Journal of Microbiology

Volume 36(4); December 1998

Mechanism of Transcriptional Activation of the Phosphate Regulon in Escherichi coli: Kozo Makino , Mitsuko Amemura , Soo-Ki Kim , Katsushi Yokoyama , Sigenobu Kimura; J. Microbiol. 1998;36(4):231-238.

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Abstract: In Escherichia coli, at least 31 genes, which are involved in the roles related to the transport and assimilation of phosphate and phosphorus compounds, are induced by phosphate starvation. They constitute a single phosphate (pho) regulon, and are under the same physiological and genetic control (30, 36, 46). Proteins PhoB and PhoR, which are regulatory systems for the transcriptional regulation of the phogenes, belong to a large family of two-component regulatory systems that respond to a variety of environmental stimuli in bacteria (23, 24, 33, 39). PhoB is the transcriptional activator, which binds to the promoters of the pho genes (21, 22). PhoR is a transmembrane protein that modulates the activity of PhoB by promoting specific phosphorylation and dephosphorylation of PhoB in response to the phosphate signal in the medium (19, 21, 37, 50). The phosphorylation of PhoB protein occurs concurrently with the acquisition of the ability to activate transcription from the pho promoters (Fig. 1). In the absence of the PhoR functions, PhoB is phosphorylated independently of the phosphate levels by PhoM, a PhoR like protein (2, 3, 26), which was renamed CreC by Wanner (45). In this article, we describe our recent studies on the mechanism of the transcriptional regulation of the pho regulon.

Assessing Survival and Detection of Catechol-bidegrading Strains in Waste Water microcosms: Soo-Jin Choi , Min-Sup Song , Soo-youn Lee , Seong-Karp Hong , Kyung-Hee Min , Jong-Ok Ka , Chi-Kyung Kim , Young-Keun Park; J. Microbiol. 1998;36(4):239-248.

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Abstract: Catechol-degrading bacteria, Escherichia coli JM101 carrying pIB1343 (strain pIB1343) and Flavimonas orizihabitans KHi (strain KH1), were chosen as model bacteria to estimate the sur vival and catechol-degradability in waste water microcosms. Before their application to ecosystem, survival and catechol degradation of these bacteria were investigated in waste water microcosms. It was found that strain pIB1343 adapted much faster to waste water environments and degraded catechol in the case of Namdong samples, whereas the strain KH1 degraded catechol much faster in Sihwa samples. When catechol was added to the microcosms, indigenous microorganisms in Sihwa samples used catechol as a carbon and energy source much better than those in Namdong samples. A modified filter extraction technique was used to obtain the high-yield purified DNA from 50 ml of waste water samples and the extracted DNA (polymorphic DNA [20~23 kb]) was of sufficient quantity and quality for the amplification. PCR was performed with catA-specicic promers, C120U and C120L, which specifically detected the catA gene encoding catechol 1,2-dioxygenase in waste water microcosms, and then the 320 bp PCR products were amplified. PCR products were quantified by densitomenter. Using the standard curve of detection limit by catA-specific PCR products, the number of catA genes for their corresponding intensities of PCR products was obtained. the number of total catA genes PCR in waste water microcosms was correalated with catechol degradation.

Distribution of Genes Coding for Aminoglycoside Avetyltransferases in gentamicin resistant Bacteria Isolated from Aquatic Environment: Lee, Young Jong , Han, Hyo Shim , Seong, Chi Nam , Lee, Hyo Yeon , Jung, Jae Sung; J. Microbiol. 1998;36(4):249-255.

