• Journal of Microbiology and Biotechnology
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• v.7 no.5
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• pp.352-355
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• 1997

The promoter region of the pupA gene of Pseudomonas putida WCS358 was fused with the structural genes for bioluminescence (luxCDABE) from Vibrio fischeri, and the resulting fusion plasmid harbored by the WCS358 host. The pup-lux fusion gene was then used for quantitative analysis of the iron-dependence of pupA promoter activity. Factors affecting bioluminescence produced by the pup-lux bioreporter were found to be cell activity, iron-chelator concentrations, Fe(III) concentrations, and nutrient components. Light production rates of the pup-lux bioreporter were inversely dependent upon iron molecules when $FeCl_3$ concentrations were between $10^{-2}$ and 1 ${\mu}M$ in nutrient-poor minimal media, and between 0.1 and 10 mM in nutrient-rich complex media.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1727-1732
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• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• Journal of Soil and Groundwater Environment
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• v.11 no.5
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• pp.43-50
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• 2006

Bioreporter bacteria, such as recombinant bioluminescent bacteria, have been used for the detection of specific compounds in complex environmental media. In this study, optimum conditions for the preparation and application of deep-freezed and Iyophilized recombinant bioluminescent strain KG1206 were investigated for the future application on contaminated environmental sites. Genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, contains TOL plasmid and the plasmid inserted $P_{m}$, promoter on the upper part of lux gone in vector pUCD615, and m-toluate and benzoate are considered direct inducers for bioluminescence. Optimum conditions determined for the preparation and application of the deep-freezed and lyophilized strain were followings: cryoprotective agent (24% sucrose), lyophilization time (12 hrs), strain concentration ($OD_{600}=0.6$), reconstitution for freezed strain (quick reconstitution at $35^{\circ}C$), reconstitution for lyophilized strain ($3{\sim}6$ hrs exposure on LB medium), carrying conditions (keep at $20^{\circ}C$ after reconstitution). These results demonstrate the feasibility of deep-freezed or lyophilized state of genetically engineered bioluminescent strain for environmental usage.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.681-688
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• 2020

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by site-directed mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

• 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 Microbiology and Biotechnology
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• v.28 no.2
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• pp.323-329
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• 2018

In Escherichia coli, the transcription of genes related to metal homeostasis is activated by the presence of target metals. The promoter regions of those genes can be fused with reporter genes to generate whole-cell bioreporters (WCBs); these organisms sense the presence of target metals through reporter gene expression. However, the limited number of available promoters for sensing domains restricts the number of WCB targets. In this study, we have demonstrated an alternative method to generate novel WCBs, based on the notion that since the sensing mechanisms of WCBs are related to metal transportation systems, their properties can be modulated by disrupting metal homeostasis. Mutant E. coli strains were generated by deleting the znt-operon genes zntA, which encodes a zinc-export protein, and zntR, which encodes a znt-operon regulatory protein, to investigate the effects on the metal-sensing properties of WCBs. Deletion of zntA increased the sensitivity but abolished the selectivity of cadmium-sensing WCBs, whereas arsenic-sensing WCBs gained sensitivity toward cadmium. When zntR was deleted, cadmium-sensing WCBs lost the ability to detect cadmium, and this was recovered by introducing exogenous zntR. In addition, the metal-binding site of ZntR was genetically engineered to modulate metal selectivity. This study provides a valuable platform for the development of novel E. coli-based WCBs.