Journal of Microbiology

Volume 33(2); June 1995

Construction of probability identification matrix and selective medium for acidophilic actinomycetes using numerical classification data: Seong, Chi Nam , Park, Seok Kyu , Goodfellow, Michael , Kim, Seung Bum , Hah, Yung Chil; J. Microbiol. 1995;33(2):95-102.

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Abstract Supplementary Material: A probability identification matrix of acidophilic Streptomyces was constructed. The phenetic data of the strains were derived from numerical classification described by Seong et al. The minimum number of diagnostic characters was determined using computer programs for calculation of different separation indices. The resulting matrix consisted of 25 clusters versus 53 characters. Theoretical evaluation of this matrix was achieved by estimating the cluster overlap and the identification scores for the Hypothetical Median Organisms (HMO) and for the representatives of each cluster. Cluster overlap was found to be relatively small. Identification scores for the HMO and the randomly selected representatives of each cluster were satisfactory. The matrix was assessed practically by applying the matrix to the identification of unknown isolates. Of the unknown isolates, 71.9% were clearly identified to one of eight clusters. The numerical classification data was also used to design a selective isolation medium for antibiotic-producing organisms. Four chemical substances including 2 antibiotics were determined by the DLACHAR program as diagnostic for the isolation of target organisms which have antimicrobial activity against Micrococcus luteus. It was possible to detect the increased rate of selective isolation on the synthesized medium. The results show that the numerical phenetic data can be applied to a variety of purposes, such as construction of identification matrix and selective isolation medium for acidophilic antinomycetes.

Characteristics of protease inhibitor produced by streptomyces fradiae SMF9: Kim, Hyoung Tae , Suh, Joo Won , Lee, Key Joon; J. Microbiol. 1995;33(2):103-108.

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Abstract: Streptomyces fradiae protease inhibitor (SFI) was purified effectively by preparative isoelectric focusing and hydroxyapatite chromatography. The molecular weight of SFI was estimated to be 1.7 kDa by SDS-PAGE and 1.8 kDa by molecular sieving HPLC. One hundred and sixty amino acid residues were determined from which molecular weight of SFI was calculated to be 17.054 Da and carbohydrate residue was not detected. SFI was calculated to be 17,064 Da and carbohydrate residue was not detected. SFI was a monomeric protein with two reactive sits, of which isoelectric point was pH 4.1. N-terminal amino acid sequence of SFI had homology with SSI (Streptomyces subsilisin inhibitor) and other protease inhibitors produced by Streptomyces.

Purification and characterization of a xylanase from alkalophilic cephalosporium sp. RYM-202: Kang, Myoung Kyu , Kwon, Tae Ik , Yang, Young Ki , Rhee, Young Ha; J. Microbiol. 1995;33(2):109-114.

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Abstract: Alkalophilic Cephalosporium sp. RYM-202 produced multiple xylanases extracellularly. One of these xylanases was purified to electrophoretical homogeneity by chromatography with DEAE-Sephadex A-50, Sephacryl S-200 HR and Superose 12 HR. The purified xylanase differed from most other microbial xylanases in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase in that it had low-molecular weight and acidic isoelectric point. The molecular weight of the xylanase was 23 kDa by SDS-polyacrylamide electrophoresis and 24 kDa by gel permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permentation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity permeation chromatography, and the isoelectric point was 4.3. The xylanase had the highest activity at pH 8.0 and 50℃. It was stable over a wide range of pH and retained more than 80% of its original activity after 24 h of incubation even at pH 12. The Km values of this enzyme on birchwood xylan and oat spelts xylan were 2.33 and 3.45 mg/ml, respectively. The complete inhibition of the enzyme of n-bromosuccinimide suggests the involvement of tryptophan in the active site. The sylanase lacked activity towards crystalline cellulose and carboxymethyl cellulose.

Purification and Characterization of an Alkaline Protease produced by a Xanthomonas sp. YL-37: Lee, Chang Ho , Kim, Hee Sik , Kwon, Gi Soek , Oh, Hee Mock , Kang, Sang Mo , Kwon, Tae Jong , Yoon, Byung Dae; J. Microbiol. 1995;33(2):115-119.

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Abstract: The alkaline protease of Xanthomonas sp. YL-37 has been purified, and the properties of the enzyme investigated. The alkaline protease of Xanthomonas sp. YL-37 was purified form crude enzyme by ammonium sulfate fractionation, CM-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. Through the series of chromatographies, the enzyme was purified to homogenecity with specific activity of 4.23 fold higher than that of the crude broth. The molecular weight of the purified protease has been estimated to be 62 KDa on SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for alkaline protease activity were 11.0 and 50℃, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to 50℃. Enzyme activity was lost up to 50% on heating at 70℃ for 30 minutes. The activity of alkaline protease was inhibited by Cu^2+, Zn^2+, Hg^2+, PMSF, and activated by Mn^2+ and Ca^2+. The K_m value for casein as a substrate was 4.0 mg/ml.

