Journal of Microbiology

Volume 61(1); January 2023

Reviews

Bacterial Crosstalk via Antimicrobial Peptides on the Human Skin: Therapeutics from a Sustainable Perspective: Seon Mi Lee , Hye Lim Keum , Woo Jun Sul; J. Microbiol. 2023;61(1):1-11. Published online January 31, 2023; DOI: https://doi.org/10.1007/s12275-022-00002-8

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Abstract: The skin’s epidermis is an essential barrier as the first guard against invading pathogens, and physical protector from external injury. The skin microbiome, which consists of numerous bacteria, fungi, viruses, and archaea on the epidermis, play a key role in skin homeostasis. Antibiotics are a fast-acting and effective treatment method, however, antibiotic use is a nuisance that can disrupt skin homeostasis by eradicating beneficial bacteria along with the intended pathogens and cause antibioticresistant bacteria spread. Increased numbers of antimicrobial peptides (AMPs) derived from humans and bacteria have been reported, and their roles have been well defined. Recently, modulation of the skin microbiome with AMPs rather than artificially synthesized antibiotics has attracted the attention of researchers as many antibiotic-resistant strains make treatment mediation difficult in the context of ecological problems. Herein, we discuss the overall insights into the skin microbiome, including its regulation by different AMPs, as well as their composition and role in health and disease.

Recent Advances in CRISPR‑Cas Technologies for Synthetic Biology: Song Hee Jeong , Ho Joung Lee , Sang Jun Lee; J. Microbiol. 2023;61(1):13-36. Published online February 1, 2023; DOI: https://doi.org/10.1007/s12275-022-00005-5

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Abstract: With developments in synthetic biology, “engineering biology” has emerged through standardization and platformization based on hierarchical, orthogonal, and modularized biological systems. Genome engineering is necessary to manufacture and design synthetic cells with desired functions by using bioparts obtained from sequence databases. Among various tools, the CRISPR-Cas system is modularly composed of guide RNA and Cas nuclease; therefore, it is convenient for editing the genome freely. Recently, various strategies have been developed to accurately edit the genome at a single nucleotide level. Furthermore, CRISPR-Cas technology has been extended to molecular diagnostics for nucleic acids and detection of pathogens, including disease-causing viruses. Moreover, CRISPR technology, which can precisely control the expression of specific genes in cells, is evolving to find the target of metabolic biotechnology. In this review, we summarize the status of various CRISPR technologies that can be applied to synthetic biology and discuss the development of synthetic biology combined with CRISPR technology in microbiology.

Journal Articles

Chryseobacterium paludis sp. nov. and Chryseobacterium foetidum sp. nov. Isolated from the Aquatic Environment, South Korea: Miryung Kim , Yong&# , Chang&#; J. Microbiol. 2023;61(1):37-47. Published online February 1, 2023; DOI: https://doi.org/10.1007/s12275-022-00008-2

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Abstract: Two novel bacterial species CJ51T and CJ63T belonging to the genus Chryseobacterium were isolated from the Upo wetland and the Han River, South Korea, respectively. Cells of these strains were Gram-stain-negative, aerobic, non-motile, rodshaped, and catalase- and oxidase-positive. Both strains were shown to grow optimally at 30 °C and pH 7 in the absence of NaCl on tryptic soy agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CJ51T and CJ63T belonged to the genus Chryseobacterium and were most closely related to Chryseobacterium piperi CTMT and Chryseobacterium piscicola VQ-6316sT with 98.47% and 98.46% 16S rRNA sequence similarities, respectively. The average nucleotide identity values of strains CJ51T and CJ63T with its closely related type strains Chryseobacterium piperi CTMT and Chryseobacterium piscicola VQ-6316sT were 81.9% and 82.1%, respectively. The major fatty acids of strains CJ51T and CJ63T were iso-C15:0, iso-C17:0 3-OH and summed feature 9 ( C16:0 10-methyl and/or iso-C17:1ω9c). Menaquinone 6 (MK-6) was identified as the primary respiratory quinone in both strains. The major polar lipids of strains CJ51T and CJ63T were phosphatidylethanolamine and several unidentified amino lipids and lipids. Based on polyphasic taxonomy data, strains CJ51T and CJ63T represent novel species of the genus Chryseobacterium, for which names Chryseobacterium paludis sp. nov. and Chryseobacterium foetidum sp. nov. are proposed respectively. The type strains are CJ51T (= KACC 22749T = JCM 35632T) and CJ63T (= KACC 22750T = JCM 35633T).

