Journal of Microbiology

Volume 35(4); December 1997

Biodegradation of Dibenzo-p-dioxin and Dibenzofuran by bacteria: Jean Armengaud , Kenneth N. Timmis; J. Microbiol. 1997;35(4):241-252.

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Abstract: Polychlorodibenzofurans and polychlorodibenzo-pdioxins are among the most toxic xenobiotics released into the biosphere and the cause of significant public concern because of their apparent ubiquityalbeit at low levels- in food and environment. Several bacteria able to degrade nonchlorinated dioxin and dibenzofuran and in some cases to attack chlorinated analogues have recently been isolated. This opens up the possibility that bioremediation processes may ultimately be developed to eliminate these toxic compounds from contaminated sites. In this review we summarize current knowledge on the genetics and biochemistry of dioxin and dibenzofruan degradation by Sphingomonas sp. RW1, a gram-negative bacterium, and highlight the unusual nature of the genetic organization of these pathways.

Phylogenetic Analysis of the Contriciacease Based on Gene Sequences of Nuclear 18S Ribosomal DNAS: Lee, Seung Shin , Jung, Hack Sung; J. Microbiol. 1997;35(4):253-258.

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Abstract: The nuclear 18S ribosomal RNA genes of seven corticioid species were sequenced. These sequences were analyzed and compared with those of 24 other species of the order Aphyllophorales and phylogenetic trees were constructed using parsimonious methods. Phylogenetic analyses showed that two species among examined members of the Corticiaceae, Resinicium bicolor and Thanatephorus praticola, are located distantly from the remaining six species. The separation of R. bicolor seems to be kphylogenetically significant because it has very unique cystidia. The independent lineage of T. practicola suggests that it is also phylogenetically distinct because it has unusual features like the homobasidium producing secondary spores and the spetal ultrastructure of pore cap. Furthermore, Auriscalpium vulgare, Bondarzewia berkeleyi, and Heterobasidion annosum from different families of the Aphyllophorales proved to be closely related to the species of the Corticiaceae. They all have amyloid spores and grouped with Aleyrodiscus amorphus, which is a member of the Corticiaceae. The amyloidity of spores seems to be an improtant character throughout the order of the Aphyllophorales.

Phylogenetic of Trichaptum Based on Mitochondrial Small Subunit rDNA Sequences: Ko, Kwan Soo , Jung, Hack Sung; J. Microbiol. 1997;35(4):259-263.

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Abstract: To study the phylogenetic relationships of species of Trichaptum and to infer intraspecific dibergence of T. abietinum, partial mitochondrial small subunit rDNA sequences were determined. Six strains of T. abietinum, two of T. biforme, and one of T. fusco-violaceum were examined. Parsimony and distance analyses showed that each Trichaptum species forms a distinct group and that T. abietinum consists of two or more subgroups. Strains from North America were distantly related to one another but the European strain formed an independent group with three Korean strains, suggesting the possibility that Korean taxa may be phylogenetically closer to European taxa than to North American taxa.

The Genetic Organization of the Linear Mitochondrial Plasmid mlp1 from Pleurotus ostreatus NFFA2: Kim, Eun Kyung , Youn, Hye Sook , Koo, Yong Bom , Roem Jung Hye; J. Microbiol. 1997;35(4):264-270.

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Abstract: The structure of plasmid mlp1, a linear 10.2kb mitochondrial plasmid of Pleurotus ostreatus NFF A2 was determined by restriction enzyme mapping and partial sequencing. The plasmid encodes at least two proteins; a putative RNA polymerase showing homology to yeast mitochondrial RNA polymerase and to viral-encoded RNA polymerases, and a putative DNA polymerase showing significant homology to the family B thpe DNA polymerases. It also contains terminal inverted repeat sequences at both ends which are longer than 274 bp. A 1.6 kb EcoRI restriction fragment of m1p1 containing the putative RNA polymerase gene did not hybridize to the nuclear or motochondrial genomes from P. ostreatus, suggesting that it may encode plasmidspecific RNA polymerase. The gene fragment also did not hybridize with the RNA polymerase gene (RPO41) from Saccaromyces cerevisiae. The relationship between genes in m1p1 and those in another linear plasmid pC1K1 of Claviceps purpurea was examined by DNA hybridization. The result indicates that the genes for DNA and RNA polymerases are not closely related with those in C. purpurea.

