Journal of Microbiology

Volume 41(1); March 2003

Genetic Relatedness within Streptococcus pneumoniae Serotype 19F and 23F Isolates in Korea by Pulsed-Field Gel Electrophoresis: Kwang Jun Lee , Song Mee Bae , Kyu Jam Hwang , Young Hee Lee , Ki Sang Kim; J. Microbiol. 2003;41(1):1-6.

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Abstract: The genetic relatedness of multidrug-resistant pneumococcal isolates of serotypes 19F and 23F was investigated. The DNA fragments digested with Sma I were resolved by pulsed-field gel electrophoresis (PFGE). PFGE analysis of 36 S. pneumoniae isolates showed 13 different patterns. Among 22 isolates of serotype 19F, 9 different PFGE patterns were present and 14 isolates of serotype 23F isolates represented 5 distinct PFGE patterns. Two isolates of serotype 19F and six isolates of serotype 23F shared the same PFGE pattern (Pattern I). Based on the genetic relatedness within the strains (one genetic cluster was defined as having more than 85% homology), we divided the pneumococcal strains into 6 genetic clusters (I, II, III, IV, V, and VI). The 22 strains of serotype 19F belonged to five distinct genetic clusters (I, II, III, IV, V and VI) and 14 strains of serotype 23F represented two genetic clusters (I and II ). These results showed that strains of serotype 19F are genetically more diverse than those of serotype 23F. Serotype 19F isolates with PFGE patterns H and I appeared to be less related to those of the remaining PFGE patterns (A to G) (less than 60% genetic relatedness), but those strains were genetically closely related with serotype 23F. These results suggest that the latter isolates originated from horizontal transfer of the capsular type 19F gene locus to 23F pneumococcal genotypes. In conclusion, the multidrug-resistant pneumococcal isolates of serotype 19F and 23F isolated in Korea are the result of the spread of a limited number of resistant clones.

Genetic and Phenotypic Diversity of Dichlorprop-Degrading Bacteria Isolated from Soils: Hae-Dong Park , Jong-Ok Ka^; J. Microbiol. 2003;41(1):7-15.

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Abstract: Nine dichlorprop-degrading bacteria and three pairs of bacteria showing syntrophic metabolism of the herbicide were isolated from soils, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genera, Sphingomonas, Herbaspirillum, and Bradyrhizobium. Twelve different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 15 isolates. The isolates were able to utilize the herbicide dichlorprop as a sole source of carbon and energy and their dichlorprop degradative pathways were induced by the presence of dichlorprop. Most of the isolates and syntrophic pairs were able to degrade both (R)- and (S)-dichlorprop, but strain DP522 exhibited enantioselective degradation of (S)-dichlorprop. The isolates degraded 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and mecoprop, in addition to dichlorprop. Oxygen uptake experiments indicated that most of the isolates degraded dichlorprop through 2,4-dichlorophenol.

Molecular Bases of High-Level Streptomycin Resistance in Pseudomonas marginalis and Pseudomonas syringae pv. actinidiae: Hyo Shim Han^ , Hye Young Nam^ , Young Jin Koh^ , Jae-Seoun Hur^ , Jae Sung Jung^; J. Microbiol. 2003;41(1):16-21.

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Abstract: We have collected eight high-level streptomycin-resistant strains of Pseudomonas marginalis and P. syringae pv. actinidiae which were isolated from kiwifruit orchards in Korea and Japan. The molecular mechanisms of resistance were investigated by the PCR, susceptibility tests, and nucleotide sequence analysis. Of the eight high-level streptomycin-resistant strains, four harbored strA-strB genes, which encode streptomycin-inactivating enzymes. While the three Korean strains of P. marginalis did not have plasmid and carried the resistant genes in the chromosomes, the Japanese strain of P. syringae pv. actinidiae had a plasmid containing strA-strB genes. The myomycin susceptibility test demonstrated that the high-level resistance to streptomycin of the remaining four strains is associated with mutations in the rpsL gene. Nucleotide sequence analyses revealed that they contain a single base-pair mutation in codon 43 of their rpsL gene.