Regulation of the Expression of nhaA Gene, Coding Na^+/H^+ Antiporter A of Escherichia coli.: Seo, Sung Yum , Lee, Seung Heon; J. Microbiol. 1995;33(2):120-125.

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Abstract: β-galactosidase activity of Escherichia coli cells containing operon fusion nhaA'-'lacZ was monitored to study the regulation of expression of nhaA gene under various conditions. The expression of the fusion was enhanced only by chemicals containing Na^+ or Li^+. This Na^+ or Li^+. This Na^+(Li^+)-specific enhancement of β-galactosidase activity represented the increase in the rate of synthesis of β-galactosidase rather than the decrease in the breakdown rate. The induction pattern was influenced by copy numbers of the gene. Induction by Na^+ or Li^+ was concentration and time dependent, reaching maximum 5-6 fold induction after 2 hours at 0.4-0.5 M for Na^+ or at 0.25-0.35 M for Li^+, Although the expression was induced at much lower concentration of Na^+ at alkaline pH values than at neutral pH in the presence of Na^+, alkaline pH itself did not induced the expression of the fusion in the absence of Na^+. Temperature shift and growth phase of culture did not affect the level of induction.

Characterization of the 5'-Flanking region upstream from the structural Gene for Zymomonas mobilis Alcohol Dehydrogenase: Yoon, Ki Hong , Park, Seung Hwan , Jung, Kyung Hwa , Pack, M. Y.; J. Microbiol. 1995;33(2):126-127.

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Abstract: A Zymomonas mobilis DNA fragment consisting of 207 nucleotides, which corresponded to the 5'-flanking region of an adhB gene encoding alcohol dehydrogenase II, was fused to the structural gene coding for a Bacillus endo-β-1, 4-glucanase. The Z. mobilis DNA fragment was identified to promote 50-fold increase in the expression of endo-β-1,4 glucanase gene in Escherichia coli.

Molecular Cloning and Nucleotide sequence of Schizosaccharomyces pombe Homologue of the Receptor for Activated Protein Kinase C Gene: Park, Seung Kiel , Yoo, Hyang Sook; J. Microbiol. 1995;33(2):128-131.

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Abstract: Using differential hybridization, we selected the prk gene fortuitously from Schizosaccharomyces pombe homologous to RACK1 of rat which encodes the receptor for activated protein kinase C. The cDNA sequence of prk was determined and its deduced amino acid sequence was 76% homologous to RACK1 and had the feature of trimeric G protein beta subunit. The specific amino acid sequences required for the protein kinase C binding were also present in Prk as in the case of RACK1 protein. From these similarities, we suggest that the Prk is protein kinase C binding protein of S. prombe. The involvement of Prk in signal transduction mediated by protein kinase C remained to be studied.

Megabase-sized DNA Isolation and Electrophoretic Karyotype of Fusarium oxysporum Schlecht: Park, Min Seon , Min, Byung Re; J. Microbiol. 1995;33(2):132-135.

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Abstract: To investigate the electrophoretic karytype of Fusarium oxysporum, intact chromosomal DNA was separated by pulsed-field gel electrophoresis (PEGE). DNA extraction from nulcei, mycelia and protoplasts were compared with one another and with the quantity and the suitability for PFGE separation in agarose gel. As a result, the most useful extracting method for intact DNA was found to be that from protoplasts. By varying the electrophoretic conditions, 8 chromosomal DNA bounds were resolved. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae as size standards, the size of Fusarium oxysporum chromosomes was estimated to range from approximately 0.6 Mb TO 6.7 Mb, and total genome size was 26.7 Mb. The suitability of electrophoretic karyotyping as a tool for strain characterization is discussed.

Nucleotide Sequence Analysis of the 5S Ribosomal RNA Gene of the Mushroom Tricholoma matsutake: Hwang, Seon Kap , Kim, Jong Guk; J. Microbiol. 1995;33(2):136-141.

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Abstract: From a cluster of structural rRNA genes which has previsouly been cloned (Hwang and Kim, in submission; J. Microbiol. Biotechnol.), a 1.0-kb Eco RI fragment of DNA which shows significant homology to the 25S and rRNA s of Tricholoma matsutake was used for sequence analysis. Nucleotide sequence was bidirectionally determined using deletion series of the DNA fragment. Comparing the resultant 1016-base sequence with sequences in the database, both the 3'end of 25S-rRNA gene and 5S rRNA gene were searched. The 5S rRNA gene is 118-bp in length and is located 158-bp downstream of 3'end of the 25S rRNA gene. IGSI and IGS2 (partial) sequences are also contained in the fragment. Multiple alignment of the 5S rRNA sequences was carried out with 5S rRNA sequences from some members of the subdivision Basidiomycotina obtained from the database. Polygenetic analysis with distance matrix established by Kimura's 2-parameter method and phylogenetic tree by UPGMA method proposed that T. matsutake is closely related to efibulobasidium allbescens. Secondary structure of 5S rRNA was also hypothesized to show similar topology with its generally accepted eukaryotic counterpart.