Isolation and characterization of the Phenotypic Revertants of Streptomyces coelicolor abs Mutant: Jung, Ho Sun , Park, Uhn Mee; J. Microbiol. 1997;35(4):271-276.

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Abstract: We isolated phenotypic suppressors of an absB (antibiotics synthesis suppression) strain. In the absB colonies, all four antibiotics including two pigmented antibiotics were blocked so that no pigmentation could be found. We assumed that in the colonies with the wuppressive(or reversive) mutation, both pigmentation would be restored so that the strains with suppressive mutation could be cisually detected. Harvested absB spores were treated with chemical mutagen along with electric shock, and were spread on specially fromulated minimal medium plates. The pigmented colonies were isolated from the unpigmented majorities. In one candidate strain, the restoration and significant overproduction of actinorhodin and undecylprodigiosin were recognized. In three other candidate strains, the overproduction of actinorhodin and restoraion of undecylprodigiosin were observed. The production of the two unpigmented antibiotics (CDA and methylenomycin) were visualized in the tested candidate strains. The strains with wuppressive mutations would be very useful in dlucidating the regulation network of antiviotics synthesis and overproduction of the antibiotics.

Isolation and Characterization of Paraquat-inducible Promoters from Escherichia coli: Lee, Joon Hee , Roe, Jung Hye; J. Microbiol. 1997;35(4):277-283.

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Abstract: Promoters inducible by paraquat, a superocide-generating agent, were isolated from Escherichia coli using a promoter-probing plasmid pRS415 with promoterless lacA gene. Twenty one promoters induced by paraquat were selected and further characterized. From sequence analysis, thirteen of the promoters were mapped to their specific loci on the Escherichia coli chromosome. Several promoters were mapped to the upstream of known genes such as usgl, katG, and mglB, whose relationships with superoxide response have not been previously reported. Other promoters were mapped to the upstream region of unknown open reading frames. Downstream of HC 96 promoter are uncharacterized ORFs whose sequences are homologous to ABC-transporter subunits. Downstream of HC84 promoter is an ORF encoding a transcriptional regulator-like protein, which contains a LysR family-specific HTH (helix-turn-helix) DNA bindign motif. We investigated whether these promoters belong to the soxRS regulon. All promoters except HC96 were found to belong to the soxRS regulon. The HC96 promoter was significantly induced by paraquat in the soxRS deletion mutant strain. The basal transcription level of three promoters (HE43, HC71, HD94) significantly increased at the stationary phase, implying that they are regulated by RpoS. However, paraquat inducibility of all promoters disappeared in the stationary phase, suggesting that SoxRS regulatory system is active only in rapidly growing cells.

Isolation of a Bacterium That Inhibits the Growth of Anabaena cylindrica: Kim, Chul Ho , Leem, Mi Hyea , Choi, Yong Keel; J. Microbiol. 1997;35(4):284-289.

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Abstract: A Gram (-), rod-shaped bacterium 2.3~2.8×0.45㎛ in size which exhibited growth-inhibiting effects against a cyanobacterium (Anabaena cylindrica) was isolated from Daechung Dam Reservoir. This isolate was identified as Moraxella sp. and designated Moracella sp. CK-1. Hollow zones formed around bacterial colonies on the cyanobacterial lawn. In a mixed-culture of A. cylindrica and the isolate, each microorganism grew inverse-proportionally, and the cyanobacterial vegetative cells completely disappeared within 24 hours. On treatment with Moraxella sp. CK-1, cell walls of A. cylindrica disappeared, but sheathes remained in a more electron dense form. The unit membrane such as thylakoidal membrane was stable to bacterial lysing activity. This bacterium showed a broad action spectrum against cyanobacteria. The growth-inhibiting activity of Moracella sp. CK-1 against A. cylindrica is believed to be performed through the excretion of active substances.

