Journal of Microbiology

Research Article

Efficiency of reverse genetics methods for rescuing severe acute respiratory syndrome coronavirus 2: Chang-Joo Park, Taehun Kim, Seung-Min Yoo, Myung-Shin Lee, Nam-Hyuk Cho, Changhoon Park; J. Microbiol. 2025;63(2):e2411023. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2411023

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Abstract PDF: Bacteria-free reverse genetics techniques are crucial for the efficient generation of recombinant viruses, bypassing the need for labor-intensive bacterial cloning. These methods are particularly relevant for studying the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This study compared the efficiency of three bacteria-free approaches—circular polymerase extension reaction (CPER) with and without nick sealing and infectious sub-genomic amplicons (ISA)—to bacterial artificial chromosome (BAC)-based technology for rescuing SARS-CoV-2. Significant differences in viral titers following transfection were observed between methods. CPER with nick sealing generated virus titers comparable to those of the BAC-based method and 10 times higher than those of the standard CPER. In contrast, ISA demonstrated extremely low efficiency, as cytopathic effects were detected only after two passages. All rescued viruses exhibited replication kinetics consistent with those of the original strain, with no significant deviation in replication capacity. Furthermore, the utility of CPER and ISA in genetically modifying SARS-CoV-2 was demonstrated by successfully inserting the gene encoding green fluorescent protein into the genome. Overall, this study underscores the potential of bacteria-free methods, such as CPER and ISA, in advancing SARS-CoV-2 research while highlighting their significant differences in efficiency.

Retracted Publication

Cryptic prophages in a blaNDM-1-bearing plasmid increase bacterial survival against high NaCl concentration, high and low temperatures, and oxidative and immunological stressors: So Yeon Kim , Kwan Soo Ko; J. Microbiol. 2020;58(6):483-488. Published online March 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9605-6

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In this study, we investigated the effect of cryptic prophage regions in a blaNDM-1-bearing plasmid, which was identified in a patient from South Korea, on the survival of bacteria against adverse environmental conditions. First, we conjugated the intact plasmid and plasmids with deleted cryptic prophages into Escherichia coli DH5α. The E. coli transconjugants carrying the plasmid with intact cryptic prophages showed increased survival during treatment with a high concentration of NaCl, high and low temperatures, an oxidative stressor (H2O2), and an immunological stressor (human serum). By contrast, the transconjugants carrying the plasmid with a single-cryptic prophage knockout did not show any change in survival rates. mRNA expression analyses revealed that the genes encoding sigma factor proteins were highly upregulated by the tested stressors and affected the expression of various proteins (antioxidant, cell osmosis-related, heat shock, cold shock, and universal stress proteins) associated with the specific defense against each stress. These findings indicate that a bacterial strain carrying a plasmid with intact carbapenemase gene and cryptic prophage regions exhibited an increased resistance against simulated environmental stresses, and cryptic prophages in the plasmid might contribute to this enhanced stress resistance. Our study indicated that the coselection of antibiotic resistance and resistance to other stresses may help bacteria to increase survival rates against adverse environments and disseminate.

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Qingxia Zhang, Lei Zhou, Yilong Zhao, Shuhong Gao, Yanjun Yang, Qingyun Chen, Wenhui Li, Qi Qi, Qiang Dong, Jiesi Lei, Xue Guo, Qun Gao, Yunfeng Yang
Journal of Hazardous Materials.2023; 457: 131763. CrossRef
Regulator of RNase E activity modulates the pathogenicity of Salmonella Typhimurium
Jaejin Lee, Eunkyoung Shin, Ji-Hyun Yeom, Jaeyoung Park, Sunwoo Kim, Minho Lee, Kangseok Lee
Microbial Pathogenesis.2022; 165: 105460. CrossRef
Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents
Anna-Ursula Happel, Christina Balle, Brandon S. Maust, Iyaloo N. Konstantinus, Katherine Gill, Linda-Gail Bekker, Rémy Froissart, Jo-Ann Passmore, Ulas Karaoz, Arvind Varsani, Heather Jaspan
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Journal Articles

The threonine-tRNA ligase gene region is applicable in classification, typing, and phylogenetic analysis of bifidobacteria: Ji&# , Chahrazed Mekadim , Radko Pechar , V&# , Eva Vlková; J. Microbiol. 2018;56(10):713-721. Published online September 28, 2018; DOI: https://doi.org/10.1007/s12275-018-8167-3

