FliO regulation of FliP in the formation of the Salmonella enterica flagellum

Clive S Barker; Irina V Meshcheryakova; Alla S Kostyukova; Fadel A Samatey

doi:10.1371/journal.pgen.1001143

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FliO regulation of FliP in the formation of the Salmonella enterica flagellum

Journal article

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FliO regulation of FliP in the formation of the Salmonella enterica flagellum

Clive S Barker, Irina V Meshcheryakova, Alla S Kostyukova and Fadel A Samatey

PLoS genetics, Vol.6(9), pp.e1001143-e1001143

09/30/2010

DOI: https://doi.org/10.1371/journal.pgen.1001143

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https://hdl.handle.net/2376/114188

PMCID: PMC2947984

PMID: 20941389

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Abstract

Bacterial Proteins - chemistry

Molecular Sequence Data

Cytoplasm - metabolism

Salmonella enterica - cytology

Structure-Activity Relationship

Genetic Complementation Test

Periplasm - drug effects

Salmonella enterica - drug effects

DNA Mutational Analysis

Periplasm - metabolism

Gene Deletion

Membrane Proteins - metabolism

Circular Dichroism

Agar - pharmacology

Protein Structure, Tertiary

Amino Acid Sequence

Leucine - metabolism

Movement - drug effects

Bacterial Proteins - genetics

Mutant Proteins - metabolism

Salmonella enterica - metabolism

Flagella - metabolism

Membrane Proteins - chemistry

Mutant Proteins - chemistry

Flagella - drug effects

Bacterial Proteins - metabolism

Cytoplasm - drug effects

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO₄₃-₁₂₅ or FliO₁-₉₅ was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO₄₃-₁₂₅, should contain beta-structure and alpha-helices. FliO₄₃-₁₂₅-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.

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Title: FliO regulation of FliP in the formation of the Salmonella enterica flagellum
Creators: Clive S Barker - Trans-Membrane Trafficking Unit, Okinawa Institute of Science and Technology, Okinawa, Japan
Irina V Meshcheryakova
Alla S Kostyukova
Fadel A Samatey
Publication Details: PLoS genetics, Vol.6(9), pp.e1001143-e1001143
Academic Unit: Chemical Engineering and Bioengineering, School of
Publisher: United States
Identifiers: 99900547438501842
Language: English
Resource Type: Journal article