The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity
The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity
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DOI：
10.1128/jb.182.23.6698-6706.2000
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发表时间：
2000-12-01
影响因子：
3.2
通讯作者：
Charon, NW
中科院分区：
文献类型：
--
作者：
Li, CH;Corum, L;Charon, NW
螺旋体周质鞭毛（PF），包括来自短螺旋体（Serpulina）、螺旋体、密螺旋体和钩端螺旋体属的那些，具有独特的结构。在大多数螺旋体中，周质鞭毛丝由至少三种蛋白（FlaB 1，FlaB 2和FlaB 3）和鞘蛋白（FlaA）组成。这些蛋白质中的每一种都由单独的基因编码。利用猪源短螺旋体作为通过等位基因交换诱变分析PF功能的模型系统，我们分析了来自先前构建的flaA：：cat、flaA：：kan和flaB 1：：kan突变体以及新构建的flaB 2：：cat和flaB 3：：cat突变体的纯化PF。我们研究了这些突变体中是否有任何一个丧失了运动性和改变了PF结构。正如先前在flaA：：cat、flaA：：kan和flaB 1：：kan突变体中发现的那样，flaB 2：：cat和flaB 3：：cat突变体仍然具有运动性，但使用菌群平板测定，所有突变体的运动性均低于野生型菌株。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹分析表明，每个突变导致组装的纯化PFs中同源基因产物的特异性丢失。与这些结果一致，北方印迹分析表明每个鞭毛丝基因是单顺反子的。cat突变体比野生型菌株和flaB 1：：kan、flaB 2：：cat和flaB 3：：cat突变体（24至25 nm）显着更薄（19.6 nm）。这些结果为FlaA在FlaB核心周围形成鞘提供了支持性遗传证据。使用高放大倍率暗视野显微镜，我们还发现，flaA：：猫和flaA：：kan突变体产生的PF具有较小的螺旋间距和螺旋直径相比，野生型菌株和flaB突变体。这些结果表明FlaA与FlaB核心的相互作用影响周质鞭毛螺旋形态。
Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF Function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs, Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic, In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.