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Volume 154, Issue 3

Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Free

Abstract

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components.

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  • Published Online:
Keyword(s): mPEG-maleimide, methoxypolyethylene glycol 5000 maleimide
SGM
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2008-03-01
2024-04-23
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Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli
Microbiology 154, 810 (2008); https://doi.org/10.1099/mic.0.2007/013854-0
/content/journal/micro/10.1099/mic.0.2007/013854-0
/content/journal/micro/10.1099/mic.0.2007/013854-0
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