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Abstract

In the modern era, molecular genetic techniques are crucial in ecological studies, as well as in the classification, typing, and phylogenetic analysis of prokaryotes. These techniques are mainly aimed at whole genome comparisons and PCRderived experiments, including amplifying the 16S rRNA and other various housekeeping genes used in taxonomy, as well as MLST (multilocus sequence typing) and MLSA (multilocus sequence analysis) of different taxonomic bacterial groups. The gene encoding threonine-tRNA ligase (thrS) is a gene potentially applicable as an identification and phylogenetic marker in bacteria. It is widely distributed in bacterial genomes and is subject to evolutionary selection pressure due to its important function in protein synthesis. In this study, specific primers were used to amplify a thrS gene fragment (~740 bp) in 36 type and 30 wild strains classified under family Bifidobacteriaceae. The full-length gene has not yet been considered as a possible identification, classification, and phylogenetic marker in bifidobacteria. The thrS sequences revealed higher sequence variability (82.7% of pairwise identities) among members of the family than that shown by 16S rRNA gene sequences (96.0%). Although discrepancies were found between the thrS-derived and previously reported whole genome phylogenetic analyses, the main phylogenetic groups of bifidobacteria were properly assigned. Most wild strains of bifidobacteria were better differentiated based on their thrS sequences than on their 16S rRNA gene identities. Phylogenetic confidence of the evaluated gene with respect to other alternative genetic markers widely used in taxonomy of bifidobacteria (fusA, GroELhsp60, pyrG, and rplB genes) was confirmed using the localized incongruence difference - Templeton analysis.

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The Type Strain of Bifidobacterium indicum Scardovi and Trovatelli 1969 (Approved Lists 1980) is ATCC 25912, not DSM 20214, and Rejection to Reclassify Bifidobacterium coryneforme as Bifidobacterium indicum
Cheng-Shan Jiang, Chun Yan Li, Chun Tao Gu
Current Microbiology.2024;[Epub] CrossRef
Bifidobacterium apicola sp. nov., isolated from the gut of honeybee (Apis mellifera)
Ting-Yu Wang, Hao Wang, Chun Tao Gu
International Journal of Systematic and Evolutionary Microbiology .2024;[Epub] CrossRef
Bifidobacterium apis sp. nov., isolated from the gut of honeybee (Apis mellifera)
Cheng-Shan Jiang, Chun Yan Li, Chun Tao Gu
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Bifidobacterium favimelis sp. nov., isolated from black comb honey
Yu Li, Qiujie Song, Haizhu Yang, Yue Wei, Bilege Menghe, Wenjun Liu
International Journal of Systematic and Evolutionary Microbiology .2024;[Epub] CrossRef
Antibacterial Activity of Rainbow Trout Plasma: In Vitro Assays and Proteomic Analysis
Toita Mizaeva, Kalimat Alieva, Eldar Zulkarneev, Stanislav Kurpe, Kseniya Isakova, Svetlana Matrosova, Ekaterina Borvinskaya, Irina Sukhovskaya
Animals.2023; 13(22): 3565. CrossRef
Bifidobacterium mizhiense sp. nov., isolated from the gut of honeybee (Apis mellifera)
Ting Ting Li, Hong Xia Zhang, Chun Tao Gu
International Journal of Systematic and Evolutionary Microbiology .2022;[Epub] CrossRef
Bifidobacterium canis sp. nov., a novel member of the Bifidobacterium pseudolongum phylogenetic group isolated from faeces of a dog (Canis lupus f. familiaris)
Vera Neuzil-Bunesova, Gabriele Andrea Lugli, Nikol Modrackova, Marie Makovska, Jakub Mrazek, Chahrazed Mekadim, Sarka Musilova, Ivona Svobodova, Roman Spanek, Marco Ventura, Jiri Killer
International Journal of Systematic and Evolutionary Microbiology .2020; 70(9): 5040. CrossRef
Glutamine synthetase type I (glnAI) represents a rewarding molecular marker in the classification of bifidobacteria and related genera
Jiří Killer, Chahrazed Mekadim, Věra Bunešová, Jakub Mrázek, Zuzana Hroncová, Eva Vlková
Folia Microbiologica.2020; 65(1): 143. CrossRef
Genetic marker-based multi-locus sequence analysis for classification, genotyping, and phylogenetics of the family Bifidobacteriaceae as an alternative approach to phylogenomics
Chahrazed Mekadim, Věra Bunešová, Eva Vlková, Zuzana Hroncová, Jiří Killer
Antonie van Leeuwenhoek.2019; 112(12): 1785. CrossRef
Prebiotic potential of natural gums and starch for bifidobacteria of variable origins
Nikol Modrackova, Marie Makovska, Chahrazed Mekadim, Eva Vlkova, Vaclav Tejnecky, Petra Bolechova, Vera Bunesova
Bioactive Carbohydrates and Dietary Fibre.2019; 20: 100199. CrossRef

