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

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.     

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H⁺–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na⁺ increased it dramatically. Phenamil, a blocker of Na⁺ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na⁺ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na⁺ channel. In wild-type cells, however, neither Na⁺ nor phenamil affected protein export, indicating that the Na⁺ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na⁺ concentration.     

FlgM Is Secreted by the Flagellar Export Apparatus in Bacillus subtilis

The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σ^D-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK.     

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.     

Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking

Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed `semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis.     

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti‐σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far‐UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.     

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

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.     

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

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH_X-FliI₆ ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues—Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83—of FliJ. A structural model of the FliI₆-FliJ ring complex suggests that they extend out of the FliI₆ ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.     

Analysis of the cytoplasmic domains of Salmonella FlhA and interactions with components of the flagellar export machinery

Most flagellar proteins are exported via a type III export apparatus which, in part, consists of the membrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR and is housed within the membrane-supramembrane ring formed by FliF subunits. Salmonella FlhA is a 692-residue integral membrane protein with eight predicted transmembrane spans. Its function is not understood, but it is necessary for flagellar export. We have created mutants in which potentially important sequences were deleted. FlhA lacking the amino-terminal sequence prior to the first transmembrane span failed to complement and was dominant negative, suggesting that the sequence is required for function. Similar effects were seen in a variant lacking a highly conserved domain (FHIPEP) within a putative cytoplasmic loop. Scanning deletion analysis of the cytoplasmic domain (FlhAc) demonstrated that substantially all of FlhAc is required for efficient function. Affinity blotting showed that FlhA interacts with several other export apparatus membrane proteins. The implications of these findings are discussed, and a model of FlhA within the export apparatus is presented.     

Interactions between flagellar and type III secretion proteins in <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis>

Background  

Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown.     

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose‐mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA^Glc is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA^Glc is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose‐rich area.     

Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus

The MS ring of the flagellar basal body of Salmonella is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliF mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in flhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the fliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.