Journal of Microbiology

Volume 38(3); September 2000

Acyl-Homoserine Lactone Quorum Sensing in Bacteria: E. Peter Greenberg; J. Microbiol. 2000;38(3):117-121.

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Abstract: Recent advances in studies of bacterial gene expression and light microscopy show that cell-to-cell communication and community behavior are the rule rather than the exception. One type of cell-cell communication, quorum sensing in Gram-negative bacteria involves acyl-homoserine lactone signals. This type of quorum sensing represents a dedicated communication system that enables a given species to sense when it has reached a critical population density, and to respond by activating expression of specific genes. The LuxR and LuxI proteins of Vibrio fisheri are the founding members of the acyl-homoserine lactone quorum sensing signal receptor and signal generator families of proteins. Acyl-homoserine lactone signaling in Pseudomonas aeruginosa is one model for the relationship between quorum sensing, community behavior, and virulence. In the P. aeruginosa model, quorum sensing is required for normal biofilm maturation and virulence. There are multiple quorum-sensing circuits that control the expression of dozens of specific genes in P. aeruginosa.

Phylogenetic Relationships of the Aphyllophorales Inferred from Sequence Analysis of Nuclear Small Subunit Ribosomal DNA: Seon Young Kim , Hack Sung Jung; J. Microbiol. 2000;38(3):122-131.

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Abstract: Phylogenetic classification of the Aphyllophorales was conducted based on the analysis of nuclear small subunit ribosomal RNA gene (nuc SSU rDNA) sequences. Based on phylogenetic groupings and taxonomic characters, 16 families were recognized and discussed. Although many of the characters had more or less homoplasies, microscopic characters such as the mitic system and clamp, spore amyloidity and rot type appeared to be important in the classification of the Aphyllophorales. Phylogenetically significant families were newly defined to improve the classification of the order Aphyllophorales.

Identification and Characterization of Leuconostoc gelidum, Isolated from Kimchi, a Fermented Cabbage Product: Bong-Joon Kim , Hye-Ja Lee , Sae-Young Park , Jeongho Kim , Hong-Ui Han; J. Microbiol. 2000;38(3):132-136.

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Abstract: We recently identified Leuconostoc gelidum, a typical psychrophile, as a microbial component from kimchi that has been laboratory-prepared and fermented at 20 C. However, it has been shown that the growth of leuconostocs in food products is highly influenced by fermenting temperature. To determine the distribution of L. gelidum species in kimchi fermented at a lower temperature, 8 C, we characterized a total of 64 dextran-forming strains isolated from kimchi using a polyphasic method including 16S rDNA sequencing and DNA-DNA hybridization. We found that 80% of the isolates were L. gelidum, which has been found mainly at chill-stored meat products. We also found that L. gelidum could be a dominant Leuconostoc species in so-called Kimjang kimchi, which is traditionally prepared at late fall to be preserved during winter in Korea. These results suggest that L. gelidum can be a predominant species in kimchi especially when fermented at low temperature.

Characterization of Isolated Lactobacillus spp. and Classification by RAPD-PCR Analysis: Oh-Sik Kwon; J. Microbiol. 2000;38(3):137-144.

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Abstract: The genetic relationships of six Lactobacillus strains and five laboratory isolates from fermented milk were determined by a random amplified polymorphic DNA (RAPD)-Polymease chan reaction (PCR) method. With 42 random primers, the results were analyzed by using the NTSYS-PC software for phenetic analysis. It revealed that all tested bacteria were divided into three distinct clusters. The clusters implied three subgenuses existed for the genus Lactobacillus, which were previously proposed by Rogosa and Sharpe. From the results, it was also possible to determine that the isolated Lactobacillus strains from fermented milk were grouped into L. acidophilus or L. bulgaricus. Interestingly, the three tested L. casei strains were divided into different clusters implying different subgenuses, i.e., Thermobacterium (L. casei YIT 9018) and Strepto-bacterium (L. casei CHR. Hansen and L. casei ATCC 4646). According to the distance matrix generated by an UPGMA program, the isolated bacteria LT01 and LT02 were determined as a subspecies of L. bulgaricus. The HK01, HK02 and HK03 were very closely related to either L. acidophilus or L. casei YIT 9018. Hence, RAPD-PCR appears to be a very practical method to determine the genetic relationships of the Lactobacillus species and to characterize the unknown Lactobacillus strains at the subspecies level.

Continuous Synthesis of Escherichia coli GroEL at a High Temperature: Young Hak Kwak , Kyong Sun Lee , Ji Yeon Kim , Dong Seok Lee , Han Bok Kim; J. Microbiol. 2000;38(3):145-149.

