (S5). Subsequently, ethyl acetate extracts of extracellular products of strain BAC (S5) were separated by a thin-layer chromatography (TLC). Based on the bioluminescence inhibitory assay, three fractions were found to have antimicrobial activity. One fraction (C) having the strongest antimicrobial activity was further purified using TLC and characterized by IR, $^1H$ NMR, mass spectrometry, SDS-PAGE, and amino acid composition analysis. The results predicted the presence of 2-pyrrolidone-S-carboxylic acid (PCA) and the octadeconic-acid-like fatty acid. Fraction C also demonstrated a broad inhibitory activity on several Gram-negative and Gram-positive bacteria. In conclusion, the Acinetobacter reporter system shows great potential to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents.