Journal of Microbiology

Volume 34(2); June 1996

Numerical classification of actinomycetes isolated from volcanic soil: Kim, Seung Bum , Lee, Soon Dong , Kim, Seon Young , Oh, Hyung Myung , Kang, Sa Ouk , Hah, Yung Chil; J. Microbiol. 1996;34(2):105-116.

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Abstract: Of actinomycetes isolated from volcanic compost soils, 115 representative strains which showed distinctive morphologicla features were numerically classified, compared with reference strains of Streptomyces. One hundred and twenty unit characters were tested and the average probability of error was 4.27%. The cluster analysis resulted in two groups: group A included strains of actinomycetes except streptomycetes. Group A was divided into 2 major clusters (over 5 strains), 10-diaminopimelic acid. Group B was divided into 5 clusters, of which 4 clusters contained mesodiminopimelic acid and 1 cluster LL-diaminopimelic acid. The major clusters of group A showed higher abilities of substrate utilization and degradation, and higher resistance to inhibitors, whereas the minor and single member clusters of group A showed relatively higher antimicrobial activities. On the other hand, all clusters of group B showed relatively lower abilities of substrate utilization and degradation and lower resistance to inhibitors.

Phylogenetic relationship of ganoderma species with the polyporaceae base don RFLP analysis of the nuclear ITS region: Park, Hong Je , Shin, Kee Sun , Yoon, Cheol Sik , Lee, Dong Hun , Bae, Kyung Sook; J. Microbiol. 1996;34(2):117-123.

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Abstract: Restriction-polymorphic patterns of nuclear-ITS were examined for the genetic relationships among 12 bisidiomycetous mushrooms to Aphyllophorales and Agaricales. The taxonomic affinity of Ganoderma species with the family Polyporaceae also was examined. With 13 restriction endonucleases, 159 restriction characters were generated form the 12 species examined. UPGMA and neighbor-joining analyses separated the 12 species into two genetically distinct groups that correspond to orders (Agaricales and Aphyllophorales) where each species is included. This result indicates that there is clear genetic demarcation between Agaricales and Aphyllophorales. Dendrograms constructed by several data analyses showed that even though Ganoderma species are somewhat in intermediate taxonomic position between the Polyporaceae and families of the Agaricales, they are genetically more related to the Polyporaceae. These results are consistent with morphological characters observed in those mushrooms. However, it is premature to conclude taxonomic status Ganoderma species in the present study employing small sample size.

Isolation and identification of infectious pancreatic necrosis virus form rainbow trout cultured in Korea: Lee, Jin Hee , Heo, Gang Joon , Lee, Chan Hee; J. Microbiol. 1996;34(2):124-130.

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Abstract: A survey was conducted to determine the prevalence of infectious pancreatic necrosis virus (IPNV) on fish farms in Korea and the epidemiology of IPNV infection in the farmed rainbow trout. In total, 43 pools of rainbow trout with apparent signs of viral infection from five provinces were obtained and analyzed. Evident cytopathic effects, including karyopycnosis and cell destruction, were observed in CHSE (chinook samlmon embyro)-214 cells infected with the virus isolates. Of these, ten viral isolates were assumed to be IPNV based on biophysical properties. RNA analysis revealed that the isolates contained two-segmented RNA genomes, further indicating that the viral isolates are IPNV. Antigenic comparison of the IPNV isolates identified three distinct serological groups separable by the cross-neutralization test. Of the ten IPNV isolates, six could be classified as strain DRT, two as strain Ab, and two as strain VR299. We were not able to isolate new strain of IPNV or any isolate serologically similar to the standard strain Sp.

Distribution and activity of sulfate-reducing bacteria in lake soyang sediments: Jin, Ho Yong , Lee, Dong Hun , Zo, Young Gun , Kang Chan Su , Kim Sang Jong; J. Microbiol. 1996;34(2):131-136.

