Journal of Microbiology

Journal Article

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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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.

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Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus
Sally W. Yousief, Nader Abdelmalek, Bianca Paglietti
Scientific Reports.2024;[Epub] CrossRef

Research Support, Non-U.S. Gov't

Translocation of Green Fluorescent Protein to Cyanobacterial Periplasm Using Ice Nucleation Protein: Wipa Chungjatupornchai , Sirirat Fa-aroonsawat; J. Microbiol. 2009;47(2):187-192. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0188-x

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Abstract

The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.

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