Journal of Microbiology

Volume 35(2); June 1997

Phylogenetic study of trichaptum inferred from nuclear ribosomal DNA sequences: Ko, Kwon Soo , Hong, Soon Gyu , Jung, Hack Sung; J. Microbiol. 1997;35(2):79-86.

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Abstract: For the phylogenetic study of the genus Trichaptum, nuclear ribosomal DNA sequences from eight strains of four Trichaptium species were examined. Phylogenetic trees were constructed using molecular data on 18 rDNA and 5.8S rDNA and thei ITSs. Parsimony analyses of the Trichaptum species showed that T. biforme and T. laricinum made a monophyletic group respectively, suggesting that each species is phylogenetically independent. However, T. abietum represented a polyphyletic group and T. fusco-violaceum formed a polytomous group, suggesting that these species could be in the process of evolutionary differentiation. Examination of base substitutions of the 18S rRNA gene reveals that the C-T transition is most predominant and that there is a stronger transition bias between closely related organisms rather than between distantly related ones.

A detection method for vibrio vulnificus using monoclonal antibodies: Chung, Mi Sun , Rim, Bung Moo , Uhm, Tae Boong , Park, Moon Kook; J. Microbiol. 1997;35(2):87-91.

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Abstract: Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10^4 to 10^7 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

Characterization of a new staphylococcal site-specific recombinase sin and genetic organization of its flanking region: Yong, Jun Hyong , Kim, Young Sun , Byeon, Woo Hyeon; J. Microbiol. 1997;35(2):92-96.

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Abstract: A new site-specific recombinase sin, as a component of a putative transposon has been cloned and its base sequence has been determined. The proposed sin shows a high degree of homology with pI9789-sin and pSK1-sin. There is a large (16 bp) inverted repeat downstream of proposed sin and the postulate dhelix-turn-helix motif is located at the extreme C-terminus of the proposed Sin. The transposase gene (tnpA) and β-lactamase gene (blaZ) are located upstream of sin and arsenate reductase gene (arsC) and arsenic efflux pump protein gene (ars B) are downstream. This genetic arrangement seems to be a part of a new putative transposon because there is no known transposon with a gene arrangement of tnpA-blaZ-sin-arsC.

Purification and charactedrization of cysteine desulfhydrase from streptomyces albidoflavus SMF301: Ryu, Jae Gon , Kang, Sung Gyun , Kim, In Seop , Rho, Young Taik , Lee, Sang Hee , Lee, Kye Joon; J. Microbiol. 1997;35(2):97-102.

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Abstract: Cysteine desulfhydrase (EC 4, 4, 1. 1. ) was purified from the culture supernatant of Streptomyces albidoflavus SMF301 by hydroxyapatite, gel filtration and Resource Q ion-exchange chromatography with a purification fold of six identical subunits. The enzyme was stabilized by dithiothreitol and pyridoxal 5'-phosphate during the purification procedures. The optimum pH and temperature were pH 8.6 and 35℃, respectively. The N-terminal amino acid sequence was identified as A-P-L-P-T-A-D-V-R-S-D-P-G-Y-E-W-L-G-E-A-V. The purified cystein desulfhydrase had a high substrate specificity toward cysteine, and exhibited no cystahionine λ-lyase activity. The K_m value for cysteine was determined to be 0.37 mM.

Staphylococcal methicillin resistance expression under various growth conditions: Lee, Yoo Nik , Poo Ha Ryoung , Lee, Young Ik; J. Microbiol. 1997;35(2):103-108.

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Abstract: To improve the detection of methicillin resistant staphylococci, lowered incubation temperature (30℃) and inclusion of sodium chloride in media have been empirically recommended. However, in this study, we found that sodium chloride in Peptone-Yeast Extract-K₂HPO₄(PYK) medium decreased methicillin minimum inhibitory concentrations. Divalent cations were shown to restore the expression of staphylococcal methicillin resistance. However, when it was determined by efficiency of plating, sodium chloride increased methicillin resistance expression on agar medium in which higher divalent cations were contained in the agar medium. The decrease of minimum inhibitory concentrations at 30℃ by sodium chloride occurred in Brain Heart Infusion but did not occur in other media investigated. Interestingly, both PYK and Brain Heart Infusion media had peptone, which contain cholic acids having detergent activities. Inclusion of sodium chloride in PYK caused a higher rate of autolysis. Penicillin binding protein 2a that has a low affinity to beta-lactam antibiotics, was highly inducible in methicillin resistant Staphylococcus epidermidis strains. In this study, we found that autolysins that are activated by the sodium chloride decreased the minimum inhibitory concentration at 30℃, and peptidoglycan is weakened due to the presence of methicillin. Peptone in the media may aggravate the fragile cells. However, stabilization due to the presence of divalent cations and production of penicilin binding protein 2a increase the survival of staphylococci.