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Abstract: In order to known the extend of contribution to the degradation of organic materials and nutrient recycling by sulfate-reducing bacteria (SRB) and methane-producing bacteria (MPB) in sediment, the distribution and activity of these two groups of microorganisms were studied montly in 1994 at two sites, one littoral (Sanggulri) and the other profundal (DAM), in Lake Soyang. In the seasonal distribution of two microorganisms, SRB were 1.07 × 10^3 - 2.42 × 10^5 cells/g-dry weight at Sanggulri, 2.40 × 10^5 - 1.29 × 10^6 at Dam and MPB were 0.52 × 10^3 cells/g-dry weight at Sangguri and 1.44 × 10^3 - 6.89 × 10^3 at Dam. In these results, the density of SRB in Lake Soyang is much higher than other lakes. These high values might be due to higher sulfate concentration, 0.69-4.05 mM, than normal freshwater, 0.01-1.2 mM. And a good correlation of SRB and chlorophyll a concentration implied that the important environmental factor on distribution of SRB might be the concentration of available organic matter. In a comparison of sulfate-reducing rate and methane producing rate in 1995, the activity of SRB for the degradation of organic matter was higher than MPB by factor of 359. Conclusively SRB superior to MPB in the distribution and activity are more important anaerobic bacteria in Lake Soyang sediments.

Regulation of fpr Gene encoding NADPH :Ferredoxin oxidoreductase by the soxRS locus in escherichia coli: Koh, Young Sang , Chouh, Jenny , Roe, Jung Hye; J. Microbiol. 1996;34(2):137-143.

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Abstract: We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of β-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of β-galactosidase was significantly reduced by introducing a Δsox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

Extracellular DNAs released form the genetically engineered E. coli CU103 during growth in different liquid media: Kim, Chi Kyung , Park, Sang Ho , Lim, Jai Yun , Kim, Young Chang , Kim, Young Soo , Min Kyung Hee , Lee, Ki Sung; J. Microbiol. 1996;34(2):144-150.

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Abstract: During growth of the genetically engineered E. coli CU103 in different media, extracellular DNAs released from the cells were studied. The extracellular DNAs released in the medium were concentrated by an ethanol precipitation method and then quantified by a fluorescence method using Hoechst 33258. The released extracellular DNAs were also examined by gel electrophoresis and identified by Southern hybridization for the cloned pcbCD genes. The chromosomal DNAs and recombinant plasmid containing the cloned genes were observed to be released in an exponential growth phase. In Luria-Bertani (LB) broth and MM2-GLUCOSE, 210 and 69 ng/ml of DNAs were detected, respectively, after 3-4 days incubation at 30℃ and at pH 7.0. But the released DNAs were measured to be about 10-15 ng/ml in filtered river water (FW) and Tris-EDTA (TE). The at both 15℃ and 4℃, but the released DNAs were more easily degraded at the higher temperature. The extracellular DNAs were produced about 2 times more at pH 7.0 than at both pH 5.0 and pH 9.0 in MM2-glucose medium at 30℃. Therefore, the extracellular DNAs were found to be released actively from the cells during growth in liquid media.

Molecular cloning and expression of shiga-like toxin II gene (slt-II) from an isolated of healthy Korean native bovine feces, fscherichia coli KSC109: Cha, In Ho , Kim kyoung Sook , Kim, Sang Hyun , Kim, Young Hwan , Lee, Young Choon; J. Microbiol. 1996;34(2):151-157.

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Abstract: By PCR amplification using the sequence of the previously cloned shiga-like toxin II DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequence s included tow open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparison of the nucleotide and predicted amino acid sequences of newly cloned gene (slt-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophabe DNA of E. coli 933 derived from a patient with hemorrhagic colities. In addition, the sequence homology of SLT-II with SLT-II variant form bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E, coli host-vector, system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxycity for Vero cells than that form original strain.

Genetic relationship between the SPT3 gene and RAS/cAMP pathway in yeast cell cycle control: Shin, Deug Yong , Yun, Jean Ho; J. Microbiol. 1996;34(2):158-165.

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Abstract: The signal transduction pathways through the RAS gene product and adenyl cyclease play a critical role in regulation of the cell cycle in yeast, Saccharomyces cerevisiae. We examined the genetic relationship between the spt3 gene and ras/cAMP pathway. A mutation in the SPT3 gene suppressed cell cycle arrest at the G1 phase caused by either an inactivation of the RAS or CYR1 gene which encodes a yeast homologue of human ras proto-oncogene or adenyl cyclase, respectively. The phenotypes such as sporulation and heat shock resistancy, that resulted from a partial inactivation of the RAS or CYR1 genes, were also suppressed by the spt3 mutation. Expression of the SSA1 gene encoding one of the heat shock proteins (Hsp70) can be induced by heat shock or nitrogen starvation. Expression of this gene is derepressed in cry1-2 and spt3 mutants. The bcy 1 mutation repressed by the bcy1 mutation, but not in spt3 mutants. These results suggest that the SPT gene is involved in expression of genes that are affected by the RAS/cAMP pathway.

