Novobiocin inhibits membrane synthesis and vacuole formation of Enterococcus faecalis protoplasts

Current Research in Microbial Sciences

2022

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Highlights More than 100 E. faecalis protoplasts were generated and microinjected into heterologous genomic DNAs. Protoplast enlargement did not stop but continued after the microinjection. Microinjection of genomic DNA into E. faecalis protoplasts frequently induced weakness in enlargement.

Section: Resultsmentioning

confidence: 99%

“…The E. faecalis protoplast enlargement requires DNA replication ( Kami et al., 2019 ; Tsuchikado et al., 2020 ). Thus, when DNA replication stops, cell enlargement also stops.…”

Section: Introductionmentioning

confidence: 99%

Effects of heterologous genome microinjection on the enlargement of Enterococcus faecalis protoplasts

Takahashi

Current Research in Microbial Sciences

2022

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Approaches for introducing large DNA molecules into bacterial cells

Journal of Applied Microbiology

2023

Engineering of the bacterial genome plays a key role in systems biology and synthetic biology. Genetic engineering of the bacterial genome involves the design and synthesis of large DNA molecules. However, functional studies of the designed and synthesized large DNA molecules are lagging. Methods for the transformation of large DNA molecules of bacterial chromosome size into bacterial cells through a single operation have not yet been established. Two major methods can be used for transferring large DNA molecules of bacterial chromosome size into bacterial cells: transformation mediated by liposomes or by microinjection. In both methods, cell wall (peptidoglycan layer)-deficient cells (L-form, protoplast, or spheroplast) should be used as the bacterial host cells. We succeeded in transferring a heterologous bacterial genome into an enlarged bacterial protoplast using a micromanipulator. This method for transferring large DNA molecules into bacterial cells through a single operation will contribute to both fundamental and applied research in microbial genome science.

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

2020

IJMS

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Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.