Purification and characterization of extracellular aspartic proteinase of candida albicans: Na, Byoung-Kuk , Lee, Seong Il , Kim, Sin Ok , Park, Young Kil , Bai, Gill Han , Kim, Sang Jae , Song, Chul Yong; J. Microbiol. 1997;35(2):109-116.

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Abstract: An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45℃. The addition of divalent cations, Ca^2+, Zn^2+ and Mg^2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe^2+, Ag^2+ and Cu^2+. With BSA as substrate, an apparent K_m was determined to be 7 × 10^7 M and K_I, using pepstatin A as an inhibitor, was 8.05 × 10^8 M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P₁position, but the enzyme activity was highly reduced when the P₂position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.

Isolation and characterization of 4-chlorophenoxyacetic acid-degrading bacteria from agricultural soils: Chung, Min Jae , Shin, Se Young , Park, Yong Keun , Min, Kyung Hee , Ka, Jong Ok; J. Microbiol. 1997;35(2):117-122.

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Abstract: Several dominant 4-CPA-degrading bacteria were isoalted from agricultural soils. Most of the isolates were identified as Burkholderia species by fatty acid methyl ester (FAME) analysis, but they were distinct in chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences. These strains were generally restricted in their substrate utilization capabilities. The 4-CPA degradative enzymes were idnducible by 4-CPA and some isolates appeared to mineralize 4-CPA via formation of 4-chlorophenol and 4-chlorocatechol as intermediates during its biodegradation pathway. Plasmid DNAs were not detected from most of the isolates and their 4-CPA genes wer on the chromosomal DAN. The 4-CPA degradation patterns in axenic cultures and natural soils varied depending on the strains and soils. The inoculation of 4-CPA degraders much improved the removal of 4-CPA from the 4-CPA treated soils.

Production of lipocortin-1_1-185 using a recombinant of escherichia coli: Lee, Kyung Il , Oh, Kyung Hee , Lee, Jung Hyun , Na, Do Sun , Lee, Kye Joon; J. Microbiol. 1997;35(2):123-126.

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Abstract: The aim of the present study was to optimize culture condition for the expression of lipocortin 1_1-185 in a recombinant of Escherichia coli using batch system. Plasmid (pHT22) carrying lipocortin-1_1-185 gene was well maintained in the recombinant with the addition of amplicillin as a selection pressures. Optimum temperature was 28℃ for seed culture and 40℃ for main culture and the optimum pH was 7.0. The production of Lipocortin-1_1-185 was closely associated with cell growth and related to plasmid amplification.

Biosynthesis of polyhydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacillus thuringiensis R-510: Park, Sang Kyu , Lee, Kang Tae , Kim, Young Baek , Rhee, Young Ha; J. Microbiol. 1997;35(2):127-133.

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Abstract: Biosynthesis of polyhydroxybutyrate and copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by Bacillus thuringiensis R-510 grown with glucose or with mixtures of glucose and propionate was investigated. n-Alkanoic acids other than propionate were not precursors of 3HV units. The fraction of 3HV unit in the copolymer increased from 0 to 84 mol% of 3HV. Polymer yield decreased as the fraction of propionate was increased but the molecular weight distribution was not affected by the composition of carbon substrate. The minimum melting temperature (around 65℃) of poly (3HB-co-3HV) copolymers was observed for the polymer bearing approximately 35 mol% of 3HV. Polyhydroxyalkanoates production by this organism was not dependent on nutritional limitation, but remarkably influenced by dissolved oxygen concentration in the culture medium. Low level of dissolved oxygen concentration prevented spore formation in the cells and stimulated the synthesis of polyhydroxyalkanoate. The composition of poly (3HB-co-3HV) produced by B. thuringiensis R-510 lyhydroxyalkanoate. The composition of poly(3HB-co-3HV) produced by B. thuringiensis R-510 varied according to the growth time. However, there was no evidence that polymers isolated from cells were mixtures of immiscible polymers.

