Jarrell K F, Bayley D P, Florian V, Klein A
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Mol Microbiol. 1996 May;20(3):657-66. doi: 10.1046/j.1365-2958.1996.5371058.x.
Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments. The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.
沃氏甲烷球菌是古菌域中一种有鞭毛的成员，它有四个鞭毛蛋白基因，排列在两个转录单元中。一个转录单元仅编码flaA，而第二个是多顺反子单元，编码三个鞭毛蛋白基因（flaB1、flaB2和flaB3）以及下游至少七个其他开放阅读框。聚合酶链反应用于扩增flaA基因的内部片段，随后将其克隆到为沃氏甲烷球菌开发的插入载体中。用该载体转化原生质体导致分离出在flaA或flaB2中发生插入的突变菌株。在flaA中发生插入的突变菌株的鞭毛，在使用电子显微镜观察时，数量和外观与野生型细胞相似。此外，在用针对沃氏甲烷球菌31 kDa鞭毛蛋白产生的抗血清进行免疫印迹时，其中一些突变菌株的图谱与野生型相同。所有flaA突变菌株和野生型细胞在33 kDa和31 kDa（对应于纯化的鞭毛蛋白）以及18 kDa处均显示免疫交叉反应条带。一些flaA突变菌株在27 kDa处也显示免疫交叉反应条带，这可能代表由插入载体产生的截短鞭毛蛋白。然而，在半群体平板实验中，这两种类型的flaA突变菌株的运动性均低于野生型。通过电子显微镜检查，在flaB2中发生插入的突变菌株无鞭毛，并且在半群体平板实验中不运动。它代表了在产甲烷菌中分离出的第一个结构突变菌株。该突变菌株缺乏在野生型和flaA突变菌株中观察到的33 kDa、31 kDa和18 kDa免疫交叉反应条带，取而代之的是在20 kDa处有一条新条带。这条带可能代表一种仍带有附着前导肽的未修饰鞭毛蛋白。如果是这样，那么多顺反子转录单元中的一个下游基因可能编码鞭毛蛋白系统的前导肽酶。
