Journal of Microbiology

Journal Article

Biophysical characterization of antibacterial compounds derived from pathogenic fungi Ganoderma boninense: Syahriel Abdullah , Yoon Sin Oh , Min-Kyu Kwak , KhimPhin Chong; J. Microbiol. 2021;59(2):164-174. Published online December 23, 2020; DOI: https://doi.org/10.1007/s12275-021-0551-8

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There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol- water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined
methods
included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, C30H54) and ergosta-5,7,22-trien-3β-ol (m/z = 396.65; ergosterol, C28H44O). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, C30H42O6), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.

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Review

REVIEW] Zika virus: An emerging flavivirus: Sang-Im Yun , Young-Min Lee; J. Microbiol. 2017;55(3):204-219. Published online February 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7063-6

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Abstract

Zika virus (ZIKV) is a previously little-known flavivirus closely related to Japanese encephalitis, West Nile, dengue, and yellow fever viruses, all of which are primarily transmitted by blood-sucking mosquitoes. Since its discovery in Uganda in 1947, ZIKV has continued to expand its geographic range, from equatorial Africa and Asia to the Pacific Islands, then further afield to South and Central America and the Caribbean. Currently, ZIKV is actively circulating not only in much of Latin America and its neighbors but also in parts of the Pacific Islands and Southeast Asia. Although ZIKV infection generally causes only mild symptoms in some infected individuals, it is associated with a range of neuroimmunological disorders, including Guillain-Barré syndrome, meningoencephalitis, and myelitis. Recently, maternal ZIKV infection during pregnancy has been linked to neonatal malformations,
result
ing in various degrees of congenital abnormalities, microcephaly, and even abortion. Despite its emergence as an important public health problem, however, little is known about ZIKV biology, and neither vaccine nor drug is available to control ZIKV infection. This article provides a brief introduction to ZIKV with a major emphasis on its molecular virology, in order to help facilitate the development of diagnostics, therapeutics, and vaccines.

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Journal Article

Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a: Bo-Kyoung Jung , Hye-Ran Kim , Gyu-Nam Park , Guangxiang Luo , Kyung-Soo Chang; J. Microbiol. 2016;54(6):451-458. Published online May 27, 2016; DOI: https://doi.org/10.1007/s12275-016-6099-3

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Abstract

Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype- dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3- specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents.

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The Role of ApoE in HCV Infection and Comorbidity
Yue Gong, Wei Cun
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Research Support, Non-U.S. Gov't

Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication: Young-Eui Kim , Mi Young Park , Kyeong Jin Kang , Tae Hee Han , Chan Hee Lee , Jin-Hyun Ahn; J. Microbiol. 2015;53(8):561-569. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5301-3

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Abstract

The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112- 113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112- 113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112- 113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112- 113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our
results
demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

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Insights into the Transcriptome of Human Cytomegalovirus: A Comprehensive Review
Janine Zeng, Di Cao, Shaomin Yang, Dabbu Kumar Jaijyan, Xiaolian Liu, Songbin Wu, Ruth Cruz-Cosme, Qiyi Tang, Hua Zhu
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The human cytomegalovirus decathlon: Ten critical replication events provide opportunities for restriction
Declan L. Turner, Rommel A. Mathias
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Degradation of SAMHD1 Restriction Factor Through Cullin-Ring E3 Ligase Complexes During Human Cytomegalovirus Infection
Seokhwan Hyeon, Myoung Kyu Lee, Young-Eui Kim, Gwang Myeong Lee, Jin-Hyun Ahn
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Primary lymphocyte infection models for KSHV and its putative tumorigenesis mechanisms in B cell lymphomas
Sangmin Kang, Jinjong Myoung
Journal of Microbiology.2017; 55(5): 319. CrossRef
Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication
Tim Schommartz, Jiajia Tang, Rebekka Brost, Wolfram Brune, Klaus Frueh
Journal of Virology.2017;[Epub] CrossRef

Journal Article

RNA Interference Targeting Nucleocapsid Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication in Marc-145 Cells: Minnan Yang , Qun Xiang , Xiaodong Zhang , Xiang Li , Seydou Sylla , Zhuang Ding; J. Microbiol. 2014;52(4):333-339. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-3419-3

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Abstract

Porcine reproductive and respiratory syndrome (PRRS) is an important disease, which leads to severe economic losses in swine-producing areas of the world. However, current antiviral strategies cannot provide highly effective protection. In this study, three theoretically effective interference target sites (71-91, 144-164, 218-238) targeting the nucleocapsid (N) gene of PRRSV were designed and selected, and then three siRNA-expressing plasmids were constructed, respectively named p2.1-N71, p2.1-N144, and p2.1-N218. The recombinant siRNA-expressing plasmids were transfected into Marc-145 cells; then the cells were infected with PRRSV (JL07SW strain); finally, after incubation for 48 h, the antiviral activity of those siRNA-expressing plasmids in Marc-145 cells was assessed by cytopathic effects, virus titers, indirect immunofluorescence, and quantitative real-time PCR. Experimental results demonstrated that these three siRNA-expressing plasmids could effectively and significantly inhibit the replication of PRRSV by 93.2%, 83.6%, and 89.2% in Marc-145 cells, respectively. Among these three siRNA-expressing plasmids, p2.1-N71 was found to be most effective, while p2.1-N144 and p2.1-N218 displayed relatively weak inhibition of virus replication. The results indicated that siRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibit PRRSV replication in Marc-145 cells. Based on our experimental results and previous reports, the 71-91, 179-197, and 234-252 sites of the N gene are good choices to effectively inhibit the replication of PRRSV, and this RNA interference technique can be a potential anti-PRRSV strategy.

