The specialised signal recognition particle family guanosine 5c-triphosphate (GTP)-binding
protein FlhF is required for the correct localisation of flagella in several bacterial
species. Here, we characterise the regions of Vibrio cholerae FlhF that are required
for its function and targeting to the old cell pole, and we present evidence for a
mechanism by which FlhF establishes flagellum polar localisation. Substitution of
residues in FlhF nucleotide-binding motifs reduced GTP binding and the efficiency
of flagellum biogenesis, and caused flagellum mislocalisation. However, replacement
of conserved putative catalytic residues (D(321), R(324), and Q(330)) had no effect,
suggesting that while GTP binding influences FlhF function, GTPase activity might
not be essential. FlhF associated with the inner membrane in the absence of other
flagellar proteins, and a functional FlhF-green fluorescent protein fusion was targeted
to the old cell pole where the flagellum is localised. FlhF targeting to the pole
was intrinsic, as no other flagellar proteins were needed. Neither the FlhF C-terminal
GTP-binding region nor the N-terminal 166-residue B-region was required for polar
localisation, though they were essential for FlhF function. Deletion of the central
108-residue N-region of FlhF, comprising alpha-helices N1-N4, did however severely
reduce the efficiency of FlhF polar targeting, as well as FlhF function. The intrinsic
localisation of FlhF to the old cell pole membrane suggested that FlhF might function
at an early stage of flagellum assembly; to test this, we assessed the effect of FlhF
on the localisation of the earliest flagellar structural component, the membrane-supramembrane
ring protein FliF. Recruitment of FliF to the pole required only FlhF and no other
flagellar proteins. FliF polar targeting was abolished in the absence of FlhF and
by deletion of the FlhF B-domain or GTP-binding region. Our data indicate that FlhF
establishes the site of flagellum assembly at the old cell pole membrane by recruiting
the earliest flagellar structural component FliF.