Journal of Microbiology

Volume 40(4); December 2002

Regulation of a Bacterial Aromatic Monooxygenase Pathway in Response to Solvent Exposure: Jerome J. Kukor , Juliana Malinverni; J. Microbiol. 2002;40(4):249-253.

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Genetic DNA Marker for A2 mating type in Phytophthora infestans: Kwon Jong Kim , Youn Su Lee; J. Microbiol. 2002;40(4):254-259.

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Abstract: The Phytophthora infestans requires two mating types for sexual reproduction. Amplified fragment length polymorphism (AFLP) was used to specifically detect different mating types of P. infestans . The AFLP primers E+AA (5'-GACTGCGTACCAATTCAA-3') and M+CAA (5'-GATGAGTCCTGAGTAACAA-3') detected a fragment that is specific in the A2 mating type of P. infestans . This fragment was cloned and sequenced. Based on the sequence data, PHYB-1 and PHYB-2 primer were designed to detect the A2 mating type of P. infestans. A single 347 bp segment was observed in the A2 mating type of P. infestans, but not in the A1 mating type of P. infestans or other Phytophthora spp. Identification of mating type was performed with phenotype (sexual reproduction) and genotype (CAPs marker) methods. Two factors, the annealing temperature and template DNA quantity, were investigated to determine the optimal conditions. Using mating type-specific primers, a unique band was obtained within annealing temperatures of 57 ℃-62℃ and DNA levels of 10pg-100 ng (data not shown).

Isolation and Identification of Biofilm-Forming Marine Bacteria on Glass Surfaces in Dae-Ho Dike, Korea: Kae Kyoung Kwon , Hyun Sang Lee , Sung-Young Jung , Joung-Han Yim , Jung-Hyun Lee , Hong Kum Lee; J. Microbiol. 2002;40(4):260-266.

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Abstract: Bacterial strains were isolated from biofilms formed on glass slides submerged in seawater in Dae-Ho Dike. Eight strains showing fast attaching ability were selected and identified. Their exopolysaccharide(EPS)-producing ability and EPS properties were characterized. Based on Microlog System, 4 among the 8 strains were identified as Micrococcus luteus and the rest were Bacillus thuringiensis, Bacillus megaterium, Staphylococcus saprophyticus and Agrobacterium vitis. A. vitis was reidentified as Sulfitobacter pontiacus based on 16S rDNA sequence data. The amount of water-soluble EPS produced by the 8 strains ranged from 0.114 to 1.329 g·l^-1 and the productivity was negatively correlated with the cell biomass. The molecular weight of the produced EPS ranged from 0.38 to 25.19x 10^4 Da. Glucose and galactose were ubiquitous sugar components. Mannose, ribose, and xylose were also major sugar components. The molecular weight and composition of the EPS showed strain-specific variation.

Microscopic Detection of Urinary Tract Infection in Nepalese Patients: Bijaya Kumar Dhakal , Bharat Mani Pokhrel , Joohong Ahnn; J. Microbiol. 2002;40(4):267-273.

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Abstract: Urinary tract infection (UTI) is one of the most common domiciliary and nosocomial bacterial infections prevalent in both males and females. UTI is diagnosed on the basis of clinical symptoms, microscopy and culture of urine. In order to evaluate the efficacy of microscopic detection for presumptive diagnosis of UTI we analyzed urine samples of Nepalese patients. We have conducted Gram staining and counting of pus cells, red blood cells (RBC) and epithelial cells. We observed that RBC and epithelial cell counts were not sensitive enough to be used for presumptive diagnosis of UTI. However, pus cell counts as well as Gram stain are sensitive and significant enough to presume UTI. When the Gram stain result was compared with the culture result, it was statistically significant. From this, we suggest that Gram stain of centrifuged urine is a very sensitive screening method to detect bacteriuria. In addition, we found that E. coli was the most predominant microorganism causing UTI and nitrofurantoin was the most effective antibiotic against the isolated urinary pathogens.

Monitoring 4-Chlorobiphenyl-Degrading Bacteria in Soil Microcosms by Competitive Quantitative PCR: Soo Youn Lee , Min Sup Song , Kyung Man You , Bae Hoon Kim , Seong Ho Bang , In Soo Lee , Chi Kyung Kim , Yong Keun Park; J. Microbiol. 2002;40(4):274-281.