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Abstract: The molecular epidemiology of the aacC1, aacC2, aacC3, and aacC4 genes encoding aminoglycoside acetyltransferases AAC(3)-I, AAC(3)-II, AAC(3)-III and AAC(3)-IV, respectively, was studied by KNA hybridization and polymerase chain reaction (PCR). One hundred twenty six moderate-level (MICs>20 ㎍/ml) and 153 high-level (MICs>2,000 ㎍/ml) gentamicin resistant strains were collected from stream water and hospital sewage. Resistant strains were screened by dot-blot hybridization for the presence of the gene encoding aminoglycoside-(3)-N-acetyl-transferase II (aacC2). The aacC2 probe hybridized with 5.4%(3/56) of the moderate-level resistant strains from stream water and 18.6% (13/70) of the strains from hospital sewage, and with 87.6% (134/153) of the high-level resistant bacteria. The presence of the genes for AAC(3)-I,-III and-IV in the strains was studied by polymerase chain reaction. The most common gene among the moderate-level resistant stratins was aacC4, in 8.9% (5/56) of the stream water strains and in 24.3%(17/70) of the hospital sewage strains, and followed by aacC3 gene, in 5.4% (3/56) and in 20.0% (14/70) of the tested strains, respectibely. None of the 126 isolates with moderate-level resistance amplified the expected DNA fragment of aacC1 gene. In contrast, 11.8% (18/153) of the high-level resistant isolates contained aacC1 gene. The frequency of aacC3 gene in the high-level resistant strains was 19.6% (30/153) while that of aacC4 was 11.1% (17/153).

Isolation and Characterization of 2,4-Dichlorophenoxyacetic Acid-degrading Bacteria from Paddy Soils: Chung, Min Jae , Ka, Jong Ok; J. Microbiol. 1998;36(4):256-261.

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Abstract: Nine numerically dominant 2,4-dichlorophenocyacetic acid (2,4-D)-degrading bacteria were isolated from rice field soils. Most of the isolates were identified as Brukholderia of Sphingomonas species by fatty acid methyl ester (FAME) analysis, and they exhibited diverse chromosomal DNA patterns in polymerase chain reaction (PCR)amplification of repetitive extragenic palindromic (REP) sequences. The isolates utilized 2,4-D as the sole source of carbon and two Sphingomonas species were capable of mineralizing both 3-chlorobenzoate (3-CB) and 4-chlorobenzoate (4-CB), in addition to 2,4-D. Plasmid DNAs were detected from all of the isolates, and conjugation analysis revealed that 2,4-D degradative genes were located on transferable plasmids in most of the isolates. PCR analysis with specific primers selected from tfd genes showed that 67% of the isolates had DNA sequences homologous to the five tfd genes of the 2,4-D degradative plasmid pJP4 of Alcaligenes eutrophus JMP134. Among the isolates, strain TFD7 appeared to be a new genotype in that it contained a transmissible 2,4-D degradative plasmid nonhomologous to the tfd genes. 2,4-D was persistent in natural paddy soils which contained no indigenous 2,4-D-degrading microorganisms, but the application of the 2,4-D-degrading isolates resulted in rapid decline of the soil 2,4-D residues.

Characterization and Identification of the Bacteriophage P4 Mutant Suppressin sir Mutations of Bacteriophage P2: Kim, Kyoung Jin , Sunshine, Melvin G. , Six, Erich W.; J. Microbiol. 1998;36(4):262-265.

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Abstract: Bacteriophage P4 ost1 was isolated as a suppressor mutant of P2 sir3 and identified by restriction enzyme site analysis. The mutant DNA turned out to be an imperfect P4 trimer containing deletions. It was suggested that the deletion resulted from int-mediated site-specific recombination. CsCl equilibrium density gradient experiment confirmed the genome size of P4 ostl.

Secondary Structure Analysis of Amino Terminal Domain in Phage Lambda Integrase: Yu, Jeong A , Nam, Chan Eun , Cho, Eun Hee; J. Microbiol. 1998;36(4):266-272.

