Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks
Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks
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DOI：
10.1128/jb.181.12.3743-3750.1999
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发表时间：
1999-06-01
影响因子：
3.2
通讯作者：
Samsonoff, WA
中科院分区：
文献类型：
--
作者：
Limberger, RJ;Slivienski, LL;Samsonoff, WA
密螺旋体弗拉操纵子由许多运动相关基因组成，然而，该操纵子的起始基因tap 1没有已知的功能。利用最近开发的一种在齿垢密螺旋体中产生特异性突变体的系统来确定Tap1是否是运动所必需的，T。鉴定、克隆和测序了齿垢菌tap 1和侧翼DNA，并构建了含有被红霉素抗性盒（ermF：和ermAM）中断的tap 1的自杀质粒。由于来自该盒的潜在极性效应，构建了第二个质粒，其含有被缺少推定的ermF转录终止子的经修饰的红霉素抗性盒中断的tap1。电穿孔介导的等位基因交换将中断的tap1基因并入T. denticola染色体，产生Tap1缺陷突变体。逆转录PCR显示，红霉素抗性盒内抽头1没有终止弗拉操纵子转录在任何突变体。此外，两个突变体的表型是不可区分的。这些突变体在液体培养中缺乏运动，不能在琼脂平板上扩散，并且缺乏鞭毛丝，如通过电子显微镜所确定的。免疫印迹显示，可检测的FlaB鞭毛丝蛋白的野生型相比，显着减少，然而，fbaB RNA很容易检测，转录水平似乎没有改变。缺乏丝蛋白表达的基础是未知的。免疫印迹还表明，鞭毛钩蛋白（FlgE）在Tap1缺陷突变体中合成，然而，电子显微镜显示，突变体具有不寻常的细长钩的可变长度。我们建议密螺旋体Tap1类似于FliK，FliK参与监测鼠伤寒沙门氏菌的鞭毛钩长度。
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, fbaB 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.