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
Restoration of wild-type motility to flagellin-knockout Escherichia coli by varying promoter, copy number and induction strength in plasmid-based expression of flagellin https://t.co/DP03dY7vJt #CRBIOTECH #Biotech #DNA— INPST (@_INPST) April 12, 2020
Other social media channels: https://t.co/WO0pc6I3Q9 pic.twitter.com/IUpo9Dn3tb
Why publish with Current Research in Biotechnology? Quality. Speed. Visibility. https://t.co/xj0ri5DgNI #CRBIOTECH #INPST pic.twitter.com/jcIVOavjjf— Atanas G. Atanasov (@_atanas_) June 26, 2019
The International Natural Product Sciences Taskforce (INPST) maintains up-to-date lists with conferences, grants and funding opportunities, jobs and open positions, and journal special issues with relevance for the area of phytochemistry and food chemistry, pharmacology, biotechnology, medicine and pharmacognosy research, and natural product science.
Join for free INPST as a member