Molecular Cloning and Characterization of theHelicobacter pylori fliD Gene, an Essential Factor in Flagellar Structure and Motility

ABSTRACT 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 ofH. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a ς28-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 thefliD 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. pyloriFliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.

Observations on the ultrastructure of the choanoflagellate Codosiga botrytis (Ehr.) Saville-Kent with special reference to the flagellar apparatus

The ultrastructure of the choanoflagellate Codosiga botrytis is described with particular reference to the flagellar appendages, the flagellar rootlet system, the transition zone, the basal body and accessory centrioles, and the stalk. The controversial early reports of flagellar appendages in this species have been confirmed and they have been detected in 2 further species, Salpingoeca frequentissima and Monosiga sp. The appendages consist of a delicate bilateral vane 2 mum wide on either side of the axis, composed of extremely fine overlapping or interwoven fibrils. The flagellar root system consists of a large number of radiating microtubules associated with bands of electron-dense material near the basal body; striated roots are absent. The microtubules extend from several separate foci, those in any one group originating near a composite electron-dense band, and for a distance of 300 nm from the basal body they are separated by blocks of interstitial material. The flagellar basal body forms one of a diplosome pair of centrioles. The triplet microtubules of the accessory centriole are embedded in amorphous electron-dense material and the whole is enveloped in a sheath of similar appearance. The existence of a third centriole close to the diplosome pair is also reported. The relatively complex structure of the flagellar transitional zone is described. The stalk is composed of a core of circular lacunae, which may or may not contain finger-like protoplasmic extensions of the posterior end of the cell, surrounded by a continuation of the sheath material which encloses the remainder of the protoplast. In the stalk only there is a further closely sheathing layer about 15 nm thick which is regularly striated, the spacing of the striations in shadowcast material and sections being about 3 times that measured by negative staining. The structure of choanoflagellates differs widely from that of the algal class Chrysophyceae, the group in which they are included in some classifications, and from the remainder of the algae; they do not appear to have a place in either the algae or the plant kingdom. The structure of Codosiga botrytis is briefly compared with that of sponge choanocytes and collared cells in the Metazoa and some of the possible phylogenetic implications of this are indicated.