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

Abstract

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.

FIG. 1

Diagram of the fla operon organization of T. denticola and 5′ upstream region. P_fla indicates the approximate location of the fla operon promoter. The ermF-ermAM cassette is indicated above the operon and is shown where it is inserted in the BglII site for creating Tap1-deficient mutants. The locations of primer pairs used for RT-PCR are indicated by arrows and are represented as follows: 1, TDW8; 2, TDW9; 3, TDW12; 4, TDW5; 5, TDWFLGEF; 6, TDWFLGER. Sequences of the primers are given in Materials and Methods.

FIG. 2

Identification of the fla operon promoter. (A) DNA sequence including the proposed −10 and −35 regions of P_fla. The large arrow indicates the start site of transcription determined by primer extension. M indicates the first amino acid of the Tap1 polypeptide. (B) Primer extension assay to determine the start site of transcription of P_fla. A, C, G, and T indicate nucleotides used to generate a size ladder with unrelated DNA. Lanes 1 and 2 contain the primer extension reaction products of 2 and 5 μl, respectively.

FIG. 2

Identification of the fla operon promoter. (A) DNA sequence including the proposed −10 and −35 regions of P_fla. The large arrow indicates the start site of transcription determined by primer extension. M indicates the first amino acid of the Tap1 polypeptide. (B) Primer extension assay to determine the start site of transcription of P_fla. A, C, G, and T indicate nucleotides used to generate a size ladder with unrelated DNA. Lanes 1 and 2 contain the primer extension reaction products of 2 and 5 μl, respectively.

FIG. 3

Comparison of the T. denticola fla promoter sequence with promoter sequences from various spirochete motility genes and consensus sigma 28 sequences.

FIG. 4

Alignment of Tap1 amino acid sequences. (A) Identical amino acids from three treponemes are boxed and shaded with SHADYBOX, which reveals the conserved region near the carboxyl terminus. The dark inverted triangle indicates the location of the point of insertion for the erythromycin resistance cassette into the T. denticola tap1 gene to generate a Tap1-deficient mutant. (B) Alignment of the conserved C-terminal region of T. denticola Tap1 with FliK of S. typhimurium (16). Identical amino acids are boxed and shaded as described above.

FIG. 5

T. denticola wild type (WT) and Tap1-deficient mutant JS97 after growth for 7 days on NOS plates containing 0.5% agarose. Approximately 0.1 μl was placed on the plate and incubated at 36°C in an anaerobic chamber.

FIG. 6

Microscopic analysis of the Tap1-deficient mutant JS97. (A) Dark-field micrograph. Bar, 5 μm. (B) Electron micrograph of a JS97 cell showing elongated hooks that do not possess flagellar filaments. The outer sheath was removed by treatment with 1% Triton X-100 reduced as indicated in the text. Bar, 100 nm. (C and D) Electron micrographs of preparations of enriched hooks from JS97. Bars, 100 nm.

FIG. 7

RT-PCR products after agarose gel electrophoresis and staining with ethidium bromide. W, wild-type T. denticola; A, Tap1-deficient mutant JS97; tap1, RT-PCR with primers 1 and 2 within tap1; tap1-flgD, RT-PCR with primers 3 and 4 downstream of the BglII site; flgE, RT-PCR with primers 5 and 6 (see Fig. 1 for the location of these primers); flaB, RT-PCR of the flagellar filament flaB gene with primers as described in Materials and Methods. Note that levels of RT-PCR products in the wild type and in JS97 are similar. Numbers at right are the molecular size markers in base pairs. Control reactions without RT did not show any bands (data not shown).

FIG. 8

Western blots with T. denticola Tap1 antiserum (A), T. pallidum FlgE antiserum (B), and T. phagedenis FlaB antiserum (C). Lanes 1, JS97; lanes 2, AS98; lanes 3, wild type. The arrow in panel A indicates the Tap1 polypeptide band. In panel B, the polypeptide ladder is a typical pattern found in treponemal hook polypeptides that may be due to cross-linking. The significance of the minor band missing in lane 3 is unknown. Numbers at right of each panel represent molecular masses in kilodaltons.