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Inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using DNA-based short antisense oligonucleotides
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Reviews

MINIREVIEW] To Peep into Pif1 Helicase: Multifaceted All the Way from Genome Stability to Repair-Associated DNA Synthesis: Woo-Hyun Chung; J. Microbiol. 2014;52(2):89-98. Published online February 1, 2014; DOI: https://doi.org/10.1007/s12275-014-3524-3

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Abstract

Pif1 DNA helicase is the prototypical member of a 5' to 3' helicase superfamily conserved from bacteria to humans. In Saccharomyces cerevisiae, Pif1 and its homologue Rrm3, localize in both mitochondria and nucleus playing multiple roles in the maintenance of genomic homeostasis. They display relatively weak processivities in vitro, but have largely non-overlapping functions on common genomic loci such as mitochondrial DNA, telomeric ends, and many replication forks especially at hard-to-replicate regions including ribosomal DNA and G-quadruplex structures. Recently, emerging evidence shows that Pif1, but not Rrm3, has a significant new role in repair-associated DNA synthesis with Polδ during homologous recombination stimulating D-loop migration for conservative DNA replication. Comparative genetic and biochemical studies on the structure and function of Pif1 family helicases across different biological systems are further needed to elucidate both diversity and specificity of their mechanisms of action that contribute to genome stability.

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MINIREVIEW] Overview: Replication of Porcine Reproductive and Respiratory Syndrome Virus: Sang-Im Yun , Young-Min Lee; J. Microbiol. 2013;51(6):711-723. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3431-z

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Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNAprocessing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.

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Research Support, Non-U.S. Gov'ts

Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1: Ming Shun Li , Jong Yul Roh , Xueying Tao , Zi Niu Yu , Zi Duo Liu , Qin Liu , Hong Guang Xu , Hee Jin Shim , Yang-Su Kim , Yong Wang , Jae Young Choi , Yeon Ho Je; J. Microbiol. 2009;47(4):466-472. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0020-2

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Abstract: Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8~25.2%) with the Rep protein coded by RCR plasmids, however. The putative double- strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. thuringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Exchange of the VP5 of Infectious Bursal Disease Virus in a Serotype I Strain with that of a Serotype II Strain Reduced the Viral Replication and Cytotoxicity: Liting Qin , Xiaole Qi , Honglei Gao , Yulong Gao , Zhigao Bu , Xiaomei Wang; J. Microbiol. 2009;47(3):344-350. Published online June 26, 2009; DOI: https://doi.org/10.1007/s12275-009-0028-7

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Abstract: Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotypeI and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotypeII group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotypeII reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.

DNA Replication is not Required in Re-establishment of HMRE Silencer Function at the HSP82 Yeast Heat Shock Locus: Lee, See Woo , Gross, David S.; J. Microbiol. 1996;34(1):30-36.

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Abstract: We have examined the re-establishment of HIMRE mediated silencing function on the transcriptional activity of yeast heat shock gene HSP82. To test whether the onset of SIR repression can occur in growing cells in the presence of a potent inhibitor of DNA replication, HMRa/HSP82 strains with SIR4^- and SIR4S^+ genetic backgrounds were arrested in S phase by incubation of a culture in 200 mM hydroxyurea for 120 min. It was clear that following a 20 minute heat shock, silencing of the HMRa/HSP82 allele in cells pretreated with hydroxyurea does occur in a SIR4-dependen fashion, even though the kinetics of repression appears to be substantially delayed. We also have tested whether re- establishment of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with α-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de novo protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 ㎍/㎖) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

Genetic Manipulation of Rhabdoviruses : New Insights to Virus Replication, Transcription and Assembly: Michael A. Whitt; J. Microbiol. 1998;36(1):1-8.

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Abstract: Rhabdoviruses, together with the other members of the Rhabdoviridae family, are one of the most widely distributed groups of viruses in nature. Rhabdoviruses have been isolated from virtually all vertebrates, several different species of insects, as well as many plant (65). It is thought that insects were the original hosts for this group of viruses and that rhabdoviruses have since adapted to grow in both vertebrates and invertebrates. This adaptation undoubtedly contributed to one of the disdinguishing features of the prototypic rhabdovirus, vesicular stomatitis virus (VSV), namely the ability to replicate in most primary cell cultures and essentially all established mammalian cell lines, as well as a number of insect and amphibian cell lines. Because VSV has a broad host range, is relatively easy to grow and replicates to high titers in cell culture it has been used extensively as a model system to study many aspects of rhabdovirus entry (32, 69, 70), replication (3, 4) and assembly(36, 55, 58).

Stable Secretion Vector Derived from the RCR (rolling-circle replication) Plasmid of Bacillus mesentericus: Seung-Soo Lee , Jeong-Sun Han , In Hyung Lee , Young- Yel l Yang , Soon-Kwang Hong , Joo-Won Suh; J. Microbiol. 2002;40(2):140-145.

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Abstract: The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMH1 turned out to have a replication origin and two open reading frames (ORFs) of the putative [gamma]-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMH1, all constructed vectors were stable over 100 generations in a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a novel secretion vector. Using the [alpha]-amylase promoter/signal sequence of B. subtilils, the novel plasmid pJSN was constructed. When [beta]-glucosidase was expressed using pJSN, we found [beta]-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

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