Dechlorination of 4-Chlorobenzoate by Pseudomonas sp. DJ-12: Chae, Jong Chan , Kim, Chi Kyung; J. Microbiol. 1997;35(4):290-294.

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Abstract: 4-Chlorobiphenyl-degrading Pseudomonas sp. DJ-12 was able to degrade 4-chlorobenzoate(4CBA), 4-iodobenzoate, and 4-bromobenzoate completely under aerobic conditions. During. the degradation of 4CBA by Pseudomonas sp. DJ-12, chloride ions were released by dechlorination and 4-hydroxybenzoate was produced as an intermediate metabolite. The NotI-KNA fragments of pKC157 containing dechlorination genes hybridized with the gene encoding 4CBA:CoA dehalogenase of Pseudomonas sp. CBS3 which is responsible for the hydrolytic dechlorination of 4CBA. These results imply that Pseudomonas sp. DJ-12 degrades 4CBA to 40hydroxybenzoate via dechlorination as the initial step of its degradativ pathway. The genes responsible for dechlorination of 4CBA were found to be blcated on the chromosomal DNA of Pseudomonas sp. DJ-12.

Characteristics of Catechol 2,3-dioxygenase Produced by 4-Chlorobenzoate-degrading Pseudomonas sp. S-47: Kim, Ki Pil , Seo, Dong In , Min, Kyung Hee , Ka, Jong Ok , Park, Yong Keun , Kim, Chi Kyung; J. Microbiol. 1997;35(4):295-299.

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Abstract: Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were 30~35℃ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

Role of the Amino Acid Residued in the Catalysis of Catechol 2,3-dioxygenase from Pseudomonas putida SU10 as Probed by Chemical Modification and Random Mutagenesis: Park, Sun Jung , park, Jin Mo , Lee, Byeong Jae , Min, Kyung Hee; J. Microbiol. 1997;35(4):300-308.

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Abstract: The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.

Molecular Cloning and Characterization of Mn-Superoxide Dismutase Gene from Candida sp.: Hong, Yun Mi , Nam, Yong Sik , Choi, Soon Yong; J. Microbiol. 1997;35(4):309-314.

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Abstract: The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on oxidative stress=responsible enzymes in the Candida sp., the gene encoding the MnSOD was isolated and examined in this study. A specific primer was designed based on conserved regions of MnSOD sequences from other organisms, and was used to isolate the gene by PCR on reverse-transcribed Candida poly(A^+) RNA. The PCR product was used to screen a Candida genomic lambda library and the nucleotide wequence of positive clone was determined. The deduced primary sequence encodes a 25kDa protein which has the conserved residues for enzyme activity and metal binding. The 28 N-terminal amino acids encoded by the Candida cDNA comprise a putatice mitochondrial transit peptide. Potential regulatory elements were identified in the 5' flanking sequences. Northern blot analysis showed that the transcription of the MnSOD gene is induced 5-to 10-fold in response to mercury, cadmium ions and hydrogen peroxide.

Effect of Polyamines on Cellular Differintiation of N. gruberi: Inhibition of Translation of Tubulin mRNA: Yoo, Jin Uk , Kwon, Kyung Soon , Cho, Hyun Il , Kim, Dae Myung , Chung, In Kwon , Kim, Young Min , Lee, Tae HO , Lee, Joo Hun; J. Microbiol. 1997;35(4):315-322.

