Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA

Abstract

For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

Distinct Roles of Highly Conserved Charged Residues at the MotA-FliG Interface in Bacterial Flagellar Motor Rotation

“…QuikChange site-directed mutagenesis was performed as described in the manufacturer's instructions (Stratagene). DNA sequencing was carried out as described previously (39). L broth (LB) and motility medium were prepared as described previously (40,41).…”

Interaction between FliJ and FlhA, Components of the Bacterial Flagellar Type III Export Apparatus

“…The bacterial strains and plasmids used in the present study are listed in Table 1. DNA manipulations, site-directed mutagenesis, and DNA sequencing were carried out as described previously (34,35). L broth (LB) and soft tryptone agar plates were prepared as described previously (13,36).…”

Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

“…Bacterial strains and plasmids used in this study are listed in Table 1. Salmonella ⌬fliH ⌬fliN and ⌬fliH ⌬flhA double null strains were constructed using the Red homologous recombination system (6) as described previously (13). L-broth (LB), Tbroth (TB), and motility agar plates were prepared as described previously (31,32).…”

“…Single cysteine and amber mutants of FliH were generated through PCR-based site-directed mutagenesis as described previously (13). All FliH substitutions were confirmed by DNA sequencing (BigDye v3.1, 3130 Genetic Analyzer; Applied Biosystems).…”

“…FlhB C acts as the export specificity switch to mediate the ordered export of the axial flagellar components during flagellar assembly (19). The specific roles of FliO, FliP, FliQ, and FliR remain unknown, although it has been shown that FliO, FliP, and FliQ interact with FlhA (24) and that FliR interacts with FlhA and FlhB (13,45). Recently, it has been shown that the fliO null mutation, which leads to a drastic reduction in motility, is bypassed by the extragenic fliP suppressor mutation, proposing that FliO has an important functional role in regulating FliP stability (2).…”