Journal of Microbiology

Volume 36(2); June 1998

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by Penicillium pinophilum: Han, Jeen Sun , Son, Young Jong , Chang, Chung Soon , Kim, Mal Nam; J. Microbiol. 1998;36(2):67-73.

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Abstract: The extracellular poly(3-hydroxybutyrate)(PHB) depolymerase of Penicilliyum pinophilum ATCC 9644 was purified and characterized. When Penicillum sp. was grown in basal salt medium with PHB as a sole carbon source, higher temperature favored fungal mycelial growth (37℃>30℃>25℃), but enzyme production was lower ar 37℃ than at any other temperatures. The PHB depolymerase was purified using Sepharose CL06B and Sephacryl S0100HR column chromatography. The isolated enzyme was found to be composed of a single polypeptide chain with a molecular weight of about 35 kDa. The optimum condition for the enzyme was pH 6.0 and 50℃, Enzyme activity decreased sharply at temperatures above 50℃. The enzyme was found to be stable in the pH range of 2.0~10.0.1mM Fe^2+ reduced the enzyme activity by 55% abd 4 mM Fe^2+ inhibited it almost completely. The PHB depolymerase (10U) degraded 47% of solvent cast PHB film, while commercial lipase (1000U) of Rhizopus arrhizus degraded 10% of the same specimen, over a period of 48 hours.

Chemical Midification of Purin Nucleoside Phosphorulase in Serratia marcescens: Choi , Hye Seon; J. Microbiol. 1998;36(2):74-79.

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Abstract: Serratia marcescens purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been previously described(Choi, H.S. 1998. Biosci. Biotechnol. Biochem. 62, 667-671). Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time dependent manner by phenylglyoxal or diethylpyrocarbonate (DEPC). There was a linear relationship between the observed rate of inactivation and the phenylglyoxal or DEPC concentration. At 30℃ the bimolecular rate constant for the modification was 0.22 mM^-1 min^-1 in 50 mM NaHCO_3 buffer, pH 7.5, for phenylglyoxal and 1.33 mM^-1min^-1 in 50 mM sodium cotrate, pH 6.0, for DEPC. Preincubation with saturated solutions of substrates protected the enzyme from inhibition by kphenylglyoxal and DEPC, indicating that reactions with these reagents were directed at arginyl and histidyl residues, respectively, which are essential for the catalytic function of the enzyme.

Identification and Characterization of Aniline-Degrading Bacteria Isolated from Soil and Wastewater: Shin, Se Young , Chung, Min Jae , Yi, Haak Rho , Kim, Chi Kyung , Min, Kyung Hee , Ka, Jong Ok; J. Microbiol. 1998;36(2):80-85.

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Abstract: Twenty two aniline-degrading bacteria were isolated from agricultural soil and wastewater. The isolates were able to utilize aniline as the sole source of carbon and nitrogen. A total of 45% of the isolates were identified to the species level by fatty acid methyl ester (FAME) analysis; the isolates were found to be strains of the Burkholderia, Nocardia, Arthrobacter, and Rhodococcus species. Their chromosomal patterns, obtained by polymerase chain reaction (PCR) amplification of repetitive extrogenic palindromic(REP) sequences, were distinct from each other. Plasmid DNAs were detected from 45% of the isolates, but only one strain, ANL5, was shown to have a transmissible, aniline degradative plasmid. The isolates were very restricted in their substrate utilization abilities. Aniline degradative enzymes were inducible by the presence of aniline, and most of the isolates appeared to mineralize aniline through an ortho-cleavage path-way using catechol 1,2-dioxygenase.

Diversity of Actinomycetes Antagonistic to Plant pathogenic Fungi in Cave and Sea-Mud Soils of Korea: Kim, Beom Seok , Lee, Jung Yeop , Hwang, Byung Kook; J. Microbiol. 1998;36(2):86-92.

