Point mutations that lock Salmonella typhimurium flagellar filaments in the straight right-handed and left-handed forms and their relation to filament superhelicity

We have determined the nucleotide sequence of two mutant and the parent fliC genes, encoding the protein flagellin (serotype i), of Salmonella typhimurium. The flagellar filaments of the two mutants, SJW1655 and SJW1660, are locked in the straight-right-handed (R) and straight-left-handed (L) conformations, respectively. Their normal, wild-type, parent strain is SJW1103. These mutant strains differ from the wild-type by only one base-pair: the mutation of SJW1655 occurs at nucleotide 1346 in the flagellin gene, changing a C.G pair to T.A (alanine 449 to valine). The mutation of SJW1660 occurs at nucleotide 1277, changing a G.C pair to C.G (glycine 426 to alanine). The resulting amino acid substitutions are near the C terminus predicted to form an alpha-helical coiled coil. The region contains six heptad repeats. Similar alpha-helical segments (three and four repeats long) are present near the N terminus. Alignment of the 17 flagellin sequences available to date confirms the generality of these segments. The mutations are within that portion of the sequence assigned, by proteolytic cleavage, to the middle flagellin domain whose length corresponds to the six heptad repeats found in the sequence (approximately 50 A). We have shown that these mutations are the sole cause of the straight phenotype by replacing the mutated segments with a wild-type one and restoring both superhelicity and motility.     

In vitro polymerization of flagellin excreted by a short-flagellum Salmonella typhimurium mutant

The culture medium of a short-flagellum mutant of Salmonella typhimurium contained a large amount of mutant flagellin and a small amount of strong inhibitors of flagellin polymerization. After being freed of the inhibitors, the mutant flagellin could be polymerized in vitro, although under nonphysiological conditions.     

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.     

Role of FliJ in flagellar protein export in Salmonella

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid Salmonella protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30 degrees C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30 degrees C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42 degrees C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.     

"Cap" on the tip of Salmonella flagella

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Conformational switching in the flagellar filament of Salmonella typhimurium.

The flagellar filament of the mutant Salmonella typhimurium strain SJW814 is straight, and has a right-handed twist like the filament of SJW1655. Three-dimensional reconstructions from electron micrographs of ice-embedded filaments reveal a flagellin subunit that has the same domain organization as that of SJW1655. Both show slight changes from the domain organization of the subunits from SJW1660, which possesses a straight, left-handed filament. This points to the possible role of changes in subunit conformation in the left-to-right-handed structural transition in filaments. Comparison of the left and right-handed filaments shows that the subunit's orientation and intersubunit bonding appear to change. The orientation of the subunit in the SJW814 filament is intermediate between that of SJW1655 and SJW1660. Its intermediate orientation may explain why the filaments of SJW1655 and SJW1660 are locked in one conformation, whereas the filament of SJW814 can be induced to switch by, for example, changes in pH and ionic strength.     

Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins.

Of the flagellar filamentless mutants of Salmonella typhimurium, the flaV, flaU, and flaW mutants, which are defective in hook-associated proteins, synthesized flagellin molecules, but flagella did not polymerize at the tips of the mutant hooks and were excreted into the culture medium as intact monomers.     

The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions

In this work we present evidence of an opposite regulation in the phytopathogenic bacteria Erwinia amylovora between the virulence-associated Type III secretion system (TTSS) and the flagellar system. Using loss-of-function mutants we show that motility enhanced the virulence of wild-type bacteria relative to a nonmotile mutant when sprayed on apple seedlings with unwounded leaves. Then we demonstrated through analyses of motility, flagellin export and visualization of flagellar filament that HrpL, the positive key regulator of the TTSS, also down-regulates the flagellar system. Such a dual regulation mediated by an alternative sigma factor of the TTSS appears to be a level of regulation between virulence and motility not yet described among Proteobacteria.     

Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin

The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.     

Ralstonia solanacearum needs motility for invasive virulence on tomato

Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.     

Purification and characterization of flagella from Roseburia cecicola, an obligately anaerobic bacterium

Flagella from Roseburia cecicola, an obligately anaerobic bacterium originally isolated from murine caecal mucosa, were purified by mechanical shearing followed by differential centrifugation. Purity of the flagellar preparation was determined by polyacrylamide gel electrophoresis, electron microscopy and chemical analysis. The flagella were composed of a single protein subunit (flagellin) with an estimated molecular weight of 42 000. The amino acid composition of the flagellin was similar to that of some facultatively anaerobic and aerobic bacteria.     

Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment

Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.     

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 approximately 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.     

Overexpression of Methanococcus voltae Flagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.     

The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.