Construction of Multiple Mutant Strains by Mating Procedures for the Cloning of pmn and pmb Genes Encoding Amino Acid Permeases in Neurospora crassa: Han, Hyo Young , Min, Kyung Hee; J. Microbiol. 1995;33(2):142-145.

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Abstract: The pmb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitr6r : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 50㎍/㎖ para-fluoro-phenylalanine (PFPA), 200㎍/㎖ canavanine, and 500㎍/㎖ histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections.

Selection of Laccase Over-secreting Mutant in Coprinus congregature: Kim, Soon Ja , Choi, Hyoung Tae; J. Microbiol. 1995;33(2):146-148.

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Abstract: Coprinus congregatus has a membrane-associated laccase which is not secreted into culture media. A mutant monokaryon obtained, by U. V. irradiation followed by protoplast generation and regeneration method, was successfully isolated. When the mutant was grown on a agar plate or in a liquid medium, it secreted laccase while the wild type did not under the same growth conditions. The laccase of the mutant was compared with that of wild type did not under the same growth conditions. The laccase of the mutant was compared with that of wild type of native PAGE analysis, and showed identical mobility.

Restriction and Transcription Maps of Mitochondrial DNA of Trimorphomyces papilionaceus: Jeoung, Won Jin , Hong, Soon Gyu , Kang, Young Won , Jung, Hack Sung; J. Microbiol. 1995;33(2):149-153.

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Abstract: Mitochondrial DNA has been isolated from Trimorphomyces papilionaceus. By analyzing DNA fragments digested by restriction enzymes, a restriction site map has been constructured. The mtDNA of T. papilionaceus amounts to 48.5 kb in size and is circular in structure. Entire mitochondrial DNA was cloned in E coli plasmids and Northern blot hybridization was done using cloned and subcloned DNAs as probes. Based on hybridization results of mitochondrial RNA transcripts, a transcription map was prepared.

Characteristics of Ustilago maydis Virus of SH14 Killer Strain Isolated in Korea: Hwang, Seon Hee , Jung, Cheong Hwan , Yue, Se Won; J. Microbiol. 1995;33(2):154-159.

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Abstract: SH-14, a novel killer strain of Ustilago maydis was isolated in Korea. It has been reported in other papers that the toxin specificity and double-stranded RNA pattern of SH-14 strain were different from other laboratory strains. In this paper, we analyzed the biochemical characteristics of U. maydis SH-14 virus. Three distinctive peaks were isolated from CsCl density gradient, designated as top (T), intermediate (I) and bottom (B) components. We found that the densities of each components, 1.285, 1.408 g/㎤, respectively, are very similar to those of other strains. As previously reported by the analysis of dsRNA in each component, the dsRNA segments are separately encapsidated. Capsid protein of SH-14 virus consists of two proteins about 70 Kd shown by SDS-PAGE analysis. Electron microscopic examination of the virus particles revealed that UmV particles are very similar in size and morphology to all isolates as well as all lab-strains. In order to test immunological cross reactivity of UmV, western blot analysis was carried out with antiserum against A8 virus. All capsid protein had positive reaction against A8 antibody which indicated that UmV are immunologically cross-reactive with all isolates from Korea. The results presented in this paper may show that UmV isolated from SH-14 strain has very similar biochemical characteristics to those of other UmV. However, the difference in the toxin specificity and the molecular weight of toxin protein from the SH-14 strain has us to conclude that U. maydis SH-14 strain is a new killer type.

Role of Mg^2+ in RNA Splicing of T4 td Intron: Sung, Jung Suk , Shin, Sookc , Park, In Kook; J. Microbiol. 1995;33(2):160-164.

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Abstract: The splicing activity of T4 phage td intron RNA has been examined with various Mg^2+ ions such as MgCl₂, MgSO₄and magnesium acetate using various splicing conditions such as different incubation time and temperature. The maximum splicing of td intron RNA occurred at the concentration of 5 mM MgCl₂. Raising the Mg^2+ concentration up to 15 mM appeared to promote P2 delection mutant to overcome the loss of some splicing activity. In both wild type and mutant, a complete hydrolysis of RNA occurred at 30 mM MgCI₂MgSO₄and magnesium acetate exhibited the rate and pattern of RNA splicing identical to MgCI₂. The optimal splicing conditions involve the incubation of RNA with 5 mM MgCI₂ at 58℃ for 15 min. The results suggest that Mg^2+ may play a key role in the catalytic mechanism of td intron RNA.

An Analysis of the Arm-type Site Binding Domain of Bacteriophage γ Integrase: Cho , Eun Hee; J. Microbiol. 1995;33(2):165-170.

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Abstract: The 356 amino acid long lambda integrase protein of bacteriophage λ constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P_tac promoter and the lacI^q gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

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