Journal of Bacteriology, June 1999, p. 3743-3750, Vol. 181, No. 12
ISSN 0021-9193
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Ronald J. Limberger,* Linda L. Slivienski, Jacques Izard, and William A. Samsonoff

David Axelrod Institute for Public Health, Wadsworth Center, New York State Department of Health, Albany,
New York 12201-2002

The treponemal fla 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 in Treponema 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 contained tap1 interrupted with an erythromycin resistance cassette
(ermF and ermAM) was constructed. Because of potential polar effects from this cassette, a second plasmid that
contained tap1 interrupted with a modified erythromycin resistance cassette that lacked the putative ermF transcription
terminator was constructed. Electroporation-mediated allelic exchange incorporated the interrupted tap1 genes into
the T. denticola chromosome, creating Tap1-deficient mutants. Reverse transcriptase PCR revealed that the erythromycin
resistance cassette within tap1 did not terminate fla 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 of
Salmonella typhimurium.

* Corresponding author. Mailing address: David Axelrod Institute for Public Health, Wadsworth Center, New York State
Department of Health, P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-4177. Fax: (518) 486-7971.
E-mail: Ron.Limberger@wadsworth.org.

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