Sorting of early and late flagellar subunits after docking at the membrane ATPase of the type III export pathway

Abstract

The bacterial flagellum assembles in a strict order, with structural subunits delivered to the growing flagellum by a type III export pathway. Early rod-and-hook subunits are exported before completion of the hook, at which point a subunit-specificity switch allows export of late filament subunits. This implies that in bacteria with multiple flagella at different stages of assembly, each export pathway can discriminate and sort unchaperoned early and chaperoned late subunits. To establish whether subunit sorting is distinct from subunit transition from the cytosol to the membrane, in particular docking at the membrane-associated FliI ATPase, the pathway was manipulated in vivo. When ATP hydrolysis by the FliI ATPase was disabled and when the pathway was locked into an early export state, both unchaperoned early and chaperoned late subunits stalled and accumulated at the inner membrane. Furthermore, a chaperone that attenuates late subunit export by stalling when docked at the wild-type ATPase also stalled at the ATPase in an early-locked pathway and inhibited export of early subunits in both native and early-locked pathways. These data indicate that the pathways for early and late subunits converge at the FliI ATPase, independent of ATP hydrolysis, before a distinct, separable sorting step. To ascertain the likely signals for sorting, the export of recombinant subunits was assayed. Late filament subunits unable to bind their chaperones were still sorted accurately, but chaperoned late subunits were directed through an early-locked pathway when fused to early subunit N-terminal export signal regions. Furthermore, while an early subunit signal directed export of a heterologous type III export substrate through both native and early-locked pathways, a late subunit signal only directed export via native pathways. These data suggest that subunits are distinguished not by late chaperones but by N-terminal export signals of the subunits themselves.

Supplementary Fig. 1

In vitro oligomerisation of FliI_E211A ATPase (FliI^EA). Purified variant FliI ATPase (0.5 μM) was incubated in cross-linking buffer (20 mM Hepes pH 8.0, 0.1 M NaCl, 0.1 mM ethylenediaminetetraacetic acid and 1 mM DTT) in the presence of E. coli liposomes, with (+) or without (−) 0.1 mM disuccinimidylglutamate). Aliquots were precipitated (10% trichloroacetic acid), subjected to electrophoresis through an SDS 4–10% acrylamide gradient gel and Coomassie stained.

Fig. 1

Membrane accumulation of early and late subunits in the pathway attenuated by enzymatically impaired FliI ATPase. (a) FliC export, assayed by immunoblotting, filtered supernatants from midexponential Luria broth (LB) cultures (A₆₀₀ = 1.0) of ΔfliIflgKflgM cells (made by P22 transduction combined with the method of Datsenko and Wanner²⁷) expressing in trans either wild-type FliI (FliI^WT) or variant FliI_E211A (FliI^EA) from pBAD33 (0.1% arabinose). A ΔfliIflgKflgM strain containing empty pBAD33 was shown to be nonmotile and attenuated in the export of early FliK subunit and late FliC subunit (data not shown). (b) Salmonella fliIflgKflgM cultures expressing wild-type FliI^WT or variant FliI^EA separated into membrane (m) and cytoplasmic (c) fractions.^, Immunoblotted for FliI ATPase, FlgN chaperone and subunits. (c) Separation of the membrane fractions into outer membrane (OMP; Coomassie stained) and inner membrane (NADH oxidase marker) by sucrose gradient ultracentrifugation (0.8–2.0 M^, top and bottom of the gradient indicated). Proteins immunoblotted using antisera described above.

Fig. 2

Membrane accumulation of early and late subunits in an early-locked pathway attenuated by stalling docked chaperone FlgN^rel. (a) Localisation of FlgN^rel (expressed in trans by 0.01% arabinose) in the whole cell (wc), membrane (m) and cytoplasm (c)^, of the ΔflgN and ΔfliI pathways, and in the isogenic (exp^E) early-locked pathways ΔflgEN and ΔflgEfliI. (b) Affinity copurification of stalled docking complexes by His–FlgN^rel bait (+) from native ΔflgN and early-locked (exp^E; ΔflgEN) pathways [(−) vector-only controls]. Cell extracts were incubated with Ni–NTA resin [20 mM tris(hydroxymethyl)aminomethane–HCl pH 8.0, 300 mM NaCl and 5 mM imidazole] before washing (10 mM imidazole) and elution in SDS sample buffer. FlgN chaperone, FlgK cognate subunit and ATPase complex components FliI and FliH were detected by immunoblotting.

Fig. 3

Attenuation of early and late subunits export by stalling FlgN^rel. Export of subunits by native ΔfliD (exp^E + L) and early-locked ΔflgE (exp^E) pathways containing FlgN^rel [expressed using 0.01% arabinose; (−) vector-only controls], assayed following precipitation from supernatants (snt) of midexponential LB cultures (wc, whole culture) by SDS-PAGE and immunoblotting for early (FliK and FlgD) and late (FliC and FlgK) subunits.

Fig. 4

Influence of subunit domains on sorting. (a) Export of chaperoned late subunits (FlgK and FliC) and their variants that cannot bind chaperone (FlgK_Δchap and FliC_Δchap) in export pathways that are either early locked (exp^E; ΔflgEKL ΔflgE, SJW1353 acquired from Ohnishi et al.²⁸) or native (exp^E + L; ΔflgKL or ΔfliC). Proteins from whole cells (wc) and supernatants (snt) were immunoblotted with FlgK or FliC antisera. (b) Export of FlgD and recombinant late subunits FlgK_Δsig and FliC_Δsig lacking amino acids 1–100 fused to residues 1–100 of early FlgD in export pathways (described above) that are either early locked (exp^E) or native (exp^E + L). Proteins from whole cells (wc) and supernatants (snt) were immunoblotted with FlgD, FlgK or FliC antisera. (c) Export of recombinant fusion proteins comprising putative early or late subunit N-terminal signal regions (FlgD_sig, FlgK_sig and FliC_sig; amino acids 1–100) fused to the signal-less SptP tyrosine phosphatase domain (SptP_phos; residues 161–543) in early locked (exp^E; ΔflgE) and native (exp^E + L; ΔfliC) pathways. Proteins were immunoblotted with SptP antisera (V. Koronakis, University of Cambridge). Genes encoding variant wild-type and variant FliC, FlgK, FlgD and SptP were amplified by overlap extension PCR using Salmonella chromosomal DNA as template. PCR products were inserted into XbaI–HindIII restriction sites of the pBAD18 expression vector. Recombinant genes were expressed (LB, 0.01% arabinose) and proteins were assayed as in Fig. 2b. Control experiments performed in isogenic ΔfliI and ΔflgEfliI strains (created by P22 transduction of ΔfliI allele into ΔflgE) showed that none of the recombinant proteins was exported (data not shown).