Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks.

Treponema denticola is an anaerobic spirochete that is associated with periodontal disease (32, 33). As with all spirochetes, the structure and motility of T. denticola are unusual and enable movement in highly viscous environments (17, 18). A unique feature of T. denticola (and all spirochetes) is the location of the flagellar filaments within the periplasmic space, although under certain conditions they may protrude from the cell (3, 15). The T. denticola flagellar filaments are complex, consisting of three core proteins (FlaB1, FlaB2, and FlaB3) and a major sheath protein, FlaA (29). However, the general structure of the flagellum appears similar to that of other bacteria, since it consists of a basal body, rod, flagellar hook, and filament (25, 29). In contrast to the structural similarities, DNA sequencing and transcription studies have clearly shown that the organization and expression of motility genes are unique in the spirochetes (7–10, 12, 13, 22). Motility of spirochetes is complex and likely requires approximately 50 genes, many of which are organized into several large operons (7–10, 12, 13, 22). The extensive treponemal fla operon in Treponema phagedenis and Treponema pallidum comprises genes directly associated with motility, including the flagellar hook gene (flgE) and flagellar motor genes (motA and motB), in addition to genes of unknown function (tap1) (12, 22). Previous studies have shown that T. phagedenis Tap1 is a soluble protein that partitions in the aqueous phase during Triton X-114 extraction and is associated with motility, because tap1 is the first gene of the fla motility operon (22). However, there has been no direct evidence for a role of tap1 in the motility of treponemes. Genetic analysis of T. denticola, like that of other spirochetes, has been hampered by a paucity of genetic tools for analysis. Recently, Li et al. showed that a gene cassette that includes ermF and ermAM in tandem (ermF-ermAM) conferred erythromycin resistance in T. denticola (ermF) and also in Escherichia coli (ermAM) (19). This cassette was inserted into flgE, and a specific mutant was created by electroporation-mediated allelic exchange of the insertionally inactivated flgE. This nonmotile mutant lacked flagellar hooks and filaments, as determined by electron microscopy. Using immunoblotting, Ruby et al. revealed that this mutant was also deficient in flagellar filament proteins (29). Specifically, FlaB polypeptides were not detected and minimal amounts of FlaA were detected in the flgE-deficient mutant. These results demonstrated the utility of the erythromycin resistance cassette in generating specific mutants for analysis. The ermF-ermAM cassette used by Li et al. was postulated by Fletcher et al. to have a potential transcription terminator located between ermF and ermAM (6). This terminator-like sequence would potentially cause polar effects when inserted within an operon, but it is not known if the terminator is functional in T. denticola. For analysis of single genes of the fla operon, it is critical to have a nonpolar cassette available for gene interruptions. Conceivably, removal of this putative terminator would permit the erythromycin resistance cassette to be used to study single genes located within operons without affecting downstream genes. Our goal was to identify the fla operon of T. denticola and interrupt the T. denticola tap1 gene with a nonpolar erythromycin resistance cassette to determine whether tap1 is involved in motility. We found that the organization of the T. denticola fla operon is similar to that for other treponemes, and interruption of T. denticola tap1 with a nonpolar cassette resulted in a nonmotile cell. The Tap1-deficient mutants possessed unusually long and variable-length flagellar hooks, suggesting that Tap1 may be involved in monitoring hook length, analogously to FliK of Salmonella typhimurium (14).