Journal of Microbiology

Volume 58(1); January 2020

Review

[Minireview]Recent advances in genetic engineering tools based on synthetic biology: Jun Ren , Jingyu Lee , Dokyun Na; J. Microbiol. 2020;58(1):1-10. Published online January 2, 2020; DOI: https://doi.org/10.1007/s12275-020-9334-x

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26 Citations

Abstract: Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. Over the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, we discuss the current genome-scale engineering technologies used for microbial engineering. Recently developed strategies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, multiplex automated genome engineering (MAGE), promoter engineering, CRISPR-based regulations, and synthetic small regulatory RNA (sRNA)-based knockdown, are considered as powerful tools for genome-scale engineering in microbiological systems. MAGE, which modifies specific nucleotides of the genome sequence, is utilized as a genome-editing tool. Contrastingly, synthetic sRNA, CRISPRi, and CRISPRa are mainly used to regulate gene expression without modifying the genome sequence. This review introduces the recent genome-scale editing and regulating technologies and their applications in metabolic engineering.

Journal Articles

[Protocol]Rapid method for chromatin immunoprecipitation (ChIP) assay in a dimorphic fungus, Candida albicans: Jueun Kim , Jung-Shin Lee; J. Microbiol. 2020;58(1):11-16. Published online June 11, 2019; DOI: https://doi.org/10.1007/s12275-020-9143-2

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5 Citations

Abstract: A chromatin immunoprecipitation (ChIP) assay is a method to identify how much a protein of interest binds to the DNA region. This method is indispensable to study the mechanisms of how the transcription factors or chromatin modifications regulate the gene expression. Candida albicans is a dimorphic pathogenic fungus, which can change its morphology very rapidly from yeast to hypha in response to the environmental signal. The morphological change of C. albicans is one of the critical factors for its virulence. Therefore, it is necessary to understand how to regulate the expression of genes for C. albicans to change its morphology. One of the essential methods for us to understand this regulation is a ChIP assay. There have been many efforts to optimize the protocol to lower the background signal and to analyze the results accurately because a ChIP assay can provide very different results even with slight differences in the experimental procedure. We have optimized the rapid and efficient ChIP protocol so that it could be applied equally for both yeast and hyphal forms of C. albicans. Our method in this protocol is also comparatively rapid to the method widely used. In this protocol, we described our rapid method for the ChIP assay in C. albicans in detail.

Paraflavitalea soli gen. nov., sp. nov., isolated from greenhouse soil: Jun Heo , Hang-Yeon Weon , Hayoung Cho , Seung-Beom Hong , Jeong-Seon Kim , Soo-Jin Kim , Soon-Wo Kwon; J. Microbiol. 2020;58(1):17-23. Published online November 23, 2019; DOI: https://doi.org/10.1007/s12275-020-9236-y

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7 Citations

Abstract: A bacterial strain designated 5GH32-13T was isolated from greenhouse soil in Yongin-city, Republic of Korea. Cells were Gram-stain-negative, strictly aerobic, motile rods of two different shapes. The strain was catalase-positive and oxidasenegative. Flexirubin-like pigments were not detected. β-Carotene was produced. The strain grew in the range of 10–37°C (optimum of 28–30°C) and pH 6–8 (optimum of pH 7) and tolerated up to 1% (w/v) NaCl (optimum of 0%). According to the 16S rRNA gene sequence comparison, strain 5GH32- 13T shared a sequence similarity of less than 96.0% with all validly named taxa, having the highest sequence similarity with Pseudoflavitalea soli KIS20-3T (95.8%), Pseudoflavitalea rhizosphaerae T16R-265T (95.4%), Flavitalea gansuensis JCN-23T (95.3%), Pseudobacter ginsenosidimutans Gsoil 221T (95.3%), and Flavitalea populi HY-50RT (95.2%). A phylogenetic tree showed that strain 5GH32-13T was not grouped consistently into any specific genus. Its only polyamine was homospermidine, and its major fatty acids (> 10% of total fatty acids) were iso-C15:0, iso-C17:0 3-OH, and iso-C15:1 G. The strain’s only respiratory quinone was MK-7, and its polar lipids were phosphatidylethanolamine, one unidentified phospholipid, six unidentified aminolipids and four unidentified lipids. Its DNA G + C content was 47.5 mol%. The results from chemotaxonomic, phenotypic and phylogenetic analyses indicated that strain 5GH32-13T represents a novel species of a novel genus of the family Chitinophagaceae, and the name Paraflavitalea soli gen. nov., sp. nov. is proposed. The type strain is 5GH32-13T (= KACC 17331T = JCM 33061T).

