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.

Native cellular architecture of Treponema denticola revealed by cryo-electron tomography

“…Cytoplasmic filaments, formed by CfpA monomers (Izard et al, 2001), can be observed in both flagellate and aflagellate strains (Fig. 5).…”

“…The alignment of the cytoplasmic filaments and the flagellar filaments may be imposed by the overall geometry of the cells and mechanical constraints. Mutagenesis experiments indicated the absence of a relationship between those two structures (Izard et al, 2001). In a mutant strain lacking the cytoplasmic filament formed by CfpA proteins, the motility of single cells was not altered (Izard et al, 2001).…”

“…Mutagenesis experiments indicated the absence of a relationship between those two structures (Izard et al, 2001). In a mutant strain lacking the cytoplasmic filament formed by CfpA proteins, the motility of single cells was not altered (Izard et al, 2001). Possible physical contact between the two structures may exist through the anchor proteins of the cytoplasmic filaments located in the inner membrane of the cell .…”

Virulence Factors of the Oral Spirochete Treponema denticola

“…Cytoplasmic filaments are cytoplasmic structures located directly beneath the PF. A CfpA-deficient mutant displayed reduced spreading ability compared with wild-type strains, implicating involvement of cytoplasmic filaments in motility (Izard et al, 2001;Vesey and Kuramitsu, 2004).…”

Dynamics of bacterial cytoskeletal elements

“…Coiled coil rich proteins (e.g., CfpA) have also been described in the cork-screw like bacterial branch of the Spirochaetes, where a bundle of four to six cytoplasmatic filaments runs along the long axis of the long and thin cells [Izard, 2006]. CfpA is a major constituent of the structure-however, its loss affects the subcellular arrangement of the genome rather than cell shape [Izard et al, 2001]. Finally, AglZ is a coiled coil rich protein which forms filamentous structures in vitro, and is involved in motility in the Gram negative proteobacterium Myxococcus xanthus [Yang et al, 2004], which is famous for its social behaviour-cells aggregate on surfaces upon starvation and build fungus-like fruiting bodies.…”