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The treponemalfla operon is comprised of numerous motility-related genes; however, the initial gene of this operon,tap1 , has no known function. A recently developed system to generate specific mutants inTreponema denticola was utilized to determine if Tap1 was essential for motility.T. denticola tap1 and flanking DNA were identified, cloned, and sequenced, and a suicide plasmid that containedtap1 interrupted with an erythromycin resistance cassette (ermF andermAM ) was constructed. Because of potential polar effects from this cassette, a second plasmid that containedtap1 interrupted with a modified erythromycin resistance cassette that lacked the putativeermF transcription terminator was constructed. Electroporation-mediated allelic exchange incorporated the interruptedtap1 genes into theT. denticola chromosome, creating Tap1-deficient mutants. Reverse transcriptase PCR revealed that the erythromycin resistance cassette withintap1 did not terminatefla operon transcription in either mutant. Moreover, the phenotypes of the two mutants were indistinguishable. These mutants lacked motion in liquid culture, were unable to spread on agar plates, and lacked flagellar filaments as determined by electron microscopy. Immunoblots revealed a marked reduction in detectable FlaB flagellar filament protein compared to that of wild type; however,flaB RNA was easily detectable, and transcription levels did not appear to be altered. The basis for the lack of filament protein expression is unknown. Immunoblotting also showed that the flagellar hook protein (FlgE) was synthesized in the Tap1-deficient mutant; however, electron microscopy revealed that the mutant possessed unusual elongated hooks of variable lengths. We propose that treponemal Tap1 is analogous to FliK, which is involved in monitoring the flagellar hook length ofSalmonella typhimurium.

Insertional Inactivation of Treponema denticola tap1 Results in a Nonmotile Mutant with Elongated Flagellar Hooks