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Abstract: The competitive quantitative PCR method targeting pcbC gene was developed for monitoring 4-chlorobiphenyl(4CB)-degrading bacteria, Pseudomonas sp. strain DJ-12, in soil microcosms. The method involves extraction of DNA from soil contaminated with 4CB, PCR amplification of a pcbC gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. To test the adequacy of the method, Pseudomonas sp. Strain DJ-12 was introduced into both contaminated and non-contaminated soil microcosms amended with 4CB. Pseudomonas sp. strain DJ-12 was monitored and quantified by a competitive quantitative PCR in comparison with 4CB degradation and the result was compared to those obtained by using the conventional cultivation method. We successfully detected and monitored 4CB-degrading bacteria in each microcosm and found a significant linear relationship between the number of 4CB-degrading bacteria and the capacity for 4CB biodegradation. The results of DNA spiking and cell-spreading experiments suggest that this competitive quantitative PCR method targeting the pcbC gene for monitoring 4Cbdegrading bacteria appears to be rapid, sensitive and more suitable than the microbiological approach in estimating the capacity of 4CB biodegradation in environmental samples.

PCR-Based RFLP Analysis of ureC Gene for Typing of Indian Helicobacter pylori Strains from Gastric Biopsy Specimens and Culture: Kanchan Kumar Mishra , Shashikant Srivastava , Prabhat P. Dwivedi , Kashi Nath Prasad , Archana Ayyagari; J. Microbiol. 2002;40(4):282-288.

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Abstract: Since culture of Helicobacter pylori is relatively insensitive and cumbersome, molecular detection and typing of H. pylori isolates are gaining importance for strain differentiation. In the present study genomic DNA of 42 gastric biopsies and H. pylori isolates from corresponding patients were analyzed and compared by PCR-based RFLP assay. The 1,132-bp product representing an internal portion of ureC gene of H. pylori was amplified by PCR and digested with restriction enzymes HindIII, AluI and PvuI. The HindIII, AluI and PvuI digestion produced 4, 7, and 2 distinguishable RFLP patterns respectively from 42-H. pylori isolates. By combining all three restriction enzyme digestions, 15 RFLP patterns were observed. However, when PCR products from 42 gastric biopsy specimens were digested by restriction enzymes HindIII, AluI and PvuI, we observed 5, 8 and 2 RFLP patterns, respectively. Patterns from 34 of 42 gastric biopsy specimens matched those of corresponding H. pylori isolates from respective patients. Patterns from the remaining eight biopsy specimens differed and appeared to represent infection with two H. pylori strains. The patterns of one strain from each of these biopsies was identical to that of the isolate from corresponding patients and the second pattern presumably represented the co-infecting strain. From the study, it appears that PCR-based RFLP analysis is a useful primary tool to detect and is distinguish H. pylori strains from gastric biopsy specimens and is superior to culture techniques in the diagnosis of infection with multiple strains of H. pylori.

Phylogenic Analysis of Alternaria brassicicola Producing Bioactive Metabolites: Dong-Sun Jung , Yeo-Jung Na , Ki Hyun Ryu; J. Microbiol. 2002;40(4):289-294.

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Abstract: The fungal strain SW-3 having antimicrobial activity was isolated from soil of crucified plants in Pocheon, Kyungki-Do, Korea. Strain SW-3 was identified as Alternaria brassicicola by its morphological characteristics, and confirmed by the analysis of the 18S gene and ITS regions of rDNA. The fungus showed a similarity of 99% with Alternaria brassicicola in the 18S rDNA sequence analysis. A. brassicicola has been reported to produce an antitumor compound, called depudecin. We found that strain SW-3 produced antimicrobial metabolites, in addition to depudecin, during sporulation under different growth conditions. The metabolite of the isolated fungus was found to have strong antifungal activity against Microsporium canis and Trichophyton rubrum, and antibacterial activity against Staphylococcus aureus and Pseudomonas aerogenes. The amount and kind of metabolites produced by the isolate were affected by growth conditions such as nutrients and growth periods.

Overexpression of Fish DRG2 Induces Cell Rounding: Jeong Jae Park , Seung Ju Cha , Myoung Seok Ko , Wha Ja Cho , Won Joon Yoon , Chang Hoon Moon , Jeong Wan Do , Sung Bum Kim , Hebok Song , Dae Kyun Chung , In Seob Han , KyuBum Kwack , Jeong Woo Park; J. Microbiol. 2002;40(4):295-300.

