• Proceedings of the Zoological Society Korea Conference
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• 1994.10a
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• pp.222.1-222
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• 1994

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.47-52
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• 1992

This study describes isolation and identification of a soil bacterium which is degradable of phosphinothricin and improvement of the isolated strain by using mutagenesis and spheroplast fusion. The experiment was performed to search for a possibility of development of a new strain which is both PPT-degradable and glyphosate-resistant by using interspecies cell fusion between the PPT-degrading bacterium. Pseudornonu.\ puucimohlis and a glyphosate -resistant strain, Pseudornonu.~ cc,pucicl. Auxotrophic mutants were obtained by the treatement of P. puucimohili.\ with ethylmethanosulfate, and used to cell fusion. Lysozyme and EDTA were used to spheroplast formation and regeneration rates :)f the spheroplast were 6.5'%1 in P. pauc.irnohili.\ and 8.8% in P.ci,j~u[,i(lr, espctively. Polyethylenglycol 5.000 was used to cell fusion as fusogen. The fusant\ulcorner F1. F2. F\ulcorner and F4 werc- obtained by the intra- and interspecies cell fusion. The fusant Fl of intraspecies cell fusion was higher to the wild type by 1 I'%l in PPT degrading ability, and the fusant F3 of inierspesis cell fusion developed plyphosatc-resistant and PPI-dcgrading ability which were propertics of two parental strains.

• Journal of Dairy Science and Biotechnology
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• v.36 no.4
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• pp.208-219
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• 2018

Emergence of drug resistant strains of Salmonella enterica threatens milk processing and related dairy industries, thereby increasing the need for development of new anti-bacterials. Developments of antibacterial drugs are largely aimed to target the bacterial envelope, but screening their efficacy on bacterial envelope is laborious. This study presents a potential biosensor for envelope-specific stress in which a gfp reporter gene fused to spy gene encoding a periplasmic chaperone protein Spy (spheroplast protein y) that can sense envelope stress signals transduced by two major two-component signal transduction systems BaeSR and CpxAR in Salmonella enterica serovars Enteritidis and S. Gallinarum. Using spy-gfp operon fusions in S. Enterititis and S. Gallinarum, we found that spy transcription in both serovars was greatly induced when Salmonella cells were forming the spheroplast and were treated with ethanol or a membrane-disrupting antibiotic polymyxin B. These envelope stress-specific inductions of spy transcription were abrogated in mutant Salmonella lacking either BaeR or CpxR. Results illustrate that induction of Spy expression can be efficiently triggered by two-component signal transduction systems sensing envelope stress conditions, and thereby suggest that monitoring the spy transcription by spy-gfp operon fusions would be helpful to determine if developing antimicrobials can damage envelopes of S. Enteritidis and S. Gallinarum.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.207-212
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• 1992

To develope the new strains of microorganisms having the degradative ability for various aromatic hydrocarbons. spheroplast cell fusions were performed with Arthrobacter spp. degrading phthalate ester and Pseudomonas putida degrading alkylbenzen sulfonate(ABS) and the characteristics of the fusants were investigated. The spheroplasts of P. putia KUD15 and Arthrobacter sp. were formed effectively by lysozyme-EDTA treatment and by Ampicillin-lysozyme-EDTA treatment. respectively. The Spheroplast formation frequency and the regeneration frequency of the strains were 98-99% and 5-8%, respectively. For cell fusion. 40% PEG6000 was used as a fusogenic agent and the formation frequencies of fusion product were $1.8{\times}10^{4}-$2.9{\times}10^{4}$ Most of the fusants, which were selected in complemented antibiotics media showed the degradative ability in minimal selective medium added phthalate ester or ABS as sole carbon source. ABS degradation by fusant strain was increased about 20% with compared with the parental strain, while the degradative ability of phthalate ester was simillilar to that of parental strain.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.175-179
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• 1986

In order to develope a protoplast fusion of the genus Cellulomonas having high assimilibility of cellulose, the optimum conditions for the protoplast formation of Cellulomonas flavigena NCIB 12901 was investigated and observed by means of Scanning Electron Microscope. The results suggested that the susceptibility of the cell wall by lysozyme treatment on protoplast formation was considerably depend on the cultural periods of the cells. Cells of C. flavigena at mid exponential phase could more efficiently convert to protoplast cells than those at late exponential phase did. The rate of the protoplast formation was 95%, even though the rate was over 99.9% on counting by indirect method after osmotic shock treatment, when cells of the organism at mid exponential phase were treated with lysozyme (400$\mu\textrm{g}$/$m{\ell}$) for 6 hours and observed by SEM. In the evaluation of protoplast formation of the genus Cellulomonas, direct method of the observation under Scanning Electron Microscope was much more reliable than the counting method of protplasts after osmotic shock treatment. Because defferences between the number of spheroplast and protoplast were not able to be figured out on counting the number of protoplast after osmotic shock treatment.

• Food Science of Animal Resources
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• v.37 no.1
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• pp.134-138
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• 2017

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (${\underline{s}}pheroplast$ ${\underline{p}}rotein$ ${\underline{y}}$). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.37-43
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• 1997

Triphenylmethane was decolorized rapidly by enterbacter cloacae MG 82 at initial reaction time. The spheroplast showed higher activity of triphenylmentane decolorization than that of intact cell suspension. The outer part of the bacterial cell envelope and the peptidoglycan are important for the function of transport barrier of triphenylmethane. In intact cell, decolorization activity was higher at 37$\circ $C than at $\circ $C, indicating that triphenylmethane decolorization is due to the enzyme reaction. Culture filtrate showed no decolorization activity, while cell-free extract appeared high activity of 1.45 units, clearly showing that decolorization activity was due to the cell-free extract. Comparing decolorization activities of cell fractions, it was found that decolorization activity was located at the compartment of cytoplasmic membrane. The enzyme activity was also shown to be Mg$^{++}$-dependent. The optimum pH and temperature of enzyme activity were 7.0 and 50$\circ $C, respectively. The thermostability of this enzyme at 35$\circ $C was kept to 58% for 3 hours.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.355-361
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• 2001

A Chromate tolerant strain of Pseudomonas aeruginosa isolated from the effluent of a tannery showed significant enzymatic activity of chromate reduction. Cells grown in chromate-supplemented medium reduced 8 $\mu\textrm{g}$ chromate/mg protein/h in the presence of NADH/NADPH. The chromate reducing activity was inducible as cells pregrown in chromate showed higher chromate reduction. In contrast, the periplasmic fraction of cells gown in chromate reduce $75\%$ chromate in 4 h and the spheroplast fraction failed to do so, indicating that chromate reductase may be located in the periplasm. The presence of a 30 kDa protein in the periplasmic extracts of cells grown in the presence of chromate, but its absence of the protein in cells grown without chromate, points out a possible role of this protein in chromate reduction.

• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1290-1296
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• 2020

Recently, it was reported that entire mammalian mtDNA genomes could be transplanted into the mitochondrial networks of yeast, where they were accurately and stably maintained without rearrangement as intact genomes. Here, it was found that engineered mtDNA genomes could be readily transferred to and steadily maintained in the mitochondria of genetically modified yeast expressing the mouse mitochondrial transcription factor A (Tfam), one of the mitochondrial nucleoid proteins. The transferred mtDNA genomes were stably retained in the Tfam-expressing yeast cells for many generations. These results indicated that the engineered mouse mtDNA genomes introduced in yeast mitochondria could be relocated into the mitochondria of other cells and that the transferred genomes could be maintained within a mitochondrial environment that is highly amenable to mimicry of the biological conditions in mammalian mitochondria.