Journal of Microbiology

Volume 38(4); December 2000

The Role of the Hsp100/Clp Family of Proteins in Prokaryotic Development: Peter Zuber; J. Microbiol. 2000;38(4):193-202.

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Abstract: Review

Human Cytomegalovirus Inhibition of Interferon Signal Transduction: Daniel M. Miller , Colleen M. Cebulla , Daniel D. Sedmak; J. Microbiol. 2000;38(4):203-208.

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Abstract: Cytomegalovirus (CMV), a beta-herpesvirus with worldwide distribution, exhibits host persistence, a distinguishing characteristic of all herpesviruses. This persistence is dependent upon restricted gene expression in infected cells as well as the ability of productively infected cells to escape from normal cell-mediated anti-viral immunosurveillance. Type I (IFN-[alpha]/[beta]) and type II (IFN-[gamma]) interferons are major components of the innate defense system against viral infection. They are potent inducers of MHC class I and II antigens and of antigen processing proteins. Additionally, IFNs mediate direct anti-viral effects through induction of effector molecules that block viral infection and replication, such as 2', 5-oligoadenylate synthetase (2, 5-OAS). IFNs function through activation of well-defined signal transduction pathways that involve phosphorylation of constituent proteins and ultimate formation of active transcription factors. Recent studies have shown that a number of diverse viruses, including CMV, EBV, HPV, mumps and Ebola, are capable of inhibiting IFN-mediated signal transduction through a variety of mechanisms. As an example, CMV infection inhibits the ability of infected cells to transcribe HLA class I and II antigens as well as the antiviral effector molecules 2, 5-OAS and MxA I. EMSA studies have shown that IFN-[alpha] and IFN-[gamma] are unable to induce complete signal transduction in the presence of CMV infection, phenomena that are associated with specific decreases in JAK1 and p48. Viral inhibition of IFN signal transduction represents a new mechanistic paradigm for increased viral survival, a paradigm predicting widespread consequences in the case of signal transduction factors common to multiple cytokine pathways.

Enzyme Activities Related to the Methanol Oxidation of Mycobacterium sp. strain JC1 DSM 3803: Youngtae Ro , Eungbin Kim , Youngmin Kim; J. Microbiol. 2000;38(4):209-217.

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Abstract: Mycobacterium sp. strain JC1 DSM 3803 grown in methanol showed no methanol dehydrogenase or oxidase activities found in most methylotrophic bacteria and yeasts, respectively. Even though the methanol-grown cells exhibited a little methanol-dependent oxidation by cytochrome c-dependent methanol dehydrogenase and alcohol dehydrogenase, they were not the key enzymes responsible for the methanol oxidation of the cells, in that the cells contained no c-type cytochrome and the methanol oxidizing activity from the partially purified alcohol dehydrogenase was too low, respectively. In substrate switching experiments, we found that only a catalase-peroxidase among the three types of catalase found in glucose-grown cells was highly expressed in the methanol-grown cells and that its activity was relatively high during the exponential growth phase in Mycobacterium sp. JC1. Therefore, we propose that catalase-peroxidase is an essential enzyme responsible for the methanol metabolism directly or indirectly in Mycobacterium sp. JC1.

Intracellular Posttranslational Modification of Aspartyl Proteinase of Candida albicans and the Role of the Glycan Region of the Enzyme: Byoung-Kuk Na , Chul-Yong Song; J. Microbiol. 2000;38(4):218-223.

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Abstract: Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAP1) of Candida albicans. Three intracellular forms of SAP1 were detected by immunoblotting using monoclonal antibody (MAb) CAP1. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAP1 and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAP1. These results show that SAP1 is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor form undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretory vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAP1 was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in part, be related to enzyme stability and activity.

Purification and Characterization of Chitinase from a Marine Bacterium, Vibrio sp. 98CJ11027: Shin Hye Park , Jung-Hyun Lee , Hong Kum Lee; J. Microbiol. 2000;38(4):224-229.

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Abstract: Chitin-degrading marine bacterial strain 98CJ11027 was isolated from bryozoa from the coastal area of Cheju Island, Korea, and identified as a member of the genus Vibrio. The molecular mass of the main extracellular chitinase (chitinase I), purified from strain 98CJ11027, was estimated to be 98 kDa. The optimal condition for chitinase I activity is pH 6.0 and 45 C. The activity was inhibited by Fe^+2 and Cu^+2. Chitinase I displayed the hydrolysis type of chitobiosidase and catalyzed reversed hydrolysis leading to the synthesis of tetraacetylchitotetraose.

Genomic Organization of Penicillium chrysogenum chs4, a Class III Chitin Synthase Gene: Yoon-Dong Park , Myung-Sook Lee , Ji-Hoon Kim , Jun Namgung , Bum Chan Park , Kyung Sook Bae , Hee-Moon Park; J. Microbiol. 2000;38(4):230-238.

