Journal of Microbiology

Volume 38(2); June 2000

Microbial Degradation of Monohydroxybenzoic Acids: Timmanagouda B. Karegoudar , Chi-Kyung Kim; J. Microbiol. 2000;38(2):53-61.

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Abstract: Hydroxybenzoic acids are the most important intermediates in the degradative pathways of various aromatic compounds. Microorganisms catabolize aromatic compounds by converting them to hydroxylated intermediates and then cleave the benzene nucleus with ring dioxygenases. Hydroxylation of the benzene nucleus of an aromatic compound is an essential step for the initiation and subsequent disintegration of the benzene ring. The incorporation of two hydroxyl groups is essential for the labilization of the benzene nucleus. Monohydroxybenzoic acids such as 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid through hydroxylation yield terminal aromatic intermediates like catechol, protocatechuic acid, gentisic acid, or pyrocatechuic acid that are susceptible for subsequent oxygenative cleavage of the benzene ring. These terminal aromatic intermediates are further degraded to cellular components through ortho-and /or meta-cleavage pathways and finally lead to the formation of constituents of the TCA cycle. Many groups of microorganisms have been isolated as degraders of hydroxybenzoic acids with diverse degradative routes and specific enzymes involved in their metabolic pathways. Various microorganisms carry out unusual non-oxidative decarboxylation of aromatic acids and convert them to respective phenols which have been documented. Further, Pseudomonas and Bacillus spp. are the most ubiquitous microorganisms, being the principal components of microflora of most soil and water environments.

Sequence Comparison of Mitochondrial Small Subunit Ribosomal DNA in Penicillium: Soon Gyu Hong , Yoon-Dong Park , Wonjin Jeong , Kyung Sook Bae; J. Microbiol. 2000;38(2):62-65.

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Abstract: Partial sequence comparisons of mitochondrial small subunit rDNA (mt SSU rDNA) were used to examine taxonomic and evolutionary relationships among seven Penicillium species: two monoverticillate species, two biverticillate species, and three terverticillate species. Amplified fragments of mt SSU rDNA highly varied among seven species in size, suggesting the existence of multiple insertions or deletions in the region. A phylogenetic tree was constructed by exhaustive search of parsimony analysis. The phylogenetic tree distinguished two statistically supported monophyletic groups, one for two monoverticillate species and the other for three terverticillate species and one biverticillate species, P. vulpinum. The phylogenetic relationship of P. waksmanii, the biverticillate species, was not clear.

PCR-RFLP and Sequence Analysis of the rDNA ITS Region in the Fusarium spp.: Lee , Young-Mi , Yong-Keel Choi , Byung-Re Min; J. Microbiol. 2000;38(2):66-73.

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Abstract: To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction (PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.

Bioluminescent Assay of Bovine Liver Riboflavin Kinase Using a Bacterial Luciferase Coupled Reaction: Ki Woong Cho; J. Microbiol. 2000;38(2):74-79.

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Abstract: For the demonstration of a novel riboflavin kinase assay method based on the bacterial bioluminescence, partially purified riboflavin kinase was prepared from bovine liver through ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. Using bacterial luciferase from Photobacterium phosphoreum and the dithionite reduction method, an easy, safe, and fast assay method was established. The optimal temperature, pH, Km values for riboflavin and ATP of bovine liver riboflavin kinase determined with this luminescence method were 35 C, pH 7, 15.3 uM and 8.3 uM, respectively. The detection limit of FMN produced by riboflavin kinase was in the range of 200 pM to 4 uM which is comparable to the HPLC-fluorescence detection method, while the detection time for each assay was less than 15 sec compared to the HPLC method which requires at least 10 min for completion.

Expression and DNA Sequence of the Gene Coding for the lux-Specific Fatty Acyl-CoA Reductase from Photobacterium phosphoreum: Chan Yong Lee , Edward A. Meighen; J. Microbiol. 2000;38(2):80-87.

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Abstract: The nucleotide sequence of the luxC gene coding for lux-specific fatty acyl-CoA reductase and the upstream DNA (325 bp) of the structural gene from bioluminescent bacterium, Photobacterium phosphoreum, has been determined. An open reading frame extending for more than 20 codons in 325 bp DNA upstream of luxC was not present in both directions. The lux gene can be translated into a polypeptide of 54 kDa and the amino acid sequences of lux specific reductases of P. phosphoreum shares 80, 65, 58, and 62% identity with those of the Photobacterium leiognathi, Vibrio fischeri, Vibrio harveyi, and Xehnorhabdus luminescens reductases, respectively. Analyses of codon usage, showing that a high frequency (2.3%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA and B genes, suggested that the AUA codon may play a modulator role in the expression of lux gene in E. coli. The structural genes (luxC, D, A, B, E) of the P. phosphoreum coding for luciferase ([alpha], [beta]) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in E. coli under the T7 phage RNA polymerase/promoter system and identification of the [^35 S]methionine labelled polypeptide products. The degree of expression of lux genes in this system, high level of luxA, B genes whereas low level of luxC, D genes, were consistent with the analyses of codon usage. High expression of the luxC gene could only be accomplished in a mutant E. coli 43R. Even in crude extracts, the acylated acyl-CoA reductase intermediate as well as acyl-CoA reductase activities could be readily detected.

