Journal of Microbiology

Volume 33(3); September 1995

Genetic Relationships among Penicillium Species by Characterizing RAPD Markers: Yoon, Cheol Sik , Bae, Kyung Sook; J. Microbiol. 1995;33(3):171-177.

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Abstract: Random amplified polymorphic DAN markers were characterized for three taxonomically problematic Penicillium species : P. aurantiogriseum var. Aurantiogriseum, P. verrucosum and P. puberulum, as well as for 25 species of mono, bi-, and terverticillate Penicillia. The relationships among mono, bi-, and terverticillate Penicillium species were determined from these RAPD markers. Eight species from mono-, eight from bi-, and nine from terverticilate Penicillia were examined. With 14 randomly chosen 10-mer primes, a 310 character by 25 species matrix was generated. Phenetic analysis separated the 25 species into three genetically distinct groups that correspond to the different arrangements of penicilli (mono-, bi-, and terverticillate). The results of this study suggest that P. aurantiogriseum var. aurantiogriseum, P. VERRUCOSUM, AND P. puberulum represent genetically distinct species, and that P. vulpinum should be included in terverticilate Penicillia. Phenogram branching patterns indicated that biverticillate species are genetically more similar to monoverticilate species than they are to terverticillate species.

Effect of Temperature on Persistence of Recombinant Plasmid pCU103 in Different Waters: Kwak, Myong Ja , Kim, Chi Kyung , Kim, Young Chang , Lim, Jae Yun , Kim, Young Soo , Lee, Ki Sung , Min, Kyung Hee; J. Microbiol. 1995;33(3):178-183.

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Abstract: The recombinant plasmid of pCU103 constructed by cloning pcbCD genes in pBluescript SK(+) was studied for the effect of temperature on its persistence in different waters by the methods of electrophoresis, Southern hybridization, quantification, and transformation. The plasmid was very rapidly degraded out in non-sterile FW water without regards to water temperature, probably due to the effect of biochemical factor such as nucleases. The pCU103 was most persistent at 4℃ in any water environments, moderately persistant at 15℃, but least stable at 30℃ such results could be explained by the facts that hydrogen bonds in double-stranded plasmid DNAs become unstable and that nucleases are activated by increasing temperature. The intact structure of pCU1-3 was generally observed by gel electrophoresis under the conditions which the plasmid should be 2.0 ng/㎕ or higher in concentration and that about 10² CFU/ml or more transformant cells should be recovered.

Effects of Hydrogen Ions on Aquatic Microbial Populations in Korea: Ahn, Young Beom , Cho, Hong Bum , Choi, Yong Keel; J. Microbiol. 1995;33(3):184-190.

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Abstract: From July 1994 to March 1995, eighteen variables of physico-chemical factors including heavy metals, and of bacteria in the four reservoirs of Kyonggi-Do were investigated to examine the effects of acidic precipitation to bacterial population. The pH range in the study area is from 6.56 to 10.24, which also showed seasonal change extensively compared to other factors. The correlation analysis showed that pH has a significant positive correlation (mean 79%) with the microbial populations in Wangsong reservoir. By multiple regression analysis on all of the seasons and stations, good explanation was obtained for the variation in total direct count of bacteria (71% and 88%, respectively), and the plate count of heterotrophic bacteria (76% and 88%, respectively). In the surface water of Wansong reservoir, the variation of total count of bacteria was affected by the S/O (soluble sugar/total organic matter ratio) value and the pH, and that of the plate count of heterotrophic bacteria was explained as 63% by pH. However, in other stations they were explained by the NO₂, total organic matter (TOM), soluble sugar (SS), temperature, and dissolved oxygen as 21~91%. On the basis of the results, the bacterial populations on the media at pH 3.0, 4.0, 5.0, 6.0 and 7.0 were compared to determine the effects of acidic proceeding. All the colonies grew the best on the media of pH 7.0, but started to decrease from pH 5.0.

Binding of IciA Protein to the dnaA Promoter Region: Kim, Hak Jung , Hwang, Deog Su; J. Microbiol. 1995;33(3):191-195.