Saccharibacillus brassicae sp. nov., an endophytic bacterium isolated from kimchi cabbage (Brassica rapa subsp. pekinensis) seeds: Lingmin Jiang , Chan Ju Lim , Song-Gun Kim , Jae Cheol Jeong , Cha Young Kim , Dae-Hyuk Kim , Suk Weon Kim , Jiyoung Lee; J. Microbiol. 2020;58(1):24-29. Published online November 25, 2019; DOI: https://doi.org/10.1007/s12275-020-9346-6

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10 Citations

Abstract: Strain ATSA2T was isolated from surface-sterilized kimchi cabbage (Brassica rapa subsp. pekinensis) seeds and represents a novel bacterium based on the polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ATSA2T formed a lineage within genus Saccharibacillus and was most closely to Saccharibacillus deserti WLG055T (98.1%) and Saccharibacillus qingshengii H6T (97.9%). The whole-genome of ATSA2T comprised a 5,619,468 bp of circular chromosome with 58.4% G + C content. The DNA-DNA relatedness values between strain ATSA2T and its closely related type strains S. deserti WLJ055T and S. qingshengii H6T were 26.0% and 24.0%, respectively. Multiple gene clusters associated with plant growth promotion activities (stress response, nitrogen and phosphorus metabolism, and auxin biosynthesis) were annotated in the genome. Strain ATSA2T was Gram-positive, endospore-forming, facultatively anaerobic, and rod-shaped. It grew at 15–37°C (optimum 25°C), pH 6.0–10.0 (optimum pH 8.0), and in the presence of 0–5% (w/v) NaCl (optimum 1%). The major cellular fatty acids (> 10%) of strain ATSA2T were anteiso- C15:0 and C16:0. MK-7 was the major isoprenoid quinone. The major polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, and three unknown glycolipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxonomic features, strain ATSA2T is proposed to represent a novel species of genus Saccharibacillus, for which the name is Saccharibacillus brassicae sp. nov. The type strain is ATSA2T (KCTC 43072T = CCTCC AB 2019223T).

Sterilization efficiency of pathogen-contaminated cottons in a laundry machine: Yoonjae Shin , Jungha Park , Woojun Park; J. Microbiol. 2020;58(1):30-38. Published online November 25, 2019; DOI: https://doi.org/10.1007/s12275-020-9391-1

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8 Citations

Abstract: Pathogenic bacteria on abiotic surfaces such as fabrics, bedding, patient wears, and surgical tools are known to increase the risk of bacterial diseases in infants and the elderly. The desiccation tolerance of bacteria affects their viability in cotton. Thus, washing and drying machines are required to use conditions that ensure the sterilization of bacteria in cotton. The objective of this study is to determine the effects of various sterilization conditions of washing and drying machines on the survival of three pathogenic bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus) commonly presented in contaminated cotton and two non-pathogenic bacteria (Bacillus subtilis and Escherichia coli) in cotton. High survival rates of A. baumannii and S. aureus in desiccated cotton were observed based on scanning electron microscope and replicate organism direct agar contact assay. The survival rates of A. baumannii and S. aureus exposed in desiccated cotton for 8 h were higher (14.4 and 5.0%, respectively) than those of other bacteria (< 0.5%). All tested bacteria were eradicated at low-temperature (< 40°C) washing with activated oxygen bleach (AOB). However, bacterial viability was shown in low temperature washing without AOB. High-temperature (> 60°C) washing was required to achieve 99.9% of the sterilization rate in washing without AOB. The sterilization rate was 93.2% using a drying machine at 60°C for 4 h. This level of sterilization was insufficient in terms of time and energy efficiency. High sterilization efficiency (> 99.9%) at 75°C for 3 h using a drying machine was confirmed. This study suggests standard conditions of drying machines to remove bacterial contamination in cotton by providing practical data.