Fast genetic variation among coliphage quasispecies revealed by a random amplified polymorphic DNA (RAPD) analysis: Kwon, Oh Sik , Lee, Jae Yung; J. Microbiol. 1996;34(2):166-171.

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Abstract: Genetic analysis was conducted on newly isolated coliphages form soil by using a RAPD assay. From the initial result, the coliphages were turned out to be different form one another but were closely related to φλ due to the fact that they shared the same RAPD maker in which other T phage testing failed to show. By using the primers EC01 or EC02, a fast genetic mutation of φC1 was found by producing specific RAPD markers on the phages from the first filial progeny to the second filial progeny. When we made a RAPD assay with combined primers (EC01, EC05 and EC08), the genetic mutation was again confirmed in φC1. The assay detection showed mutations in other coliphages such as φC2 and φC3 by revealing specific RAPD bands among different progeny phages, where genetic instability of the coliphages in implied.

Effect of copper on the growth and methanol dehydrogenase activity of methylobacillusd sp. strain SK1 DSM 8269: Kim, Si W. , Kim, Young M.; J. Microbiol. 1996;34(2):172-178.

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Abstract: Methylobacillus sp. strain SK1, which grows only on methanol, was found to grow in the absence of added copper. The doubling time (t_d = 1.3 h) of the bacterium growing at the exponential growth phase at 30℃ in the absence of copper was the same as that of the cell growing in the presence of copper. The bacterium growing after the exponential phase in the absence of copper, however, grew faster than the cell growing in the presence of copper. Cells harvested after thee early stationary phase in the presence of copper were found to exhibit no methanol dehydrogenase (MDH) activity, but the amount and subunit structure of the enzyme in the cells were almost the same as that in cells harboring active MDH. Pellets of the cells harvested after the early stationary phase in the presence of copper were pale green. Cell-free extracts prepared from cells harvested at the early stationary phase in the presence of copper were pink and exhibited MDH activity, but it turned dark-green rapidly from the surface under air. The green-colored portions of the extracts showed no MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH proteins in the green portions were found to have antigenic sites identical to those of the active one as the inactive MDHs in cells grown in the presence of copper. The bacterium was found to accumulate copper actively during the exponential growth phase. MDH prepared from cells grown in the presence or absence of copper was found to be more stable under nitrogen gas than under air. Methanol at 10 mM was found to enhance the stability of the MDH under air.

Optimization of culture conditions for production of pneumococcal capsular polysaccharide type I: Kim, Su Nam , Min, Kwan Ki , Kim, Seung Hwan , Choi, In Hwa , Lee, Suhk Hyung , Pyo, Suhk Noung , Rhee, Dong Kwon; J. Microbiol. 1996;34(2):179-183.

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Abstract: Streptoccus Pneumoniae (pneumococcus), the most common cause of bacterial pneumonia, has an ample polysaccharide (PS) capsule that is highly antigenic and is the source of PS vaccine. This investigation was undertaken to optimize the culture conditions for the production of capsulard PS by type 1 pneumococcus. Among several culture media, brain heart infusion (BHI) and Casitone based media were found to support luxuriant growth of pneumococcus type 1 at the same level. Because BHI medium is rather expensive and more complex than the Casitone based media, the Casitone based media was uwed to study optimization of the culture condition. The phase of growth which accomodated maximum PS production was logarithmic phase. Concentrations of glucose greater than 0.2% did not ehnahce growth or PS production. Substitution of netrogen sources with other resources or supplementation of various concentrations of metal ion (with the exception of calcium ion) had adverse affects on growth and PS production. On the other hand, low level aeration was beneficial for increased PS production. Addition of 3 mg/l concentration of methionine, phenylalanine, and threonine were found to enhance growth and PS production. The synerigistic effect of all the favorable conditions observed in pneumococcal growth assays provided a two-fold cummulative increase in capsular PS production.

Lipid analysis of streptomycetes isolated form volcanic soil: Kim, Seung Bum , Kim, Min Young , Seong, Chi Nam , Kang Sa Ouk , Hah, Yung Chil; J. Microbiol. 1996;34(2):184-191.