Effects of Continuous Straw Returning on Soil Functional Microorganisms and Microbial Communities: Yunpeng Guan , Meikang Wu , Songhao Che , Shuai Yuan , Xue Yang , Siyuan Li , Ping Tian , Lei Wu , Meiying Yang , Zhihai Wu; J. Microbiol. 2023;61(1):49-62. Published online January 26, 2023; DOI: https://doi.org/10.1007/s12275-022-00004-6

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Abstract: This study examined the changes in soil enzymatic activity, microbial carbon source metabolic diversity, and straw decomposition rates in paddy fields treated with 1, 2, or 3 years of straw returning (SR1–SR3). The soil’s ability to decompose straw and cellulolytic bacteria increased with the number of treatment years (1: 31.9% vs. 2: 43.9% vs. 3: 51.9%, P < 0.05). The numbers of Azotobacter, Nitrobacteria, cellulolytic bacteria, and inorganic phosphate bacteria increased progressively with the numbers of straw returning years. Cellulolytic bacteria and inorganic phosphate bacteria were significantly positively correlated with the decomposition rate (r = 0.783 and r = 0.375, P < 0.05). Based on 16S sequencing results, straw returning improved the microbial diversity of paddy soils by increasing unclassified bacteria and keeping dominant soil microorganism populations unchanged. The relative importance of individual microbial taxa was compared using random forest models. Proteobacteria, ammoniating bacteria, and potassium dissolving bacteria contributed to peroxidase activity. The significant contributors to phosphate monoesterase were Acidobacteriota, Desulfobacterota, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria. Proteobacteria, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria contributed to urease activity. Desulfobacterota, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria contributed to the neutral invertase activity. In conclusion, soil microbial community structure and function were affected within 2 years of straw returning, which was driven by the combined effects of soil organic carbon, available nitrogen, available potassium, and pH. With elapsing straw returning years, soil properties interacted with soil microbial communities, and a healthier soil micro-ecological environment would form.

Diversity and Dynamics of Marine Arenicolous Fungi in Three Seasides of the Korean Peninsula: Jun Won Lee , Chang Wan Seo , Wonjun Lee , Ji Seon Kim , Ki Hyeong Park , Yoonhee Cho , Young Woon Lim; J. Microbiol. 2023;61(1):63-82. Published online January 30, 2023; DOI: https://doi.org/10.1007/s12275-023-00011-1

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Abstract: Various arenicolous fungal species have been detected from the beach sand in the coastal area. However, little has been revealed regarding their distribution and dynamics. To investigate the overall diversity of marine arenicolous fungi (MAFs) in Korea and whether the composition of MAFs is affected by ocean currents, we isolated and analyzed the fungal community from the western, southern, and eastern seasides of the Korean Peninsula. In total, 603 strains were isolated and identified as 259 species based on appropriate molecular markers for each genus (ITS, BenA, CaM, tef1, and act). The composition of MAFs showed differences among the seasides. Our results indicate that many MAFs inhabit the beach sand on the Korean Peninsula, and the composition of MAFs is also affected by ocean currents flowing along each coast.

Genome Sequencing Highlights the Plant Cell Wall Degrading Capacity of Edible Mushroom Stropharia rugosoannulata: Mengpei Guo , Xiaolong Ma , Yan Zhou , Yinbing Bian , Gaolei Liu , Yingli Cai , Tianji Huang , Hongxia Dong , Dingjun Cai , Xueji Wan , Zhihong Wang , Yang Xiao , Heng Kang; J. Microbiol. 2023;61(1):83-93. Published online February 1, 2023; DOI: https://doi.org/10.1007/s12275-022-00003-7

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Abstract: The basidiomycetous edible mushroom Stropharia rugosoannulata has excellent nutrition, medicine, bioremediation, and biocontrol properties. S. rugosoannulata has been widely and easily cultivated using agricultural by-products showing strong lignocellulose degradation capacity. However, the unavailable high-quality genome information has hindered the research on gene function and molecular breeding of S. rugosoannulata. This study provided a high-quality genome assembly and annotation from S. rugosoannulata monokaryotic strain QGU27 based on combined Illumina-Nanopore data. The genome size was about 47.97 Mb and consisted of 20 scaffolds, with an N50 of 3.73 Mb and a GC content of 47.9%. The repetitive sequences accounted for 17.41% of the genome, mostly long terminal repeats (LTRs). A total of 15,726 coding gene sequences were putatively identified with the BUSCO score of 98.7%. There are 142 genes encoding plant cell wall degrading enzymes (PCWDEs) in the genome, and 52, 39, 30, 11, 8, and 2 genes related to lignin, cellulose, hemicellulose, pectin, chitin, and cutin degradation, respectively. Comparative genomic analysis revealed that S. rugosoannulata is superior in utilizing aldehyde-containing lignins and is possible to utilize algae during the cultivation.