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Abstract: The amino-terminal domain of bacteriophage λ integrase recognizes specific DNA sequences called arm-type sites. To study the structural and functional relationships of the integras armtype DNA binding domains were confirmed by gel mobility-shift assay. The polypeptides were subjected to circular dichroism spectroscopy to estimate the amount of secondary structures they contain Based upon analyses of circular dichroism spectra and comparison with predicted secondary structural compositions, it was estimated that the amino terminal domain of integrase in an aqueous solution was composed of a little α-helical region. The helical content increased with an increasing amount of ethanol, an α-helix inducer. This indicates that its conformation can be changed to a form with higher content of α-helical structure under a certain condition.

Production of Stress-shock Proteins in Pseudomonas sp. DJ-12 Treated with 4-Hydroxybenzoate: Park, Sang Ho , Oh, Kye Heon , Lee, Kil Jae , Kim, Chi Kyung; J. Microbiol. 1998;36(4):273-279.

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Abstract: Pseudomonas sp. DJ-12 can grow on 4-hydroxybenzoate (4HBA) at concentration of 5 mM or lower by degrading 4HBA for carbon and energy sources. The organisms were found to produce DnaK stress-shock protein when treated with several aromatic hydrocarbons including 4HBA. Those cells treated with 5 mM 4HBA exhibited increased tolerance to 10 mM concentration. In this study, the production of other stress-shock kproteins besides KnaK was examined in Pseudomonas sp. DJ-12 exposed to various concentrations of 4HBA, compraing the production of the proteins with their survival and degradation of 4HBA. The organisms could degrade 4HBA at 0.5 to 5 mM concentrations after 60 to 90 minutes of incubation. The survival rate of the organism decreased when treated with 4HBA at 10 mM or higher concentrations. The stress-shock proteins of DnaK, GroEL, and GroES were produced in the cells which were treated with 4HBA at 0.5 mM or higher concentrations for 10 minutes. Fifteen additional stress-shock proteins were produced in the cells which were treated with 5 mM 4HBA for 40 minutes. The DnaK and GroEL proteins in the cells gradually decreased upto 6 hours after the stress was removed from the culture.

Purification of an Immunosuppressor Produced by Penicillium urticae: Lee, Jae Jung , Yoo, Seoung Ku , Kim, Eui Joong , Yu, Ju Hyun , Bai, Dong Hoon , Yoon, Sung Sik , Kim, Wook Sung; J. Microbiol. 1998;36(4):280-282.

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Abstract: An immunosuppressive chemical produced by Penicilium urticae (KFCC 11611) was purified from the supernatant of a culture broth by ethyl acetate extraction, silica gel column chromatography, and precipitation by adding cold n-hexane followed by crystalization. The chemical effectively inhibited the mouse mixed lymphocyte reaction (MLR) at a concentration of 1.25ⅹ10^3 ㎍/ml, which was lower than the effective concentration of cyclosporin A. The chemical was identified as the antibiotic patulin (MW 154.1 Da) by NMR and mass spectroscopy.

Expression of Human Protease Inhibitor Nexin-I in Escherichia coli: Ha, Sang Deuk , Kim, Ji Ha , Park, Hey Lyoun , Hyun, Hyung Hwan , Chung, Hyung Min , Kim Kwon, Yun Hee , Seo, Seong Yum , Ko, Jung Jae , Lee, Hyun Hwan; J. Microbiol. 1998;36(4):283-288.

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Abstract: Human protease inhibitor nexin-I(NX-I) cDNA(1.2Kb) was isolated from human lung cDNA library and expressed under the control of T7 promoter as a fused protein in Escherichia coli BL21 and E. coli GJ1158 by addition of IPTG and Nacl as inducers. For GJ1158, 300 mM NaCl was added for induction after the cell reached A_600=0.6. As a result, E. coli GJ1158 showed higher expression level than BL2l with lesser extent of inclusion bodies. The optimum concentration of NaCl exerting no induction effect but shortening the time to reach A_600=0.6 was 50 mM. All the results suggested that E. coli GJ1158 was a useful host for efficient expression of NX-I using NaCl as an inducer. The expressed NX-1 showed an inhibitory effect on thrombin activity. The expressed protein was purified by immobilized metal affinity column chromatography (IMAC) and characterized by digestion with enterokinase (EK).