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Abstract: The effects of a polyamine, spermine, on the differentiation of Naegleria gruberi amebas into flagellates were tested. Addition of spermine at early stages of differentiation (until 40 min after the initiation of differentiation) completely inhibited the differentiation. To understand the inhibition mechanism, we examined the effect of spermine treatment on the transcription and translation of differentiation-specific genes during differentiation. Addition of spermine at early stages did not inhibit the accumulation of two differentiation-specific mRNAs, α-tubulin and Class I mRNA, significantly, but rather prevented the rapid degradation of the mRNAs in later overall protein synthesis partially and gradually. However, translation of the α-tubulin mRNA was completely inhibited. These data suggest that the inhibition of differentiation of N. gruberi by spermine treatment did not result from the inhibition of transcription of differentiation-specific genes but from the specific inhibition of translation of the mRNAs during the differentiation.

Biochemical Characteristics of a Killer Toxin Produced by Ustilago maydis Virus SH14 Isolated in Korea: Ham Eun Soo , Yie, Se Won , Choi, Hyung Tae; J. Microbiol. 1997;35(4):323-326.

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Abstract: Toxin protein from Ustilago maydis virus SH14 isolated in Korea was purified using ethanol precipitation, cation exchange, gel filtration and anion exchange chromatography. The molecular weight of the purified protein was estimated to be 8.3 kDa by SDS-PAGE analysis. The Nterminal sequence of the protein is L-G-I-N-C(K)-R-G-S-S-Q--C(K)-G-L-S-G which is highly homologous with that of P4 toxin, but the amino acid composition and electrophoretic mobility in a native PAGE of the toxin protein were totally different from those of P4 toxin respectively. The SH14 toxin was shown to have immunological cross-reactivity about 50% with P4 toxin when examined by Western hybridization.

The Schizosaccharomyces pombe Proteins that Bind to the Human HnRNPA1 Winner RNA: Kim , Jeong Kook; J. Microbiol. 1997;35(4):327-333.

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Abstract: Although extensively characterized in human cells, no heterogeneous nuclear ribonucleoprotein(hnRNP) has been found in the fission yeast Schizosaccharomyces pombe which is amenable to genetic studies and more similar to mammals than Saccharomyces cerevisiae is in terms of RNA processing. As a first step to characterize hnRNPs from S. pombe, attempt was made to find human hnRNP A1 homologs from S. pombe. The RNA molecule (A1 winner) containing the consensus high-affinity hnRNP A1 binding site (UAGGGA/U) was synthesized in vitro and used in an ultraviolet(UV) light-induced protein-RNA cross-linking assay. A number of S, pombe proteins bound to the A1 winner RNA. An approximately 50-kDa protein(p50) cross-linked more efficiently to the A1 winner RNA than other proteins. The p50 protein did not cross-link to a nonspecific RNA, but rather to the A1-5’ SS RNA in which the consensus 5’ splice junction sites of S. pombe introns were abolished. This suggests that the p50 protein, however, did not bind to the single-stranded DNA to shich the human hnRNP A1 could bind and be eluted with 0.5M NaCl. Further analysis should reveal more features of this RNA-binding protein.

Isolation and characterization of pre-tRNA^Val splicing Mutants of Schizosaccharomyces pombe: Hwang, Ku Chan , Kim, Dae Myung; J. Microbiol. 1997;35(4):334-340.

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Abstract: A collection of 132 temperature sensitive (ts) mutants was generated by the chemical mutagenesis of Schizosaccharomyces pombe wild type strain and screened for tRNA splicing defects on Northern blots by hybridization with an oligonucleotide that recognizes the exon of the S. pombe tRNA^Val as a probe. We identidied 6 mutants which accumulate precursor tRNA^Val. Among them, 2 mutants exhibited remarkable morphological differences compared to wild type cells. One tRNA splicing mutant showed elongated cell shape in permissive as well as non-permissive cultures. The other mutant exhibited shortened cell morphology only in nonpermissive culture. The total RNA pattern in the splicing mutants appeared to be normal. Genetic analysis of four tRNA^Val splicing mutants demonstrated that the mutation reside in different genes.

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