Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis

The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the hooks are composed of a covalently cross-linked polypeptide.