Journal of Microbiology

Volume 36(3); September 1998

Biomass and Productivity of Bacterioplankton Related to Surface Water Divergence in the Northeast Equatorial Pacific Ocean: Hyun, Jung Ho , Choi, Joong Ki , Yang, Eun Jin , Kim, Kyeong Kong; J. Microbiol. 1998;36(3):151-158.

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Abstract: Bacterial biomass and productivity together with physico-chemical parameters in the northeast equatorial kPacific Ocean (7~10.5˚N) were investigated during a KODOS(Korea Deep Ocean Study) 96-1 cruise. Surface water dibvergence occurred at the boundary (8˚N) of the north equatorial current and the north equatorial counter current, and largely determined the horizontal and vertical distribution of physico-chemical and microbiological parameters. The diverging area (7~9˚N) was characterized by a shallower thermocline depth, higher nutrient and chlorophyll-a (Chl-a)concentrations, and a higher cyanobacterial cell number than those of the KODOS area (10~10.5˚N). Bacterial communities in the diverging area showed a high productivity but low biomass. Protozoa grazing seeded to be responsible for the low bacterial biomass. High bacterial turnover rate but low biomass suggested that bacteria in the diverging area may be a trophic link between a photosynthetic carbon source and protozoa, and thus accelerate the microbial loop. Relatively lower bacterial turnover but higher biomass in the KODOS area indicated that resources (organic carbon) limit the bacterial growth, and thus bacteria may play as a sink for photosynthetically ficed organic carbon. The results imply that major ecological role of hererotrophic bacteria in the microbial food web process in the two adjacent waters is different.

Adaptaion of Azomonas agilis PY101 Exposed to Cadmium vua Production of Cadmium-Binding Pigment Promoted by Cd^2+: You, Kyung Man , Park, Yong Keun; J. Microbiol. 1998;36(3):159-163.

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Abstract: Azomonas agilis PY101 produced a fluorescent yellow-green pigment promoted by cadmium. The amount of extracellular pigment produced during the growth of A. agilis PY101 increased to approximately 6 times its initial value after the addition to 1.0 mg/ml of CdCl_2. The pigment peak(peak II) was observed when the supernatant solution acquired from the cells cultivated in the presence of cadmium was fractionated on a column of Superdex 75. Peak II contained about 70% of extracellular cadmium in the supernatant solution. This cadmium-binding pigment contained several sulfur-containing groups. The dramatic decrease (97%) of sulfate ion (SO_4^-2)concentration in the cytoplasm from 9.60 to 0.25 ㎍/ml during the growth of A. agilis PY101 under cadmium stress was confirmed by ion chromatography. Moreover, transmission electron microscopic analysis showed that Z. agilis PY101 actively accumulated cadmium in the interior of the cells. It appears that the cadmium adaptation of A. agilis PY101 is achieved by the microbial binding of the sulfur-containing pigment to cadmium.

Purification and Characterization of Dehydroascorbate Reductase from Pleurotus ostreatus: Kim, Yeon Ran , Kang, Sa Ouk; J. Microbiol. 1998;36(3):164-170.

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Abstract: Dehydroascorbate reductase was purified 93-fold relative to the crude cell extracts from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield of 5%. The molecular mass of the native enzyme determined by gel filtration chromatography was 86 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the enzyme is a single polypeptide. Dehydroascorbate kreductase from P. ostreatus contained relatively quite a lot of lysine and a relatively small amount of glutamate/glutamine. The enzyme was optimally active at pH 7.5 and at 45℃. Apparent K_m values of dehydroascorbate reductase were 2.5 mM and 0.7 mM for dehydroascorbate and glutathione. The enzyme was significantly unstable under acidic and highly alkaline conditions. The absorption spectrum of the purified enzyme showed an unusual flavin peak, the result of which suggests that the enzyme might form flavin adduct.

Response of CuZnSOD and HP1 conjugated gene in Escherichia coli double mutants to oxidative stress: Park, Hye Young , Kim, Young Gon; J. Microbiol. 1998;36(3):171-178.

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Abstract: In Escherichia coli, the counterbalances between superoxide dismutase (SOD) and hydroperoxidase are more important for overall sensitivity to oxidative stress such as paraquat (PQ, 0.1 mM), H_2O_2 (1mM), CuSO_4(0.1 mM), CuSO_4 and heat shock (37℃→42℃) than the level of either SOD or HP1 alone. To evaluate the relative coordinate defense, E. coli SOD and/or hydroperoxidase double mutants were transformed with plasmids expressing SOD^+HP1^-, SOD^-HP1^+ and SOD^-HP1^-, and their physiological response to oxidative stess was measured. SOD was found to be more important than hydroperoxide in preventing oxygen-mediated stress except for the case of H_2O_2 treatment in the presence of SOD. Nevertheless, a balance between both enzymes are necessary for an effective defense against oxygen mediated radicals. The average ratio of SOD:HP1 on activity of various E. coli strains under oxidative stress except copper sulphate treatment show about 1.4 in the wild type. When the ratio, however, was reversed in mutant cells encoded with Photobactrium leiognathi CuZnSOD gene, their sensitivities were more increased. This suggests that although HP1 could reduce the H_2O_2 or hydroxyl radicals, it also inhibited the SOD function against ixygen radicals.