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Abstract: IciA protein has been shown as an inhibitor for the initiation of E. coli chromosomal DNA replication at oriC. IciA protein binds the AT-rich region in oriC and then blocks the initiation of chromosomal DNA replication. Two binding sites for IciA protein were identified in dnaA gene, encoding the initiator for the E. coli chromosomal replication, promoter region by gel-shift assay and DNase I footprinting, One, named as IciA site I, is located upstream of the dnaA promoter 1P. The other, named as IciA site II, is located downstream of the dnaA promoter 2P. The sequence comparison of the regions protected from the DNase I cleavage did not result in a clear consensus sequence for the binding of IciA protein, suggesting that IciA protein may be a member of multimeric complex dsDNA binding proteins. This study provided information about the binding mode of IciA protein. Even though the IciA site II and IciA binding site in oriC seem to be composed of two IciA binding units, one binding unit is likely enough to cause the binding of IciA protein to the IciA site I. The binding of IciA protein to the dna4 promoter implies that IciA protein may involve not only the control of the initiation of chromosomal DNA replication but also the control of the dna4 gene expression.

Heat Inducible Expression of the CDC70 Gene under the Control of Heat Shock Element in Saccharomyces cerevisiae: Lee, Seok Jae , Jahng, Kwang Yeop , Lee Young Hoon , Chae, Keon Sang; J. Microbiol. 1995;33(3):196-200.

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Abstract: In order to express the CDC70 gene of Saccharomyces cerevisiae by heat shock, we have designed heat inducibe hybrid promoters using the Drosophila melanogaster heat shock elements (HSEs). A 220 bp-long upstream fragment of the D. melanogaster hsp70 gene comprised of four HSEs was placed upstream of the putative proximal TATA box of the CDC70 gene. Hybrid promoters containing different fusion joints were tested for their ability to drive the CDC70 gene expression by heat shock. The results showed that the HSEs of D. melanogaster conferred the heat-induced CDC70 gene expression, but the heat inducibility was much lower than that in D. melanogaster.

Overexpression of the SPP2 Gene of Saccharomyces cerevisiae and Production of Antibodiesd to Spp2p: Park, Kwang Hark , Lea, Ho Zoo , Woolford, John L. , Kim, Kyung Hoon; J. Microbiol. 1995;33(3):201-207.

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Abstract: We have previously reported that SPP2 gene product of yeast Saccharomyces cerevisiae is involved in the pre-mRNA splicing. To investigate the rol ein the splicing pathway of the Spp2p protein, the SPP2 gene was overexpressed in Escherichia coli and polyclonal antibodies to Spp2p were generated from rabbits. First, a DNA fragment containing the SPP2 GENE without its promoter was subcloned into an E. coli expression vector, pKK233-3. The resulting recombinant plasmid pBQ14 contained an IPTG inducible tac promoter and the SPP2 structural gene. Overexpression of the SPP2 gene was achieved by additionof 0.1 to 1.0 mM IPTG to a logarithmic culture of E. coli JM103(pBQ14) for 90 min at 37℃. Sequence of N-terminal 15 amino acids of the overproduced protein was well matched to the deduced one from the SPP2 reading frame. Then, polyclonal antibodies were generated from rabbits immunized with gel-purified Spp2p protein. These antibodies reacted specifically with the Spp2p protein extracted from yeast cells expressing the SPP2 gene to a great extent. The antibodies could also block the activity of yeast splicing extracts.

Effects of Genetically Different 2.4-D-degradative Plasmids on Degradation Phenotype and Competitiveness of Soil Microorganisms: Hong, Seok Myeong , Ahn, Young Joon , Park, Yong Keun , Min, Kyung Hee , Kim, Chi Kyung , Ka, Jong Ok; J. Microbiol. 1995;33(3):208-214.