Overexpression of Insecticidal Protein Gene of Bacillus thuringiensis var. kurstaki HD1: Hwang, Sung Hei , Yoo, Kwan Hee , Moon, Eui Sik , Cha Soung Chul , Lee, Hyung Hoan; J. Microbiol. 1998;36(4):289-295.

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Abstract: The Insecticidal protein (ICP) gene from Bacillus thuringiensis var, kurstaki HD-1 was cloned in pBluescript SK(+) vector and charicterized by overexpression in Escherichia coli XL1-blue. Total plasmids in the B. thuringiensis were isolated and digested with restriction enzyme BamHI. Then, southern blot was performed with a probe to locate the gene in the fragments. The hybridized 3.8 kb NdeI DNA fragment was cloned into the SmaI site of pBluescript SK(+) and named pHLN1-80 in forward orientation to the lacZ gene promoter and pHLN2-80 in reverse orientation to the lacZ gene promoter. Determination of 153 bp nucleotide sequence of 5'-end of the NdeI fragment in the pHLN1-80 clone revealed that there are-80 bp region of the ICP gene promoter and +73 bp region of the ICP gene at the 5'end of the ICP gene. In addition, the-80 bp promoter of the ICP gene contained transcription initiation point G at-77 bp point and BtI promoter and Shine-Dalgarno sequence at-14 to-4 bp region. The two clones showed strong insecticidal activity against 3rd the instar Bompyx mori larvae. SDS-PAGE analysis revealed that the pHLN2-80 clone clearly produces distinguishable amount (27 times more) of the 130 kDa ICP band and 100 times the insecticidal activity than that of the clone pHLN1-80. These marked differences in production and toxicity due to different orientations of the gene in the vectior provide us valuable points for further study on the ICP gene transcription at the molecular level.

Isolation and Characterization of the New Conditional-lethal Mutations in byr4 of Schizosaccharomyces pombe by in vitro Mutagenesis: Song, Ki Won , Shin, Se Jong , Albright, Charles F.; J. Microbiol. 1998;36(4):296-302.

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Abstract: Coordination of nuclear division, cytokinesis, and septation is essential for maintaining the genomic stability during the cell division cycle. byr4 in fission yeas schizosaccharomyced pombe encodes an essential gene that regulates the timing of cytokinesis and septation in a dosage-de-pendent manner (Song et al., 1996). The knock-out of byr4 causes cell cycle arrest in late mitosis with multiple cytokinesis and septation, while byr4 is an essential gene, characterization of the byr4 null phenotypes and inbestigation of its genetic interactions with other mutants entail technical limitations. To better characterize the functional mechanisms of byr4 through phenotypic and genetic analyses, we generated five temperature-sensitive byr4 mutant alleles. A truncated byr4 with a deletion corresponding to the N-terminal 29 amino acids was randomly mutagenized by hydrocylamine in vitro. The mutagenized byr4 with an N-treminal truncation was integrated into the byr4 locus of S. pombe genome. Cells that formed colonies at the permissive temperature, 25℃, but could not grow at the restrictive temperatures, 18℃ or 35℃, were isolated. We successfully isolated five temperature-sensitive byr4 alleles (KSY1-5) that could not grow at 35℃. In the restrictive temperature, KSY1, KSY3, and KSY5 alleles arrested cells with multiple septation while chromosome segregation was normal in these alleles. KSY2 and KSY4 alleles exhibited two phenotypes at the restrictive temperature: cells were arrested with multiple nuclei due to the inhibition of cytokinesis or with multiple nuclei that were separated by septum. These newly isolated byr4 conditional alleles will be useful for the deduction of cellular processes where byr4 functions. Genetic studies and suppressor screens of the conditional alleles can provide useful tools for the isolation of interacting proteins with Byr4p.