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Abstract: To isolate actinomycetes antagonistic to plant pathogenic fungi, soil samples were collected from caves and sea-shores in Korea. The 481 actinomycetes were isolated from the soil samples examined, representing more than 50% of total counts. Nocardioform actinomycetes were rare actinomycete genera. Saccharomonospora could be isolated only in 3 cave soil samples from Cheondong, Kosoo, and Nodong, but was not present in all the sea-mud soils examined. Dactylosporangium, Saccharomonospora, and Streptosporangium were very rare in both cave and sea-mud soils. The 311 of 481 actinomycete isolates inhibited the mycelial growth of at least one of the tested fungi. The isolation rates of antagonistic actinomycetes from cave soils ranged from 45.7% to 78%, and those of sea-mud soils were from 59.1% to 66.0%. The 96 of 136 Streptomyces isolates from cave soils, and 93 of 133 isolates from sea-mud soils showed antifungal activity. The proportion of antagonistic isolates of Nocardioform actinomycetes (13.6%) was lower than that of other genera. Among the actinomycetes from sea-mud soils, Dactylosporangium and Streptosporangium had highest proportions of actinomycete antagonists of 85.7% and 80%, respectively. The isolation rate of Nocardioform antagonist from sea-mud soils was 11.1% similar in the cabve soils. Streptomyces strains showed higher antifungal activities against plant pathogenic fungi than did other rare actinomycete antagonists.

Cellular Response of Pseudomonas sp. DJ-12 to the Stresses of Several Aromatic Pollutants: Park, Sang Ho , Ko, Yeon Ja , Oh, Kye Heon , Kim, Chi Kyung; J. Microbiol. 1998;36(2):93-98.

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Abstract: Pseudomonas sp. DJ-12 is capable of degrading biphenyl, 4-chlorobiphenyl (4CB), and 4-hydroxybenzoate (4HBA) at relatively low concentrations. The degradability and cellular responses of the organism to the aromatic pollutants at higher concentrations have not been studied. In this study, the survival of the cells, production of stress proteins, and induction of tolerance were examined by exposing cells to various concentrations of the aromatics. The survival rates of the organism were reversely proportional to the concentration of each aromatic during incubation for 6 hs. Stress protein specifically reacting with anti-DnaK monoclonal antibody was commonly produced when cells were treated with each aromatic. Those cells pretreated with each aromatic at lower concentrations exhibited a certain degree of tolerance to higher concentrations of the aromatics. Such cellular responses of the organism to water-soluble 4HBA were more clearly distinguished than those to insoluble 3CB and biphenyl. Therefor, induction of DnaK stress protein in the cells by exposure to the aromatiocs could be used to develop an indicator for pollution by the aromatics.

Cloning and Expression in E. coli of the Genes Responsible for Degradation of 4-Chlorobenzoate and 4-Chlorocatechol drom Pseudomonas sp. Strain S-47: Kim, Ki Pil , Seo, Dong In , Lee, Dong Hun , Kim, Young Soo , Kim, Chi Kyung; J. Microbiol. 1998;36(2):99-105.

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Abstract: Pseudomonas sp. strain S-47 can grow on 4-chlorobenzoate (4CBA) and transform 4CBA to 4-chlorocatechol (4CC) under aerobic conditions, which is subsequently degraded to produce 2-hydroxypent-2, 4-dienoate (2H-2,4DA). The upper steps for conversion of 4CBA to 4CC are recognized to be conducted by the benzoate-1,2-dioxygenase (B12O) system encoded by benABC and benD. The ensving meta-cleabage reaction of 4CC is catalyzed by catechol 2,3-dioxygenase(C23O) encoded by the xylE gene. The benABCD and the xylE genes were cloned from the chromosome of Pseudomonas sP. S-47 into pCS1(48.7kb), pCS101(24.4kb), pCS201(17.7kb), and pCS202(6.7kb) recombinant plasmids, and were well ecpressed in E. coli XL1-Blue host cells. The PstI-insert (4.0kb) of pC202 was found to contain the benABCD and cylE genes and to have 2 EcoRV, 1 SphI, and 3 SacII restriction sites.

Laboratory Developed fluoroquinolone Resistant Escherichia coli Has a new Missense Mutation in QRDR of PartC: Lee, Soon Deuk , Lee, Yeon Hee; J. Microbiol. 1998;36(2):106-110.

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Abstract: The fluoroquinolone resistance mechanism of four laboratory developed fluorquinolone resistant strains of Escherichia coli was studied. Fluoroquinolone concentrations inside the resistant cells were similar to the concentrations in the susceptible cells. DNA sequencing of the quinolone resistance determining regions (QRDR) in gyrA and parC revealed the presence of Ser 83Leu and Asp87Gly mutations in GyrA, and Gly78Cys and Ser80Arh mutations in ParC of the ofloxacin, norfloxacin, and HK3140 resistant strains, while the ciprofloxacin resistant strain had Ser83Leu and Aasp87Tyr mutations in GyrA, and Gly78Cys and Ser80Ile mutations in ParC. A Gly78Cys substitution in ParC was newly detected in this work and seemed to be responsible for the extremely high MICs to fluroquinolones.