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Abstract: Previously, we reported induced expression of developmentally regulated GTP-binding protein 2 (DRG2) in fish cells at the late stage of rhabdovirus infection. To investigate the biological role of fish DRG2 (fDRG2), we transfected CHSE-214 cells with an expression vector containing complete fDRG2 fused to the N-terminal end of an enhanced green fluorescent protein (EGFP). Low level expression of fDRG2-EGFP did not induce morphological change or cell death. However, a high level expression of fDRG2-EGFP induced cell rounding and caused depletion of the cell population in FACS analysis. Several truncated fragments were fused to EGFP. FACS analysis was conducted to determine the presence of cells expressing high levels of the resulting chimera. While cells expressing a high level of N-terminus were detected, those expressing high levels of the C-terminal fragment 243-290 containing the G4 motif were absent in FACS analysis. Based on these observations, we propose that overexpression of fDRG2 may induce cell rounding, a representative cytopathic effect of virus-infected cells in the late stage of infection and the C-terminus of the fDRG2 is essential for this function.

The Value of Submitting Multiple Sputum Specimens for Accurate Diagnosis of Pulmonary Tuberculosis: Ozgul Kisa , Ali Albay , Orhan Baylan , Levent Doganci; J. Microbiol. 2002;40(4):301-304.

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Abstract: Is a multiple number of sputum specimens necessary for the diagnosis of pulmonary tuberculosis? To answer this question, 6844 respiratory specimens obtained from previously untreated patients suspected of having pulmonary tuberculosis between 1998 and 2001 were evaluated retrospectively. All of the specimens were evaluated by acid fast bacilli smear and BACTEC 460 TB culture system. A total of 785 (11%) specimens from 353 patients were positive for Mycobacterium tuberculosis complex. For 76% (270/353) of these patients the organism was detected from sputum specimens collected sequentially for daily basis. Mycobacterium tuberculosis was isolated in the first, second and third samples of the majority (98%, 195/199) of patients who had three or more sputum samples sent to the laboratory. Our results indicate that, we could carry out Mycobacterium tuberculosis isolation in the first, second and third sputum samples of the overwhelming majority of the patients and the diagnostic value of four or more sputum specimens submitted to the laboratory was very low (2%). We recommend that, for definitive and cost-effective diagnosis of pulmonary tuberculosis at least three sequential sputum specimens be collected for all patients suspected pulmonary tuberculosis.

Polyamine Stimulation of arcA Expression in Escherichia coli: Mun Su Rhee , Young Sik Kim , Seon Young Park , Myung Hun Choi , Bo Min Kim , Seong Uk Kang , Kui Joo Lee , Jong Ho Lee; J. Microbiol. 2002;40(4):305-312.

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Abstract: The effects of two natural polyamines (putrescine and spermidine) on the synthesis of ArcA, a response regulator of the Arc two-component signal transduction system, were studied using an E. coli mutant deficient in polyamine biosynthesis. Endogenous polyamine deficiency of the mutant resulted in marked reduction in the ArcA level determined by Western blot analysis. Putrescine supplement to the growth medium effectively increased the ArcA level of the mutant in a concentration-dependent manner. Spermidine also stimulated the ArcA level in the mutant to a greater degree than putrescine. Expression of arcA'::lacZ operon fusion in the mutant was stimulated 6-fold and 10-fold by putrescine and spermidine at a 1mM concentration, respectively, indicating that the stimulatory effect of the polyamines on ArcA synthesis is due to transcriptional induction, and that spermidine is a more potent arcA inducer than putrescine. The polyamine-dependent arcA'::lacZ induction was growth-phase-dependent and independent of either arcA or fnr which are two regulators involved in anaerobic stimulation of the ArcA level. These results suggested that putrescine and spermidine polyamines may be potential intracellular signal molecules in the control of arcA expression, and thereby may play an important role in cellular metabolism.

Production and Prophylactic Efficacy Study of Human Papillomavirus-like Particle Expressing HPV16 L1 Capsid Protein: Jie-Yun Park , Hyun-Mi Pyo , Sun-Woo Yoon , Sun-Young Baek , Sue-Nie Park , Chul-Joong Kim , Haryoung Poo; J. Microbiol. 2002;40(4):313-318.