Genetic variation and phylogenetic relationships of the ectomycorrhizal Floccularia luteovirens on the Qinghai-Tibet Plateau: Rui Xing , Qing-bo Gao , Fa-qi Zhang , Peng-cheng Fu , Jiu-li Wang , Hui-ying Yan , Shi-long -Chen; J. Microbiol. 2017;55(8):600-606. Published online July 4, 2017; DOI: https://doi.org/10.1007/s12275-017-7101-4

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Abstract

Floccularia luteovirens, as an ectomycorrhizal fungus, is widely distributed in the Qinghai-Tibet Plateau. As an edible fungus, it is famous for its unique flavor. Former studies mainly focus on the chemical composition and genetic structure of this species. However, the phylogenetic relationship between genotypes remains unknown. In this study, the genetic variation and phylogenetic relationship between the genotypes of F. luteovirens in Qinghai-Tibet Plateau was estimated through the analysis on two protein-coding genes (rpb1 and ef-1α) from 398 individuals collected from 24 wild populations. The sample covered the entire range of this species during all the growth seasons from 2011 to 2015. 13 genotypes were detected and moderate genetic diversity was revealed. Based on the results of network analysis, the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) analyses, the genotypes H-1, H-4, H-6, H-8, H-10, and H-11 were grouped into one clade. Additionally, a relatively higher genotype diversity (average h value is 0.722) and unique genotypes in the northeast edge of Qinghai- Tibet plateau have been found, combined with the results of mismatch analysis and neutrality tests indicated that Southeast Qinghai-Tibet plateau was a refuge for F. luteovirens during the historical geological or climatic events (uplifting of the Qinghai-Tibet Plateau or Last Glacial Maximum). Furthermore, the present distribution of the species on the Qinghai-Tibet plateau has resulted from the recent population expansion. Our findings provide a foundation for the future study of the evolutionary history and the speciation of this species.