Identification and characterization of a novel light-induced promoter for recombinant protein production in Pleurotus ostreatus: Chaomin Yin , Xiuzhi Fan , Kun Ma , Zheya Chen , Defang Shi , Fen Yao , Hong Gao , Aimin Ma; J. Microbiol. 2020;58(1):39-45. Published online November 4, 2019; DOI: https://doi.org/10.1007/s12275-020-9230-4

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Abstract: A lectin gene (plectin) with a high level of expression was previously identified by comparative transcriptome analysis of Pleurotus ostreatus. In this study, we cloned a 733-bp DNA fragment from the start codon of the plectin gene. Sequence analysis showed that the plectin promoter (Plp) region contained several eukaryotic transcription factor binding motifs, such as the TATA-box, four possible CAAT-box, light responsiveness motifs and MeJA-responsiveness motifs. To determine whether the Plp promoter was a light-regulated promoter, we constructed an expression vector with the fused egfp-hph fragment under the control of the Plp promoter and transformed P. ostreatus mycelia via Agrobacterium tumefaciens. PCR and Southern blot analyses confirmed the Plpegfp- hph fragment was integrated into the chromosomal DNA of transformants. qRT-PCR, egfp visualization, and intracellular egfp determination experiments showed the Plp promoter could be a light-induced promoter that may be suitable for P. ostreatus genetic engineering. This study lays the foundation for gene homologous expression in P. ostreatus.

Development of a neutralization assay based on the pseudotyped chikungunya virus of a Korean isolate: Woo-Chang Chung , Kwang Yeon Hwang , Suk-Jo Kang , Jae-Ouk Kim , Moon Jung Song; J. Microbiol. 2020;58(1):46-53. Published online November 25, 2019; DOI: https://doi.org/10.1007/s12275-020-9384-0

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Abstract: The Chikungunya virus (CHIKV) belongs to the Alphavirus genus of Togaviridae family and contains a positive-sense single stranded RNA genome. Infection by this virus mainly causes sudden high fever, rashes, headache, and severe joint pain that can last for several months or years. CHIKV, a mosquito- borne arbovirus, is considered a re-emerging pathogen that has become one of the most pressing global health concerns due to a rapid increase in epidemics. Because handling of CHIKV is restricted to Biosafety Level 3 (BSL-3) facilities, the evaluation of prophylactic vaccines or antivirals has been substantially hampered. In this study, we first identified the whole structural polyprotein sequence of a CHIKV strain isolated in South Korea (KNIH/2009/77). Phylogenetic analysis showed that this sequence clustered within the East/ Central/South African CHIKV genotype. Using this sequence information, we constructed a CHIKV-pseudotyped lentivirus expressing the structural polyprotein of the Korean CHIKV isolate (CHIKVpseudo) and dual reporter genes of green fluorescence protein and luciferase. We then developed a pseudovirus-based neutralization assay (PBNA) using CHIKVpseudo. Results from this assay compared to those from the conventional plaque reduction neutralization test showed that our PBNA was a reliable and rapid method to evaluate the efficacy of neutralizing antibodies. More importantly, the neutralizing activities of human sera from CHIKVinfected individuals were quantitated by PBNA using CHIKVpseudo. Taken together, these results suggest that our PBNA for CHIKV may serve as a useful and safe method for testing the neutralizing activity of antibodies against CHIKV in BSL-2 facilities.

Human cytomegalovirus IE86 protein aa 136–289 mediates STING degradation and blocks the cGAS-STING pathway: Jun-Kyu Lee , Jung-Eun Kim , Bang Ju Park , Yoon-Jae Song; J. Microbiol. 2020;58(1):54-60. Published online January 2, 2020; DOI: https://doi.org/10.1007/s12275-020-9577-6

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9 Citations

Abstract: We previously reported that human cytomegalovirus (HCMV) 86 kDa immediate-early 2 gene product (IE86) promotes proteasome-dependent degradation of STING. In the present study, we determined the specific residues of IE86 responsible for STING degradation using a STING-firefly luciferase fusion protein expression system for quantitative measurement of STING protein levels. IE86 amino acids (aa) 136–289 were sufficient to promote STING degradation and further induced down-regulation of 2􍿁3􍿁-cyclic GMP-AMP (cGAMP)-mediated IFN-β promoter activation. Interestingly, transactivation domains (TAD) of the IE86 protein located at the N- and C-termini were required for down-regulation of Toll/interleukin-1 receptor (TIR) domain-containing adaptor- inducing interferon β (IFN-β) (TRIF)-mediated IFN-β- and p65/RelA-induced NF-κB-dependent promoter activation while amino acids (aa) 136–289 had no significant effects. Our collective data suggest that the IE86 protein utilizes the aa 136–289 region to promote STING degradation and inhibit the cGAS-STING pathway.

