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- A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae
- Kiwon Rhee , Hyungseok Choi , Keun P. Kim , Jeong H. Joo
- J. Microbiol. 2023;61(11):939-951. Published online December 11, 2023
- DOI: https://doi.org/10.1007/s12275-023-00094-w
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Abstract
- Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while twodimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.
- Whole genome and RNA sequencing of oral commensal bacterium Streptococcus anginosus subsp. anginosus with vancomycin tolerance
- Kyu Hwan Kwack , Jae-Hyung Lee , Ji-Hoi Moon
- J. Microbiol. 2022;60(2):167-176. Published online January 7, 2022
- DOI: https://doi.org/10.1007/s12275-022-1425-4
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Abstract
- “Antibiotic tolerance” promotes the rapid subsequent evolution of “antibiotic resistance,” however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.
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