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Abstract: The effects of various 2, 4-D-degradative plasmids on the axenic growth patterns, the degradation phenotypes, and the competitiveness of different host bacteria were evaluated in liquid cultures; the organisms and plasmids used were Alcaligenes eutrophus JMP134/pJP4, Alcaligenes paradoxus/p2811, Pseudomonas pickettii/p712, pJP4, and p712 or p 2811 exhibited very different restriction fragment profiles in restriction endonuclease digests. These plasmids were transferred to the recipients (P. cepacia and Alcaligenes JMP228) at relatively high frequencies ranging from 8.9 × 10^-3 to 1.6 × 10^-5 per donor cell. In the axenic liquid cultures the fast-growing strains, such as P. pseudomallei/p745 and P. cepacia/pJP4, exhibited short lag periods, high specific growth rates, and high relative fitness coefficients, while the slow-growing strains, such as P. pickettii/p712 and A. paradoxus/p2811, had long lag periods, low specific growth rates, and low relative fitness coefficients. Depending on the type of plasmid containing the genes for the 2, 4-D pathway, some transconjugants exhibited intermediate growth patterns between the fast-growing strains and the slow-growing strains. The plasmid and plasmid-host interactions determined specific growth rate and lag time, respectively, which were shown to be principal determinants of competitiveness among the strains, but relative fitness coefficient derived from the axenic culture was not always predictive for the mixed culture condition.

Isolation and Genetic Mapping of Paraquat-Resistant Sporulating Mutants of Streptomyces Coelicolor: Chung, Hye Jung , Kim, Eun Ja , Park, Uhn Mee , Roe, Jung Hye; J. Microbiol. 1995;33(3):215-221.

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Abstract: S. coelicolor A3(2) cells were treated with various redox-cycling agents on nutrient agar plates and examined for their effect on the growth and differentiation. When treated with plumbagin, severe effect on cell viability was observed at concentrations above 250 uM. However, the surviving colonies differentiated normally. When treated with 100 uM paraquat, growth rate was decreased and morphological differentiation was inhibited, while the survival rate was maintained at about 100% even at 5 mM paraquat. Menadione or lawsone did not cause any visible changes at concentrations up to 1 mM. The effect of paraquat was also observed when it was added to nutrient agar plate before spore inoculation. Paraquat had also observed when it was added to nutrient agar plate before spore inoculation. Paraquat had no effect on colonies growing on R2YE agar plates. Among the components of R2YE medium selectively added to nutrient agar medium, CaCl₂was found to have some protective function from the inhibitory effect of paraquat. As a first step to study the mechanism of the inhibitory effect of paraquat on differentiation, resistant mutants which sporulate well in the presence of paraquat were screened following UV mutagenesis. Three paraquat-resistant mutants were isolated with a frequency of 3 × 10^-5. Their mutation sites were determined by genetic crossings. All three mutations were mapped to a single locus near argA at about 1 o'clock on the genetic map of S. coelicolor A3(2).

Alteration of chromosomal structure within β-Tubulin and flagellar calmodulin genes during differentiation of Naegleria gruberi Amebae into Flagellates: Bok, Jin Woong , Lee, Joo Hun; J. Microbiol. 1995;33(3):222-227.

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Abstract: We have examined DNase I sensitivity of β-tubulin and flagellar calmodulin genes which are transiently and coordinately activated differentiation of Naegleria gruberi amebae into flagellates. The DNase I sensitivity of β-tubulin and flagellar calmodulin genes changed in parallel with the changes in transcriptional activity of the respective genes during differentiation. The two genes were resistant to DNase I inamebae stage when transcription of the two genes was inactive. Forty minutes after initiation of differentiation, when the two genes were most actively being transcribed, the two genes showed the highest sensitsivity to DNase I. One hundred and twenty minutes after initiation, the differentiation was completed and transcriptional activity of the two genes decreased to a low level. At this stage, the two genes were resistant to DNase I treatment like the ones at the ameba stage. This change in the DNase I sensitivity of the two genes was not observed when transcription of the two genes was blocked by adding cycloheximide at the beginning of differentiation.

Binding Sites for Lead Ion in Staphylococcus epidermidis: Kim, Mal Nam , Sung, Hye Yoon; J. Microbiol. 1995;33(3):228-233.

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Abstract: As S. epidermidis cell was fractionated into cell wall, cell membrane, and cytoplasm, the cell membrane proved to be the most efficient absorbent for lead ion. Ultrasonication was effective, when the cells were treated during their exponential growth. The amount of the lead ion adsorbed in cell membrane decreased as hydrogen ion concentration of solution increased. Protein purified from the cell membrane showed higher adsorption capacity for the lead ion than peptidoglycan, teichoic acid from cell wall, or cell membrane lipid. Modification of carboxyl groups in the membrane protein with ethylenediamine and 1-ethyl-3-carbodiimide hydrochloride resulted in a considerable decrease of lead ion adsorption capability, suggesting that the main binding site for lead ion was the carboxyl groups of protein in cell membrane.

