• International Journal of Industrial Entomology and Biomaterials
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• v.10 no.2
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• pp.119-123
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• 2005

Benomyl-resistant mutants of entomopathogenic fungus, Metarhizium anisopliae were isolated and their physiological characteristics were investigated. These militants were obtained spontaneously or by UV irradiation in benomyl-treated media. Four spontaneous (S-2, S-11, S-18, S-19) and four UV-induced (UV-4, UV-5, UV-19, UV-24) mutants, which grow stably and normally were selected. No significant differences in conidia or hyphal shape, conidia viability, mycelial biomass, or virulent to the diamondback moth were observed between the wild type and their mutants. But differently from the mycelial growth of other benomyl-resistant mutants which was slower than that of the wild type on a modified Czapek-Dox, SDAY, $4\%$ chitin, or $1\%$ skim milk medium, that in the spontaneous mutants, S-18 and S-19, did not show any difference from the wild type. Especially, S-18 and S-19 grew well at benomyl concentrations up to 50 times or higher than that which inhibits wild type proliferation. These results suggested that S-18 and S-19 could potentially be used with the fungicide, benomyl.

• Korean Journal of Food Science and Technology
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• v.32 no.2
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• pp.431-438
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• 2000

Properties of acid tolerance of an adipic acid-resistant mutant, Leuconostoc paramesenteroides (ANaP100) were studied and compared with those of its paired wild type of Leu. paramesenteroides (LPw). The value of protons permeability of LPw after an acid shock at pH 5.0 was 4.3 min, while the value of ANaP100 was 4.8 min at the same pH. The maximal specific activities of ATPase of LPw and ANaP100 were 0.59 unit/mg protein and 0.63 unit/mg protein at pH 6.0, respectively. The release of magnesium ion from the mutant strain was about 27.3% at pH 4 after 2 hrs, while the wild strain was about 52.2% under the same conditions. The contents of $C_{19:0,cyclo}$ and $C_{18:1}$ in a membrane fatty acid of ANaP100 and LPw were higher and lower, respectively, than that of LPw. These results indicated that acid tolerance of ANaP100 was improved in comparison with that of its wild type, LPw.

• Journal of Microbiology and Biotechnology
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• v.23 no.9
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• pp.1293-1303
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• 2013

Antibiotic resistance of soilborne Acinetobacter species has been poorly explored. In this study, norfloxacin resistance of a soil bacterium, Acinetobacter oleivorans DR1, was investigated. The frequencies of mutant appearance of all tested non-pathogenic Acinetobacter strains were lower than those of pathogenic strains under minimum inhibitory concentration (MIC). When the quinolone-resistance-determining region of the gyrA gene was examined, only one mutant (His78Asn) out of 10 resistant variants had a mutation. Whole transcriptome analysis using a RNA-Seq demonstrated that genes involved in SOS response and DNA repair were significantly up-regulated by norfloxacin. Determining the MICs of survival cells after norfloxacin treatment confirmed some of those cells were indeed persister cells. Ten colonies, randomly selected from among those that survived in the presence of norfloxacin, did not exhibit increased MIC. Thus, both the low mutation frequency of the target gene and SOS response under norfloxacin suggested that persister formation might contribute to the resistance of DR1 against norfloxacin. The persister frequency increased without a change in MIC when stationary phase cells, low growth rates conditions, and growth-deficient dnaJ mutant were used. Taken together, our comprehensive approach, which included mutational analysis of the target gene, persister formation assays, and RNA sequencing, indicated that DR1 survival when exposed to norfloxacin is related not only to target gene mutation but also to persister formation, possibly through up-regulation of the SOS response and DNA repair genes.

• Korean Journal of Microbiology
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• v.23 no.1
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• pp.9-12
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• 1985

As a part of the host cell development for a amplified recombinant trp operon, $aroP^-$ mutation was introduced in a E. coli host strain. $aroP^-$ mutation was induced by transposon Tn10 and transduced into the E. coli host cell by bacteriophage P1Kc. The effect of $aroP^-$ mutation on the excretion of tryptophan in E. coli $trpR^{-ts}/ColE_1 -trp^+$ cells was investigated. Mutant lacking the general aromatic transport system was resistant to ${\beta}-2-thienylalanine\;(2{\times}10^{-4}\;M)$, p-fluorophenylalanine $(2{\times}10^{-4}M)$, or 5-methyltryptophan $(2{\times}10^{-4}\;M.)[^3H]-tryptophan$ uptake of the $aroP^-$ mutant strain was reduced considerably as compared with $aroP^+$ counterpart. The rate of $[^3H]-tryptophan$ uptake of the $aroP^-$ mutant strain treated with $NaN_3(3{\times}10^{-2}\;M)$ was much less affected than that of $aroP^+$ counterpart. The $aroP^-$ transductants increased the tryptophan excretion from E. coli $trpR^{-ts}/ColE_1 -trp^+$ four times more than $aroP^+$ counterpart.