Purification and characterization of an exo-polygalacturonase from botrytis cinerea: Lee, Tae Ho , Kim, Byung Young , Chung, Young Ryun , Lee, Sang Yeol , Lee, Chang Won , Kim, Jae Won; J. Microbiol. 1997;35(2):134-140.

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Abstract: Botrytis cinerea T81-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as Ca^2+, Mg^2+, and Cu^2+. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was 50℃. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 30℃.

Characterization of biphenyl biodegradation, and regulation of biphenyl catabolism in alcaligenes xylosoxydans: Lee, Na Ri , On, Hwa Young , Jeong, Min Seon , Kim, Chi Kyung , Park, Young Keun , Ka, Jong Ok , Min, Kyung Hee; J. Microbiol. 1997;35(2):141-148.

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Abstract: Alcaligenes xylosoxydans strain SMN3 capable of utilizing biphenyl grew not only on phenol, and benzoate, but also on salicylate. Catabolisms of biphenyl and salicylate appear to be interrelated since benzoate is a common metabolic intermediate of these compounds. Enzyme levels in the excatechol 2,3-dioxygenas which is meta-cleavage enzyme of catechol, but did not induce catechol 1, 2-dioxygenase. All the oxidative enzymes of biphenyl and 2,3-dihydroxybiphenyl (23DHBP) were induced when the cells were grown on biphenyl and salicylate, respectively. Biphenyl and salicylate could be a good inducer in the oxidation of biphenyl and 2, 3-dihydroxybiphenyl. The two enzymes for the degradation of biphenyl and salicylate were induced after growth on either biphenyl or salicylate, suggesting the presence of a common regulatory element. However, benzoate could not induce the enzymes responsible for the oxidation of these compounds. Biphenyl and salicylate were good inducers for indigo formation due to the activity of biphenyl dioxygenase. These results suggested that indole oxidation is a property of bacterial dioxygenase that form cis-dihydrodiols from aromatic hydrocarbon including biphenyl.

A plasmid vector faciliting gene expression in both yeast and mammalian cells: Lee , Tae Ho; J. Microbiol. 1997;35(2):149-151.

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Abstract: A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The human cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced β-galactosidase, dimonstrating its dual host usage.

Nitric oxide in human cytomegalovirus replication and gene expression: Lee, Jee Yeon , Lee, Chan Hee; J. Microbiol. 1997;35(2):152-157.

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Abstract: Infection of human fibroblast (HF) cells with human cytomegalovirus (HCMV) result in changes in the intracellular level of second messengers. Since nitric oxide (NO) production has been known to be related with other second messengers, it is probable that HCMV infection of HF cells may involve NO. To test this possibility, the amount of NO was measured following HCMV infection but we were not able to detect significant change in the production of NO. Exogenous addition of NO generators such as sodium nitroprusside (SNP) or S-nitroso-N-a-cetylpenicillamine (SNAP) immediately after HCMV infection, however, inhibited virus multiplication. Furthermore, immunoblot experiment using monoclonal antibody to HCMV major immediate early (MIE) proteins or CAT assay using pCMVIE/CAT (plasmid containing CAT gene driven by HCMV MIE promoter) revealed that SNP or SNAP blocked the MIE gene expression. SNP was more effective than SNAP in hibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP in inhibiting HCMV multiplication or MIE gene expression. SNP produced more NO than SNAP. Although the mechanism for the inhibition of HCMV multiplication and MIE gene expression by NO is still elusive some correlation with NO-mediated inhibition of HCMV-induced increase in cytosolic free Ca^2+ concentration ([Ca^2+]) was observed. The increase of [Ca^2+] following HCMV infection was inhibited by SNP, and less effectively by SNAP. Raising [Ca^2+ with bromo-A23187 partially reversed the SNP block of MIE gene expression. Thus, there appear to e some relationships among NO. [Ca^2+], and HCMV MIE gene expression.

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