Morphological changes of biomembranes by amphiphilic basic peptides mastoparan B and 4₃: Park, Nam Gyu , Kim, Chan Hee , Chung, Joon Ki , Huh, Min Do , Park, Jang Su , Kang, Shin Won; J. Microbiol. 1998;36(3):179-183.

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Abstract: We investigated the modes interaction of mastoparan B, model peptide Ac-(Leu-Ala-Arg-Leu)_3-NHCH_3 (4_3) and gramicidin S, amphiphilic peptides with biomembranes. As a result, we observed morphological changes in erythrocytes and bacterial membranes. Mastoparan B caused a change in the shape of erythrocytes from normal discoid to a crenated form (named echinocytes). The major morphological changes of Staphylococcus aureus 209P induced by 4_3 and gramicidin S were the destruction of the cell wall and the accumulation of large enectron-opaque structures in the ccytoplasm. The damage was initiated by gap formation in the cell wall. In the freeze-fracture method, heavy damages occurred in the cell membrane. The cell wall became thin and finally ruptured. The protoplasts prepared from the 4_3-treated bacteria were unstable in an osmotically controlled vuffer and lysed within 2 hr, whereas those prepared from control cell and 4_3treated protoplasts were stable for more than 3 hr. These structural changes were not specific for 4_3 but were also found in gramicidin S-treated cells. Therefore, we expect that the bactericidal mechanism of 4_3, an amphiphilic linear peptide, and gramicidin S, an amphiphilic cyclic peptide, on Staphylococcus aureus 209P will by basically the same. In the case of erythrocytes, their transformation to echinocytes by mastoparan B should be similar to the way in which the transformation is caused by amphiphilic basic peptides such 4_3 and gramicidin S.

rpoS mutation relieves biosynthesis of flagella in hns mutants of salmonella typhimurium UK1: Cho, Mi Ook , Bang, Ile Soo , Hong, Seong Karp , Bang, Seong Ho , Park, Yong Keun; J. Microbiol. 1998;36(3):184-188.

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Abstract: The biosynthesis and function of flagella is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex and the nucleoid protein H-NS. In this report, we show that nonmotile Salmonella typhimurium hns mutants could recover its motility by introducing the rpoSmutation. In a swarm plate assay, rpoS/hns double mutants could woim while hns mutants could not. This regeneration of motility resulted from the flagella synthesis. Transmission electron microscopy analysis showed the capability of rpoS/hns double mutants for flagella synthesis. And rpoS mutation derepressed the transcription of flhD, the flagella master gene, in hns mutants.

Characterization of an Attenuated Japanese Encephalitis Virus Adapted to African Green Nomkey Kidney Cells, Vero: Chung, Yong Ju , Hong, Sun Pyo , Moon, Sang Beom , Shin, Young Cheol , Kim, Soo Ok; J. Microbiol. 1998;36(3):189-195.

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Abstract: Live attenuated Japanese encephalitis (JE) virus SA14-14-2 produced in primary dog kidney cells (PDK) was adapted to African green monkey kidney cells, Vero. In an effort to gain insight into the molecular basis of the biological characteristics of the isolated SA14-14-2 (Vero) strain, the 1,500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and dompared with the sequences of two other attenuated JE virus strains, SA14-14-2 (PHK) and SA14-14-2 (PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) of the SA14-14-2 (Vero) E gene was found to be identical to those of strains SA14-14-2 (PHK) and SA14-14-2 (PDK), while the N-terminal region (a.a. 1-279) showed sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a.138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA14-14-2 (Vero). Animal testing showed that SA14-14-2 (Vero) has a neurovirulence phenotype similar to that of the parent SA14-14-2 (PDK) strain in suckling mice. The SA14-14-2 (Vero) grew very efficiently in Vero cells enough to support vaccine production. The growth characteristics of SA14-14-2 (Vero) in Vero cell and conservation of attenuation determinant of neurovirulence support that SA14-14-2 (Vero) could be developed as a new vaccine strain for human use.

Factors affecting pheromone induction of schizosaccharomyces pomba and isolation of pheromone induction mutants: Jun, Jai Hyun , Kim, Young Min , Lee, Joo Hun , Chung, In Kwon , Kim, Dae Myung; J. Microbiol. 1998;36(3):196-202.

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Abstract: The mating pheromones of Schizosaccharomyces pombe are induced by nutritional starvation. However, this nutritional signaling pathway is largely unknown. For a complete understanding of pheromone induction, we examined the environmental factors affecting the induction afer cells were transferred to a nitrogen-starved medium. It appeared that the induction of mfm2 transcription was affected by the general environmental stress including incubation time, incubation temperature, and the growth phase of the cells. We identified 7 pheromone induction mutants by screening temperature sensitive mutant bank. Three of these mutants showed elongated cell shapes and one mutant exhibited swollen cell morphology in permissive culture, suggesting that their cell cycles were also impaired. Characterization of the pheromone induction mutants may elucidate the components required in nutritional signaling pathway leading to pheromone induction.

