Restoration of wild-type motility to flagellin-knockout Escherichia coli by varying promoter, copy number and induction strength in plasmid-based expression of flagellin

Abstract

Flagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. However, gene complementation studies often fail to report whether true wild-type motility was restored by expressing flagellin from a plasmid. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially (~50%) restored using the tightly regulated rhamnose promoter due to weak flagellin gene expression, but wild-type motility was regained with the T5 promoter, which, although leaky, allowed titration of induction strength. The endogenous E. coli flagellin promoter also restored wild-type motility. However, flagellin gene transcription levels increased 3.1–27.9-fold when wild-type motility was restored, indicating disturbances in the flagellar regulatory mechanisms. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

Read full text: Nicholas M. Thomson, Mark J. Pallen, Restoration of wild-type motility to flagellin-knockout Escherichia coli by varying promoter, copy number and induction strength in plasmid-based expression of flagellin, Current Research in Biotechnology, Volume 2, 2020, Pages 45-52, https://doi.org/10.1016/j.crbiot.2020.03.001

Keywords: Flagella, Motility, Promoter, Tuneable, Plasmid, Copy number, Rhamnose promoter, Flagellin-knockout, Escherichia coli, #Biotechnology, #GeneEditing.

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