Quantitative Analysis of Expressed Genes in Aspergillus Oryzae by Sequencing 3'-directed cDNA Clones: Hwang, Hyun Ah , Lee, Dong Whan , Kim, Jong Hwa , Lee, Tae Kyoo , Yang, Moon Sik , Chae, Keon Sang; J. Microbiol. 1998;36(2):111-117.

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Abstract: Sequence analysis of randomly selected 3'-directed cKNA clones has been known to be one of the most powerful methods of examining the genes highly expressed in a tissue or cell type. We constructed a 3'-directed cDNA libraty from Aspergillus oryzae mycelia, and sequenced 345 randomly selected 3'-directed cDNA clones. Determined nucleotide sequences, not shorter than 30nt, were compared with one other to generate gene signatures (GSs) and were then compared with GenBank entries to analyze sequence similarity to known genes. A GS for the most highly expressed gene appeared six times, one GS five times, five GSs four times, five GSs three times and 22 GSs twice. In total, 324 clones yielded 268 GSs consisting of 34 redundant GSs appeaning at least twice and 234 solitary ones. Forty-three GSs showed similarities ranging from 60% to 99% with known sequences from Genbank. A considerable number of A. oryzae GSs mateched those obtained from the sexual structures of A. nidulans suggests that A. oryzae may not be phylogentically distant from A. nidulans and that A. oryzae may have a sexual life cycle from the ancient period.

Utilization of Inorganic Nitrogen by a Phototrophic Bacterium, Chromatium sp. at Mid-Depth in Lake Kaiike: Moon, Sang Wook , Michrio Matruyama; J. Microbiol. 1998;36(2):118-123.

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Abstract: Two bacterial species, Chromatium sp. and Macromonas sp. formed dense populations (bacterial plate) at an upper boundary of the H_2S layer of Lake Kaiike, Kamikoshiki island, Kagoshima Profecture. To obtain information on the utilization of inorganic nitrogen by the phototrophic bacterium Chromatium sp. in relation to light and H_2S, field observation was conducted in Lake Kaiike, in October, 1997. A dense population of Chromatium sp. was found from between 5 to 6 m in the lake, and the maximum number, 1.01×10^6cell/ml, was found at 5.5m. Average daytime light intensities at 5 and 5.5 m, expressed as photosynthetically available radiation (PAR), were 66.7 and 1.2 ㎛ole/m^2/s, respectively. The relative abundances of H_2S and NH_4^+ below the bacterial plate of the lake was established on their molar bases. The molar ratio of △H_2S to △NH_4^+ was 12.2 The bacterial N_2 fication is considered to have occurred when the bacterial requirement for nitrogen increased due to an elebated light level. It is suggested that the high H_2 concentration found at the upper part of the bacterial plate might be attrivuted to the bacterial N_2 fixation.

A Simple and Rapid Molecular Typing of Mycobacterium tuberculosis by Polyberase Chain Reaction: Lee, Hye Young , Bangm Hye Eun , Lee, Jin Hee , Myung, Han Jung , Kim, Joo Deuk , Cho, Sang Nae; J. Microbiol. 1998;36(2):124-129.

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Abstract: As an attempt to evaluate a molecular tool fingerprinting clinical isolates of Mycobacterium tuberculosis, a PCR-based typing method, so-called outward-PCR, was employed in this study. Outward-PCR used in this study was designed to amplify the wequences in-between two IS6110 elements. A total of 81 M. tuberculosis isolates including 73 Korean and 8 Philippine isolates were subjected to PCR amplification and the profiles of the agarose gel electrophoresis were analyzed. In brief, under the PCR conditions used in this study, the 81 clinical isolates were classified into 33 distinctive sub-groups. Among these, 5 sub-groups represented major clusters with 7 to 11 clinical isolates belonging to each suv-group. The banding patterns were clear and reproducible, implying that this repid and simple PCR-based typing method can be a valuable tool for typing clinical isolates of M. tuberculosis.