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Abstract: Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5?lanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at ?6 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for post-translational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P. chr ysogenum and genus Aspergillus.

Analysis of the Dual Promoters and the H 2 O 2 -responsive Element of the catA Gene Encoding Catalase A in Streptomyces coelicolor: You-Hee Cho , Ji-Sook Hahn , Jung-Hye Roe; J. Microbiol. 2000;38(4):239-244.

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Abstract: The catA gene encodes the major catalase in Streptomyces coelicolor, whose production increases upon H_2O_2 treatment. Besides the previously identified primary promoter (catAp1), a minor promoter (catAp2) was newly assigned by S1 nuclease mapping. The catAp2 transcript was observed transiently upon entry into the stationary phase in liquid culture and upon differentiation on solid plates, whereas the level of catAp1 transcription did not change significantly during this growth transition. The catAp1 promoter was transcribed by the major vegetative RNA polymerase holoenzyme containing [sigma]^HrdB , whereas the catAp2 was transcribed in vitro by the holoenzyme containing [sigma]^R that is activated under oxidative conditions. The cis-element regulating the H_2 O_2 -inducibility of catAp1 was identified within the 23 bp inverted repeat sequence located between -65 and -43 of the catAp1 promoter. We named this sequence HRE (H_2O_2 -responsive element). The distal half of the inverted repeat was more crucial for H_2 O_2 ?pendent induction of the catAp1 transcript than the proximal half. HRE most likely serves as a binding site for the H_2 O_2 -responsive repressor CatR.

Roles of the meta- and the ortho-Cleavage Pathways for the Efficient Utilization of Aromatic Hydrocarbons by Sphingomonas yanoikuyae B1: Jeongmin Song , Junghee Sung , Young Min Kim , Gerben J. Zylstra , Eungbin Kim; J. Microbiol. 2000;38(4):245-249.

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Abstract: Catabolic pathways for the degradation of various aromatics by Sphingomonas yanoikuyae B1 are intertwined, joining at the level of substituted benzoates, which are further degraded via ring cleavage reactions. The mutant strain EK497, which was constructed by deleting a large DNA region containing most of the genes for biphenyl, naphthalene, m-xylene, and m-toluate degradation, was unable to grow on all of the aromatics tested except for benzoate as the sole source of carbon and energy. S. yanoikuyae EK497 was found to possess only catechol ortho-ring cleavage activity due to deletion of the genes for the meta-cleavage pathway. Wild-type S. yanoikuyae B1 grown on benzoate has both catechol ortho-and meta-cleavage activity. However, m-xylene and m-toluate, which are metabolized through methylbenzoate, and biphenyl, which is metabolized through benzoate, induce only the meta-cleavage pathway, suggesting the presence of a substrate-dependent induction mechanism.

Simultaneous Utilization of Two Different Pathways in Degradation of 2,4,6-Trinitrotoluene by White Rot Fungus Irpex lacteus: Hyoun-Young Kim , Hong-Gyu Song; J. Microbiol. 2000;38(4):250-254.

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Abstract: This study confirmed that white rot fungus Irpex lacteus was able to metabolize 2,4,6-trinitrotoluene (TNT) with two different initial transformations. In one metabolic pathway of TNT a nitro group was removed from the aromatic ring of TNT. Hydride-Meisenheimer complexes of TNT (H^- -TNT), colored dark red, were confirmed as the intermediate in this transformation by comparison with the synthetic compounds. 2,4-Dinitrotoluene as a following metabolic product was detected, and nitrite produced by denitration of H^- -TNT supported this transformation. In the other TNT pathway, nitro groups in TNT were successively reduced to amine groups via hydroxylamines. Hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes were identified as the intermediates. The activity of a membrane-associated aromatic nitroreductase was detected in the cell-free extract of I. lacteus. This enzyme catalyzed the nitro group reduction of TNT with NADPH as a cofactor. Enzyme activity was not observed in the presence of molecular oxygen.

Measurement of Antiviral Activities Using Recombinant Human Cytomegalovirus: Byung-Hak Song , Gyu-Cheol Lee , Chan-Hee Lee; J. Microbiol. 2000;38(4):255-259.

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Abstract: For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/pp28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

Detection of Marine Birnavirus (MBV) from Rockfish Sebastes schlegeli Using Reverse Transcription and Nested PCR: Seong-Joon Joh , Doo-Won Kim , Jeong-Ho Kim , Gang-Joon Heo; J. Microbiol. 2000;38(4):260-264.