Identification and Functional Analysis of Acyl‑Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica: Ruigang Yang , Hui Wang , Lingyun Zhu , Lvyun Zhu , Tianzhong Liu , Dongyi Zhang; J. Microbiol. 2023;61(1):95-107. Published online January 31, 2023; DOI: https://doi.org/10.1007/s12275-022-00001-9

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Abstract: The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Metformin Regulates Gut Microbiota Abundance to Suppress M2 Skewing of Macrophages and Colorectal Tumorigenesis in Mice: Linfeng Fan , Xiangfu Zeng , Guofeng Xu; J. Microbiol. 2023;61(1):109-120. Published online January 26, 2023; DOI: https://doi.org/10.1007/s12275-022-00010-8

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Abstract: The correlation of imbalanced gut microbiota with the onset and progression of colorectal cancer (CRC) has become clear. This work investigates the effect of metformin on gut microbiota and genesis of CRC in mice. Human fecal samples were collected from healthy control (HC) donors and CRC patients. Compared to HC donors, CRC patients had reduced abundance of gut microbiota; however, they had increased abundance of detrimental Bacteroidetes. Mice were injected with azomethane (AOM) to induce colorectal tumorigenesis models. Treatment of CRC patients-sourced fecal microbiota promoted tumorigenesis, and it increased the expression of Ki67, β-catenin, COX-2, and Cyclin D1 in mouse colon tissues. Further treatment of metformin blocked the colorectal tumorigenesis in mice. Fecal microbiota from the metformin-treated mice was collected, which showed decreased Bacteroidetes abundance and suppressed AOM-induced colorectal tumorigenesis in mice as well. Moreover, the metformin- modified microbiota promoted the M1 macrophage-related markers IL-6 and iNOS but suppressed the M2 macrophage-related markers IL-4R and Arg1 in mouse colon tissues. In conclusion, this study suggests that metformin-mediated gut microbiota alteration suppresses macrophage M2 polarization to block colorectal tumorigenesis.

Potential Use of Mycobacterium paragordonae for Antimycobacterial Drug Screening Systems: Ga-Yeong Cha , Hyejun Seo , Jaehun Oh , Byoung-Jun Kim , Bum-Joon Kim; J. Microbiol. 2023;61(1):121-129. Published online January 31, 2023; DOI: https://doi.org/10.1007/s12275-022-00009-1

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Abstract: Our recent genome-based study indicated that Mycobacterium paragordonae (Mpg) has evolved to become more adapted to an intracellular lifestyle within free-living environmental amoeba and its enhanced intracellular survival within Acanthamoeba castellanii was also proved. Here, we sought to investigate potential use of Mpg for antimycobacterial drug screening systems. Our data showed that Mpg is more susceptible to various antibiotics compared to the close species M. marinum (Mmar) and M. gordonae, further supporting its intracellular lifestyle in environments, which would explain its protection from environmental insults. In addition, we developed two bacterial whole-cell-based drug screening systems using a recombinant Mpg stain harboring a luciferase reporter vector (rMpg-LuxG13): one for direct application to rMpg-LuxG13 and the other for drug screening via the interaction of rMpg-LuxG13 with A. castellanii. Direct application to rMpg-LuxG13 showed lower inhibitory concentration 50 ( IC50) values of rifampin, isoniazid, clarithromycin, and ciprofloxacin against Mpg compared to Mmar. Application of drug screening system via the interaction of rMpg-LuxG13 with A. castellanii also exhibited lower IC50 values for rifampin against Mpg compared to Mmar. In conclusion, our data indicate that Mpg is more susceptible to various antibiotics than other strains. In addition, our data also demonstrate the feasibility of two whole cellbased drug screening systems using rMpg-LuxG13 strain for the discovery of novel anti-mycobacterial drugs.

Recombinant Protein Mimicking the Antigenic Structure of the Viral Surface Envelope Protein Reinforces Induction of an Antigen‑Specific and Virus‑Neutralizing Immune Response Against Dengue Virus: Ju Kim , Tae Young Lim , Jisang Park , Yong&#; J. Microbiol. 2023;61(1):131-143. Published online February 1, 2023; DOI: https://doi.org/10.1007/s12275-023-00021-z

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Abstract: Dengue virus (DENV), belonging to the family Flaviviridae, is the causative agent of dengue and comprises four serotypes. A second heterologous DENV infection is a critical risk factor for severe dengue, and no effective vaccine is available to prevent infection by all four DENV serotypes. Recombinant DENV vaccines are primarily based on the envelope proteins, prM and E. The E protein and its envelope domain III (EDIII) have been investigated as candidate antigens (Ags) for recombinant subunit vaccines. However, most EDIII-based Ags are monomers that do not display the cognate antigenic structure of E protein, which is essential for induction of virus-neutralizing immunity. Here, we developed recombinant DENV-2 envelope domain (r2ED) protein as an Ag that mimics the quaternary structure of E protein on the DENV surface. We confirmed that r2ED retained the conformational epitope displayed at the E-dimer interface, which reportedly exhibits broad virus-neutralizing capacity, without displaying the fusion loop epitope that causes antibody (Ab)-dependent enhancement. Furthermore, compared with EDIII alone, r2ED elicited stronger Ag-specific and cross-reactive neutralizing Ab and T cell-mediated immune responses in mice. This Ag-specific immunity was maintained at an elevated level 6 months after the last immunization, suggesting sustained Ag-specific immune memory. Taken together, these observations suggest that r2ED could be used to develop an improved subunit vaccine capable of inducing a broadly cross-reactive and long-lasting immune response against DENV infection.

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