Phylofenetic relationships of Amanita species based on ITS1-5.8S rDNA-ITS2 region sequences: Lim, Young Woon , Jung, Hack Sung; J. Microbiol. 1998;36(3):203-207.

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Abstract: To determine the phylogenetic relatedness of Amanita species, internal transcribed spacers (ITSs) and the 5.8S ribosomal RNA gene were amplified by polymerase chain reaction and then sequenced according to the dideoxy chain termination method using an automatic DNA sequencer. The ITS region provided sufficient variability for phylogenetic analyses within the species. Analyses of the ITS sequence data by distance and parsimony methods revealed that the Amanita species are composed of three distinct groups whose main branch is strongly supported by bootstrap analysis. The Singerian system did not fully correspond to present phylogenetic results based on molecuar data. The amyloidd nature of the spores was still phylogenetically significant and the type of volva as well as the cap color were proved to be additional important characters in Amanita phylogenetics.

Identification of Critical Amino Acids in the Core RNA Polynerase Binding Region of Yease Mtflp: Yang, Jae Sub , Jang, Sei Heon; J. Microbiol. 1998;36(3):208-213.

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Abstract: Yeast mitochondral RNA polymerase specificity factor encoded by the nuclear MTF1 gene is required for a selective transcription on nonanucleotide mitochondral promoter by core RNA polymerase. Although there is a little amino acid sequence similarity of Mtf1p with bacterial sigma factors, the mode of transcriptional initiation of mitochondrial RNA polymerase is identical to that of E. coli RNA polymerase. To study the interaction of mtf1p with core polymerase, we carried out region-directed random mutagenesis of the core binding domain with the pool of mutant oligonucleotide. Out of 4,000 transformants screened for petite phenotype on glycerol media by plasmid shuffling, six alleles of the MTF1 gene were isolated. The positions of amino acid replacements that resulted in mtf1 mutants were limited to amino acids 53-54 and 65-67. Among mutant forms of Mtf1p overproduced in E. coli, Mtf1p with either L53H or Y65Dmutation was unable to produce a selective transcript in run-off transcription reaction, suggesting that amino acids L53 and Y65 are crucial for promoter recognition and/or contact with core polymerase.

cDNA cloning and expresion of human rotavirus outer capsid protein VP4: Kang, Seok W. , Yang, Jai M.; J. Microbiol. 1998;36(3):214-221.

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Abstract: cDNA for the VP4-coding RNA segment 4 of human rotavirus isolated from Korean patients Was synthesized and cloned (HRV-k41), and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequence of JRV-k41 showed 90.6%, 86.6%, 74.6%, 66%, 70.1% and 65.4% homology to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M), and P[14](PA169) genotypes respectively. The deduced amino acid sequence homology of HRV-k41 to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M), and P[14](PA169) genotypes respectively. The deduced amino acid sequence homology of HRV-k41 to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M) and P[14](169) was 92.9% 89.7%. 75.8%, 64.1%, 70.6% and 64.3% respectively. These results suggest that HRV-k41 is closely related to the P1A genotype. Two trypsin cleavage sites (arginine 240 and arginine 246) and four cysteine residues (215, 317, 379, and 773) conserved in VP4 of all rotavirus strains were also found in JRV-k41. Similar to other virulent human rotaviruses, an additional trypsin cleavage site(lysine 245) was also detected in this strain. The cDNA of the VP4-coding RNA segment was cloned into pGEX-4T-3, an Escherichia coli expression vector, and it's expression was confirmed by Western-blot analysis.

The invariant region I sequence of the adenovirus serotype 2 DNA polymerase influences template specificity during DNA synthesis: Houng , In Sil; J. Microbiol. 1998;36(3):222-230.

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Abstract: Mutants in highly conserved region I (YGDTDS) of the adenovirus serotyope 2 DNA polymerase (Ad Pol) have been shown previously to be defective in assays for initiation and elongation of adenovirus DNA replication in vitro. A selected subset of these mutants was characterized in a number of assays to determine in more detail the nature of the defect that they cause in Ad Pol. The single amino acid substitution in this sequence motif had no detectable effect on binding either to factors required for viral DNA replication or to Ad DNA origin. However, in the deletion mutant mimicking a similar sequence found in the Klenow fragment and in RNA polymerases, binding to Ad DNA origins was reduced. When the nucleotide and template specificity of partially purified mutant Ad Pol proteins was checked there were no significant differences between mutant and wild-type Ad Pols in RNA polymerase assays both on DNA templates and on RNA templates. In reverse transcription assays, both wild type and mutant Ad Pol were inactive, with the exception of a Gly to Met replacement mutant which mimicked the sequence found in reverse transcriptase; this mutant showed low but reproducible levels of reverse transcriptase activity when compared to wt Ad Pol. Taken together, these results suggest that region I may influence template specificity during DNA synthesis, although the single replacement is not sufficient to convert Ad Pol to a reverse transcriptase or an RNA polymerase. Region I probably acts independently of other regions of Ad Pol that are responsible for DNA binding, since mutations in this sequence did not significantly alter adenovirus DNA origin interactions in gel shift assays.

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