Journal of Microbiology

Volume 37(4); December 1999

Biodegradation of Phenanthrene by Sphingomonsa sp. Strain KH3-2: Su-Kyuong Shin , Young-Sook Oh , Sang-Jin Kim; J. Microbiol. 1999;37(4):185-192.

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Abstract: A phenanthrene-degrading bacterium was isolated from an oil-spilled intertidal sediment sample and identified as Sphingomonas sp. KH3-2. The strain degraded polycyclic aromatic compounds such naphthalene, fluorene, biphenyl, and dibenzothiophene. When strain KH3-2 was cultured for 28 days at 25C, a total of 500 ppm of phenanthrene was degrated with a concomitant production of biomass and Folin-Ciocalteau reactive aromatic intermediates. Analysis of intermediates during phenanthrene degradation using high-performance liquid chromatography and gas chromatography/mass spectrometry indicated that Sphingomonas sp. KH3-2 primarily degrades phenanthrene to 1-hydroxy-2-naphthoic acid (1H2NA) and further metabolizes 1H2NA through the degradation pathway of naphthalene.

Bacterial Diversity of Culturable Isolates from Seawater and a Marine Coral, Plexauridae sp., near Mun-Sum, Cheju-Island: Jung-Hyun Lee , Hyun-Hee Shin , Deuk-Soo Lee , Kae Kyung Kwon , Sang-Jin Kim , Hong Kum Lee; J. Microbiol. 1999;37(4):193-199.

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Abstract: Fifty-eight strains showing different colony morphological characteristics on various media were isolated from marine coral (Plexauridae sp.) and ambient seawater near Mun-Sum, Cheju-Island in 1998. Bacterial diversity was studies by phylogenetic analysis of the partial 16S rRNA gene sequences. All isolates representing the bacterial domain included affiliates of the high G+C (59%) and los G+C (3%) subdivision of Gram positive bacteria, and the alpha (33%) and gamma (5%) subdivision of the Proteobacteria. The 16S rDNA sequence similarity of the isolates was in the 88.3 to 100% range (average, 95.6%) to reported sequence data. In the comparison of the isolates from marine coarl and ambient seawater, more diverse groups belonging to alpha-proteobacteria were preferentially obtained from seawater.

Isolation of Novel Pseudomonas diminuta KAC-1 Strain Producing Glutaryl 7-Aminocephalosporanic Acid Acylase: Dae-Weon Kim , Sang-Mo Kang , Ki-Hong Yoon; J. Microbiol. 1999;37(4):200-205.

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Abstract: 7-Aminocephalosporanic acide (7-ACA) is the initial compound in preparation of cephalosporin antibiotics widely used in clinical treatment. Bacteria producing glyutaryl 7-ACA acylase, which convert cephalosporin C to 7-ACA, has been screened in soil samples. A bacterial strain exhibiting high glutaryl 7-ACA acylase activity, designated KAC-1, was isolated and identified as a strain of Pseudomonas diminuta by characterizing its morphological and physiological properties. The screening procedures include culturing on enrichment media containing glutaric acid, glutamate, and glutaryl 7-aminocephalosporanic acid as selective carbon sources. To enhance enzyme production, optimal cultivation conditions were investigated. This strain grew optimally at pH 7 to 9 and in temperatures of 20 to 40 C, but acylase production was higher when the strain was grown at 25 C. Glutaric acid, glutamate and glucos also acted as inducers for acylase production. In a jar fermenter culture, P. diminuta KAC-1 produce acylase in a growth-associated manner. The substrate specificity of KAC-1 acylase by cell extract showed that this enzyme had specificity toward glutaryl 7-ACA, glutaryl 7-ADCA, but not cephalosporin C.

Isolation and Identification of an Anaerobic Dissimilatory Fe(III)-Reducing Bacterium, Shewanella putrefaciens IR-1: Moon-Sik Hyun , Byung-Hong Kim , In-Seop Chang , Hyung-Soo Park , Hyung-Joo Kim , Gwang-Tae Kim , Mi-a Kim , Doo-Hyun Park; J. Microbiol. 1999;37(4):206-212.