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Abstract: The cellular fatty acids and quinones of streptomycetes isolated from volcanic soils were analysed. The strains contained fatty acids of 14 to 17 carbon chains, and 12-methyltetradecanoic acid and 14 methylpentadecanoic acid were dominant in most strains. The total profiles consisted of 74% branched fatty acid family, 16.8% linear family and 8.2% unsaturated family. The largest cluster of grey spore masses defined by numerical classification was separated from the remainders in the principal component analysis, but the other clusters were overlapped with one another. In the analysis of respiratory quinones, all of the strains contained either the menaquinone of 9 isoprene units with 6 hydrogenations of 8 hydrogenations as the major species. The distribution of menaquinones among the clusters could provide an important key in the chemotaxonomy of streptomycetes.

Detection of L-xylosone and its physiological effects in saccharomyces cerevisiae: Seok, Yeong Jae , Yang, Kap Seok , Kang, Ju Gyeong , Kim, Seong Tae , Huh, Won Ki , Kang, Sa Ouk; J. Microbiol. 1996;34(2):192-197.

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Abstract: L-Xylosone was detected as its quinoxaline derivative in the degradation solution of dehydro-L-ascorbic acid. The production rate of L-xylosone was much faster in aerated phosphate-cirate buffer (pH 5. 6) than in pure water. L-Xylosone and dehydro-L-ascorbic acid were identified in the crude extracts of Saccharomyces cerevisiae. The concentration of L-xylosone in the crude cell extracts was calculated to be about 0.2 nmol (mg protein)^-1. When L-xylosone was added to asynchronous culture of S. cerevisiae, it inhibited primarily the synthesis of protein and RNA. Examination of the effect of L- xylosone on synchronous culture of the yeast indicated that L-xylosone inhibited the initiation of DNA replication and that the cells were arrested at G₁stage of cell division cycle. These results suggested a possibility that L-xylosone can act as an inhibitor of cell growth.

Partial characterization of proteases from culture filtrate of mycobacterium tuberculosis: Na, Byoung Kuk , Song, Chul Yong , Park, Young Kil , Bai, Gill Han , Ki, Sang Jae; J. Microbiol. 1996;34(2):198-205.

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Abstract: Two proteases were partially characterized from culture filtrate of Mycobacterium, tuberculosis KIT110. Their molecular weights were approximately 200 and 180 kDa, respectively and they exhibited similar enzymatic characteristics. These enzymes were inhibited significantly by EDTA and to some extent by EGTA. Their activity was enhanced by Ca^2+ and Mg^2+ to some degree. However, Cu^2+ and Ag^2+ completely inhibited the enzyme activity at the concentration of 2.5 and 5 mM, respectively. The optimal pH was 7.0 and optimal temperature was around 40℃. These enzymes were rapidly inactivated at 80℃. Therefore, they were heat-labile, neutral metalloproteases. These enzymes exhibited antigenicity shown by their reacting with sera from the partients with pulmonary tuberculosis. These enzymes were able to degrade serum proteins including hemoglobin, bovine serum albumin, lysozyme and immunoglobulin G and structural matrix protein such as type I collagen. Therefore, these enzymes may be thought to contribute to tissue necrosis and pathogenesis during infection.

An FMN-containing NADH-quinone reductase from streptomyces sp: Youn, Hong Duk , Lee, Jin Won , Youn, Hwan , Lee, Jeong Kug , Hah, Yung Chil , Kang, Sa Ouk; J. Microbiol. 1996;34(2):206-213.

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Abstract: NADH-quinone reductase was purified 22-fold from the cytosolic fraction of Streptomyces sp. Imsnu-1 to apparent hemogenity, with an overall yield of 9%, by the purification procedure consisting of ammonium, sulfate precipitation and DEAE Sephacryl S-200 and DEAE 5 PW chromatographies. The molecular mass of the enzyme determined by gel filtration chromatography was found to be 110 kDa. SDS-PAGE revealed that the enzyme consists of two sugunits with a molecular mass of 54 kDa. The enzyme contained 1 mol of FMN per subunit as a cofactor. The A_272/A_457 ratio was 6.14 and the molar extinction coefficients were calculated to be 20, 800 and 25, 400M/sup -1/cm/sup -1/ AT 349 AND 457 nm, respectively. The N-terminal sequence of the enzyme contained the highly conserved fingerprint of ADP-binding domain. The enzyme used NADH as an electron donor and various quinones as electron acceptors. Cytochrome c was practically inactive. Air-stable flavin semiquinone was produced by the addition of NADH to the enzyme. Also, naphthosemiquinone was detected in the reaction mixture containing the enzyme.

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