Purification and Characterization of Cold Active Lipase from Psychrotrophic Aeromonas sp. LPB 4: Han-Ki Lee , Min-Jung Ahn , Sung-Ho Kwak , Won-Ho Song , Byeong-Chul Jeong; J. Microbiol. 2003;41(1):22-27.

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Abstract: A lipase from Aeromonas sp. LPB 4, a psychrotophile isolated from a sea sediment was purified and characterized. The lipase was purified 53.5 fold to a homogeneous state by acetone precipitation and QAE sephadex column chromatography and its molecular weight was determined to be 50 kDa by SDS-PAGE. The enzyme exhibited maximum activity at 10oC and was stable at temperatures lower than 50℃. This lipase favored substrates containing medium carbon chain of acyl group, while too low and high carbon chain decreased its activity. The lipolytic activity of purified lipase was slightly increased by the addition of 0.1% detergent, but decreased by 1% of detergent. Butanol severely decreased the lipase activity while methanol increased the activity about 15%.

Examination of the Antioxidant Potential of Pycnogenol under Conditions of Oxidative Stress in Escherichia coli Mutants Deficient in HP1 and Superoxide Dismutase Activities: Jeong A Youm , Young Gon Kim; J. Microbiol. 2003;41(1):28-33.

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Abstract: Pycnogenol (PYC) is believed to have potential as a therapeutic agent against free radical-mediated oxidative stress. It is important, therefore, to understand the interactions between PYC and cellular defenses against oxidative stress. Toward this end, we analyzed the survival rates on the gene expression responses of E. coli sod katG mutants to PYC after pre-treatment of PQ or H_2O_2-mediated stress under aerobic conditions. We identified SOD induced by PYC, but not HP1 in sod katG mutants. A striking result was the PYC induction of SOD with antioxidant property in single katG mutant cells, particularly MnSOD and CuZnSOD. These inductions were further increased with oxidative stress, while HP1 was not induced in these conditions. The effects of pycnogenol treatment on these cells depend in part on its concentration on the stress response. Protective effects of PYC exposure which affected gene expression in cells were consistent with cell survival rates. Our results demonstrate that pycnogenol may alter the stress response gene expression in a specific manner such as SoxRS because PYC induction of single mutant only worked under increased PQ stress. All together our data indicate that SOD activity is essential for the cellular defense against PQ-mediated oxidative stress, suggesting that PYC may not be effective as an antioxidant in only oxidative stress conditions. On the other hand, it was expected that PYC may play a role as a pro-oxidant and if it is available for use, it should be evaluated carefully.

Environmental factors affecting development of Aspergillus nidulans: Kap-Hoon Han , Dong-Beom Lee , Jong-Hak Kim , Min-Su Kim , Kyu-Yong Han , Won-Shin Kim , Young-Soon Park , Heui-Baik Kim^ , Dong-Min Han^; J. Microbiol. 2003;41(1):34-40.

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Abstract: Aspergillus nidulans, a homothalic ascomycete, has a complete sexual reproductive cycle as well as an asexual one. Both sexual and asexual development are known to be genetically programmed, but are also strongly affected by environmental factors including nutrients, light, temperature and osmolarity. We have examined these factors to define favored conditions for fruiting body (cleistothecium) formation. In general, fruiting body formation was enhanced where carbon and nitrogen sources were sufficient. Limitation of C-source caused predominant asexual development while inhibiting sexual development. When higher concentrations of glucose were supplied, more cleistothecia were formed. Other carbon sources including lactose, galactose and glycerol made the fungus develop cleistothecia very well, whereas acetate caused asexual sporulation only. Organic nitrogen sources like casein hydrolysate and glycine, and an increase in nitrate or ammonium concentration also enhanced sexual development. In addition to nutrient effects, low levels of aerobic respiration, caused either by platesealing or treatment with various chemicals, favored sexual development. Carbon limitation, light exposure and a high concentration of salts promoted asexual development preferentially, suggesting that stress conditions may drive the cell to develop asexual sporulation while comfortable and wellnourished growth conditions favored sexual development.

Energy Status of Neurospora crassa Mutant nap in Relation to Accumulation of Carotenoids: Tatyana A. Belozerskaya^ , Tatyana V. Potapova^† , Elena P. Isakova , Eugene I. Shurubor , Ludmila V. Savel'eva , Renata A. Zvyagilskaya; J. Microbiol. 2003;41(1):41-45.

