Function of the conserved FHIPEP domain of the flagellar type III export apparatus, protein FlhA

Abstract

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.

Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

“…Barker et al recently carried out a mutational study targeted to the CD-1 domain (Barker et al , 2016). Their study explored the probable role of the domain in regulating the sorting of substrates into the export pore, and although the focus was not on the mechanism of coupling to the gradient, their observations are consistent with the present results and with the proposal that the R147/R154/D158 group is involved in coupling.…”

“…One of the suppressing mutations here (R85H) was also found in the recent study of Barker et al . (Barker et al , 2016). This implies that a non-protonatable group is not essential at position 208, provided certain other mutations are present some distance away (R85 and A257 are both predicted to lie near the middles of membrane segments) (Fig.…”

“…(Hara et al , 2011) undertook a mutational analysis of eight conserved protonatable residues in the trans-membrane segments of FlhA and found that FlhA residue D208 is important for function. A recent mutational analysis by Barker et al (Barker et al , 2016) was focused on a cytoplasmic domain of FlhA, and although the question of proton coupling was not addressed explicitly, several potentially proton-binding residues were found to be important. In the present study, we sought to identify functionally important proton-binding residues of the T3S apparatus by systematic mutational analysis of the conserved acidic and basic residues in all of the membrane components.…”

Molecular Models for the Core Components of the Flagellar Type-III Secretion Complex

“…This region is well conserved and has been the subject of genetic studies [59, 60]. In particular, ASP 208 is juxta-membrane on helix 5 and has been hypothesised to bind a proton as part of the proton influx pathway.…”

“…Further from the membrane, LYS 203 may interact with the first cytoplasmic loop of FliR as suggested by suppressor mutations which partially restore motility [59]. VAL 151, also in the loop, may have a role in substrate selection [60]. …”