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Abstract: GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37 C. However, GroEL production increased 3.4-fold at 42 C. GroEL synthesis was not transient but continuous at 42 C, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly, while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42 C were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42 C.

Bioluminescent Assay of Phospholipase C Using A Luminescent Marine Mutant Bacterium Vibrio harveyi M-17: Ki Woong Cho , SangJun Mo , Hyi-Seung Lee , Jung-Rae Rho , Jongheon Shin; J. Microbiol. 2000;38(3):150-155.

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Abstract: A bioluminescent assay method for detecting the activity of phospholipase C (PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2-dimyristoyl phosphatidyl choline (DMPC) as a substrate were used in the demonstration, and the produced sn-1,2-dimyristoyl glycerol was further hydrolyzed with lipase from Candida cylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hydrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the amount of enzyme added. Activity measurement conditions (at 25 C, pH 6.5, 10 min fixed time assay) were established to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.

Subcellular Localization of Catalase Encoded by the ctt1^+ Gene in Schizosaccharomyces pombe: Sang-il Lee , Joon Lee , Jung-Hye Roe; J. Microbiol. 2000;38(3):156-159.

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Abstract: The ctt1^+ gene in Schizosaccharomyces pombe encodes a catalase responsible for H_2O_2 -resistance of this organism as judged by the H_2O_2 -sensitive phenotype of the ctt1[delta] mutant. In this study, we investigated the subcellular localization of the Ctt1 gene product. In wild type cells catalase activity was detected in the organelle fraction as well as in the cytosol. The ctt1[delta] mutant contained no catalase activity, indicating that both cytosolic and organellar catalases are the products of a single ctt1^+ gene. Western blot analysis revealed two catalase bands, both of which disappeared in the ctt1[delta] mutant. The major, faster-migrating band existed in the cytosol whereas the minor, slower-migrating band appeared to be located in organelles, most likely in peroxisomes. These results suggest that the ctt1^++ gene product targeted to the peroxisome is a modified form of the one in the cytosol.

Streptomyces griseus HH1, An A-factor Deficient Mutant, Produces Diminished Level of Trypsin and Increased Level of Metalloproteases: Jung-Mee Kim , Soon-Kwang Hong; J. Microbiol. 2000;38(3):160-168.

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Abstract: A-factor is a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. To identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by S. griseus IFO 13350 and its A-factor deficient mutant strain, S. griseus HH1, as well as S. griseus HH1 transformed with the afsA gene were studied. In general, S. griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of S. griseus IFO 13350 was greatly enhanced more than twice compared with that of S. griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of S. griseus HH1 was greatly enhanced more than twice compared with that of S. griseus IFO 13350, and this observation was reversed in the presence of thiostreptone. However, S. griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between S. griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-aminopeptidase activity was 2 times higher in S. griseus HH1 than in strain IFO 13350. S. griseus HH1 harboring afsA showed a similar level of enzyme activity, however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morphological differentiation, the formation of aerial mycelium and spores was delayed by two or three days.

Inhibitory Effects of Lactic Acid Bacteria (LAB) on the Azoxymethane-induced Colonic Preneoplastic Lesions: Sang-Myeong Lee , Wan-Kyu Lee; J. Microbiol. 2000;38(3):169-175.

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Abstract: Epidemiological and experimental studies provide evidences that diet and intestinal microflora play an important role in colon carcinogenesis. In recent years, it has been suggested that lactic acid bacteria (LAB) used to ferment dairy products have an inhibitory effect on the colon cancer. This study was designed to determine the effect of Bifidobacterium longum HY8001 (Bif) and Lactobacillus acidophilus HY2104 (Lac) of Korean origin on azoxymethane (AOM)-induced colonic preneoplastic lesions such as aberrant crypt foci (ACF) formation and cecal pH. At five weeks of age, Spraque-Dawley rats were divided at random into four (AOM alone, Bif, Lac, and Bif+Lac) groups. Animals were weighed weekly and oral administration of LAB cultures were performed daily until the termination of the study. Two weeks later, all animals were given a subcutaneous injection of AOM dissolved in normal saline at a dose of 15 mg/kg of body weight once per week for 2 weeks. All rats were necropsied 7 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons. The total number of aberrant crypt in Bif, Lac, and Bif+Lac groups were significantly lower than that of the AOM alone group and the percentage of inhibitions weas 35.0, 45.4 and 45.0%, respectively. Significant inhibition (p<0.001) in the total number of ACF was also observed in LAB treated groups (Bif, Lac, and Bif+Lac group by 30.3, 38.6, and 41.2%, respec-tively). Furthermore, cecal pH appeared to significantly decrease by LAB administration. The results of present study provide some evidences for potential colon tumor-inhibitory properties of lactic cultures and fermented dairy products.

