Journal of Microbiology

Observation and Enumeration of Attached Bacteria on Cellulos Film: Ahn, Tae Seok , Choi, Seung Ik , Byeon, Myeong Seop; J. Microbiol. 1995;33(1):1-4.

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Abstract: The DNA specific fluorochorome, Hoechst 33258, was used to enumerate the attached bacteria on cellulose film by epifluorescence microscope. Stainning with Hoechst dye gave fine features, whereas staining with acridine orange and DAPI gave poor contrast between background and bacterial cells. In Lake Uiam, Morphologically different two bacteria were detected after 3 days submerging in lake water. In Lake Soyang, the bacterial colonies appeared after 16 hours. This staining method had distinct advantages to enumerate the attached bacteria on surface.

Sequence Analysis of the Cytochrome b Gene of Trimorphomyces papilionaceus Mitochondria: Kim, Young Hyun , Kang, Young Won , Jung, Hack Sung; J. Microbiol. 1995;33(1):5-9.

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Abstract: The DNA sequence of the cytochrome b (cob) gene region of Trimorphomyces papilionaceus mitochondrial DNA has been determined. The cob gene is interrupted by an intron of 1267 bp, which has an open reading frame of 897 bp contiguous to to the upstream exon. The intron belongs to group IB and contains a reading frame with a GIY-10-YVG motif. The deduced amino acid sequence shows 52~60% homology with those of other reported fungi. The phylogenetic relationship of T. papilionaceus with Neurospora crassa, Podospora adserina, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Homo sapiens were inferred through sequence alignment and homology comparison of amino acids deduced from cob genes. When Homo sapiens was set as an outgroup, two ascomycetous yeasts S. cerevisiae and S. pombe made one group and three ascomycetous fungi N. crassa, P. anserine, and a basidiomycetous fungus T. papilionaceus the other group, suggesting that the former group evolved first and then the latter group separated into ascomycetous and basidiomycetous fungi right after that.

Isozyme Analysis of Spiny Species of Scenedesmus in Korea: Chang, Yoon Kyung , An, Seon Sook , Yoo, Soon Ae , Lee, Ki Sung; J. Microbiol. 1995;33(1):10-15.

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Abstract: We present the results of electrophoretic analysis on eight species of the genus Scenedesmus for the first time. According to the phenogram, S. opoliensis and A. armatus are clustered in one froup and S. intermedius, S. communis, S. bicaudatus, S. elliposoideus, S. spinosus are separated into another group. The two groups are distinguished by cell shape and cell wall ultrastructure although there are distinct morphological characteristics which demarcate each species. The isozyme analysis indicates that genetic similarity between species of Scenedesmus is low but the similarity within a species is very high. Therefore, isozyme banding patterns can be useful tools in Scenedesmus taxonomy.

Characterization of the Deletion Endpoints in Yeast Saccharomyces cerevisiae Mitochondrial oxi3 Gene Large-Deletion Mutants: Nam, Su Gil , Kim, Bok Whan , Kim, Sang Ho; J. Microbiol. 1995;33(1):16-20.

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Abstract: Previous results in our laboratory showed that there are site-specific large deletions in yeast Saccharomyces cerevisiae 777-3A mitochondrial oxi3 gene and most of them fall into A and B class only. The B-class mutants were analyzed by PCR and sequencing. About 420 bp deletion junction fragments were successfully amplified and subjected to direct sequencing. Based on the sequencing results, the deletion endpoints of class B-deletions were identified at a distance of 8,222 bp; interestingly, one end(position 26180 coincided with the 3’ splice site(intron/exon junction) of first intron aI1, the other end(position 10,839) was located in the middle of the fifth intron (aI5) of the gene, especially in an open reading frame(ORF).

Identification of the Genes Involved in Stationary-Phase Specific Acid Resistance of Salmonella typhimurium: Bang, Iel Soo , Lee, In Soo , Lee, Yung Nok , Park, Yong keun; J. Microbiol. 1995;33(1):21-27.