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Abstract: In order to isolate a Fe(III)-reducer from the natural environment, soil samples were collected from various patty fields and enriched with ferric citrate as a source of Fe(III) under anaerobic condition. Since the enrichment culture was serially performed, the Fe(III)-reduction activity was serially diluted and cultivated on an agar plate containing lactate and ferric citrate in an anaerobic glove box. A Gram negative, motile, rod-shpaped and facultative anaerobic Fe(III)-reducer was isolated based on its highest Fe(III)-reduction activity, Bacterial growth was coupled with oxidation of lactate to Fe(III)-reduction, but the isolate fermented pyruvate without Fe(III), The isolate reduced an insoluble ferric iron (FeOOH) as well as a soluble ferric iron (ferric citrate). Using the BBL crystal enteric/non-fermentor identification kit and 16S rDNA sequence analysis, the isolate was identifice as Shewanella putrefaciens IR-1.

Characterization of Bacillus cereus SH-7 Extracellular Protease: Hak Kyu Yi , Young Jin Chum , Han Bok Kim; J. Microbiol. 1999;37(4):213-217.

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Abstract: An extracellular endopeptidase from Bacillus cereus SH-7 was purified to homogeneity. The protease was most active at pH 8 and 40 C, respectively. The molecular mass of the protease was 40 kDa on SDS-PAGE, and 120 kDa by gel filtration, suggesting that the native enzyme is composed of three homogeneous subunits. The K_m and V_max values of the protease for N-succinyl-(Ala)_2-Pro-Phe-p-nitroanilide were 11.11 mM and 170 nmol/mg of protein/min, respectively. The protease was also identified as a metalloprotease. The bioactivity of the SH-7 protease will need further study in the future.

Cloning and Sequencing of the rph Gene Encoding RNase PH from Legionella pneumophila: Se Jin Kim , Jong-Seok Lim , Nicholas P. Cianciotto , Yong-Kyung Choe; J. Microbiol. 1999;37(4):218-223.

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Abstract: Legionella pneumophila, the cause of Legionnaires disease, is able to survive intracellularily in eukaryotic cells such as monocytes, macorphages, and protozoan ogranisms. During protein biosynthesis, the rph gene encodes ribonuclease (RNase) PH which functions as a phosphorolytic nuclease that removes nucleotides following the CCA terminus of tRNA and as a nucleotidyl-transferase which adds nucleotides to the ends of RNA molecules by usingnucelside diohosphates as substrates.In this sutdy, the rph gene was screened in pUC19 library employing a DNA probe whcich was constructed from PCR based on a consensus pattern of multiple alignment of RNas PH. The encoded protein consists of 235 amino acid residues with a calculated molecualr weight of 26,112 Daltons. The RNase PH signature domains are completely conserved.

DNA Sequence of the phnN Gene for Benzaldehyde Dehydrogenase form Pseudomonas sp. DJ77 and Its Substrate Preference: Seong-Jae Kim , Soonyoung Hwang , Young-Chang Kim; J. Microbiol. 1999;37(4):224-228.

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Abstract: Benzaldehyle dehydrogenase (BZDH), an enzyme involved in the degradation of toluene and xylenes, is encoded by the phnN gene of Pseudomonas sp. strain DJ77. We determined the nucleotide sequence of a DNA fragment of 1,803 base pairs which included the phnN gene. The fragment contained an open reading frame of 1,506 base pairs to accommodate th 55 kDa sized enzyme encoding BZDH. The enzyme efficiently oxidized benzaldehyde, salicylaldehyde, m-tolualdehyde and ps-tolualdehyde.

Function of mORF1 Protein as a Terminal Recognition Factor for the Linear Mitochondrial Plasmid pMLP1 from Pleurotus ostreatus: Eun-Kyoung Kim , Jung-Hye Roe; J. Microbiol. 1999;37(4):229-233.

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Abstract: The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

DNA Sequencing and Expression of the Circumsporozoite Protein of Plasmodium vivax Korean Isolate in Escherichia coli: Hyeong Woo Lee , Jong Soo Lee , Won Ja Lee , Ho Sa Lee; J. Microbiol. 1999;37(4):234-242.

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Abstract: To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyunggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no corss reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagonis of malaria patients and seroepidemiology.

Suppressive Effects of Divalent Cations on Self-splicing Inhibition by Spectinomycin of Group 1 Intron RNA: In Kook Park; J. Microbiol. 1999;37(4):243-247.

