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- [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.
- Paenibacillus lycopersici sp. nov. and Paenibacillus rhizovicinus sp. nov., isolated from the rhizosphere of tomato (Solanum lycopersicum)
- Shin Ae Lee , Tae-Wan Kim , Mee-Kyung Sang , Jaekyeong Song , Soon-Wo Kwon , Hang-Yeon Weon
- J. Microbiol. 2020;58(10):832-840. Published online September 29, 2020
- DOI: https://doi.org/10.1007/s12275-020-0258-2
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Abstract
- Two Gram-stain-positive, rod-shaped, endospore-forming bacteria, designated 12200R-189T and 14171R-81T were isolated from the rhizosphere of tomato plants. The 16S rRNA gene sequence similarity between strains 12200R-189T and 14171R-81T were 97.2%. Both strains showed the highest 16S rRNA gene sequence similarities to Paenibacillus sacheonensis SY01T (96.3% and 98.0%, respectively). The genome of strain 12200R-189T was approximately 6.7 Mb in size with 5,750 protein-coding genes (CDSs) and the G + C content was 58.1 mol%, whereas that of strain 14171R-81T comprised one chromosome of 7.0 Mb and two plasmids (0.2 Mb each) with 6,595 CDSs and the G + C content was 54.5 mol%. Comparative genome analysis revealed that average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among 12200R-189T, 14171R-81T, and other closely related species were below the cut-off levels 95% and 70%, respectively. Strain 12200R-189T grew at a temperature range of 15–40°C, pH 6.0–9.0, and 0–3% NaCl (w/v), whereas strain 14171R-81T grew at a temperature range of 10–37°C, pH 6.0– 8.0, and 0–1% NaCl (w/v). Menaquinone-7 (MK-7) was the only isoprenoid quinone detected in both strains. The predominant cellular fatty acids (> 10%) were iso-C15:0, anteiso- C15:0, and iso-C16:0. The polar lipids of strain 12200R- 189T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), aminophospholipid (APL), phospholipid (PL), phosphatidylglycolipid (PGL), and four aminophosphoglycolipids (APGLs) and those of strain 14171R-81T were DPG, PG, PE, APL, three PLs, two PGLs, and three APGLs. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic analyses, strains 12200R- 189T and 14171R-81T represent two novel species of the genus Paenibacillus, for which the names Paenibacillus lycopersici sp. nov. and Paenibacillus rhizovicinus sp. nov. are proposed. The type strains are 12200R-189T (= KACC 19916T = CCTCC AB 2020027T) and 14171R-81T (= KACC 19915T = CCTCC AB 2020026T).
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