• Tuberculosis and Respiratory Diseases
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• v.82 no.2
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• pp.143-150
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• 2019

Background: The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto). Methods: A total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay. Results: The katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations. Conclusion: It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

• 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.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.461-465
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• 2006

We investigated the protein productivity of the promoters for genes showing prolonged induction upon cold shock in Escherichia coli. Six low temperature inducible genes (frdA, glpB, hypB, katG, nupG, ompT) were selected based on the previously reported cDNA microarray based global transcription profiling of Escherichia coli Kl2 in response to cold shock. Their promoter regions were isolated from the genomic DNA of E. coli JM109 and expression levels induced by the promoters were examined by using green fluorescence protein (GFP) as a reporter at $15^{\circ}C$ and $37^{\circ}C$. Among the six promoters, the promoter for nupG showed the highest and prolonged expression at both temperatures and the cold inducibility of nupG promoter was not observed.

• Biomedical Science Letters
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• v.11 no.4
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• pp.455-463
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• 2005

Resistance to isoniazid (INH), which is one of the most important drugs in Mycobacterium tuberculosis chemotherapy, has been associated with mutations in genes encoding the mycobacterial catalse-peroxidase (katG), the enoyl acyl carrier protein (ACP) reductase (inhA), alkyl hydroperoxide reductase (ahpC), beta-ketoacyl acyl carrier protein synthase (kasA), and NADH dehydrogenase (ndh). In this study, we examined INH-resistance related genes in 50 INH-resistant and 24 INH-susceptible isolates by PCR-sequence analysis. In brief, mutations at the katG gene were found at codon 315 alone (2/50), at codon 463 alone (19/50), and both at 315 and 463 (29/50). However, while mutations at codon 315 were only detected in INH-resistant isolates, mutations at codon 463 were also detected in INH-susceptible isolates indicating mutations at 463 alone do not seem to confer resistance to INH. Similar to the case of katG 463, some of inhA mutations were also found among INH-susceptible isolates. For example, whereas mutations at 8 upstream of the start codon (UPS) and 15 UPS of the inhA gene were detected only in INH-resistant isolates, mutations at 101, 115, and 125 UPS were detected only in INH-susceptible isolates. Many different kinds of mutations were detected in INH­resistant isolates at ahpC, oxyR gene, and intergenic region of the oxyR-ahpC genes. Howerver, the mutations were not found oxyR and the intergenic regions in INH-susceptible isolates. No mutations were found at either kasA or at ndh gene among INH-resistant isolates. In conclusion, some of mutations such as katG 315, inhA promotor region, and oxyR-ahpC seem to be strongly related to INH-resistance. Currently we are developing a molecular diagnostic method based on these results.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1390-1393
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• 2007

In this study, three of the representative EDCs, $17{\beta}$-estradiol, bisphenol A, and styrene, were employed to find their mode of toxic actions in E. coli. To accomplish this, four different stress response genes, recA, katG, fabA, and grpE genes, were used as a representative for DNA, oxidative, membrane, or protein damage, respectively. The expression levels of these four genes were quantified using a real-time RT-PCR after challenge with three different EDCs individually. Bisphenol A and styrene caused high-level expression of recA and katG genes, respectively, whereas $17{\beta}$-estradiol made no significant changes in expression of any of those genes. These results lead to the classification of the mode of toxic actions of EDCs on E. coli.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.187-190
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• 2000

Effects of gamma ionizing radiation on recombinant Escherichia coli cells containing stress promoters, recA, fabA, grpE, or katG, fused to luxCDABE originated from Vibrio fischeri were characterized by monitoring transcriptional responses reflected by bioluminescent output. Quantification of gamma-ray intensity may be possible using the recA and fabA promoter fusion since a linear enhancement of bioluminescence emission with increasing gamma-ray intensity was observed. Other strains sensitive to either oxidative stress (DPD2511, katG::luxCDABE) or protein-damaging stress (TV1061, grpE::luxCDABE) were also irradiated by gamma-rays, and resulted in no noticeable bioluminescent output while DPD2794 with recA promoter and DPD2540 with fabA promoter irradiated by the same intensities of gamma-rays gave a significant bioluminescent output. This indicates that the main stresses in the recombinant bacteria caused by ionizing radiation are DNA and membrane-damage, not protein- or oxidative-damage. In addition, in this study, to investigate the relationship between the radiation dose rate and bacterial responses, two recombinant Escherichia coli strains, DPD2794 and GC2, containing lac promoter fused to luxCDABE originating from Photorhapdus luminescences, were used for detecting DNA damage and cellular toxicity under various radiation dose rates. Throughout this study, it was found that these bacteria showed quantitative stress responses to DNA damage and general toxicity caused by gamma rays, depending on the radiation dose rates, indicating that the bacterial stress responses and general toxicity were seriously influenced according to radiation dose rates.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.101-105
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• 2003

Recombinant bioluminescent bacteria were used to monitor and classify the to xicity of azo dyes. Two constitutive bioluminescent bacteria, Photobacterium phosphoreum and Es-Cherichia coli, E, coli GC2 (lac::luxCOABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminestent E. coli, DPD2794 (recA::luxCDABE), a DNA damage Sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classific ation of some azo dyes, In the field, may be possible using these recombinant bioluminescent bacteria.

• Journal of Microbiology
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• v.35 no.4
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• pp.277-283
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• 1997

Promoters inducible by paraquat, a superocide-generating agent, were isolated from Escherichia coli using a promoter-probing plasmid pRS415 with promoterless lacA gene. Twenty one promoters induced by paraquat were selected and further characterized. From sequence analysis, thirteen of the promoters were mapped to their specific loci on the Escherichia coli chromosome. Several promoters were mapped to the upstream of known genes such as usgl, katG, and mglB, whose relationships with superoxide response have not been previously reported. Other promoters were mapped to the upstream region of unknown open reading frames. Downstream of HC 96 promoter are uncharacterized ORFs whose sequences are homologous to ABC-transporter subunits. Downstream of HC84 promoter is an ORF encoding a transcriptional regulator-like protein, which contains a LysR family-specific HTH (helix-turn-helix) DNA bindign motif. We investigated whether these promoters belong to the soxRS regulon. All promoters except HC96 were found to belong to the soxRS regulon. The HC96 promoter was significantly induced by paraquat in the soxRS deletion mutant strain. The basal transcription level of three promoters (HE43, HC71, HD94) significantly increased at the stationary phase, implying that they are regulated by RpoS. However, paraquat inducibility of all promoters disappeared in the stationary phase, suggesting that SoxRS regulatory system is active only in rapidly growing cells.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.6
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• pp.395-399
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• 2000

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.