Journal of Microbiology

Volume 58(10); October 2020

Journal Articles

[PROTOCOL]A Signature-Tagged Mutagenesis (STM)-based murine-infectivity assay for Cryptococcus neoformans: Kwang-Woo Jung , Kyung-Tae Lee , Yong-Sun Bahn; J. Microbiol. 2020;58(10):823-831. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0341-8

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Abstract: Signature-tagged mutagenesis (STM) is a high-throughput genetic technique that can be used to investigate the function of genes by constructing a large number of mutant strains with unique DNA identification tags, pooling them, and screening them for a particular phenotypic trait. STM was first designed for the identification of genes that contribute to the virulence or infectivity of a pathogen in its host. Recently, this
method
has also been applied for the identification of mutants with specific phenotypes, such as antifungal drug resistance and proliferation. In the present study, we describe an STM
method
for the identification of genes contributing to the infectivity of Cryptococcus neoformans using a mutant library, in which each strain was tagged with a unique DNA sequence.

Paenibacillus lycopersici sp. nov. and Paenibacillus rhizovicinus sp. nov., isolated from the rhizosphere of tomato (Solanum lycopersicum): Shin Ae Lee , Tae-Wan Kim , Mee-Kyung Sang , Jaekyeong Song , Soon-Wo Kwon , Hang-Yeon Weon; J. Microbiol. 2020;58(10):832-840. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0258-2

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Abstract: Two Gram-stain-positive, rod-shaped, endospore-forming bacteria, designated 12200R-189T and 14171R-81T were isolated from the rhizosphere of tomato plants. The 16S rRNA gene sequence similarity between strains 12200R-189T and 14171R-81T were 97.2%. Both strains showed the highest 16S rRNA gene sequence similarities to Paenibacillus sacheonensis SY01T (96.3% and 98.0%, respectively). The genome of strain 12200R-189T was approximately 6.7 Mb in size with 5,750 protein-coding genes (CDSs) and the G + C content was 58.1 mol%, whereas that of strain 14171R-81T comprised one chromosome of 7.0 Mb and two plasmids (0.2 Mb each) with 6,595 CDSs and the G + C content was 54.5 mol%. Comparative genome analysis revealed that average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among 12200R-189T, 14171R-81T, and other closely related species were below the cut-off levels 95% and 70%, respectively. Strain 12200R-189T grew at a temperature range of 15–40°C, pH 6.0–9.0, and 0–3% NaCl (w/v), whereas strain 14171R-81T grew at a temperature range of 10–37°C, pH 6.0– 8.0, and 0–1% NaCl (w/v). Menaquinone-7 (MK-7) was the only isoprenoid quinone detected in both strains. The predominant cellular fatty acids (> 10%) were iso-C15:0, anteiso- C15:0, and iso-C16:0. The polar lipids of strain 12200R- 189T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), aminophospholipid (APL), phospholipid (PL), phosphatidylglycolipid (PGL), and four aminophosphoglycolipids (APGLs) and those of strain 14171R-81T were DPG, PG, PE, APL, three PLs, two PGLs, and three APGLs. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic analyses, strains 12200R- 189T and 14171R-81T represent two novel species of the genus Paenibacillus, for which the names Paenibacillus lycopersici sp. nov. and Paenibacillus rhizovicinus sp. nov. are proposed. The type strains are 12200R-189T (= KACC 19916T = CCTCC AB 2020027T) and 14171R-81T (= KACC 19915T = CCTCC AB 2020026T).

Fungal diversity in deep-sea sediments from Magellan seamounts environment of the western Pacific revealed by high-throughput Illumina sequencing: Shuai Yang , Wei Xu , Yuanhao Gao , Xiaoyao Chen , Zhu-Hua Luo; J. Microbiol. 2020;58(10):841-852. Published online September 2, 2020; DOI: https://doi.org/10.1007/s12275-020-0198-x

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Abstract: There are lots of seamounts globally whose primary production is disproportionally greater than the surrounding areas. Compared to other deep-sea environments, however, the seamounts environment is relatively less explored for fungal diversity. In the present study, we explored the fungal community structure in deep-sea sediments from four different stations of the Magellan seamounts environment by using high-throughput sequencing of the ITS1 region. A total of 1,897,618 ITS1 sequences were obtained. Among these sequences, fungal ITS1 sequences could be clustered into 1,662 OTUs. The majority of these sequences belonged to Ascomycota. In the genera level, the most abundant genus was Mortierella (4.79%), which was reported as a common fungal genus in soil and marine sediments, followed by Umbelopsis (3.80%), Cladosporium (2.98%), Saccharomycopsis (2.53%), Aspergillus (2.42%), Hortaea (2.36%), Saitozyma (2.20%), Trichoderma (2.12%), Penicillium (2.11%), Russula (1.86%), and Verticillium (1.40%). Most of these recovered genera belong to Ascomycota. The Bray-Curtis analysis showed that there was 37 to 85% dissimilarity of fungal communities between each two sediment samples. The Principal coordinates analysis clearly showed variations in the fungal community among different sediment samples. These results suggested that there was a difference in fungal community structures not only among four different sampling stations but also for different layers at the same station. The depth and geographical distance significantly affect the fungal community, and the effect of depth and geographical distance on the structure of the fungal community in the Magellan seamounts is basically same. Most of the fungi were more or less related to plants, these plant parasitic/symbiotic/endophytic fungi constitute a unique type of seamounts environmental fungal ecology, different from other marine ecosystems.