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Abstract: Salmonella encounters variables pH fluctuation during its life cycle and has been developed adaptative systems such as acid tolerance response (ATR) to survive at severe acidic environment. As part of on going investigation of stationary-phase specific acid resistance, we have searched for acid sensitive mutations in virulent Salmonella typhimurim UK-1 usin the MudJ fusion technique and two lethal selection procedures including DNP(dinitrophenol) selection media and microtiterplate selection method. Two acid sensitive mutations have been identified and designated, spatrK2, spatrK5. These mutations removed both stationary-phase acid tolerant effect and stationary-phase specific acid resistance. Non-specific histone like protein, H-NS and stationary-phase specific sigma factor, RpoS made little contribution to that system at respective single mutation(5-10 fold decrease). But, when both mutations were combined together, no acid resistance was achieved while acid tolerance response was still effective. Two dimensional SDS polyacrylamide gel electrophoresis showed new stationary-phase specific acid shock proteins as well as proteins already known. Not expectedly, the gels from acid adapted samples of both rpoS and hns mutation showed that double mutation of those regulators does not make change of the standard acid shock proteins. Only four acid shock proteins were regulated by these regulators, while fifteen proteins were newly identified as the members of acid shock response system by these regulators. These results implicate that stationary-phase acid resistance of that organism has RpoS/H-NS soubly dependent acid protective system and independent acid tolerance response system.

cloning of Gene Encoding for Siderophore biosynthesis in Fluorescent Pseudomonas sp.: Koh, Han Cheol , Ha, Sung Cheol , Na, Jung A , Kim, Ho Sang , Yeo, Myeong Gu , Lee, Jung Sup , Kim, Sung Jun , Park, yeal; J. Microbiol. 1995;33(1):28-33.

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Abstract: Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

Cloning of the Gene Responsible for Dechlorination of 4-Chlorobenzoate from Pseudomonas sp. DJ-12: Chae, Jong Chan , Kim, Young Chang , Kim, Young Soo , Kim, Chi Kyung; J. Microbiol. 1995;33(1):34-39.

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Abstract: The gene responsible for dechlorination of 4-chlorobenzoate(4CBA) was cloned from chromosomal DNA of Pseudomonas sp. DJ-12 in Escherichia coli XL1-Blue by using pBluescript SK(+) phagemid vector. Two recombinant plasmids of pCJ1 and pCJ2 carrying dechlorinase gene were constructed. The inserted DNAs in the pCJ1 and pCJ2 were found to have the fragment of 9.5 kb carrying dechlorinase gene, but they were oriented in opposite direction. The inserted DNA of 3.4kb in the pCJ101 subclone carrying dechlorinase gene had two restriction sites for AccI and each one site for HincII, KpnI, PstI, and SacIi but the dechlorinase gene in pCJ101 was found to be placed over the HincII site. The dechlorinase fenes in the recombinant cells of E. coli CJ1 and CJ101 were well expressed to show the dechlorination activity of 4CBA. The proteins encoded by dechlorinase genes in E. coli CJ1 and CJ101 were about 49 kDa in molecular weight. But this protein was not produced by E. coli CJ102, CJ103 and E. coli XL1-Blue. Therefore, the proteins produced by Pseudomonas sp. DJ-12 and the cloned cells of E. coli CJ1 and CJ101 were thought to be the dechlorinase enzyme which was the product of dechlorinase gene.

Cloning and sequence determination of α-tubulin, β-tubline and Flagellar Calmodulin cDNAs of Naegleria gruberi: Choi, Youn Jeong , Park, Hye Lee , Lee, Joo Hun; J. Microbiol. 1995;33(1):40-45.

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Abstract: Five cDNAs encoding two α-tubulins(α13 and α15), two β-tubulins(β5 and β5), and one flagellar calmodulin (Cal-1) were cloned from naegleria gruberi NB-1 and their nt sequences were determined. The α13(EMBL number X81049) and β1(EMBL number X81050) contained a complete open reading frame for α-tubulin and β-tubulin, respectively. The other three clones (α15, β5 and Cal-1) had a part of coding region and a 3’ untranslated region of the respective genes. The α13, β1 and Cal-1 had no homologous sequences in the coding regions and in the 3’ untranslated sequences. However, the α13 and β1 shared an eight nucleotide (AATACAAA) sequence in front of the respective initiation codons. The AATACAAA sequence was also found in N. gruberi strain NEG α-tubulin cDNA clone(αpT1) at the same position. Comparison of the α13 to the αpT1 revealed another stretch of identical sequence, which is 30 nts long, in the 5’ untranlated region.