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Abstract: Reverse transcription (RT)-PCR and nested PCR methods (2-step PCR) were tested for their ability to detect marine birnavirus (MBV) in cultured rockfish, Sebastes schlegeli. One set of primers for RT-PCR was designed, based on a gene of infectious pancreatic necrosis virus (IPNV), and another set of primers for nested PCR was designed based on the VP2/NS junction region of MBV. This 2-step PCR method was specific for MBV and sensitivity was heightened when nested PCR was combined to RT-PCR. This 2-step PCR method was useful for detecting MBV not only in diseased fish, but also in asymptomatic fish. These results indicate that this 2-step PCR method is useful for detecting MBV in rockfish.

Iron Increases Susceptibilities of Pseudomonas aeruginosa to Ofloxacin by Increasing the Permeability: Sookyoung Kim , Jinsook Kim , Hyeran Nam , Yusun Jung , Yeonhee Lee; J. Microbiol. 2000;38(4):265-269.

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Abstract: Iron increased the susceptibilities of clinical isolates of Pseudomonas aeruginosa to quinolones. In the presence of iron, increased susceptibilities to ofloxacin were observed in twenty-six out of thirty isolates and with no change in four isolates. In the case of norfloxacin, iron increased susceptibilities of twelve isolates but did not render any change in eighteen isolates. In the case of ciprofloxacin, iron decreased the MICs (Minimal Inhibitory Concentration) of twenty isolates, increased the MIC of one isolate, and did not change the MICs of nine isolates. To find out how iron increased susceptibility to ofloxacin, bacterial cells were grown in Muller Hinton (MH) media and succinate minimal media (SMM) to induce iron acquisition systems and the intracellular ofloxacin concentrations were assayed in the presence of iron. The addition of iron to the media decreased the MICs of cells whether they were grown in MH or SMM. Siderophores, carbonyl cyanide m-chlorophenylhydrazone (an inhibitor of proton motive force), and ouabain (an inhibitor of ATPase) did not decrease the effect of iron. Results suggested that the increase in the intracellular ofloxacin concentration by iron is accomplished not by decreasing the efflux but by increasing the ofloxacin permeability.

Penetration of HEp-2 and Chinese Hamster Ovary Epithelial Cells by Escherichia coli Harbouring the Invasion-Conferring Genomic Region from Salmonella typhimurium: Jeong Uck Park , Sang-Gu Hwang , Ja-Young Moon , Yong-Kweon Cho , Dong Wan Kim , Yong Kee Jeong , andKwang-Ho Rhee; J. Microbiol. 2000;38(4):270-274.

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Abstract: Pathogenic Salmonella typhimurium can invade the intestinal epithelium and cause a wide range of diseases including gastroenteritis and bacteremia in human and animals. To identify the genes involved in the infection, the invasion determinant was obtained from S. typhimurium 82/6915 and was subcloned into pGEM-7Z. A subclone DH1 (pSV6235) invaded HEp-2 and Chinese hamster ovary epithelial cells and contained a 4.4 kb fragment of S. typhimurium genomic region. Compared with the host strain E. coli DH1, the subclone DH1 (pSV6235) invaded cultured HEp-2 and Chinese hamster ovary cells at least 75- and 68-fold higher, respectively. The invasion rate of E. coli DH1 for the cells significantly increased by harbouring the genomic region derived from pathogenic S. typhimurium 82/6915.

Cloning and Sequence Analysis of the xylL Gene Responsible for 4CBA-Dihydrodiol Dehydrogenase from Pseudomonas sp. S-47: Dong-Woo Park , Youngsoo Kim , Sang-Mahn Lee , Jong-Ok Ka , Chi-Kyung Kim; J. Microbiol. 2000;38(4):275-280.

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Abstract: Pseudomonas sp. S-47 is capable of catabolizing 4-chlorobenzoate (4CBA) as carbon and energy sources under aerobic conditions via the meta-cleavage pathway. 4CBA-dioxygenase and 4CBA-dihydrodiol dehydrogenase (4CBA-DD) catalyzed the degradation of 4CBA to produce 4-chlorocatechol in the pathway. In this study, the xylL gene encoding 4CBA-DD was cloned from the chromosomal DNA of Pseudomonas sp. S-47 and its nucleotide sequence was analyzed. The xylL gene was found to be composed of 777 nucleotide pairs and to encode a polypeptide of 28 kDa with 258 amino acid residues. The deduced amino acid sequence of the dehydrogenase (XylL) from strain S-47 exhibited 98% and 60% homologies with those of the corresponding enzymes, Pseudomonas putida mt-2 (XylL) and Acinetobacter calcoaceticus (BenD), respectively. However, the amino acid sequences show 30% or less homology with those of Pseudomonas putida (BnzE), Pseudomonas putida F1 (TodD), Pseudomonas pseudoalcaligenes KF707 (BphB), and Pseudomonas sp. C18 (NahB). Therefore, the 4CBA-dihydrodiol dehydrogenase of strain S-47 belongs to the group I dehydrogenase involved in the degradation of mono-aryls with a carboxyl group

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