Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects

Abstract

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.

FIG. 1

Immunoblot using monoclonal antibody M416 against T. phagedenis CfpA. (A) T. phagedenis cell extract. (B) Lane 1, T. denticola cell extract; lane 2, T. denticola cfpA mutant cell extract. The positions of size markers are shown (in kilodaltons).

FIG. 2

Macroscopic motility test in dense medium. (A) Plate after 3 days of incubation. A liquid culture (0.1 μl) of each sample was spotted on a 0.5% agarose–NOS plate and incubated at 36°C. 1, T. denticola wild type; 2, T. denticola CfpA-deficient mutant; 3, T. denticola fliK flagellar filament-less mutant (30). (B) T. denticola wild-type from a 3-day plated culture spread in agarose and propagated. Vertical section of the plate with side illumination. Bar, 1.5 mm. (C) Plate after 7 days of incubation, same strains as in panel A. (D) Vertical section of the plate with side illumination. Bar, 1 mm. T. denticola CfpA-deficient mutant from a 7-day plated culture does spread in the agarose but in a more limited volume. (E) Vertical section of the plate with side illumination. Bar, 1 mm. T. denticola fliK flagellar filament-less mutant from a 7-day plated culture was nonmotile and did not spread in the agarose. The vertical section photographs of the plate with side illumination were obtained using an SZ40 stereo microscope with a 35-mm camera mounted on a PM-20 exposure control unit (Olympus, Melville, N.Y.).

FIG. 3

Macroscopic measurement of cell spreading over time in dense medium. A liquid culture (0.1 μl) was spotted on the center of a 0.5% agarose–NOS plate and incubated at 36°C. The colony diameter was measured using a caliper. ●, T. denticola wild-type colony; ▴, T. denticola cytoplasmic filament-less mutant colony; ○, T. denticola fliK flagellar filament-less mutant colony (30). Error bars show the range of measured colony diameters.

FIG. 4

T. denticola wild-type and CfpA-deficient cells from a mid-log-phase NOS broth culture as observed by dark-field microscopy. (A) Representative single cell of wild-type T. denticola. (B) Wild-type T. denticola undergoing cell division. (C) A rarely observed single cell of T. denticola CfpA-deficient mutant. (D) Representative cell of T. denticola CfpA-deficient mutant. Bars, 5 μm.

FIG. 5

Electron micrograph of wild-type T. denticola (A) and cytoplasmic filament-less mutant (B) cells. The outer membrane has been removed by detergent treatment of the cell using sodium deoxycholate 1% for 10 min, and the periplasmic flagellar filaments are freed. No cytoplasmic filaments were detected in the CfpA-deficient mutant. PFF, periplasmic flagellar filament; CF, cytoplasmic filament. Bar, 1 μM.

FIG. 6

Chromosomal DNA visualization and localization with Hoechst 33342 dye. (A) Dark-field microscopy of wild-type T. denticola and (B) the corresponding image by fluorescence. (C) Group of wild-type T. denticola cells showing a similar uniform DNA distribution. (D) Dark-field microscopy of T. denticola CfpA-deficient cells and (E) corresponding image by fluorescence. Arrows indicate condensed DNA areas. (F) T. denticola CfpA-deficient mutant cells, showing a typical pattern of distribution. Arrows indicate condensed DNA areas. Bars, 10 μm.

FIG. 7

Schematic representation of the distribution of condensed chromosomal DNA in the T. denticola CfpA-minus mutant. (A) Distribution in a single cytoplasmic cylinder (142 cytoplasmic cylinders). E1 and E2, extremities of the cytoplasmic cylinder; C, center region. For example, in Fig. 5F, the upper right corner cell has one cytoplasmic cylinder and bears the pattern E1-C-E2. (B) Distribution in two adjacent cytoplasmic cylinders (55 cytoplasmic cylinder pairs). The bar between the two schematic cytoplasmic cylinders indicates that they are under the same outer membrane. E1 to E4, extremities of the cytoplasmic cylinder; C1 and C2, center regions. For example, in Fig. 5F, the lower left corner cell bears two patterns, E4-C1 and C1-C2.