Repositioning of a mucolytic drug to a selective antibacterial against Vibrio cholerae: In-Young Chung† , Bi-o Kim† , Hye-Jeong Jang† , You-Hee Cho; J. Microbiol. 2020;58(1):61-66. Published online January 2, 2020; DOI: https://doi.org/10.1007/s12275-020-9590-9

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Abstract: Drug repositioning, the approach to explore existing drugs for use in new therapeutic indications, has emerged as an alternative drug development strategy. In this study, we found that a mucolytic drug, N-acetylcysteine (NAC) showed antibacterial activity against Vibrio cholerae. NAC can provide acid stress that selectively inhibited the growth of V. cholerae among other bacterial pathogens. To address the antibacterial mechanism of NAC against V. cholerae, six acr (acetylcysteine- resistant) mutants were isolated from 3,118 random transposon insertion clones. The transposon insertion sites of the six mutants were mapped at the five genes. All these mutants did not display NAC resistance under acidic conditions, despite their resistance to NAC under alkaline conditions, indicating that the NAC resistance directed by the acr mutations was independent of the unusual pH-sensitivity of V. cholerae. Furthermore, all these mutants displayed attenuated virulence and reduced biofilm formation, suggesting that the acr genes are required for pathogenesis of V. cholerae. This study validates the relevance of drug repositioning for antibacterials with new modes of action and will provide an insight into a novel antibacterial therapy for V. cholerae infections to minimize side effects and resistance emergence.

Zur-regulated lipoprotein A contributes to the fitness of Acinetobacter baumannii: Eun Kyung Lee , Chul Hee Choi , Man Hwan Oh; J. Microbiol. 2020;58(1):67-77. Published online January 2, 2020; DOI: https://doi.org/10.1007/s12275-020-9531-7

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11 Citations

Abstract: Acinetobacter baumannii is a notorious nosocomial pathogen that commonly infects severely ill patients. Zinc (Zn) is essential to survive and adapt to different environment and host niches in A. baumannii. Of the Zinc uptake regulator (Zur)-regulated genes in A. baumannii, the A1S_3412 gene encoding a Zur-regulated lipoprotein A (ZrlA) is critical for cell envelope integrity and overcoming antibiotic exposure. This study investigated whether ZrlA contributes to the fitness of A. baumannii in vitro and in vivo using the wildtype A. baumannii ATCC 17978, ΔzrlA mutant, and zrlAcomplemented strains. The ΔzrlA mutant showed reduced biofilm formation, surface motility, and adherence to and invasion of epithelial cells compared to the wild-type strain. In a mouse pneumonia model, the ΔzrlA mutant showed significantly lower bacterial numbers in the blood than the wildtype strain. These virulence traits were restored in the zrlAcomplemented strain. Under static conditions, the expression of csuCDE, which are involved in the chaperone-usher pili assembly system, was significantly lower in the ΔzrlA mutant than in the wild-type strain. Moreover, the expression of the bfmR/S genes, which regulate the CsuA/BABCDE system, was significantly lower in the ΔzrlA mutant under static conditions than in the wild-type strain. Our results indicate that the zrlA gene plays a role in the fitness of A. baumannii by regulating the BfmR/S two-component system and subsequently the CsuA/BABCDE chaperone-usher pili assembly system, suggesting it as a potential target for anti-virulence strategies against A. baumannii.

Published Erratums

Erratum]Vibrio parahaemolyticus cqsA controls production of quorum sensing signal molecule 3-hydroxyundecan-4-one and regulates colony morphology: Kui Wu , Yangyun Zheng , Qingping Wu , Haiying Chen , Songzhe Fu , Biao Kan , Yongyan Long , Xiansheng Ni , Junling Tu; J. Microbiol. 2020;58(1):78-78.; DOI: https://doi.org/10.1007/s12275-020-9721-3

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Abstract: In the article by Wu et al. published in Journal of Microbiology 2019; 57, 1105–1114, the figure 8 is unfortunately incorrect. The figure 8 should be corrected as below. We apologize for any inconvenience that this may have caused.

Erratum]Methylobacterium terrae sp. nov., a radiation-resistant bacterium isolated from gamma ray-irradiated soil: Jiyoun Kim , Geeta Chhetri , Inhyup Kim , Hyungdong Kim , Myung Kyum Kim , Taegun Seo; J. Microbiol. 2020;58(1):79-79.; DOI: https://doi.org/10.1007/s12275-020-9722-2

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Abstract: In the article by Kim et al. published in Journal of Microbiology 2019; 57, 959–966, The NBRC accession number NBRC 112879T on 33th line of 2nd paragraph in the section of ‘Description of Methylobacterium terrae sp. nov.’ on page 964 should be corrected in NBRC 112873T. The sentence in abstract should have read: The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genome sequences of the type strain 17Sr1-28T (= KCTC 52904T = NBRC 112873T) are KY939566 and CP029553, respectively. We apologize for any inconvenience that this may have caused.

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