Journal of Microbiology

Volume 37(3); September 1999

Catabolism of 4-Hydroxybenzoic Acid by Pseudomonas sp. DJ-12: Karegoudar, Timmanagouda B. , Chae, Jong Chan , Kim, Chi Kyung; J. Microbiol. 1999;37(3):123-127.

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Abstract: A Pseudomonas sp. strain DJ-12 isolated by 4-cholrobiphenyl enrichment culture technique is capable of utilizing 4-hydroxybenzoic acid as a sole source of carbon and energy. The bacterium catabolized 4-hydroxybenzoic acid through the intermediate formation of protocatechuic acid, which was further metabolized. The cell free extracts of pseudomonas sp. DJ-12, grown on 4-hydroxybenzoic acid showed higher activities of 4-hydroxyenzoate 3-hydroxylase and protocatechuate 4,5-dioxygenase, but the activity of catechol 2,3-dioxygenase was lower. The results suggest that 4-hydroxybenzoic acid is catabolized via protocatechuic acid rather than catechol or gentisic acid in this bacterium and that the protocatechuic acid formed was metabolized through a metacleavage pathway by protocatechuate 4,5-dioxygenase.

Identification and Characterization of an Oil-degrading Yeast, Yarrowia lipolytica 180: Kim, Tae Hyun+ , Lee, Jung-Hyun , Oh, Young Sook , Bae, Kyung Sook , Kim, Sang Jin; J. Microbiol. 1999;37(3):128-135.

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Abstract: Among oil-degrading microorganisms isolated from oil-polluted industrial areas, one yeast strain showed high degradation activity of aliphatic hydrocarbons. From the analyses of 18S rRNA sequences, fatty acid, coenzyme Q system, G+C content of DNA, and biochemical characteristics, the strain was identified as Yarrowia lipolytica 180. Y. lipolytica 180 degraded 94% of aliphatic hydrocarbons in minimal salts medium containing 0.2% (v/v) of Arabian light crude oil within 3 days at 25℃. Optimal growth conditions for temperature, pH, NaCl concentration, and crude oil concentration were 30℃, pH 5-7, 1%, and 2% (v/v), respectively. Y. lipolytica 180 reduced surface tension when cultured on hydrocarbon substrates (1%, v/v), and the measured values of the surface tension were in the range of 51 to 57 dynes/cm. Both the cell free culture broth and cell debris of Y. lipolytica 180 were capable of emulsifying 2% (v/v) crude oil by itself. They were also capable of degrading crude oil (2%). The strain showed a cell surface hydrophobicity higher than 90%, which did not require hydrocarbon substrates for its induction. These results suggest that Y. lipolytica has high oil-degrading activity through its high emulsifying activity and cell hydrophobicity, and further indicate that the cell surface is responsible for the metabolism of aliphatic hydrocarbons.

Antimutagenicity of Phellinus linteus in Salmonella typhimurium: Shon, Yun Hee , Lee, Jae Sung , Lee, Hang Woo , Kim, Joong Wan , Lim, Jong Kook , Kim, Cheorl Ho , Nam, Kyung Soo; J. Microbiol. 1999;37(3):136-140.

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Abstract: The mutagenicities and antimutagenicities of butanol (PL I) and water (PL II) extracts from the filtrate of the cultured broth of Phellinus linteus were examined using the Ames/Salmonella test. No mutagenic activity of PL I and PL II was found in Salmonella typhimurium strains TA98 and TA100, either with or without S9 activation. In contrast, PL I and PL II showed inhibitory effects on the mutagenic activities induced by the directly-acting mutagens, 4-nitro-o-phenylenediamine (NPD) using the tester strain TA98 and sodium azide (NaN₃) using the tester strain TA 100 in the absence of S9 mix. PLI and PL II also showed inhibitory effects on the mutagenicities of the indirectly-acting mutagens, 2-aminofluorene (2-AF) using the tester strain TA98 and benzo[a]pyrene (B[a]P) using the tester strain TA 100 in the presence of S9. These results suggest that P. linteus has an antimutagenic activity and may play a role in the prevention of cancer.

Transcriptional Induction of a Carbon Starvation Gene during Other Starvation and Stress Challenges in Pseudomonas putida MK1: A Role of a Carbon Starvation Gene in General Starvation and Stress Responses: Chitra, Subramanian+ , Lee, Ho Sa , Kim, Young Jun; J. Microbiol. 1999;37(3):141-147.

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Abstract: Thirteen transcriptionally-fused carbon starvation mutants, derived from Pseudomonas putida ATCC 12633, were analyzed for their survivability and transcriptional induction profiles upon carbon starvation. One of these mutants, MK114, which exhibited the lowest survivability and the highest induction rate, was selected and further examined under different starvation (nitrogen and phosphate) and stress (osmolarity, H₂O₂, salts, alcohol, and heat) conditions. Under all tested conditions MK114 induced β-galactosidase activity, implying that the interrupted gene (cst114) is a general starvation and stress response gene. The rate of induction ranged from 2.6-fold for phosphate starvation to 3.7-fold for osmotic shock. The mini-Tn5 flanking DNA was cloned from the chromosome of MK114. The cloned DNA fragment exhibited carbon starvation activity, indicating that this fragment contains a carbon starvation-related promoter region. This region was partially sequenced. Possible physiological roles of Cst114 in a carbon sensing mechanism and in other stress responses are also discussed.

