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- CA‑CAS‑01‑A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus
- Seung-Chul Lee , Yongkwan Kim , Ji-Won Cha , Kiramage Chathuranga , Niranjan Dodantenna , Hyeok-Il Kwon , Min Ho Kim , Weonhwa Jheong , In-Joong Yoon , Joo Young Lee , Sung-Sik Yoo , Jong-Soo Lee
- J. Microbiol. 2024;62(2):125-134. Published online March 13, 2024
- DOI: https://doi.org/10.1007/s12275-024-00116-1
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Abstract
- African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ ml assay, TCID50/ ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.
- Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains of Escherichia coli BL21 (DE3)
- Yinfeng Wang , Guanhua Xuan , Houqi Ning , Jiuna Kong , Hong Lin , Jingxue Wang
- J. Microbiol. 2023;61(5):559-569. Published online May 22, 2023
- DOI: https://doi.org/10.1007/s12275-023-00048-2
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Abstract
- Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.
- Activation of the SigE-SigB signaling pathway by inhibition of the respiratory electron transport chain and its effect on rifampicin resistance in Mycobacterium smegmatis
- Yuna Oh , Hye-In Lee , Ji-A Jeong , Seonghan Kim , Jeong-Il Oh
- J. Microbiol. 2022;60(9):935-947. Published online August 1, 2022
- DOI: https://doi.org/10.1007/s12275-022-2202-0
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Abstract
- Using a mutant of Mycobacterium smegmatis lacking the major aa3 cytochrome c oxidase of the electron transport chain (Δaa3), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa3 mutant, leading to upregulation of the SigB-dependent genes in the mutant. The phoU2 (MSMEG_1605) gene implicated in a phosphatesignaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa3 mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain.
- Pikeienuella piscinae gen. nov., sp. nov., a novel genus in the family Rhodobacteraceae
- Jeeeun Park , Young-Sam Kim , Seong-Jin Kim , Sang-Eon Kim , Hyun-Kyoung Jung , Min-Ju Yu , Young Jae Jeon , Kyoung-Ho Kim
- J. Microbiol. 2021;59(6):546-551. Published online April 20, 2021
- DOI: https://doi.org/10.1007/s12275-021-0678-7
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Abstract
- A novel bacterium, designated strain RR4-56T, was isolated from a biofilter of a seawater recirculating aquaculture system. The 16S rRNA gene sequence analysis showed that the isolate was closely related to Halovulum dunhuangense YYQ- 30T (92.6%), Albimonas donghaensis DS2T (91.3%), Pontivivens insulae GYSW-23T (91.3%), and Monaibacterium marinum C7T (90.9%), belonging to the family Rhodobacteraceae. The strain was aerobic, Gram-negative, rod-shaped, oxidasepositive, and catalase-negative. Its optimum temperature, pH, and salinity for growth were 25–30°C, pH 8.5, and 2–3% NaCl (w/v), respectively. Its growth occurred at 15–35°C, pH 5.0–9.5, and 0–7% NaCl (w/v). It contained ubiquinone-10 (Q-10), a respiratory quinone, and the major cellular fatty acids were 11-methyl C18:1 ω7c (31.9%), C18:1 ω6c (30.4%), and C19:0 cyclo ω8c (16.1%). The polar lipids present in the strain were phosphatidylglycerol, an unidentified phospholipid, and an unidentified aminolipid. The strain had one 4,373,045 bp circular chromosome with G + C contents of 65.9 mol% including 4,169 genes, 4,118 coding sequences (CDSs), 3 rRNAs, and 45 tRNAs. Genome annotation predicted some gene clusters related to the degradation of several types of organic matter such as protocatechuate, catechol, and phthalate. Based on the polyphasic characteristics, RR4-56T represents a novel genus and species in the family Rhodobacteraceae, for which the name Pikeienuella piscinae gen. nov., sp. nov. was proposed. The type strain is RR4-56T (= KCTC 52648T = DSM 107918T).
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