Human Antibody Responses to Capsular Polysaccharides of Streptococcus pneumoniae 6B, 14, and 19F: Kim, Ji Hye , Kim, Kyung Hyo , Kim, Jung Soo , Song, Jae Ho , Park, Moon Kook; J. Microbiol. 1998;36(4):303-307.

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Abstract: Human antibody responses to Streptococcus pneumoniae 6B, 14, and 19F capsular polysaccharide were analyzed. Thirty-one healthy young adults were immunized with the pneumococcal 23-valent PS vaccine. serum samples were obtained from them before and 1 month after vaccination. The amounts of total antibody, heavy chain and light chain isotypes were determined by enzyme-linked immunosorbent assay (ELISA). Vaccination increased the total lebvels of anti6B, anti-14, and anti-19F PS antibodies by 3.4-fold, 3.8-fold and 4.1-fold, respectively. Some inantibody was predominant in the responses to the three PSs, and most of the IgG anti-PS antibodies were IgG2 isotype. There was no significant difference in the k and λresponses.

Studies on the Inhibition of HIV Replication with a Number of RRE Decoy Derivatives: Lee , Seong Wook; J. Microbiol. 1998;36(4):308-315.

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Abstract: RRE decoys are short RNA oligonucleotides corresponding to the HIV Rev response element (RRE) sequence, which protect cells from HIV replication by inhibiting the binding of the HIV regulatory protein Rev to the authentic HIV RRE region. Previously minimal RRE decoy containing the 13-nucleotide primary Rev binding domain of RRE was described to be a potent inhibitor of HIV in CEM cells. In this report, we analyzed and compared the ability of a series of RRE decoy derivatioves to inhibit HIV replication in CEM cells to develop increasingly effective RRE decoy. Using an improved tRNA cassette to express high level of RRE transcripts in cells, we found that a variant form of stem-loop II(SLII) binding domain of wild type RRE termed RRE40 was more potent than any other RRE decoys previously developed or tested here and protected all cells most effectively from HIV. RRE40 was previously selected in vitro which binds to Rev protein 10-fold better than wild type RRE. CEM cells expressing RRE40 decoy RNAE40 decoys inhibit HIV specifically by sequestering Rev binding to the authentic RRE target in HIV RNA and indicated that RRE40 RNA identified by using in vitro binding studies also binds Rev in cells. These obserbations have important implications for experiments involving optimization of clinical application of RNA decoy based gene therapy protocol against HIV.

Isolation and Characterization of Fatty Acid Derivatives from an Actinomycetes and Examination of the Effects on Activities of Phospholipase C and Protein Kinase C: Ko, Hack Ryong , Kim, Bo Yeon , Lee, Hyun Sun , Kang, Dae Ook , Ryu, Sung Ho , Suh, Pann Ghill , Mheen, Tae Ick , Ahnm Jong Seog; J. Microbiol. 1998;36(4):316-321.

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Abstract: In our screening to search inhibitors of phosphoinositide(PI)-specific phospholipase C (PI-PLC), two inhibitors, MT965-A and-B were isolated from a culture broth of an actinomycetes. MT965-A and-B were identified as fatty acid deribatives, 14-methylpentadecanoic acid and 16-methyllinoleic acid methyl ester, respectively, based on the spectral data including NMR and MS. Both inhibitors directly inhibited not only in vitro PLCγ1 activity but also the platelet-derived growth factor(PDGF)-induced inositol phosphates(IPt) formation in NIH 3T3γ1 cells ocerexpressing PLCγ1. However, the inhibitors enhanced in vitro protein kinase C (PKC) activity. On examination of the effects of various fatty acids(FAs) on activities of PLC, PKC, and PDGF-induced IPt formation, the unsaturated FAs(UFAs) showed the same activities like the inhibitors, but the saturated FAs(SFAs) did not show similar activities. It was inferred that the chain length, degree of unsaturation, methyl esterification, branching with a methyl group, and cis-configuration were important for their activity.

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