Role of the flaR gene in flagellar hook formation in Salmonella spp.

Flagellar filaments were reconstituted by polymerization with exogenously supplied flagellin monomers at the tips of normal hooks on Salmonella cells which were missing the filaments because of mutations in either the flaL or flaU gene or the flagellin genes H1 and H2. Reconstitution did not occur at the tips of polyhooks of the flaR mutant cells. Thus, the absence of flagellar filaments in the flaR mutant cells was probably caused by the inability of the polyhooks to work as polymerization nuclei for flagellin. A Phf+ mutant which produced polyhooks with flagellar filaments was isolated from a flaR polyhook mutant. Genetic analysis of the Phf+ mutant showed that it carried an intracistronic suppressor mutation of the original flaR mutation. This result indicated that the flaR gene regulates hook length and initiates flagellin formation.     

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.     

Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase

A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli. The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined. There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only. The last, soluble-fraction-only, class was predominant in fla-lac gene fusions. In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions. These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.     

Defining functional domains within PF16: a central apparatus component required for flagellar motility

Mutations affecting the assembly and stability of the central apparatus result in flagellar paralysis. Chlamydomonas cells with mutations at the PF16 locus have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. On the basis of its mutant phenotype, sequence, and localization, PF16, a member of the armadillo repeat containing family of proteins, is involved in protein-protein interactions required for stability of the C1 microtubule and flagellar motility. Previous biochemical analysis of flagella isolated from pf16 cells demonstrated that assembly of the PF16 protein is either dependent on, or required for, the assembly of at least two other flagellar components. As a first step toward identifying functional domains in the PF16 protein that are essential for these interactions, we have characterized three mutations at the PF16 locus. In addition, we have generated deletion constructs of the PF16 gene and tested for their ability to assemble and rescue motility upon transformation of mutant pf16 cells. Our results demonstrate that the first armadillo repeat is necessary but not sufficient for assembly; that the C-122 amino acids are not required for assembly or motility; and that the repeats appear to form a single functional unit required for PF16 assembly.     

Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.     

Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor.

FliG, FliM, and FliN are three proteins of Salmonella typhimurium that affect the rotation and switching of direction of the flagellar motor. An analysis of mutant alleles of FliM has been described recently (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992). We have now analyzed a large number of mutations in the fliG and fliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively). The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate. However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly. Missense mutations in fliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene. CW-bias and CCW-bias mutations tended to segregate into separate subclusters. The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes. Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias. The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.     

Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes.

The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.     

Formation of flagella lacking outer rings by flaM, flaU, and flaY mutants of Escherichia coli.

Among flagellar mutants of Escherichia coli, flaM or flaU mutants form basal bodies lacking the outer P and L rings, whereas flaY mutants predominantly form basal bodies lacking the L ring. In these mutants, hooks and filaments are occasionally assembled onto these incomplete basal bodies. When the hook protein gene, flaFV, of Salmonella typhimurium was cloned on the multicopy plasmid pBR322 and introduced into these mutants, the efficiency with which cells assembled hooks and filaments onto the incomplete basal bodies increased significantly. Such cells formed characteristic dotted swarms on semisolid plates, indicating that cells carrying flagella without the outer rings are weakly motile because of poor function of their flagella, a low flagellar number per cell, or both of these defects. FlaV mutants also produced incomplete basal bodies lacking the outer rings, but assembly of hooks and filaments did not occur in these mutants even after introduction of the plasmid carrying flaFV of S. typhimurium. The failure in the case of flaV mutants was attributed to their inability to modify the rod tip to the structure competent for assembly of hook protein.