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Abstract: To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG[alpha]-HIR525 under the control of GAL10 promoter. The transformation of YEG[alpha]-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity

Fluorescence Microscopy of Condensed DNA Conformations of Bacterial Cells: Erhan Suleymanoglu; J. Microbiol. 2002;40(4):319-326.

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Abstract: Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 uM MgCl_2. Under these conditions, the nucleoids were compacted to volumes of around 2㎛^3 or comparable sizes with that of living cells.

Determination of Enteric Bacteria at Microbiologically Risky Points by Multiplex Polymerase Chain Reaction: Mahir Gulec , Bilal Bakir , Recai Ogur , Omer Faruk Tekbas; J. Microbiol. 2002;40(4):327-330.

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Abstract: The purpose of this research was to test multiplex polymerase chain reaction in investigating the microbiological quality of the risky surfaces in social living places of a military base where over 15 thousand people live together. In 22 samples of 99, there were no bacteria. Only four of the samples contained Shigella, and one of them contained Salmonella, but 77 of the samples contained thermotolerant coliform organisms. There was no statistically significant difference among the microbiological quality of different sites and different equipment surfaces (p>0.05).

Characterization and Cytotoxic Activities of Nonadecanoic Acid Produced by Streptomyces scabiei subsp. chosunensis M0137 (KCTC 9927): Jin-Cheol Yoo , Ji-Man Han , Seung-Kwan Nam , Ok-Hyun Ko , Cheol-Hee Choi , Keun-Hong Kee , Jae-Kyung Sohng , Jung-Sun Jo , Chi-Nam Seong; J. Microbiol. 2002;40(4):331-334.

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Abstract: The substance 0116p, which exhibits cytotoxicity against human macrophage cell line THP-1, was isolated from a mycelial extract of Streptomyces scabiei subsp. chosunensis M0137. The cytotoxic substance was purified by Diaion-HP20 adsorption, solvent extraction, Sephadex LH-20 column chromatography, and silica-gel column chromatography. The molecular formula is C_19 H_38 O_2 (MW 301.10) based on elemental and spectrometric analysis. It was identified as nonadecanoic acid by NMR spectral data. It exhibits cytotoxic activities in various human cancer cell lines, including A549, SK-OV-3, SK-MEL-2 and HCT-15. In addition, 0116p also inhibits IL-12 production in lipopolysaccharide-activated macrophages.

Inferring the Molecular Phylogeny of Chroococcalian Strains (Blue-green algae/Cyanophyta) from the Geumgang River, Based on Partial Sequences of 16S rRNA Gene: Wook Jae Lee , Kyung Sook Bae; J. Microbiol. 2002;40(4):335-339.

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Abstract: Partial sequences of 16S rRNA gene of five chroococcalian blue-green algal strains, Aphanothece nidulans KCTC AG10041, Aphanothece naegelii KCTC AG10042, Microcystis aeruginosa KCTC AG10159, Microcystis ichthyoblabe KCTC AG10160, and Microcystis viridis KCTC AG10198, which were isolated from water from the Geumgang River, were determined and were inferred their phylogenetic and taxonomic positions among taxa of order Chroococcales. Most taxa of Chroococcales whose partial 16S rRNA gene sequences were aligned in this study, are clustered with other related taxa. Aphanothece nidulans KCTC AG10041 and Aphanothece naegelii KCTC AG10042 made a cluster with other European species of these genera, which supported 100% of the bootstrap trees with a very high sequence similarity (97.4-99.4%) in this study. Three strains, Microcystis aeruginosa KCTC AG10159, M. ichthyoblabe KCTC AG10160, and M. viridis KCTC AG10198, formed a cluster with other Microcystis spp. supported 100 % of the bootstrap trees with a similarity of 97.0-99.9% except for two strains. However, this phylogentic tree made no resolution among the species of Microcystis spp. The topology of the tree reconfirmed the taxonomic status of three species of Microcystis, identified in this study based on the morphology, as three colonial types of Microcystis aeruginosa com. nov. Otsuka et al. (1999c). The genera of chroococcalian cyanophytes are heterogeneously clustered in these sequence analyses. We suggest that more molecular studies on the genera of Chroococcales with reference strains, widely collected from restricted geographic or environmental ranges, get accurate taxonomic or phylogenetic determinations.

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