• Korean Journal of Microbiology
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• v.31 no.4
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• pp.318-325
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• 1993

The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at $37^{\circ}C$ for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were $2.0{\times}10^{-5}~3.6{\times}10^{-5}$. All tested recombinant clones were very stable on further propagation.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.198-204
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• 1987

Biparental clones and recombinant clones were obtained by spheroplast fusion of Pseudomonas putida KU218R-3 and P.putida KU428. Formation of the fusion product was the most effective when the Pseudomonas spheroplast mixture were treated with 40% plyethyleneglycol(PEG) 6000 for 10min at room temperature, The fusants which selected by indirect method were obtained at an average frequency of 10.8%. Most of the fusants were biparental clones (10.4%) and the recombinant clones were produced in low yield (0.42%). Fusants, at the frequency of 4% were obtained without PEG 6000, which shows that fusion is not strictly dependant on PEG. The stability of fusants were examined. Most of the biparental clones were segregated to parental form amd late recombinants were formed on further propagation of biparental clone but the recombinant clones were nery stable.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.298-304
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• 1988

Antibiotic resistance plasmids (RP4, pMG1, R679 and R91-5) were used as primary selective markers to detect the fusants in Pseudomonas spp. By using plasmid marker, clones containing both plasmids of parental strains were obtained as fusants by direct selection. The spheroplast fusion was occured not only between strains of interspecies, but also between strains of intergenus such as Pseudomonas and E. coli. The frequencies of fusant formation were variable from $2.8\times 10^{-1}$ to $6.0\times 10^{-4}$ -4/. In addition, chromosomal recombinants were formed among the clones with both parental plasmids in frequencies of 0.44-1.3%. The fusant formation frequency between interspecies of intraspecies was not different markedly, but stability of plasmids in fusants correlated with the phylogenic smilarity of the parental strains.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.259-266
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• 2000

Spheroplast cell fusions were performed with Flavimonas oryzihabitans degrading aniline and Pseudomonas sp. degrading 4-chlorobiphenyl to develope the new fusant degrading aniline and 4-CBP and its characters were investigated. F. oryzihabitans was induced to antibiotic marker ($Cm^r$ by NTG treatment for the fusants selection. The results of spheroplast formation and regeneration frequencies of the strains treated with lysozyme-EDTA were 99% and 5.0~6.6%, respectively. Fusion products were treated with 40% (v/v) PEG 6000 and fusion frequency was $3.16{\times}10^{-4} $. The DNA content of fusant, F22 was approximately 2-fold compared with parents. The fusant was stable, and showed the mixed biochemical characteristics of the parent strains. F22 was similar to parent for cell growth pattern and degrading capacity on 5 mM aniline but cell growth rate of F22 was 1.5-fold higher than that of the parent on 10mM aniline. However 4-CBP degrading ability of F22 was slightly lower than that of parental strain.

• 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 Yeungnam Medical Science
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• v.2 no.1
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• pp.175-181
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• 1985

Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called Ti plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow Ti plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of Ti plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts and Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high $Ca^{2+}$ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high $Ca^{2+}$ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observations suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.

• 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

• 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.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.117-122
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• 1991

In order to improve the removal ability of phosphate, Spheroplast fusions were performed among auxotrophic mutants of Aeromonas hydrophila isolated from waste water, named A13 and A14, Aci37 auxotrophic mutant of Acinetobactercalcoaceticus, and auxotrophic E. coli HR262/pCE27 carring pit gene. Eight fusants obtained from this experiment showed different biochemical characteristics. When the rate of phosphate uptake among fusants (F1-F8) was investigated in Phosphate Uptake Medium (PUM), F8 strain showed the highest rate for phosphate removal, 7 times as much as control after two hours incubation. The role of cations ($Mg^{++}$ ,$Ca^{++}$ , $K^{+}$ in phosphate uptade by F8 was also investigated in PUM without each salt. $K^{+}$ seemed to be crucial. Being compared with phosphate untake rate in PUM, that in PUM without $K^{+}$ was reduced 1.5 times. Therefore, by applying F8 strain and $K^{+}$ in practical environmental system, the increased efficiency in phosphate removal can be derived.

• 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.