Journal of Microbiology

Volume 35(3); September 1997

Phylogenetic study of penicillium chrysogenum based on the amino acid sequence analysis of chitin synthase: Park, Bum Chan , Lee, Dong Hun , Bae, Kyung Sook , Park, Hee Moon; J. Microbiol. 1997;35(3):159-164.

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Abstract: The phylogenetic study of Penicilium chrysogenum was performed based on amino acid sequence comparison of chitin synthase. Phylogenetic trees were constructed with the deduced amino acid sequences of the highly conserved region of chitin synthase gene fragments amplified by PCR. The BlasP similarity search and the bootstrap analysis of the deduced amino acid sequences of chitin synthase from P. chrysogenum with those form other fungi showed a close evolutionary relationship of Penicillium to ascomycetous fungi, especially to genus Aspergilus. The result from bootstrap analysis of the deduced amino acid sequences of the Class II chitin synthase from ascomyceteous fungi supported the usefulness of the Class II chitin synthease for phylogenetic study of filamentous fungi.

Isolation, characterization, and phylogenetic position of a new sulfur-oxidizing bacterium: Chang, So Youn , Yoon, Joon Sik , Shin, Yong Kook , Park, Yong Ha , Park, Jin Yeol , Yang, Song Suk , Koh, Moon Joo , Yoon, Seong Myeong , Lee, Jung Sup , Lee, In Hwa , Kim, Si Wouk; J. Microbiol. 1997;35(3):165-171.

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Abstract: A sulfer-oxidizing bacterium was isolated from mine wastewater and characterized. The isolate was gram-negative, rod (0.2 × 1.2-1.5㎛), nonmotiloe, catalase positive, and oxidase positive. The optimal pH and temperature for growth were 7.0 and 30℃, respectively. The optimum thiosulfate concentration was 70 mM and the maximum growth rate was 0.081 hr. The major ubiquinone contained in the isolate was Q-8. The cellular fatty acid composition was C_16:0, C_18:1, C_17cyc, and C_19cyc as nonpolar fatty acids, and 3-OH C10 : 0 and 3-OH C_12:0 as hydroxylated fatty acids. The isolate was a facultative chemolithoautotroph which can grow autotrophically on sodium thiosulfate and sodium sulfide and which can grow heterotrophically on yeast extract. It can also grow mixotrophically on sodium thiosulfate and yeast extract. Comparison of the 16S rRNA gene sequence of the isolate with that of Thiobacillus species and Paracoccus thiocyanatus revealed that it is closely related to T. caldus which belongs to the β-subclass of the class Proteobacteria. However, the isolated could not grow at extremely low pH (pH 1-3.5). On the basis of the phenotypic, chemotaxonomic and phylogenetic data, the isolate was tentatively named Thiobacillus sp. strain C.

Genomic polymorphism in clinical mycobacterial strains analyzed by pulsed-field gel electrophoresis: Kim, Jeong Ran , Kim, Cheorl Ho; J. Microbiol. 1997;35(3):172-176.

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Abstract: The Mycobacterium tuberculosis clinical isolates in Korea, showing different drug resistances, were analyzed by comparing large restriction fragment (LRF) patterns produced y digestion of genomic DNA with infrequent-cutting endonucleases of SpeI, AsnI and pulsed-field gel electrophoresis (PFGE). SpeI and AsnI allowed with AsnI and SpeI, strains yielded an absolutely identical pattern for Korean type's mycobacteria even though they showed different drug resisstance. However, when three M. tuberculosis strains, showing drug resistance, were digested with XbaI, patterns were different from those of the other M. tuberculosis strians which are susceptible to drugs. This study reveals that the comparison of chromosomal restriction patterns is very useful as an additional aid for the differentiation and identification of M. tuberculosis strains showing drug resistances.