Induced Level of CIN2 Gene Transcripts in yeast by Cycloheximide Treatment: Lee , Myeong Sok; J. Microbiol. 1995;33(1):46-50.

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Abstract: The interrelationship of expression of the two adjacent genes, HSP82 and CIN2 in Saccharomyces cerevisiae was investigated. The two genes are very close each other, such that the poly(A) addition site of the HSP82 gene is likely to be immediately followed by the promoter region of the CIN2 gene. Thus, transcription of the upstream HSP82 gene might affect expression of the CIN2 gene via promoter occlusion. Based on HSP82 and CIN2 mRNA analysis of the wild type, disruption mutant of the HAP82 gene, and conditional RNA polymerase II mutant strains, I conclude that CIN2 transcription is not affected by HSP82 transcription. However, surprisingly the level of CIN2 gene transcripts, but not HSP82, is heightened by cycloheximide treatment, presumably due to an increase in its stability. No change is observed in kinetics of HSP82 RNA induction by cycloheximide treatment.

Isolation of Glucose Utilizing Mutant of Alcaligenes eutrophus, its Substrate Selectivity, and Accumulation of Poly-β-hydroxybutyrate: Kim, Hye Yeon , Park, Jin Seo , Shin, Hyun Dong , Lee, yong Hyun; J. Microbiol. 1995;33(1):51-58.

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Abstract: A glucose utilizing mutant was selected from parent strain Alcaligenes eutrophus H16, and named as Glu-9. The mechanisms of glucose utilization of the mutant Glu-9 was investigated by measuring the D-[1-¹⁴C] glucose transport activity and the activities of key enzymes related to glucose and fructose uptake via facilitated diffusion. The uptaken glucose seems to activate key enzymes related to glucose matabolism. The selectivity between glucose and fructose of mutant Glu-9 was also analyzed by measuring glucose transport activity and enzyme activities under the various cultivation conditions using different carbon sources. Mutant Alcaligenes eutrophus Glu-9 preferentially consumed fructose from mixed substrates of glucose and fructose due to the inhibition of fructose to glucose transport activity. The characteristics of cell growth and PHB accumulation of Alcaligenes eutrophus Glu-9 were examined under various cultural conditions. Mutant strain Glu-9 showed tolerance in high concentration of glucose and increased yield of PHB production.

Biosynthesis of Poly-β-Hydrozyalkanoates by Bacillus thuringiensis R-510: Lee, Kang Tae , Kim, Jeong Yoon , Rhee, young Ha , Bae, Kyung Sook , Kim, Young Baek; J. Microbiol. 1995;33(1):59-65.

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Abstract: Synthesis and accumulation of Poly-β-Hydrozyalkanoates (PHA) in Bacillus thuringiensis R-510 isolated from soil were investigatd. This organism was resistant to relatively higj concentration of propionate and had a capability of accumulatinf copolymers consisting of 3-hydroxybutyrate(3HB) and 3-hydroxyvalerate(3HV) when the medium was supplemented with propionate as a precursor, The PHA content maximally reached up to 44.5% of dry cell weight in the presence of 0.1% propionate. The molar fraction of 3HV in the copolymer was increased from 19.4 to 80,2 mol% by adding 0.05 to 0.5% propionate to glucose medium. The addition of propionate during exponential or stationary phase of cell growth was less effective for the enhancement of 3HV content in the copolymer, although cell mass and PHA content were not affected by the time of propionate addition. PHB homopolymer and copolymer produced by B. thuringiensis R-510 were measured to have number average molecular weights in the range of 53,000 to 65,000. Polydispersity indices were between 1.5 and 2.2. Some of the produced polymers had bimodal molecular weight distribution.

Osmotic Tolerance Response of Salmonella typhimurium with Respect ro Growth-Phase and Identification of otr201, a rpoS-Related Gene: Lim, Si Keun , Bang, Soo Iel , Bang, Seong Ho , Lee, Yung Nok , Park, Yong Keun; J. Microbiol. 1995;33(1):66-73.