Roles of Dhh1 RNA helicase in yeast filamentous growth: Analysis of N-terminal phosphorylation residues and ATPase domains: Eunji Lee , Daehee Jung , Jinmi Kim; J. Microbiol. 2020;58(10):853-858. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0431-7

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Abstract: In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1- mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEADbox RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous- inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.

Intervention with kimchi microbial community ameliorates obesity by regulating gut microbiota: Seong-Eun Park , Sun Jae Kwon , Kwang-Moon Cho , Seung-Ho Seo , Eun-Ju Kim , Tatsuya Unno , So-Hyeon Bok , Dae-Hun Park , Hong-Seok Son; J. Microbiol. 2020;58(10):859-867. Published online September 2, 2020; DOI: https://doi.org/10.1007/s12275-020-0266-2

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Abstract: The objective of this study was to evaluate anti-obesity effects of kimchi microbial community (KMC) on obesity and gut microbiota using a high fat diet-induced mouse model compared to effects of a single strain. Administration of KMC decreased body weight, adipose tissue, and liver weight gains. Relative content of Muribaculaceae in the gut of the KMCtreated group was higher than that in the high-fat diet (HFD) group whereas relative contents of Akkermansiaceae, Coriobacteriaceae, and Erysipelotrichaceae were lower in KMCtreated group. Metabolic profile of blood was found to change differently according to the administration of KMC and a single strain of Lactobacillus plantarum. Serum metabolites significantly increased in the HFD group but decreased in the KMC-treated group included arachidic acid, stearic acid, fumaric acid, and glucose, suggesting that the administration of KMC could influence energy metabolism. The main genus in KMC was not detected in guts of mice in KMC-treated group. Since the use of KMC has advantages in terms of safety, it has potential to improve gut microbial community for obese people.

Stenotrophomonas maltophilia outer membrane protein A induces epithelial cell apoptosis via mitochondrial pathways: Xin Wang , Yan Li , Xueping Tang , Xueyi Shang , Zunquan Zhao , Yongqiang Jiang , Yan Li; J. Microbiol. 2020;58(10):868-877. Published online September 2, 2020; DOI: https://doi.org/10.1007/s12275-020-0235-9

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Abstract: Stenotrophomonas maltophilia (S. maltophilia) is a common opportunistic pathogen in intensive care units and causes infections most often after surgeries in immune-compromised patients such as those undergoing chemotherapy. Outer membrane protein A (OmpA) is the most abundant of the outer membrane proteins in S. maltophilia. Previous studies on OmpA usually focus on its interaction with the host cells and its role in vaccine development. However, the impact of OmpA on the virulence of S. maltophilia to host cells and the effects on apoptosis remain unclear. In this study, we exposed purified recombinant S. maltophilia OmpA (rOmpA) to HEp-2 cells and investigated the effects of OmpA on epithelial cell apoptosis. Morphologic and flow cytometric analyses revealed that HEp-2 cells stimulated with rOmpA multiple apoptosis features, including nuclear roundness and pyknosis, chromatin aggregation, and phosphatidylserine eversion. We found that rOmpA regulated the protein levels of Bax and Bcl-xL in HEp-2 cells, leading to changes in mitochondria permeability and the release of cytochrome c and apoptosis-inducing factors into the cytoplasm. These subsequently activate the caspase-9/caspase-3 pathway that promote apoptosis. We also observed that rOmpA enhanced the generation of reactive oxygen species and increased intracellular Ca2+ levels in HEp-2 cells. Collectively, our data suggested that rOmpA induced epithelial cells apoptosis via mitochondrial pathways.

Anti-inflammatory and anti-oxidative effect of Korean propolis on Helicobacter pylori-induced gastric damage in vitro: Moon-Young Song , Da-Young Lee , Eun-Hee Kim; J. Microbiol. 2020;58(10):878-885. Published online September 2, 2020; DOI: https://doi.org/10.1007/s12275-020-0277-z

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Abstract: Helicobacter pylori, present in the stomach lining, is a Gramnegative bacterium that causes various gastrointestinal diseases, including gastritis and peptic ulcers. Propolis is a natural resinous substance collected from a variety of plants, and contains several natural bioactive substances. The aim of this study was to investigate the anti-inflammatory and antioxidative effects of Korean propolis on H. pylori-induced damage in the human adenocarcinoma gastric cell line. The propolis used in this study was obtained from the Korea Beekeeping Association in South Korea. The expression of pro-inflammatory interleukins (ILs), such as IL-8, IL-12, IL-1β, tumor necrosis factor alpha, cyclooxygenase-2, and inducible nitric oxide synthase, which was increased after H. pylori infection, significantly decreased in a dose-dependent manner upon pretreatment with Korean propolis, because of the suppression of mitogen-activated protein kinases and nuclear factor κB pathway. The anti-oxidative activity of propolis was assessed using the 2,2-diphenyl-1-picrylhydrazyl hydrate free radical assay. Korean propolis showed significant anti-oxidative effects via reactive oxygen species scavenging. In addition, pretreatment with Korean propolis upregulated the expression of anti-oxidant enzymes through Nrf2 signaling activation. These findings indicate that the use of Korean propolis, which has anti-inflammatory and anti-oxidative effects, can be promising for the prevention of H. pylori-induced gastric damage.

Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity: Young-Il Kim , Mark Anthony B. Casel , Se-Mi Kim , Seong-Gyu Kim , Su-Jin Park , Eun-Ha Kim , Hye Won Jeong , Haryoung Poo , Young Ki Choi; J. Microbiol. 2020;58(10):886-891. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0335-6

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Abstract: Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The
results
substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful
method
for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

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