Eveluation of line probe assay in detecting rifampicin resistance of mycobacterium tuberculosis: Park, Young Kil , Cho, Snag Hyun , Na, Nyoung Kuk , Song, Chul Yong , Bai, gill Han , Kim, Sang Jae; J. Microbiol. 1997;35(3):177-180.

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Abstract: The purpose of this study was to evaluate the efficiency of Line Probe Assay (LiPA) in detecting the rpoB gene mutation of clinically isolated Mycobacterium tuberculosis (MTB) and to compare the level of resistance to the various rifamycins with their mutation sites. The mutation in the rpoB gene was found in 84 (97.6%) out of 86 rifampicin (RMP) resistant strains as determined by LiPA. No mutation was observed in 2 RMP resistant strains and in any of 38 RMP susceptible strains tested. Only one of 3 strains with Δ5/R5, one of 2 strains with Δ3, and one of 3 strains with Δ2/R2 LiPA profile showed a slightly lower level of resistance to the rifapentine than the other strains. Although we could not find correlations between mutation sites in the rpoB gene and the level of susceptibility to the various rifamycins, the LiPA is recommended as a fast screening tool for detection of RMP resistant MTB.

Optimal condition for efficient DNA transfer in filamentous cyanobacteria by electroporation: Poo , Ha Ryoung; J. Microbiol. 1997;35(3):181-187.

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Abstract: Filamentous cyanobacteria are an ecologically important group of bacteria because they are able to provide both organic carbon fixed nitrogen that can support the nutritional requirements for other microorganisms. Because of their prokaryotic nature, they can also be used as potentially powerful model systems for the analysis of oxygenic photosynthesis and nitrogen fixation. Gene transfer is an indispensable procedure for genetic analysis of filamentous cyanobacteria. Electroporation was used to introduce foreign DNA into cyanobacterial cells. In experiments designed to optimize the electroporation technique, the effects of the field strength (amplitude of pulse) and time constant (duration of pulse), DNA concentration and host restriction/modification of DNA on the efficiency of electro-transformation were investigated. The results of this research revealed that a high voltage pulse of short duration was effective for the electro-transformation of Anabaene sp. M131. The maximal number of transformants was obtained at 6 kV/cm with a pulse duration of 5 msec. The efficiency of electro-transformation was also sensitive to concentration of DNA; even small amounts of DNA (0.01 ㎍/ml) were able to give a large number of transformants (1.0 × 10³ cfu/ml).

Isolation of pseudomonas sp. S-47 and its degradation of 4-chlorobenzoic acid: Seo, Dong In , Lim, Jai Yun , Kim, Young Chang , Min, Kyung Hee , Kim, Chi Kyung; J. Microbiol. 1997;35(3):188-192.

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Abstract: The strain of S-47 degrading 4-chlorobenzoic acid (4CBA) was isolated from Ulsan chemical industrial complex by enrichment cultivation with 1 mM 4CBA. The strain was Gram-negative rod and grew optimally at 30℃ and pH 7 under aerobic condition, so that the organism was identified as a species of Pseudomonas. Pseudomonas sp. S-47 degraded 4-chlorobenzoic acid to produce a yellow-colored meta-cleavage product, which was confirmed to be 5-chloro-2-hydroxymuconic semialdehyde (5C-2HMS) by UV-visible spectrophotometry. 5C-3HMS was proved trometry. This means that Pseudomonas sp. S-47 degraded 4CBA via 4-chlorocatechol to 5C-2HMS by meta-cleavage reaction and then to 5C-2HMA by 5C-2HMS dehydrogenase.

Characterization of BTX-degrading bacteria and identification of substrate interactions during their degradation: Oh, Young Sook , Choi, Sung Chan; J. Microbiol. 1997;35(3):193-199.

