• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• The Journal of Korean Physical Therapy
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• v.10 no.2
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• pp.113-125
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• 1998

The neurotropic psudorabies virus(PRV) to replicate within neurons is very useful pathogen for neuronal tracing. I carried out this study to investigate the parametric analysis on the viral infection in the rat circadian control center with two genetically engineered strains out of PRV. The two strains are isogenic with the attenuated Bartha strain of PRV ; in one strain a lacZ reporter gene was inserted into the gC locus (PRV-BaBlu ; $4.75\times10^8pfu/ml$) and the other strain contained a PRV envelope glycoprotein gene(PRV-D ; $2.5\times10^8pfu/ml$) theat is absent in PRV-BaBlu. simultaneous or temporally separated sequential injection of$2{\mu}l$ of each strain into the vetreous body of eye produced a course of transsynaptic infection of retinohypothalamic circuitry. The results were as follows; 1. PRV-BaBlu and PRV-D infected the suprachiasmatic nucleus in hypothalamus and intergeniculate leaflet in lateral geniculate nucleus of thalamus. 2. The rate of PRV infection was dependent upon PRV strain. 3. Pre-infected neurons by PRV-D were interfered with the replication of PRV-BaBlu. 4. Dual injection of PRV-D and PRV-BaBlu showed more virulent than the parental strain.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.3
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• pp.202-206
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• 2000

The genetically-engineered Escherichia coli strain, DPD2540, which contains a fabA:::luxCDAbefusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more pactical application of this strain in the filed as biosensor, freezedrying was adopted. A 12% surcrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be most most effective composition for lyophilization solution among various compositions testitons tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at-7$0^{\circ}C$ and -2$0^{\circ}C$. The biosensing activities of the cells showed a greater sensitivity when the cells from the expontial phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biodencor field was determined.

• Journal of Korean Society of Environmental Engineers
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• v.27 no.5
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• pp.507-512
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• 2005

This research focuses on the development and application of a method for the detection of m-toluate in soils using a genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 produces light by direct (m-toluate and benzoate) and indirect (toluene analogs) inducers. For detection of m-toluate in soil system, 9.9 mL strain was amended with 0.1 mL soil ethanol extractant. A high correlation ($r^2>0.97$) was observed between bioluminescence and m-toluate concentration. The unknown concentrations of m-toluate in soil samples were pre-determined using a method developed based on bioluminescence activity of strain with extracted inducers. Values between by LC analysis and bioluminescence activity show moderate statistical results. These results demonstrate the feasibility of recombinant bioluminescent microorganism, engineered to generate a quantifiable bioluminescence signal in response to specific pollutants, may serve as combined sensing and reporting tools in environmental monitoring.

• Journal of Microbiology
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• v.34 no.4
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• pp.320-326
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• 1996

To analyze the effects of host versus plasmid on survival of 2, 4-degrading bacteria in environmental samples, strains Pseudomonas cepacia/pJP4, Alcaligenes JMP228/pJP4, P. cepacia/p712, and Alcaligenes JMP228/p712 were separately inoculated into samples of field soil, paddy soil, lake water, and river water, and then the changes of their populations were measured. The strains used contained a 2, 4-D degradative plasmid, either pJP4 conferring fast-growing property to the host or p712 conferring slow-growing property, and were resistant to antibiotics such that the inoculated strains could be enumerated against the indigenous microbial populations. In sterile environmental samples, these strains were stably maintained at the levels used for inoculation, except in sterile paddy soil where Alcaligenes JMP228 strains died drapidly. In natural soil samples for four strains declined steadily with time, but in naturla water samples their polulations fell rapidly at the early phase and then remained almost constant. When the environmentla samples were treated with 2, 4-D, P. cepacia/pJP4 and P. cepacia/p712 maintained significant numbers, while Alcaligenes JMP228/pJP4 and Alcaligenes JMP228/p712 declined significantly in most of the samples. The results indicated that the survivability of genetically modified microorganisms could vary depending on the environments and that their abundance in the environments under s2, 4-D selection was markedly influenced by the nature of the 2, 4-D degradative plasmid as well as type of the host strain.

• Journal of Periodontal and Implant Science
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• v.52 no.2
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• pp.141-154
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• 2022