Purification and Properties of Laccase of the White-rot Basidiomycete Coriolus hirsutus: Lee, Yeo Jin , Shin, Kwnag Soo; J. Microbiol. 1999;37(3):148-153.

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Abstract: Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, Sephacryl S-2000 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q HR 5/5 chromatography. The purification of laccase was 46.6-fold with an overall yield of 23.7%. Laccase from this fungus was a monomeric glycoprotein with 16% carbohydrate content, and has an isoelectric point of 4.2, and molecular mass of 78 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed significant homology to hoste of laccases from Coriolus versicolor, Pycnoporus cinnabarius, and an unidentified basidiomycete, PM1. The highest rate of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation by laccase was reached at 45℃, and the pH optima of the enzyme varied depending on the substrate in the range of 2.0 to 4.5. The enzyme was stable at 60℃ for 5 h and lost 80% activity at 80℃ in 30 min. The enzyme oxidized a variety of usual laccase substrates including lignin-related phenol, and had the highest affinity toward ABTS. Under standard assay conditions, the apparent Km value of the enzyme toward ABTS was 8.1 μM. The enzyme was completely inhibited by L-cysteine and sodium azide, but not by potassium cyanide, SDS, ad thiourea.

Arg243, Invariably Critical for the Transcriptional Activation of Yeast Gcn4p: Cho, Gyu Chull , Lee, Jae Yung , Kim, Joon; J. Microbiol. 1999;37(3):154-158.

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Abstract: The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

Effects of Selected Environmental Conditions on Biomass and Geosmin Production by Streptomyces halstedii: Kevin K. Schrader , Willard T. Blevins; J. Microbiol. 1999;37(3):159-167.

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Abstract: The effects of bicarbonate concentration, atmospheric carbon dioxide level, and reduced atmospheric oxygen on biomass and geosmin production and geosmin/biomass (G/B) values for Streptomyces halstedii, a producer of the off-flavor compound geosmin, were determined. In addition, a study was performed to determined possible synergistic relationships between a cyanobacterium, Oscillatoria tenuis UTEX #1566, and S. halstedii in the enhancement of actinomycete growth and/or geosmin production. These studies took into consideration those conditions that can occur during cyanobacterial bloom die-offs. Increasing bicarbonate concentration caused slight decreases in geosmin production and G/B for S. halstedii. Increasing atmospheric oxygen promoted geosmin production and G/B while lower oxygen levels resulted in a decrease in geosmin production and G/B by S. halstedii. Biomass production by S. halstedii was adversely affected by reduced oxygen levels while changes in bicarbonate concentration and atmospheric carbon dioxdie levels had little effect on biomass production. Sonicated cells of O. tenuis UTEX #1566 promoted biomass production by S. halstedii, and O. tenuis culture (cells and extracellular metabolites) and culture supernatnat (extracellular metabolites) each promoted geosmin and G/B yields for S. halstedii. In certain aquatic systems, environmental conditions resulting from cyanobacterial blooms and subsequent bloom die-offs could favor actinomycete growth and off-flavor compound production by certain actinomycetes.

Cloning and Nucleotide Sequence Analysis of Verotoxin Gene from Escherichia coli O157 KNIH317 Isolated in Korea: Park, Yong-Chjun , Shin, Hee Jung , Kim, Young Chang; J. Microbiol. 1999;37(3):168-173.

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Abstract: Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bacteriphage 933W.

Mutatioal Analysis of the Role of vir-box in the Expression of the virE Gene: Han, Seong-Su , Sim, Woong-Seop; J. Microbiol. 1999;37(3):174-179.

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Abstract: To elucidate the role of vir-box in the expression of the virE gene, the vir-box was modified by site-directed mutagenesis and tested for β-galactosidase activities. A, C, T T, A, C substitutions at -62, -63, and -65 positions, destroying the 5'-region of the vir-box and A T at position -55, destroying the 3'-region of the vir-box respectively, showed only 17% promoter activity. When the vir-box was modified to contain perfect dyad symmetry structure (DSR) by the substitutions T, G A, T at -60 an d-61 positions, β-glactosidase activity increased 302%. These results indicate that the 5' and 3'-region of vir-box as well as the imperfect DSR of the vir-box itself may play a very important role in the regulation of virE gene expression.

Generation of Isotype Switch Variants form Hybridoma cells Producing anti-Streptococcus penumoniae 6B Polysaccharide Antibody: Kim, Ji Hye , Ryu, Eun Ja , Park, Moon Kook; J. Microbiol. 1999;37(3):180-184.

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Abstract: Hybridoma cells producing IgM anti-pneuococcal 6B polysaccharide antibodies were induced to switch to IgG-producing cells in vitro by treating with acridine orange. Treating 0.5 ㎍/ml of acridine orange for 24 hours generated maximal number of variant cells. The maximal isotype switch frequency was 3×10^-5, which is about 30-fold higher than the frequency of spontaneous switching. Resulting IgG-producing variants were enriched by sib selection and ELISA spot assay. Two IgG3-producing variant cells were finally cloned by limiting dilution. The variant cells produced similar amounts of antibodies as their parental cells did. The two switched antibodies had similar reactivity to pneumococcal 6B polysaccharide. When compared to their parental IgM antibodies, the switched IgG3 than that of IgM antibody. The antibodies will be useful as essential tools for comparative study of the role of heavy chain isotypes in protection against Streptococcus pneumoniae.

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