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Abstract: From several industrial wastewaters, 14 bacterial strains which degrade benzene, toluene, o-xylene, m-xylene, or p-xylene (BTX) were obtained. These strains were characterized as to their species composition and the substrate range, kinetic parameters and the substrate interactions were investigated. Although BTX components have a similar chemical structure, isolated strains showed different substrate ranges and kinetic parameters. None of the strains could degrade all of BTX components and most of them showed an inhibition (Haldane) kinetics on BTX, BTX mixtures were removed under inhibitory substrate interactions with variation in the intensity of inhibition. For a complete degradation of BTX, a defined mixed culture containing three different types of pathways was constructed and all of the BTX components were simultaneously degraded with the total removal rate of 225.69 mg/g biomass/h Judging from the results, the obtained mixed culture seems to be useful for the treatment of BTX-contaminated wastewater or groundwater as well as for the removal of BTX from the contaminated air stream.

Kinetic and spectral investigations on Ca^2+ and Sr^2+ containing methanol dehydrogenases: Kim, Si Wouk , Kim, Chung, Sung , Lee, Jung Sup , Koh, Moon Joo , Yang, Song Suk , Duine, Johannis A. , Kim, Young Min; J. Microbiol. 1997;35(3):200-205.

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Abstract: Both Ca^2+ and Sr^2+ containing methanol dehydrogenases (MDH) were purified to homogeneity with yields of 48% and 42%, respectively, from Methylabacillus methanolovorus sp. strain SK5. Most of the biochemical and structural properties were similar to each other. However, some differences were found: (1) although the overall shape of the absorption spectrum of Sr^2+ MDH was very similar to that of Ca^2+ MDH, the absorption intensity originating from the cofactor in Sr^2+. MDH was higher than that in Ca^2+-MDH. Small blue shift of the maximum was also observed. These are probably due to a difference in redox state of the cofactors in Ca^2+ and Sr^2+ -MDH; (2)Sr^2+ -MDH was more heat-stable than Ca^2+-MDH above 56℃; (3) the V_max values for the methanol-dependent activities of Sr^2+ Ca^2+ -MDH in the presence of 3 mM KCN were 2.038 and 808 nmol/mg protein/min, respectively. In addition, the K_m values of Sr^2+ and Ca^2+ MDH for methanol were 12 and 21 uM, respectively; (4) the endogenous activity of Ca^2+ -MDH was more sensitive than that of Sr^2+ -MDH in the presence of cyanide; (5) Diethyl pyrocarbonate treatment increased the enzyme activities of Ca^2+ and Sr^2+ MDH 4.2 and 1.4 folds, respectively. These results indicate that Sr^2+ stabilizes the structural conformation and enhances the activity of MDH more than Ca^2+.

Isolation and characterization of a noval membrane-bound cytochrome C_553 from the strictly anaerobic phototroph, heliobacillus mobilis: Lee, Woo Yiel , Blankenship, Robert E. , Kim, Seung Ho; J. Microbiol. 1997;35(3):206-212.

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Abstract: Heliobacillus mobilis is a strictly anaerobic Gram-positive bacterium which contains a primitive Photosystem I-type reaction center. The membrane-bound cytochrome c_553 from the heliobacterium suggested to be the immediate electron donor to the photooxidized pigment (P798+) has been isolated and characterized. The heme protein was visualized as a major component with an apparent molecular size of 17kDa in TMBZ-staining analysis of the membrane preparation and showed characteristic α (552.5 nm), β (522nm), and Soret absorption (416 nm) peaks of a typical reduced c-type cytochrome in the partially purified sample. The internal 43 amino acid sequence of the electron donor was obtained by chemical agent and protease treatments followed by N-terminal sequencing of the resulting fragments. The internal sequence carries lots of lysine residues and a Cys-X-X-Cys-His sequence motif which are the characteristics of typical c-type cytochromes. The analysis of the sequence by FAST or FASTA program, however, did not show any significant similarity to other known heme proteins.

A possible mechanism responsible for translocation and secretion an alkaliphilic bacillus sp. S-1 pullulanase: Shim, Jae Kyoung , Kim, Kyoung Sook , Kim, Cheorl Ho; J. Microbiol. 1997;35(3):213-221.