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Abstract: Salmonella typhimurium can stand against and survive under lethal osmotic exposure. Two systems of osmotic tolerance response(OTR) were found to be utilized by that organism, which were possibly overlapping with each other. The first system is an induction in response to non-lethal high osmoshock(0.3~0.7 M NaCl) at log-phase. The second system is induced during famine condition of stationary-phase. The viability of wild types(UK1, LT2) under these unfavorable conditions was increased by both systems. The viability of stationary-phase cells was approximately 5-fold that of the cells adapted at log-phase. In addition, a few regulatory fenes(rpoS, fur, crp, atp), one carbonstarvation-inducible(cstA104), and an osmotic-inducible gene(proU) were found to play an important role in osmotic tolerance at both growth phases. RpoS, a putative alternative sigma factor (σ^38), was found to participate in OTR systems regardless of growth-phase, but rpoS-defective mutant could still develope the adaptive tolerance. Thus, we concluded that there is rpoS-defective and rpoS-independent systems for osmotic tolerance at both growth-phase. Of the possible otr mutants newly isolated using MudJ(Km, lac) operon fusion techniques, YK3092 (otr201::MudJ) was most sensitive to osmotic challenge regardless of growth phase. It was mapped nearby at 57 min on chromosome and showed rpoS-negative phenotypes such as no catalase activity and inability to accumulate glycogen : but was not linked to rpoS. Therefore, this result strongly suggest that otr201 might be a rpoS-related regulatory gene not gound before.

Incorpotation and production of glucose in Lake Soyang: kwag, No Tae , Choi, Seung Ik , Ahn, Tae Young , Ahn, Tae Seok; J. Microbiol. 1995;33(1):74-79.

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Abstract: Kinetics of heterotrophic activity (glucose uptake) and extracellular enzyme activity(β-glucosidase, cellobiohydrolase) and cell numbers were measured in Lake Soyang during phytoplankton bloom development and after its breakdown. V_max for glucose was lower during Diatom bloom and that was higher after its breakdown. But the increase ion β-glucosidase activity was detected in late of Diatom bloom. Glucose uptake did not associated with β-glucosidase activity. The tight relationship between β-glucosidase and the incorporation of glucose by bacteria was not shown and the significance of depolymerization on the incorporation of glucose in lake water are discussed.

Purification and Characterization of α-amylase from Aspergillus sp. JP-1: Park, Hyung Nam , Yoo, Jin Cheol , Yang, Young Ki; J. Microbiol. 1995;33(1):80-84.

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Abstract: The α-amylase was purified from Aspergillus sp. JP-1 and some enzyme characteristics were studied. The enzyme waw approzimately purified 80-fold and an overall yield was 16.5% from the culture medium by ammonium sulfate fractionation, Sephadex G-150 gel permeation chromatography, and DEAE-Sephadex A-50 ion exchange column chromatography in order. The molecular weight of the purified α-amylase has been estimated to be 56 KDa on SDS-polyacrulamide gel electrophoresis and Sephadex G-150 chromatography. The purigied enzyme functions optimally at pH5.5 and 40℃, respectively. The Km value for soluble starch was 2.5 mg/ml. The enzymatic activity increased in the presence of Ca^2+, Co^2+, EDTA, Mg^2+, Mn^2+ and Zn^2+ and was inhibited by adding Cu^2+, Fe^2+, and Ni^2+.

Expression of human hypoxanthine phosphoribosyltransferase in Insect Cells Using a Baculovirus Vector: Lee, Chong Ho , Yang, Ji Won , Baek, Sang Ki , Yang, jai Myung; J. Microbiol. 1995;33(1):85-90.

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Abstract: The hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to the mononucleotide, IMP and GMP, respectively. For construction of recombinant AcNPV carrying human HPRT, a transfer vector p918 constructed by cloning full-length cDNA for human HPRT into pVL1393 and AnNPV genomic DNA were co-transfected into Sf21 cells. The tissue culture fluid containing extracellular virus was plaque assayed and a recombinant virus with occlusion minus phenotype was obtained by three rounds of plaque purification. Southern blot analysis and PRC results confirmed the insertion of the human HPRT cDNA within the recombinant virus(AcHPRT918). SDS-PAGE and Western blot analysis of the Sf21 cell extracts infected with AcHPRT918 indicated that human HPRT was expressed in insect cell. Large quantities of functional HPRT expressed in insect cells would facilitate characterization of the biological properties of this enzyme.

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