The effect of oxygen on NAD breakdown in Salmonella typhimurium: Park , Uhnmee; J. Microbiol. 1995;33(3):234-238.

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Abstract: The breakdown rate of NAD in Salmonella typhimurium was investigated both in aerobic and anaerobic conditions. After NAD is broken down to nicotinamide ring containing moiety, almost all the nicotinamide ring containing moiety recycles back to NAD pool. However almost none of the adenine containing moiety recycles back. We pulse-label the endogeneous NAD with [¹⁴C]-adenine and [³H]- niacin. The remaining [¹⁴C]-radioactivity in NAD pool at each time was regarded as unbroken portion of NAD, Where ad that of [³H] was served as a total amount of NAD to start with. Under aerobic condition, the half-life of NAD was around 2 hours. However, the breakdown rate was significantly reduced (around 3-5 fold) under anaerobic condition. The observation that under aerobic conditions, NAD turnover is considerably faster than under anaerobic conditions suggests that oxygen has important effect in NAD breakdown.

Purification and Characterization of Catalase-3 of Deinococcus radiophilus: Lee, In Jeong , Lee, Young Nam; J. Microbiol. 1995;33(3):239-243.

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Abstract: Deinococcus radiophilus, an UV resistant bacterium seemed to contain three issoenzymes of catalase. Among them, the samllest and most abundant species in cell-free extract, catalase-3 which also exhibited peroxidase activity was purified to electrophoretic homogeneity (145-fold purification) by chromatographic procedures. Its molecular weight was 155 kDa composed of four 38 kDa subunits. The K_m value of catalase-3 for H₂O₂was approximately 0.5 mM. This enzyme showed a typical ferric heme spectrum with maximum absorption at 405 nm. Upon binding to cyanide, the 405 nm peak shifted to 420 nm. Catalase-3 was very sensitive to inhibitors of heme proteins, such as cyanide, azide and hydroxylamine. A ratio of A_405/A_28O was 0.5 Catalase-3 was active over a wide range of pH, between pH 7 and 10. The enzyme was rather heat-labile and partially sensitive to ethanol-chloroform treatment, but resistant to 3-amino-1, 2, 4-triazole. Catalase-3 of D. radiophilus, which is a bifunction catalatic peroxidatic enzyme seemed to share certain molecular properties with the typical catalase and the catalase-peroxidase along with its own unique features.

Purification and Characterization of an Extracellular Protease from Culture Filtrate of Salmonella schttmulleri: Na, Byoung Kuk , Song, Chul Yong; J. Microbiol. 1995;33(3):244-251.

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Abstract: An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca^2+, Zn^2+, Fe^2+, Mg^2+ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40℃. It was stable at least for 1 week at 40℃ and maintained its activity for 24 hours at 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium 50℃, but it was rapidly inactivated at 65℃. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

Genetic Variation of BIV Isolates Characterized by PCR Using Degenerate Primers: Kwon, Oh Sik , Sninsky, John J.; J. Microbiol. 1995;33(3):252-259.

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Abstract: The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the Lentivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.

Sequence Analysis of NS4 Region of HCV Isolated from Korean Patient: Paik, Sang Hoon , Lee, Young Ik , Kim, Won Bae , Yang, Jai Myung; J. Microbiol. 1995;33(3):260-266.

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Abstract: Hepatitis C virus (HCV) has been considered as a major causative agent of post-transfusion related non-A, non-B hepatitis. In this study, the cDNA sequence of NS4 region of HCV (HCV-S) obtained from a Korean patient's plasma was determined. Comparative nucleotide sequence analysis between to type II. 67.2% homology to type III, and 66.4% homology to type IV. The putative amino acid sequence homologies to types I, II, III, and IV were 82.8-84.7%, 92.5-95.1%. 72.5% and 71.1% respectively. This data strongly suggests that HCV-S should be classified as type II. Significant similarities of hydrophobicity profiles and putative transmembranous domains were found in HCV-S and four major prototypes, indicating that the protein structure is similar in spite of the heterogeneities of intertype homologies at the level of the primary nucleotide and amino acid sequences.

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