Sequence Analysis of the Latent Membrane Protein 1 Genes of Epstein-Barr Virus isolataes in Korea: Cho, Shin , Cho, Sung Gyu , Shim, Young Shik , Lee, Won Keun; J. Microbiol. 1998;36(2):130-138.

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Abstract: The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is essential for Blymphocyte transformation, and activates NF-kB transcription factior in lymphocytes. LMP1 genes were isolated and sequenced from three type 1 isolates (SNU-321, SNU-538, and SNU-1103) and a type 2 isolate (SNU-20), all derived from Korean cancer pstients, to assess sequence variations in the LMP1 of Korean EBV isolates. Sequence analysis revealed that the SNU-1103 and SNU-20 LNP1 genes were nearly identical to that of the prototype B95-8 type 1 EBV strain, with 98% and 96% identities at the nucleotide and protein level, respectively. The SNU-321 and SNU-538 type 1 LMP1 genes both had a G to T substitution at nucleotide position 169,426, resulting in the loss of a XhoI site, and a carboxy-termina 30 base pair deletion (position 168,287-168,256), indicating they were variant LMP1 genes, as initially described in a Chinese nasopharyngeal carcinoma-derived EBV isolate (CAO). These two variant LMP1 genes shared more sequence variations than the SNU-20 and SNU-1103 IMP1 geres presumably associated with the LMP1 XhoI polymorphism, and showed 96% and 94% sequence identities, respectively, at the nucleotide and amino acid level to respective sequences of B95-8. There were consistent variations between all four isolates and B95-8, including 8-amino acid changes (B95-8 residues 85, 122, 129, 222, 309, 312, 334, 338, and 366) and a 5-amino acid deletion in the carboxy-terminal third 11-amino acid repeat. Transfection of each of these cloned LMP1 genes into Jurkat cells resulted in tenfold stimulation of NF-kB activity, confirming functionality of LMP1 proteins expressed from these genes. Taken together, these results indicate that there is a high degree of overall conservation in sequences of LMP1 between different EBV isolates, yet the distinct sequence variation patterns are consistent with the notion that there are at least two distinct LMP1 variants.

High Dosage of Rok1p, a Putative ATP-dependent RNA Helicase, Leads to a Cell Cycle Arrest at G1/S Stage in Saccharomyces cerevisiae: jeong, Hyun Sook , Oh, Jae Young , Kim, Jin Mi; J. Microbiol. 1998;36(2):139-144.

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Abstract: The ROK1 gene encodes a putative ATP-dependent RNA helicase which is essential for mitotic cell growth. ROK1 has been thought to affect microtubule and spindle pole body (SPB) functions in Saccharomyces cerevisiae. To investigate the intracellular functions of ROK1, we varied the Rok1 protein dosage in a cell and analyzed its phenotypic effects. Overexpression of the ROK1 gene by using a strong GAL1 promoter was lethal, leading cells to arrest at the unbudded stage. This arrest phenotype is very similar to that of the rok1 null mutation. Indirect immunofluorescence revealed that the majority of arrested cells contained a single SPB. Normas development of microtubules between the duplicated SPSs was rarely observed. Multinuclear cells with abnormal microtubule array were detected in small fraction. Taken together with the phenotype of the rlk1 null mutation, these results imply that ROK1 is required for cell cycle progression at the G1/S stage.

Effect of β-Glucosidase on the Heteroprophic Bacteria in Cheonho Reservoir: Kwag, No Tae , Han, Suk Kyun , Go, You Seok , Ahn, Tae Young; J. Microbiol. 1998;36(2):145-150.

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Abstract: Cheonho reservoir is a small and eutrophicated lake. The variation of BOD in the reservoir was correlated to the variation of chlorophyll-a. BOD was maximum (39.1㎍/L) at site 1 in July of 1995 when chlorophyll-a was the highest. β-Glucosidase activities ranged from 9 to 241 nM/h. β-Glucosidase activity varied in the following order:site 3>site 2>site 1>site 4. The β-Glucosidase activity of the site 4 was less than 50% of the other three sites. Organic matter was produced by algae at all sites but was not directly related with the increase of heterotrophic bacteria. β-Glucosidase activity showed a close correlation with the number of heterotrophic bacteria, indicating a tight coupling between algae and heterotrophic bacteria through agal organic production.

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