Journal of Microbiology

Journal Articles

Mycobacterium tuberculosis PE_PGRS45 (Rv2615c) Promotes Recombinant Mycobacteria Intracellular Survival via Regulation of Innate Immunity, and Inhibition of Cell Apoptosis: Tao Xu , Chutong Wang , Minying Li , Jing Wei , Zixuan He , Zhongqing Qian , Xiaojing Wang , Hongtao Wang; J. Microbiol. 2024;62(1):49-62. Published online February 9, 2024; DOI: https://doi.org/10.1007/s12275-023-00101-0

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Abstract: Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.

[Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract: Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Review

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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2 Citations

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.

Journal Articles

Assessing the microcystins concentration through optimized protein phosphatase inhibition assay in environmental samples: Kyoung-Hee Oh , Kung-Min Beak , Yuna Shin , Young-Cheol Cho; J. Microbiol. 2022;60(6):602-609. Published online April 30, 2022; DOI: https://doi.org/10.1007/s12275-022-2020-4

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5 Citations

Abstract: Protein phosphatase (PPase) inhibition assay (PPIA) is widely used to analyze the concentration of microcystins (MCs) because it is comparatively less expensive and faster than other assays. This study aimed to optimize the PPIA by determining a suitable reaction terminator and an optimal methanol concentration in the sample. The most suitable reaction time was 90 min, with the corresponding methanol concentration in the sample being 15% or less. When p-nitrophenyl phosphate (pNPP) was used as a substrate, copper chloride solution was suitably used as a reaction terminator, and when 4- methylumbelliferyl phosphate (MUP) was used, a glycine buffer not only increased the measurement sensitivity of the reaction product but also terminated the enzymatic reaction. When PPase 1 and MUP were used as an enzyme and a substrate, respectively, the limit of quantitation for MC-leucine/ arginine (LR) was 0.02 μg/L, whereas it was 0.1 μg/L when pNPP was used as a substrate. The proposed method facilitated the measurement of MC-LR concentration without additional pretreatments, such as concentration or purification; therefore, this method was suitable and feasible for the continuous monitoring of MCs in drinking water.

[PROTOCOL] Flow cytometric monitoring of the bacterial phenotypic diversity in aquatic ecosystems: Jin-Kyung Hong , Soo Bin Kim , Seok Hyun Ahn , Yongjoo Choi , Tae Kwon Lee; J. Microbiol. 2021;59(10):879-885. Published online September 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1443-7

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Abstract: Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using nextgeneration 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems.

Raman spectroscopy reveals alteration of spore compositions under different nutritional conditions in Lysinibacillus boronitolerans YS11: Youngung Ryu , Minyoung Hong , Soo Bin Kim , Tae Kwon Lee , Woojun Park; J. Microbiol. 2021;59(5):491-499. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0679-6

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6 Citations

Abstract: Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation mediumTM; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8–2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (~1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates.

Patterns and drivers of Vibrio isolates phylogenetic diversity in the Beibu Gulf, China: Xing Chen , Hong Du , Si Chen , Xiaoli Li , Huaxian Zhao , Qiangsheng Xu , Jinli Tang , Gonglingxia Jiang , Shuqi Zou , Ke Dong , Jonathan M. Adams , Nan Li , Chengjian Jiang; J. Microbiol. 2020;58(12):998-1009. Published online October 23, 2020; DOI: https://doi.org/10.1007/s12275-020-0293-z

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Abstract: Members of the genus Vibrio are ubiquitous in aquatic environments and can be found either in a culturable or a viable but nonculturable (VBNC) state. Despite widespread concerns as to how to define the occurrence and dynamics of Vibrio populations by culture-independent approaches, further physiological research and relevant biotechnological developments will require the isolation and cultivation of the microbes from various environments. The present work provides data and perspectives on our understanding of culturable Vibrio community structure and diversity in the Beibu Gulf. Finally, we isolated 1,037 strains of Vibrio from 45 samples and identified 18 different species. Vibrio alginolyticus, V. cyclitrophicus, V. tasmaniensis, V. brasiliensis, and V. splendidus were the dominant species that had regional distribution characteristics. The correlation between the quantitative distribution and community structure of culturable Vibrio and environmental factors varied with the Vibrio species and geographical locations. Among them, salinity, nitrogen, and phosphorus were the main factors affecting the diversity of culturable Vibrio. These results help to fill a knowledge gap on Vibrio diversity and provide data for predicting and controlling pathogenic Vibrio outbreaks in the Beibu Gulf.

Phenotypic characterization of a conserved inner membrane protein YhcB in Escherichia coli: Chul Gi Sung , Umji Choi , Chang-Ro Lee; J. Microbiol. 2020;58(7):598-605. Published online April 22, 2020; DOI: https://doi.org/10.1007/s12275-020-0078-4

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8 Citations

Abstract: Although bacteria have diverse membrane proteins, the function of many of them remains unknown or uncertain even in Escherichia coli. In this study, to investigate the function of hypothetical membrane proteins, genome-wide analysis of phenotypes of hypothetical membrane proteins was performed under various envelope stresses. Several genes responsible for adaptation to envelope stresses were identified. Among them, deletion of YhcB, a conserved inner membrane protein of unknown function, caused high sensitivities to various envelope stresses and increased membrane permeability, and caused growth defect under normal growth conditions. Furthermore, yhcB deletion resulted in morphological aberration, such as branched shape, and cell division defects, such as filamentous growth and the generation of chromosome- less cells. The analysis of antibiotic susceptibility showed that the yhcB mutant was highly susceptible to various anti-folate antibiotics. Notably, all phenotypes of the yhcB mutant were completely or significantly restored by YhcB without the transmembrane domain, indicating that the localization of YhcB on the inner membrane is dispensable for its function. Taken together, our results demonstrate that YhcB is involved in cell morphology and cell division in a membrane localization-independent manner.

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