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Diversity and Multigene Phylogeny of the Genus Floccularia (Agaricales, Basidiomycota)
Zai-Wei Ge, Hua Qu, Malka Saba, Tian Gao, Martin Ryberg
Journal of Fungi.2025; 11(1): 74. CrossRef
Metabolomic Profiling of Floccularia luteovirens from Different Geographical Regions Proposes a Novel Perspective on Their Antioxidative Activities
Chuyu Tang, Yuejun Fan, Tao Wang, Jie Wang, Mengjun Xiao, Min He, Xiyun Chang, Yuling Li, Xiuzhang Li
Antioxidants.2024; 13(5): 620. CrossRef
Selection and validation of reference genes for normalization of gene expression in Floccularia luteovirens
Yanqing Ni, Qin Zhang, Wensheng Li, Luping Cao, Rencai Feng, Zhiqiang Zhao, Xu Zhao
Fungal Biology.2024; 128(1): 1596. CrossRef
Study on Enzyme Activity and Metabolomics during Culture of Liquid Spawn of Floccularia luteovirens
Yanqing Ni, Qiuhong Liao, Siyuan Gou, Tongjia Shi, Wensheng Li, Rencai Feng, Zhiqiang Zhao, Xu Zhao
Journal of Fungi.2024; 10(9): 618. CrossRef
The Research Status and Prospects of Floccularia luteovirens: A Mycorrhizal Fungus with Edible Fruiting Bodies
Yanqing Ni, Luping Cao, Wensheng Li, Qin Zhang, Rencai Feng, Zhiqiang Zhao, Xu Zhao
Journal of Fungi.2023; 9(11): 1071. CrossRef
Polysaccharides of Floccularia luteovirens Alleviate Oxidative Damage and Inflammatory Parameters of Diabetic Nephropathy in db/db Mice
Huan Wang, Yang Yang, Shumin Wang, Changtian Li, Changbao Chen, Xilin Wan, Dan Li, Yu Li
Frontiers in Bioscience-Landmark.2023;[Epub] CrossRef
Structure, stability, and potential function of groundwater microbial community responses to permafrost degradation on varying permafrost of the Qinghai-Tibet Plateau
Sining Zhong, Bin Li, Bowen Hou, Xuming Xu, Jinyun Hu, Rong Jia, Shanqing Yang, Shungui Zhou, Jinren Ni
Science of The Total Environment.2023; 875: 162693. CrossRef
Fine-scale genetic diversity and genet dynamics of the fairy ring fungus Floccularia luteovirens on the Qinghai–Tibet plateau
Rui Xing, Yan-fang Deng, Yu Yao, Qing-bo Gao, Fa-qi Zhang, Jiu-li Wang, Hai-rui Liu, Shi-long Chen
Fungal Ecology.2022; 60: 101194. CrossRef
The Molecular Mechanism of Yellow Mushroom (Floccularia luteovirens) Response to Strong Ultraviolet Radiation on the Qinghai-Tibet Plateau
Jing Guo, Zhanling Xie, Hongchen Jiang, Hongyan Xu, Baolong Liu, Qing Meng, Qingqing Peng, Yongpeng Tang, Yingzhu Duan
Frontiers in Microbiology.2022;[Epub] CrossRef
Chemical Constituents and Molecular Mechanism of the Yellow Phenotype of Yellow Mushroom (Floccularia luteovirens)
Xiaolong Gan, Xuemei Bao, Baolong Liu, Yun Li, Dong Cao, Hg Zhang, Yuan Zong
Journal of Fungi.2022; 8(3): 314. CrossRef
The Genomic and Transcriptomic Analyses of Floccularia luteovirens, a Rare Edible Fungus in the Qinghai–Tibet Plateau, Provide Insights into the Taxonomy Placement and Fruiting Body Formation
Zhengjie Liu, Hongyun Lu, Xinglin Zhang, Qihe Chen
Journal of Fungi.2021; 7(11): 887. CrossRef
Genetic relationships and low diversity among the tea-oil Camellia species in Sect. Oleifera, a bulk woody oil crop in China
Huasha Qi, Xiuxiu Sun, Wuping Yan, Hang Ye, Jiali Chen, Jing Yu, Dai Jun, Chunmei Wang, Tengfei Xia, Xuan Chen, Dongliang Li, Daojun Zheng
Frontiers in Plant Science.2020;[Epub] CrossRef
Draft Genome Assembly of Floccularia luteovirens, an Edible and Symbiotic Mushroom on Qinghai-Tibet Plateau
Xiaolong Gan, Dong Cao, Zhenyu Zhang, Shu Cheng, Le Wei, Shiming Li, Baolong Liu
G3 Genes|Genomes|Genetics.2020; 10(4): 1167. CrossRef
Genetic diversity and its conservation implications ofVitex rotundifolia(Lamiaceae) populations in East Asia
Yiqi Sun, Hong Yang, Qiaoyan Zhang, Luping Qin, Pan Li, Joongku Lee, Shichao Chen, Khalid Rahman, Tingguo Kang, Min Jia
PeerJ.2019; 7: e6194. CrossRef
Microbial communities inhabiting the fairy ring of Floccularia luteovirens and isolation of potential mycorrhiza helper bacteria
Rui Xing, Hui‐ying Yan, Qing‐bo Gao, Fa‐qi Zhang, Jiu‐li Wang, Shi‐long Chen
Journal of Basic Microbiology.2018; 58(6): 554. CrossRef

Molecular epidemiology of norovirus in asymptomatic food handlers in Busan, Korea, and emergence of genotype GII.17: Hee Soo Koo , Mi Ok Lee , Pyeong Tae Ku , Su Jeong Hwang , Dong Ju Park , Hyung Suk Baik; J. Microbiol. 2016;54(10):686-694. Published online September 30, 2016; DOI: https://doi.org/10.1007/s12275-016-6312-4

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Abstract

The molecular epidemiology of norovirus infections was studied in food handlers without any symptoms from January to December 2015 in Busan city, Korea. A total of 2,174 fecal specimens from asymptomatic food handlers were analyzed, and 2.3% (49/2,174) were norovirus-positive. Fourteen of 335 samples (4.2%) were positive in January; fifteen of 299 samples (5.0%) in February, and seven of 189 samples (3.7%) in December. However, norovirus was rarely detected in other months. From sequencing analysis, 11 genotypes (five GI and six GII genotypes) were detected. Among the 42 capid gene sequences identified, 14 were from the GI genogroup, while 28 were from the GII genogroup. The most commonly detected genotype was GII.17, comprising 15 (35.7%) of positive samples. From January 2012 to December 2015, 5,138 samples were collected from gastroenteritis patients and outbreaks in Busan. The most detected genotype in 2012, 2013, and 2014 was GII.4 (121, 24, and 12 cases, respectively), but in 2015, GII.17 (25 cases) was the most common. The GII.4 genotype was the major cause of acute gastroenteritis from 2012 to 2014, but the GII.17 genotype became the most prevalent cause in 2015. Continued epidemiological surveillance of GII.17 is needed, together with assessment of the risk of norovirus infection.