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Abstract: Effects of divalent cations on self-splicing inhibition by the antibiotic spectinomycin of the phage T4 thymidylate synthase intro (td) have been investigated. Ca^2+ ion at 1mM concentration suppressed splicing inhibition of spectinomycin by 10% and 50 uM Co^2+ ion also suppressed splicing inhibition of specinomycin by 10%. Mg^2+ ion at 6 mM concentration decreased splicing inhibition of spectinomycin by 42% while Mn^2+ ion decreased the splicing inhibition by 10%. Zn^2+ ion at 10 uM concentration lowered the splicing inhibition by spectinomycin of 15%. Of all divalent cations tested, Mg^2+ ion was the most effective in suppressing splicing inhibition by specinomycin whereas Ca^2+ ion was the least effective. The results suggest that spectinomycin may interact with specific and functional Mg^2+ -binding sites within intron RNA that lead to a displacement of Mg^2+ essential for catalytic activity.

A Yeast MRE3/REC114 Gene is Essential for Normal Cell Growth and Meiotic Recombination: Sun-Hee Leem; J. Microbiol. 1999;37(4):248-255.

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Abstract: We have analyzed the MRE3/REC114 gene of Saccharomyces cerevisiae, previously detected in isolation of mutants defective in meiotic recombination. We cloned the MRE3/REC114 gene by complementation of the meiotic recombination defect and it has been mapped to chormosome XIII. The DNA sequence analysis revealed that the MRE3 gene is identical to the REC114 gene. The upstream region of the MRE3/REC114 gene contains a T_4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. Coincidentally, northern blot analysis indicated that the three sizes of MRE3/REC114 transcripts, 3.4, 1.4 and 1.2 kb, are induced in meiosis. A less abundant transcript of 1.4 kb is detected in both mitotic and meiotic cells, suggesting that it is needed in mitosis as well as meiosis. To examine the role of the MRE3/REC114 gene, we constructed mre3 disruption mutants. Strains carrying an insertion or null deletion of the MRE3/REC114 gene showed slow growth in nutrient medium and the doubing time of these cells increased approximately by 2-fond compared to the wild-type strain. Moreover, the deletion mutant (delta-mre3) displayed no meiotically induced recombination and no viable spores. The mre3/rec114 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass reductional division. The double-stranded breaks (DSBs) which are involved in initiation of meiotic recombination were not detected in the analysis of meiotic chromosomal DNA from the mre3/rec114 disruptant. From these results we suggest that the MRE3/REC114 gene product is essential in nomal growth and in early meiotic stages involved in meiotic recombination.

Deletion Analysis of the Major NF-[kappa]B Activation Domain in Latent Membrane Protein 1 of Epstein-Barr Virus: Shin Cho , Won-Keun Lee; J. Microbiol. 1999;37(4):256-262.

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Abstract: Latent membrane protein 1 (LMP1) of the Epstein-Barr virus (EBV) is an integral membrane protein with six transmembrane domains, which is essential for EBV-induced B cell transformation. LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) like membrane receptor, whose signaling requires recruitment of TNFR-associated factors (TRAFs) and leads to NF-[kappa]B activation. NF-[kappa]B activation by LMP1 is critical for B cell transformation and has been linked to many phenotypic changes associated with EBV-induced B cell transformation. Deletion analysis has identified two NF-[kappa]B activation regions in the carboxy terminal cytoplasmic domains of LMP1, termed CTAR1 (residues 194-232) and CTAR2 (351-386). The membrane proximal C-terminal domain was precisely mapped to a PXQXT motif (residues 204-208) involved in TRAF binding as well as NF-[kappa]B activation. In this study, we dissected the CTAR2 region, which is the major NF-[kappa]B signaling effector of LMP1, to determine a minimal functional sequence. A series of LMP1 mutant constructs systematically deleted for the CTAR2 region were prepared, and NF-[kappa]B activation activity of these mutants were assessed by transiently expressing them in 293 cells and Jurkat T cells. The NF-[kappa]B activation domain of CTAR2 appears to reside in a stretch of 6 amino acids (residues 379-384) at the end of the carboxy terminus.

Optimal Protocol for Enumeration of Attached Bacteria on Glass Slides: Hyun Sang Lee , Kae Kyoung Kwon , Jong Ho Lee , Hong Kum Lee; J. Microbiol. 1999;37(4):263-266.

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Abstract: In examining bacterial growth on glass surfaces immersed in sea water, we found serious differences between enumeration methods. Therefore, we compared various methods and found sonication and direct count methods were superior to other methods. Since the direct count method was not suitable for long-term investigation, we chose the sonication method and confirmed that sonication periods 8 times for 30 seconds was optimal for the detachment of bacteria from glass surfaces.

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