• Bulletin of the Korean Chemical Society
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• v.32 no.8
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• pp.2549-2552
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• 2011

In this study, the kinetic properties of wild-type and C117D mutant H. influenzae MurA (Hi MurA), which catalyzes the first reaction in the biosynthetic pathway of the cell wall, were characterized. Purified recombinant Hi MurA was active at pH values ranging from pH 5.5 to pH 10, and its $K_m$ (UNAG), $K_m$ (PEP), and $k_{cat}$ values were measured to be 31 ${\mu}M$, 24 ${\mu}M$, and 210 $min^{-1}$, respectively. Hi MurA activity was effectively inhibited by fosfomycin with an $IC_{50}$ value of 60 ${\mu}M$. Hi MurA contains a cysteine residue (C117) at the loop region near the PEP binding, whereas MurA from fosfomycin resistant Mycobaterium tuberculosis or Chlamydia trachomatis contain an aspartate residue instead of the cysteine at the corresponding site. Aspartate substitution of Cys117 in Hi MurA shifted its optimum pH from 7.8 to 6.0. In addition, the $K_m$ values for UNAG and PEP were increased to 160 ${\mu}M$ and 150 ${\mu}M$, respectively, and the $k_{cat}$ value was significantly reduced to 41 $min^{-1}$. Furthermore, the C117D mutant form of Hi MurA was not inhibited by 1 mM fosfomycin. These results indicate that the Cys117 of Hi MurA is the binding site of fosfomycin and plays an important role in the fast turnover of the catalytic reaction.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.304-312
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.51-56
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• 2017

Cellulosic biomass is a renewable source for biofuel production from non-edible biomass. An optimized pretreatment process is required for the efficient utilization of cellulosic biomass. Among various pretreatment processes, the use of ionic liquids has been reported recently. However, the residual ionic liquid after pretreatment acts as an inhibitor of microbial fermentation. Recently, we isolated mutant Saccharomyces cerevisiae strains resistant to the ionic liquid 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) by using a directed evolutionary approach. When 3% [EMIM][Ac] was added to a medium containing 80 g/l of glucose, mutants D452-B2 and D452-S3 produced 35.6 g/l and 36.3 g/l of ethanol, respectively, for 18 h while the parental strain (S. cerevisiae D452-2) produced 1.3 g/l of ethanol. Thus, these mutant S. cerevisiae strains might prove advantageous when ionic liquids are used for biofuel production from cellulosic biomass.

• Korean Journal of Food Science and Technology
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• v.32 no.2
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• pp.424-430
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• 2000

The purpose of the study was to investigate effect of addition of mutant strains of both Leuconostoc mesenteroides and Leu. paramesenteroides on extending shelf-life of Kimchi. The mutant strains have an increased adipic acid resistance in comparison with that of their paired wild types. Addition of both strains was more effective than that of one strain alone to extend shelf-life of Kimchi. The optimal amount of inoculation was determined as 0.005% of the two mixed mutant strains with a ratio of 1 : 10, Leu. mesenteroides : Leu. paramesenteroides based on the results of acidification and organoleptic tests.

• Bulletin of the Korean Chemical Society
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• v.22 no.5
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• pp.514-518
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• 2001

We have explored the importance of two ATP binding domains of Hsp104 protein in protection of yeast cells from cadmium exposure. In the previous study we have discovered that the presence of two ATP binding sites was essential in providing heat sh ock protection as well as rescuing cells from oxidative stress. In this paper we first report wild type cell with functional hsp104 gene is more resistant to cadmium stress than hsp104-deleted mutant cell, judging from decrease in survival rates as a result of cadmium exposure. In order to demonstrate functional role of two ATP binding sites in cadmium defense, we have transformed both wild type (SP1) and hyperactivated ras mutant (IR2.5) strains with several plasmids differing in the presence of ATP binding sites. When an extra copy of functional hsp104 gene with both ATP binding sites was overexpressed with GPD-promoter, cells showed increased survival rate against cadmium stress than mutants with ATP binding sites changed. The degree of protection in the presence of two ATP binding sites was similarly observed in ira2-deleted hyperactivated ras mutant, which was more sensitive to oxidative stress than wild type cell. We have concluded that the greater sensitivity to cadmium stress in the absence of two ATP binding sites is attributed to the higher concentration of reactive oxygen species (ROS) produced by cadmium exposure based on the fluorescence tests. These findings, taken all together, imply that the mechanism by which cadmium put forth toxic effects may be closely associated with the oxidative stress, which is regulated independently of the Ras-cAMP pathway. Our study provides a better understanding of cadmium defense itself and cross-talks between oxidative stress and metal stress, which can be applied to control human diseases due to similar toxic environments.

• Applied Biological Chemistry
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• v.9
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• pp.91-96
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• 1968

1. Two strains of Aspergilli producing acid resistant amylase were isolated from air. The strain $U^{-1}$ and $U^{-2}$ were similar to Asp. usamii tan type mutant in the morphological characteristics. 2. The influence of cultural pH for production of amylase were investigated in the suface culture, it was different each other in the cultural optimum initial pH. The activity of acid resistant amylase produced by strain $U^{-1}$ was higher than that of strain $U^{-2}$. The acid resistance was increased by culturing in lower pH. 3. The lower initial pH ($U^{-1}$ pH 4.0, $U^{-2}$ pH 2.0) was better than neutral (pH 6.0) for the production of amylase and growth of them. The effect of low initial pH for the production of amylase was not to accelerate but caused by change of pH in the cultural process, in the range of this experiment.