Tohru Minamino

Interactions among components of the Salmonella flagellar export apparatus and its substrates

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild‐type cells and inhibition of the export of substrates such as the hook protein FlgE. Co‐overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His‐FLAG‐tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook‐type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament‐type export substrates FlgK and FlgL (hook–filament junction proteins) and FliC (flagellin). We tested for protein–protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook‐type substrates were strong, whereas those with filament‐type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP‐dependent process mediated by FliI.

FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase activity

Both FliH and the ATPase FliI are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N‐terminus of FliI interacts with FliH and that this interaction is important for the ATPase function of the C‐terminal domain of FliI. N‐terminally histidine‐tagged, wild‐type FliI retarded untagged FliH in a Ni‐NTA affinity chromatography assay, as did N‐His‐tagged versions of FliI carrying catalytic mutations. In contrast, N‐His‐tagged FliI carrying the double mutation R7C/L12P did not, further indicating that the N‐terminus of FliI is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and FliI form a complex. Analytical gel filtration with in‐line multiangle light scattering indicated that FliH alone forms a dimer, FliI alone remains as a monomer, and FliH and FliI together form a (FliH)2FliI complex. Ni‐NTA affinity chromatography using N‐His‐tagged FliH and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH–FliI complex was about 10‐fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.

FLIK, THE PROTEIN RESPONSIBLE FOR FLAGELLAR HOOK LENGTH CONTROL IN SALMONELLA, IS EXPORTED DURING HOOK ASSEMBLY

In wild‐type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at ≈ 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild‐type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook‐filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N‐terminal region of FliK, but not by C‐terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook‐length control and the switching of export substrate specificity.

Molecular dissection of Salmonella FliH, a regulator of the ATPase FliI and the type III flagellar protein export pathway

FliH is a soluble component of the flagellar export apparatus that binds to the ATPase FliI, and negatively regulates its activity. The 235‐amino‐acid FliH dimerizes and interacts with FliI to form a hetero‐trimeric (FliH)2FliI complex. In the present work, the importance of different regions of FliH was examined. A set of 24 scanning deletions of 10 amino acids was constructed over the entire FliH sequence, along with several combined deletions of 40 amino acids and truncations of both N‐ and C‐termini. The mutant proteins were examined with respect to (i) complementation; (ii) dominance and multicopy effects; (iii) interaction with wild‐type FliH; (iv) interaction with FliI; (v) inhibition of the ATPase activity of FliI; and (vi) interaction with the putative general chaperone FliJ. Analysis of the deletion mutants revealed a clear functional demarcation between the FliH N‐ and C‐terminal regions. The 10‐amino‐acid deletions throughout most of the N‐terminal half of the sequence complemented and were not dominant, whereas those throughout most of the C‐terminal half did not complement and were dominant. FliI binding was disrupted by C‐terminal deletions from residue 101 onwards, indicating that the C‐terminal domain of FliH is essential for interaction with FliI. FliH dimerization was abolished by deletion of residues 101–140 in the centre of the sequence, as were complementation, dominance and interaction with FliI and FliJ. The importance of this region was confirmed by the fact that fragment FliHC2 (residues 99–235) interacted with FliH and FliI, whereas fragment FliHC1 (residues 119–235) did not. FliHC2 formed a relatively unstable complex with FliI and showed biphasic regulation of ATPase activity, suggesting that the FliH N‐terminus stabilizes the (FliH)2FliI complex. Several of the N‐terminal deletions tested permitted close to normal ATPase activity of FliI. Deletion of the last five residues of FliH caused a fivefold activation of ATPase activity, suggesting that this region of FliH governs a switch between repression and activation of FliI. Deletion of the first 10 residues of FliH abolished complementation, severely reduced its interaction with FliJ and uncoupled its role as a FliI repressor from its other export functions. Based on these data, a model is presented for the domain construction and function of FliH in complex with FliI and FliJ.

Structural Properties of FliH, an ATPase Regulatory Component of the Salmonella Type III Flagellar Export Apparatus

FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation.

BIOGENESIS OF FLAGELLA: EXPORT OF FLAGELLAR PROTEINS VIA THE FLAGELLAR MACHINE

The flagellum is a motility apparatus that many bacteria use for responding to various environmental stimuli. The major part of the flagellum is a helical filament, which can thrust the cell body through liquid when it rotates. The rotary force (torque) is generated by the basal structure (therefore called the flagellar motor), which is anchored both in the outer and inner membranes. The basal structure is made of more than 20 different proteins. The bacterial flagellum, a complicated complex made of more than 10 different proteins, is constructed through both the membranes and cell wall towards the outside of the cell. Since most of the component proteins do not retain signal peptides recognized by the general secretion pathway, they have to be secreted by their own export system. The export apparatus consists of a channel and a gate. Flagellar proteins to be exported are transported to the gate with the help of chaperones and pushed into the channel by a flagella-specific ATPase. There are two modes in substrate specificity at the gate: one for rod/hook-type proteins and one for filament-type proteins. The switching of the two modes is triggered by hook completion, which seems to be necessary for maintaining an ordered and thus economic secretion.

Assembly and Activation of the MotA/B Proton Channel Complex of the Proton-Driven Flagellar Motor of Salmonella enterica

Salmonella enterica can swim by rotating multiple flagella, which arise randomly over the cell surface (Fig. 1A). The flagellum consists of at least three parts: the basal body, the hook, and the filament. The basal body is embedded within the cell membranes and acts as a bidirectional rotary motor powered by an electrochemical potential gradient of protons across the cytoplasmic membrane (Fig. 1B, C). The hook and filament extend outwards in the cell exterior. The filament acts as a helical propeller. The hook exists between the basal body and filament and functions as a universal joint to smoothly transmit torque produced by the motor to the filament. When the motors rotate in counterclockwise direction, the cells can swim smoothly. By quick reversal rotation of the motor to clockwise direction, the cells tumble and change their swimming direction to move toward more favorable environments (Fig. 2) (Berg, 2003; Blair, 2003; Minamino et al., 2008).