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Abstract: The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly but quantitatively released into the medium during growth of the cells in medium containing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M_r pullulanase to a 140,000M_r polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M_r extracellular form. Processing of the 180,000 M_r protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

Cloning and phylogenetic analysis of chitin synthase gene from entomopathogenic fungus, beauveria brongniartii: Nam, Jin Sik , Lee, Dong Hun , Park, Ho Yong , Bae, Kyung Sook; J. Microbiol. 1997;35(3):222-227.

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Abstract: DNA fragments homologous to chitin synthase gene were amplified from the genomic DNA of Beauveria brongniartii by PCR using degenerate primers. Cloning and sequencing of the PCR-amplified fragments led to the identification of a gene, designated BbCHS1. Comparison of the deduced amino acid sequence of BbCHS1 with those of other Euacomycetes revealed that quence of BbCHS1 displayed the highest rate of similarity, 95.8%, with CHS2 or Metarhizium anisopliae. Phylogenetic analysis of the amino acid sequences confirmed the taxonomic and evolutionary position of B. brongiartii, which was previously derived by traditional fungel clasification based on morphological features.

Amino acid substitutions conferring cold-sensitive phenotype on the yeast MTF1 gene: Jang , Sei Heon; J. Microbiol. 1997;35(3):228-233.

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Abstract: The MTF1 gene of Saccharomyces cerevisiae encodes a 43 kDa MITOCHONDRIAL RNA polymerase specificity factor which recognizes mitochondrial promoters to initiate correct transcription. To better understand structure-function of the MTF1 gene as well as the transcription mechanism of mitochondrial RNA polymerase, two cold-sensitive alleles of the MTF1 mutation were isolated by plasmid shuffling method after PCR-based random mutagenesis of the MTF1 gene. The mutation sites were analyzed by nucleotide sequencing. These cs phenotype mtf1 mutants were respiration competent on the nonfermentible glycerol medium at the permissive temperature, but incompetent at 13℃. The cs phenotype allele of the MTF1, yJH147, encoded an L146P replacement. The other cs allele, yJH148, contained K179E and K214M double replacements. Mutations in both alleles were in a region of Mtflp which is located between domains with amino acid sequence similarities to conserved regions 2 and 3 of bacterial s factors.

A conditional lethal mutation of a nucleoporin gene, NUP49 in saccharomyces cerevisiae: Lee, Youn Soo , Song, Young Ja , Hwang, Mi Kyung , Lee, Woo Bok , Kim, Jin Mi; J. Microbiol. 1997;35(3):234-238.

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Abstract: Conditional lethal mutation nup49-1 of a nuclear pore complex component gene was constructed in Saccharomyces cerevisiae. This mutation deleted one third of the essential NUP49 gene at the carboxy-terminal, but retained 13 repeats of the highly conserved GLFG domain. The nup49-1 mutant strain was viable with a slow-growth phenotype, indicating that the C-terminal is dispensable at normal growth temperature. This strain exhibited both temperature-sensitivity at 37℃ and cold-sensitivity at 16℃. Temperature shift experiments revealed that the arrest phenotype at 37℃ was random in the cell division cycle. The nup49-1 mutation was tested to be recessive and is expected to be useful for the functional analysis of nuclear pore complex proteins as well as for studies of nuclear transport systems.

Increased Activities of SOD and Catalase on Aerobic Growth in Arcobacter nitrofigilis: Park, Young Bok , han, Yeong Hwan; J. Microbiol. 1997;35(3):239-240.

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Abstract: A free-living nitrogen fixing Arcobacter nitrofigilis exhibuted the typical characteristics of aerobic growth in which the maximal cell growth was shown under an ambient air atmospjere, whereas no cell growth was shown umder an anaerobic condition. When oxygen concentration was increased, the activities of SOD and catalase were increased. These suggest that the aerobic nature of A. nitrofigilis might be due to the increased levels of both enzymes that scavenge toxic forms of oxygen.

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