• Journal of Life Science
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• v.21 no.10
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• pp.1370-1376
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• 2011

To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyltransferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acidocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.243-250
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• 1986

Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

• The Korean Journal of Mycology
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• v.15 no.2
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• pp.80-86
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• 1987

The author isolated high cellulolytic fungi from natural sources and determined optimal condition of protoplast formation and fusion as fundamental step for improvement of the isolated it's cellulolytic ability. Three different cellulolytic fungi, such as Aspergillus sp., Penicillium sp. and Trichoderma sp., were isolated from soil. Their cellulolytic activities were compared with that of Aspergillus niger which was useful industrially and had cellulase activity. It was Aspergillus sp. that showed the highest activity of all these four fungi. And then it was followed by Penicillium sp., Trichoderma sp., and Aspergillus niger in order. An auxotrophic mutant of Aspergillus sp. was obtained by UV mutagenesis method. Having try to produce protoplast from mycelia, the author found that ${\beta}-glucuronidase$, at pH 6.0, was effective cell-wall lytic enzyme. And the optimal concentration of this enzyme was 5,000 unit/ml. Regeneration rates of wild type, met. auxotroph and arg. auxotroph, in presence of osmotic stabilizer, were 7. 0%, 7. 5% and 5.2%, respectively. PEG with M.W. 6,000 was effective stimulator for protoplast fusion in the concentration of 30% (W IV). In such a condition, we obtained 1.2% cell fusion rate.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.45-49
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• 1998

Two kinds of Mutants of Corynebacterium glutamicum, which were resistant to branched chain amino acid analogues, were obtained for L-leucine production; C. glutamicum LT26 resistant to 4-azaleucine and $\alpha$-amino-$eta$-hydroxyvaleric acid, and from which C. glutamicum LT3811-70 resistant to DL-4-thiaisoleucine were derived. Accumulation of L-leucine in the culture broths of these mutant strains, C. glutamicum LT26 and LT3811-70, were much higher than those of their parent strains even though they were non-auxotrophic mutants. Enzymatic analyses were performed to measure the activities of $\alpha$-acetohydroxy acid synthase (AHAS) and $\alpha$-isopropylmalate synthase (IPMS), which were the key enzymes for the L-isoleucine, L-valine and L-leucine biosynthetic pathways branching from a common precursor. In C. glutamicum LT26 and LT3811-70, AHAS and IPMS were found to be derepressed and desensitized to L-leucine. In addition, in C. glutamicum LT3811-70, IPMS was further more derepressed by L-leucine and AHAS was more desensitized by L-isoleucine and L-valine compared to its parent strain, C. gIEitamicum LT26.

• The Journal of the Korean Society for Microbiology
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• v.14 no.1
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• pp.49-61
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• 1979

Two hundred and ninety-five strains of Peudomonas aeruginosa isolated from clinical sources were tested for drug resistance and demonstration of R plasmids by intraspecies conjugation system. Sixty strains were found highly resistant to two or more of drugs. The rate of resistant strains were 38.9% to kanamycin(km), 33.2% to streptomydn(sm), 22.7% to sulfisomidine(Sa), 14.2% to chloramphenicol(Cp), 13.8% to tetracycline(Tc), 3.0% to carbenicillin(Cb), and to gentamicin(Gm), respectively. But no strains was resistant to nalidixic acid and colistine. They were resistant to per milliliter to more than $400{\mu}g$ per ml. of Tc, $800{\mu}g$ per ml of Cp and of Sm, $6,400{\mu}g$ per ml. of Sa, $200{\mu}g$ per ml. of Cb, $100{\mu}g$ per ml. of Gm, and $25{\mu}g$ per ml. of colistine. Forty-three strains of Pseudomonas aeruginosa could be transferred their resistance to Pseudomonas aeruginosa 2-70, 1005 rifampin resistant FP-auxotrophic mutant. Of sixty multiple resistant strains, forty-three(71.6%) demonstrated R plasmids; nineteen carried resistance to(Tc Cp Sm Sa), six to(Tc Cp Sm), three to(Tc Cp Sa), and Cp, five to(Tc Sm Sa), two to(Tc Sa), (Cp Sm) and Tc, and one to(Cp Sm Sa). Degree of resistance of recipients recieving R plasmids from donors were almost the same level of resistance as the donor in regardless of mating temperature at $25^{\circ}C$ and $37^{\circ}C$. Resistance to Tc, Sm, and Sa were transferred to a very few of recipient cells at five minutes after mating with donor and recipient cells but resistance to Cp were transferred to the majority of recipient cells. The transfer frequency of Tc, Cp, Sm, and Sa resistance from donors to recipients were from $1.0^{-1.4}\;to\;1.0^{-3.5}$ at $25^{\circ}C$ for 18 hours of incubation and were from $1.0^{-1.5}\;to\;1.0^{-3.5}$ at $37^{\circ}C$ for 18 hours of incubation.