Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects
Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects
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DOI：
10.1128/jb.183.3.1078-1084.2001
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发表时间：
2001-02-01
影响因子：
3.2
通讯作者：
Limberger, RJ
中科院分区：
文献类型：
--
作者：
Izard, J;Samsonoff, WA;Limberger, RJ
在齿密螺旋体中，在细胞分裂过程的所有阶段，细胞质细丝的带状结构横跨细胞质。插入失活是确定细胞质细丝功能的第一步。构建了含有部分CFPA的自杀质粒，并在基因开头附近插入了非极性红霉素抗性盒(ermF和ermAM)。将该质粒电击入齿纹夜蛾中，筛选出染色体拷贝插入失活的双交叉重组体。免疫印迹和电子显微镜证实该突变体缺乏细胞质细丝，进一步用暗视野显微镜分析细胞形态，并通过两种荧光染料与DNA的结合来评估细胞核酸的分布。与野生型细胞中DNA在细胞质中的均匀分布相比，细胞质细丝蛋白缺失突变体表现出多效性缺陷，包括高度浓缩的染色体DNA。此外，细胞质细丝缺陷突变体形成了细胞链，这些细胞在琼脂糖凝胶中的伸展减少，这可能是由于细胞长度异常所致。细胞链和高度浓缩的染色体DNA表明，胞质细丝可能参与了密螺旋体的染色体结构、分离或细胞分裂过程。
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.