P22-Based Challenge Phage Constructs to Study DNA-Protein Interactions between the [sigma]^54-Dependent Promoter, dctA, and Its Transcriptional Regulators: Eungbin Kim , Daeyou Kim , Joon Haeng Lee; J. Microbiol. 2000;38(3):176-179.

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Abstract: A challenge phage system was used to study the DNA-protein interaction between C_4 -dicarboylic acid transport protein D (DCTD) or [sigma]^54 , and a [sigma]^54 -dependent promoter, dctAp. R. meliloti dctA promoter regulatory region replaced the O_mnt site on the phage. S. typhimurium strains overproducing either DCTD or [sigma]^54 directed this challenge phage towards lysogeny, indicating that DCTD or E[sigma]^54 recognized the dctA promoter on the phage and repressed transcription of the ant gene. These challenge phage constructs will be useful for examining interactions between DCTD (or [sigma]^54 ) and the dctA promoter region.

Inhibitory Effect of Nitrate on Fe(III) and Humic Acid Reduction in Shewanella putrefaciens DK-1: Il-Gyu Lee , Sang-Jin Kim , Tae-Young Ahn; J. Microbiol. 2000;38(3):180-182.

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Abstract: The inhibitory effects of nitrate on Fe(III) and humic acid reduction were examined in Shewanella putrefaciens DK-1. There is no difference in Fe(III) reduction until 25 hours between cultures using Fe(III) alone as an electron acceptor and using Fe(III) and nitrate as electron acceptors, but after 25 hours Fe(II) production was decreased drastically when Fe(II) and nitrate were used as electron acceptors. The production of AHQDS (2,6-anthrahydroquinon disulfonate) showed similar patterns when AQDS alone and both AQDS and Fe(III) were used as electron acceptors. When AQDS (2,6-anthraquinon disulfonate) and nitrate were used as electron accepors, the production of AHQDS was completely inhibited.

Construction of a Bioluminescent Reporter Using the luc Gene and meta-Cleavage Dioxygenase Promoter for Detection of Catecholic Compounds: Sang-Ho Park , Dong-Hun Lee , Kye-Heon Oh , Chi-Kyung Kim; J. Microbiol. 2000;38(3):183-186.

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Abstract: Several types of bioluminescent reporter strains have been developed for the detection and monitoring of pollutant aromatics contaminating the environment. In this study, a bioluminescent reporter strain, E. coli SHP3, was constructed by fusing the luc gene of firefly luciferase with the promoter of pcbC responsible for the meta-cleavage of aromatic hydrocarbons. The bioluminescence expressed by the luc gene in the reporter was well triggered by the promoter when it was exposed to 2,3-dihydroxybiphenyl (2,3-DHBP) at 0.5 to 1 mM concentrations. The bioluminescent response was more extensive when the reporter strain was exposed to 5 mM catechol and 2 mM 4-chlorocatechol. These different types of bioluminescent responses by E. coli SHP3 appeared to be characterized by the nature of the aromatics to stress. Since E. coli SHP3 responded to 2,3-DHBP quite sensitively, this reporter strain could be applied for detecting some catecholic pollutants.

Solvent/Detergent Inactivation and Chromatographic Removal of Human Immunodeficiency Virus During the Manufacturing of a High Purity Antihemophilic Factor Ⅷ Concentrate: In Seop Kim , Yong Woon Choi , Hang Sang Woo , Chong E. Chang , Soungmin Lee; J. Microbiol. 2000;38(3):187-191.

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Abstract: A validation study was conducted to determine the efficacy of solvent/detergent (S/D) inactivation and Q-Sepharose column chromatographic removal of the human immunodeficiency virus (HIV) during the manufacturing of a high purity antihemophilic factor VIII (GreenMono) from human plasma. S/D treatment using the organic solvent, tri (n-butyl) phosphate, and the detergent, Triton X-100, was a robust and effective step in eliminating HIV-1. The HIV-1 titer was reduced from an initial titer of 8.3 log 10 TCID 50 to undetectable levels within one minute of S/D treatment. HIV-1 was effectively partitioned from factor VIII during Q-Sepharose column chromatography with the log reduction factor of 4.1. These results strongly assure the safety of GreenMono from HIV.

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