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Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi).2017; 58(1): 12. CrossRef
Occurrence of norovirus infection in an asymptomatic population in Indonesia
Takako Utsumi, Maria Inge Lusida, Zayyin Dinana, Rury Mega Wahyuni, Laura Navika Yamani, Juniastuti, Soetjipto, Chieko Matsui, Lin Deng, Takayuki Abe, Yen Hai Doan, Yoshiki Fujii, Hirokazu Kimura, Kazuhiko Katayama, Ikuo Shoji
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Research Support, Non-U.S. Gov'ts

Development of a Chimeric Strain of Porcine Reproductive and Respiratory Syndrome Virus with an Infectious Clone and a Korean Dominant Field Strain: Jung-Ah Lee , Nak-Hyung Lee , Sang-Won Lee , Seung-Yong Park , Chang-Seon Song , In-Soo Choi , Joong-Bok Lee; J. Microbiol. 2014;52(4):345-349. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4074-4

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Abstract

The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 106 TCID50/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.

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NOTE] Two Novel Talaromyces Species Isolated from Medicinal Crops in Korea: Hyunkyu Sang , Tae-Jin An , Chang Sun Kim , Gyu-Sub Shin , Gi-Ho Sung , Seung Hun Yu; J. Microbiol. 2013;51(5):704-708. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-3361-9

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Abstract: Two novel biverticillate Talaromyces species, T. angelicus and T. cnidii, were collected from the medicinal crops Angelica gigas and Cnidium officinale, respectively, in Korea. Phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and the β-tubulin gene as well as morphological analyses revealed that the two species differ from any known Talaromyces species. Talaromyces angelicus is related to T. flavovirens in the phylogeny of the ITS region, but the new species is grouped together with Penicillium liani and T. pinophilus in terms of its β-tubulin phylogeny, and its growth rate on Czapek yeast autolysate differs from that of T. flavovirens. Talaromyces cnidii is phylogenetically similar to T. siamensis, but exhibits differences in the morphologies of the colony margin, metulae, and conidia.

NOTE] Penicillium daejeonium sp. nov., a New Species Isolated from a Grape and Schisandra Fruit in Korea: Hyunkyu Sang , Tae-Jin An , Chang Sun Kim , Young Phil Choi , Jian-Xin Deng , Narayan Chandra Paul , Gi-Ho Sung , Seung Hun Yu; J. Microbiol. 2013;51(4):536-539. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3291-6

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Abstract: Two isolates of monoverticillate Penicillium species were collected from a grape and schisandra fruit in Korea. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and genes encoding β-tubulin (benA) and calmodulin (cmd), as well as morphological analyses revealed that the two isolates are members of the P. sclerotiorum complex in Penicillium subgenus Aspergilloides, but different from species of the P. sclerotiorum complex. The isolates are closely related to P. cainii, P. jacksonii, and P. viticola in terms of their multigene phylogeny, but their colony and conidiophore morphologies differ from those of closely related species. The name P. daejeonium is proposed for this unclassified new species belonging to the P. sclerotiorum complex in subgenus Aspergilloides.

Methyl Coenzyme M Reductase (mcrA) Gene Based Phylogenetic Analysis of Methanogens Population in Murrah Buffaloes (Bubalus bubalis): Prem Prashant Chaudhary , Sunil Kumar Sirohi , Dheer Singh , Jyoti Saxena; J. Microbiol. 2011;49(4):558-561. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1052-y

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Abstract: The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant methanogenic communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.

Molecular Pathogenesis of Vibrio vulnificus: Paul A. Gulig , Keri L. Bourdage , Angela M. Starks; J. Microbiol. 2005;43(1):118-131.

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Abstract: Vibrio vulnificus is an opportunistic pathogen of humans that has the capability of causing rare, yet devastating disease. The bacteria are naturally present in estuarine environments and frequently contaminate seafoods. Within days of consuming uncooked, contaminated seafood, predisposed individuals can succumb to sepsis. Additionally, in otherwise healthy people, V. vulnificus causes wound infection that can require amputation or lead to sepsis. These diseases share the characteristics that the bacteria multiply extremely rapidly in host tissues and cause extensive damage. Despite the analysis of virulence for over 20 years using a combination of animal and cell culture models, surprisingly little is known about the mechanisms by which V. vulnificus causes disease. This is in part because of differences observed using animal models that involve infection with bacteria versus injection of toxins. However, the increasing use of genetic analysis coupled with detailed animal models is revealing new insight into the pathogenesis of V. vulnificus disease.

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