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Abstract: N. crassa mutant strain nap showed reduced growth rate, decreased electric membrane potential, and elevated intracellular ATP content in comparison to the wild type. Blue light induced a hyperpolarization of the membrane potential in both strains. The analysis of oxidative and phosphorylation activities of mitochondria isolated from the two strains has revealed that nap utilized more efficient oxidative pathways. The higher intracellular ATP content in the nap was presumably due to impaired transport systems of the plasma membrane, and to a lesser extent to the functioning of the fully competent respiratory chain. The excess ATP possibly accounts for carotenoid accumulation in the mutant.

Identification and Cloning of jipA Encoding a Polypeptide That Interacts with a Homolog of Yeast Rad6, UVSJ in Aspergillus nidulans: Jae-Han Cho^ , Seok-Soong Yun^ , Young-Kug Jang^ , Mee-Jeong Cha , Nak-Jung Kwon , Suhn-Kee Chae^; J. Microbiol. 2003;41(1):46-51.

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Abstract: RAD6 in yeast mediates postreplication DNA repair and is responsible for DNA-damage induced mutations. RAD6 encodes ubiquitin-conjugating enzyme that is well conserved among eukaryotic organisms. However, the molecular targets and consequences of their ubiquitination by Rad6 have remained elusive. In Aspergillus nidulans, a RAD6 homolog has been isolated and shown to be an allele of uvsJ. We screened a cDNA library to isolate UVSJ-interacting proteins by the yeast two-hybrid system. JIPA was identified as an interactor of UVSJ. Their interaction was confirmed in vitro by a GST-pull down assay. JIPA was also able to interact with mutant UVSJ proteins, UVSJ1 and the active site cysteine mutant UVSJ-C88A. The N- and the C-terminal regions of UVSJ required for the interaction with UVSH, a RAD18 homolog of yeast which physically interacts with Rad6, were not necessary for the JIPA and UVSJ interactions. About 1.4 kb jipA transcript was detected in Northern analysis and its amount was not significantly increased in response to DNA-damaging agents. A genomic DNA clone of the jipA gene was isolated from a chromosome I specific genomic library by PCR-sib selection. Sequence determination of genomic and cDNA of jipA revealed an ORF of 893 bp interrupted by 2 introns, encoding a putative polypeptide of 262 amino acids. JIPA has 33% amino acid sequence identity to TIP41 of Saccharomyces cerevisiae which negatively regulates the TOR signalling pathway.

Mutation Spectrum of Manganese (II) Peroxidase Gene in the Pleurotusostreatus Mutants Induced by Gamma Radiation: Hwa-Hyoung Chang^ , Young-Keun Lee^ , Jae-Sung Kim^ , Ki-Sung Lee^ , Kyu Seong Cho^; J. Microbiol. 2003;41(1):52-57.

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Abstract: The mutational spectra in the manganese (II) peroxidase gene (mnp) of the Pleurotus ostreatus mutants induced by gamma radiation (Co^60) give evidence to prove the effect of gamma radiation on the gene. mnp of each mutant was cloned, sequenced and analyzed. Among the 1941 base pairs of the sequenced region of the mnp genes of 4 mutants (PO-5, -6, -15 and -16), nine mutational hotspots on which the same base was mutated simultaneously were found, additionally 6 mutations were also found at different positions in the mnp gene. These mutation-spectra were predominantly A:T_G:C transitions (50.1%). By the analysis of putative amino acid sequences, PO-5 and PO-16 mutants have 3 and 1 mutated residues, respectively. Since the mutational spectra reported herein are specific to the mnp gene, we propose that the mutational hotspots for the gamma radiation could be in the gene(s) within cells.

Multicatalytic Alkaline Serine Protease from the Psychrotrophic Bacillus amyloliquefaciens S94: Eui-Sun Son , Jong-Il Kim; J. Microbiol. 2003;41(1):58-62.

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Abstract: An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45℃ (protein substrate) and pH 8, 45℃ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyzed substrates with Leu or Lys residues at P1 site. The protease had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15℃ to 45℃, specially at low temperature.

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