Expression and Characterization of the Human rpS3 in a Methylotrophic Yeast Pichia pastoris: Jae Yung Lee , Sang Oun Jung , BuHyun Youn , Oh Sik Kwon , Joon Kim; J. Microbiol. 2000;38(2):88-92.

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Abstract: A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme (UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was previously characterized, and this activity of mammalian rpS3 was found to be non-specific upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.

Characterization of an Escherichia coli O157:H7 Strain Producing Verotoxin 2 Isolated from a Patient in Korea: Chang-Kyu Sohn , Wan Huh , Byung-Chun Kim , Wan Park; J. Microbiol. 2000;38(2):93-98.

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Abstract: Nine hundred patients diagnosed with diarrhea or hemorrhagic uremic syndrome in the Kyungpook Province, Korea, were examined from November 1998 to February 2000. One patient in Kumi appeared to possess the Escherichia coli O157:H7 strain, which is very important in clinical decision making and public health action. The isolated strain, an E. coli O157:H7 KM, contained a 60 MDa plasmid and typical virulence genes including the verotoxin 2 gene, ehxA gene (encoding enterohemorrhagic hemolysin), and eae (encoding attaching and effacing protein-intimin) gene. This strain produced only verotoxin 2. Pulsed field gel electrophoretic analysis showed that the genomic organization of the E. coli O157:H7 KM strain may differ greatly from those of representative strains previously reported in the United States and Japan.

Monitoring and Characterization of Bacterial Contamination in a High-Purity Water System Used for Semiconductor Manufacturing: In Seop Kim , Geon-Hyoung Lee , Kye Joon Lee; J. Microbiol. 2000;38(2):99-104.

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Abstract: Hydrogen peroxide has been used in cleaning the piping of an advanced high-purity water system that supplies ultra-high purity water (UHPW) for 16 megabyte DRAM semiconductor manufacturing. The level of hydrogen peroxide-resistant bacteria in UHPW water was monitored prior to and after disinfecting the piping with hydrogen peroxide. Most of the bacteria isolated after hydrogen peroxide disinfection were highly resistant to hydrogen peroxide. However, the percentage of resistant bacteria decreased with time. The hydrogen peroxide-resistant bacteria were identified as Micrococcus luteus, Bacillus cereus, Alcaligenes latus, Xanthomonas sp. and Flavobacterium indologenes. The susceptibility of the bacteria to hydrogen peroxide was tested as either planktonic cells or attached cells on glass. Attached bacteria as the biofilm on glass exhibited increased hydrogen peroxide resistance, with the resistance increasing with respect to the age of the biofilm. These results indicate that bacteria resistant to hydrogen peroxide play an important role in biofilm regrowth on piping after hydrogen peroxide treatment. In order to optimize the cleaning strategy for piping of the high-purity water system, the disinfecting effect of hydrogen peroxide and peracetic acid on the bacteria was evaluated. The combined use of hydrogen peroxide and peracetic acid was very effective in killing attached bacteria as well as planktonic bacteria.

Rapid PCR Method for Detecting Candida albicans Using Primers Derived from the Integrin-like Protein Gene [alpha]INT1 of Candida albicans: Young-Hee Lim , Do-Hyun Lee; J. Microbiol. 2000;38(2):105-108.

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Abstract: Oligonucleotide primers amplifying a 344 bp fragment on the integrin-like protein alpha-INT1p gene ([alpha]INT1) of Candida albicans were synthesized for screening of C. albicans from clinicalsamples by the polymerase chain reaction (PCR). The PCR specifically amplified DNA from C. albicans and none from any other Candida, fungal, or human DNA in standard strains used here. The PCR assay showed that the primers (LH1 and LH2) were specific for 26 isolates of C. albicans from clinical samples, whereas the positive fragment, 344 bp, was not amplified from 15 clinical isolates including 14 other medically important Candida species and an isolate of Saccharomyces cerevisiae. PCR was conducted on the urine samples of 20 patients and 4 samples were C. albicans positive. The detection limit of the PCR assay for C. albicans was shown to be approximately 10 cells/ml saline. The PCR system using 344 bp [alpha]INT1 as a target is more specific and rapid than the conventional culture method, and the sensitive detection method is applicable to clinical diagnosis of C. albicans infections.

A Recombinant Mouse GM-CSF Protein Expressed as an Inclusion Form Shows Colony Stimulating Activity: Jin-Kyoo Kim , Eun-Jung Sohn , Soo-O Lee , Choon-Taek Lee , Ah Young Lee , Hye Kyung Chung , Bong Whan Sung , Hyun Joo Youn; J. Microbiol. 2000;38(2):109-112.

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Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor and an activator of mature myeloid cells, and recombinant GM-CSF is increasingly under clinical studies for the treatment of various diseases including cancer, infectious diseases and hematopoietic diseases. We constructed a recombinant mouse GM-CSF expression plasmid with pelB leader sequence and His.Tag under T7 promoter control, and showed that the construct produced a 20 kDa recombinant protein in 8M urea. We also showed that the 20 kDa recombinant protein prepared in 8M urea stimulated colony formation in vitro, indicating that the recombinant mGM-CSF can be renatured to its native form to show the colony stimulating activity.

Use of the Yeast 1.5-Hybrid System to Detect DNA-Protein-Protein Interactions: Sook-Kyung Kim , Jin Hee Han; J. Microbiol. 2000;38(2):113-116.

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Abstract: Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find an additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The result indicates that this system is a promising one, capable of selecting an interacting component.

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