Purpose: C57BL/6 mice, which are among the most common backgrounds for genetically engineered mice, are resistant to the induction of periodontitis by oral infection with periodontal pathogens. This study aimed to develop a periodontitis model in C57BL/6 mice using coaggregation between human pathogens and the mouse oral commensal Streptococcus danieliae (Sd). Methods: The abilities of Porphyromonas gingivalis ATCC 33277 (Pg33277), P. gingivalis ATCC 49417 (Pg49417), P. gingivalis KUMC-P4 (PgP4), Fusobacterium nucleatum subsp. nucleatum ATCC 25586 (Fnn), and F. nucleatum subsp. animalis KCOM 1280 (Fna) to coaggregate with Sd were tested by a sedimentation assay. The Sd-noncoaggregating Pg33277 and 2 Sd-coaggregating strains, PgP4 and Fna, were chosen for animal experiments. Eighty C57BL/6 mice received oral gavage with Sd once and subsequently received vehicle alone (sham), Fna, Pg33277, PgP4, or Fna+PgP4 6 times at 2-day intervals. Mice were evaluated at 5 or 8 weeks after the first gavage of human strains. Results: Fnn, Fna, and PgP4 efficiently coaggregated with Sd, but Pg33277 and Pg49417 did not. Alveolar bone loss was significantly higher in the PgP4 group at both time points (weeks 5 and 8) and in all experimental groups at week 8 compared with the sham group. The PgP4 group presented greater alveolar bone loss than the other experimental groups at both time points. A higher degree of alveolar bone loss accompanied higher bacterial loads in the oral cavity, the invasion of not only PgP4 but also Sd and Fna, and the serum antibody responses to these bacteria. Conclusions: Periodontitis was successfully induced in C57BL/6 mice by oral infection with a P. gingivalis strain that persists in the oral cavity through coaggregation with a mouse oral commensal bacterium. This new model will be useful for studying the role of human oral bacteria-host interactions in periodontitis using genetically engineered mice.

• The Journal of Korean Physical Therapy
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• v.23 no.5
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• pp.35-41
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• 2011

Purpose: This study was carried out to investigate the spatiotemporal localization of the amygdaloid nucleus innervating the rat stomach using PRV-BaBlu, which has been known to be an excellent type of neurotracer with the ability to transpass the neuronalsynaptic cleft. Methods: Ninety Sprague-Dawley rats (250~300 g) that were injected with PRV-BaBlu into the stomach were randomly divided into 3, 4 and 5 day groups (each group n=30). $2{\mu}l$ of PRV-BaBlu, a genetically modified strain of PRV-Bartha with the lac-Z gene,was injected into the rat stomach and immunostained with a mouse anti-${\beta}$-galactosidase at 3, 4 and 5 days after the virus injection. Results: The PRV-BaBlu infected the neurons in the amygdaloid nucleus, and the degree of viral infection in experimental animals showed a tendency to increase significantly with time (p<0.05). The neurons between the left and right amygdaloid nucleus significantly differ (p<0.05). Conclusion: This showed that PRV-BaBlu was an excellent neurotracer for localizing the amygdaloid nucleus, and the amygdaloid nucleus has a sensory input and motor output on stomach movement, influencing emotional behavior.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.607-612
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• 1990

The biodegradation of Aroclor 1242 was investigated by the mixed cultivation of the natural bacterial isolates and a genetically engineered microorganism (GEM). The natural strain of MS-1003 degraded the Aroclor 1242 through the ortho-cleavage pathway, while the other strains through the meta-cleavage pathway. When the MS-1003 strain was additionally inoculated into the 1 day culture of the DJ-26 strain and then cultivated for 2 days, the Aroclor was degraded up to 86% and resulted in increase of the meta-cleavage product. But in the MS-1003 culture inoculated with the DJ-26, degradation of the Aroclor was limited to the level of each pure culture. By the mixed cultivation of the DJ-26 strain together with the DJ-12 or its GEM strain of DF-10, which degrades the Aroclor through the meta-cleavage pathway, degradation of the Aroclor as well as production of the meta-cleavage compound were lower than those of each pure culture. The degradation of Aroclor 1242 by the GEM strain was not improved over the parental strain. Therefore, a form of cometaboiism of Aroclor 1242 was found in the mixed culture of the DJ-26 and MS-1003 strains which degrade the Aroclor through the different metabolic pathway, but in the mixed culture of the DJ-26 and DJ-12 strains degrading Aroclor 1242 through the same pathway, a kind of competetion for the substrate was observed.

• 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.

• Toxicological Research
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• v.13 no.1_2
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• pp.167-173
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• 1997

LBD-001, a recombinant human interferon $\gamma$ produced by genetically engineered yeast as a host system, was intravenously administered to pregnant female rabbits (New Zealand White strain) from day 6 to 18 of gestation at dose levels of $0.35 \times 10^6$, $0. 69 \times 10^6$, and $1.38 \times 10^6$ I.U./kg/day. Hydrocortisone sodium succinate (0.3 mg/kg/day) was also given in the same way. Teratological effects of the test agents on the organogenesis of fetuses and the development of offsprings (F1 rabbits) were investigated. The results were as followings: (1) No significant changes by the treatment of LBD-001 or hydrocortisone sodium succinate were observed in the body weights, the food and water consumption, the lactating or nurshing behaviors, and the autopsy of the pregnant rabbits. (2) No significant changes in the resorption rate, the fetal organogenesis, and the normal develpoment of offsprings (F1) by the treatment of LBD-001 or hydrocortisone sodium succinate were detected. The results show that LBD-001 at the dose of $1.38 \times 10^6$ I.U./kg/day or less and hydrocortisone sodium succinate at the dose of 0.3 mg/kg/day are neither teratogenic in the organogensis of the fetuses and the development of the offsprings (F1) nor toxic to the mother rabbits.