Journal of Microbiology

Journal Articles

Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11: Wonsik Lee; J. Microbiol. 2022;60(12):1123-1129. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2476-2

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Abstract: Transposon mutant libraries are an important resource to study bacterial metabolism and pathogenesis. The fitness analysis of mutants in the libraries under various growth conditions provides important clues to study the physiology and biogenesis of structural components of a bacterial cell. A transposon library in conjunction with next-generation sequencing techniques, collectively named transposon sequencing (Tnseq), enables high-throughput genome profiling and synthetic lethality analysis. Tn-seq has also been used to identify essential genes and to study the mode of action of antibacterials. To construct a high-density transposon mutant library, an efficient delivery system for transposition in a model bacterium is essential. Here, I describe a detailed protocol for generating a high-density phage-based transposon mutant library in a Staphylococcus aureus strain, and this protocol is readily applicable to other S. aureus strains including USA300 and MW2.

Lysobacter arenosi sp. nov. and Lysobacter solisilvae sp. nov. isolated from soil: Kyeong Ryeol Kim† , Kyung Hyun Kim† , Shehzad Abid Khan , Hyung Min Kim , Dong Min Han , Che Ok Jeon; J. Microbiol. 2021;59(8):709-718. Published online June 1, 2021; DOI: https://doi.org/10.1007/s12275-021-1156-y

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Abstract: Two Gram-stain negative, yellow-pigmented, and mesophilic bacteria, designated strains R7T and R19T, were isolated from sandy and forest soil, South Korea, respectively. Both strains were non-motile rods showing catalase- and oxidase-positive activities. Both strains were shown to grow at 10–37°C and pH 6.0–9.0, and in the presence of 0–1.5% (w/v) NaCl. Strain R7T contained iso-C14:0, iso-C15:0, iso-C16:0, and summed feature 9 (comprising C16:0 10-methyl and/or iso-C17:1 ω9c), whereas strain R19T contained iso-C11:0 3-OH, C16:1 ω7c alcohol, iso-C11:0, iso-C15:0, iso-C16:0, and summed feature 9 (comprising C16:0 10-methyl and/or iso-C17:1 ω9c) as major cellular fatty acids (> 5%). Both strains contained ubiquinone- 8 as the sole isoprenoid quinone and phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid as the major polar lipids. The DNA G + C contents of strains R7T and R19T calculated from their genomes were 66.9 mol% and 68.9 mol%, respectively. Strains R7T and R19T were most closely related to Lysobacter panacisoli C8-1T and Lysobacter niabensis GH34-4T with 98.7% and 97.8% 16S rRNA sequence similarities, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains R7T and R19T formed distinct phylogenetic lineages within the genus Lysobacter. Based on phenotypic, chemotaxonomic, and molecular features, strains R7T and R19T represent novel species of the genus Lysobacter, for which the names Lysobacter arenosi sp. nov. and Lysobacter solisilvae sp. nov. are proposed. The type strains of L. arenosi and L. solisilvae are R7T (= KACC 21663T = JCM 34257T) and R19T (= KACC 21767T = JCM 34258T), respectively.

Full-repertoire comparison of the microscopic objects composing the human gut microbiome with sequenced and cultured communities: Edmond Kuete Yimagou , Jean-Pierre Baudoin , Rita Abou Abdallah , Fabrizio Di Pinto , Jacques Yaacoub Bou Khalil , Didier Raoult; J. Microbiol. 2020;58(5):377-386. Published online April 11, 2020; DOI: https://doi.org/10.1007/s12275-020-9365-3

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Abstract: The study of the human gut microbiome is essential in microbiology and infectious diseases as specific alterations in the gut microbiome might be associated with various pathologies, such as chronic inflammatory disease, intestinal infection and colorectal cancer. To identify such dysregulations, several strategies are being used to create a repertoire of the microorganisms composing the human gut microbiome. In this study, we used the “microscomics” approach, which consists of creating an ultrastructural repertoire of all the cell-like objects composing stool samples from healthy donors using transmission electron microscopy (TEM). We used TEM to screen ultrathin sections of 8 resin-embedded stool samples. After exploring hundreds of micrographs, we managed to elaborate ultrastructural categories based on morphological criteria or features. This approach explained many inconsistencies observed with other techniques, such as metagenomics and culturomics. We highlighted the value of our cultureindependent approach by comparing our microscopic images to those of cultured bacteria and those reported in the literature. This study helped to detect “minimicrobes” Candidate Phyla Radiation (CPR) for the first time in human stool samples. This “microscomics” approach is non-exhaustive but complements already existing approaches and adds important data to the puzzle of the microbiota.

Impact of small RNA RaoN on nitrosative-oxidative stress resistance and virulence of Salmonella enterica serovar Typhimurium: Sinyeon Kim , Yong Heon Lee; J. Microbiol. 2020;58(6):499-506. Published online April 11, 2020; DOI: https://doi.org/10.1007/s12275-020-0027-2

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Abstract: RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In
conclusion
, small regulatory RNA RaoN controls nitrosativeoxidative stress resistance and is required for virulence of Salmonella in mice.

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