Blank C E, Kessler P S, Leigh J A
Department of Microbiology, University of Washington, Seattle 98195.
J Bacteriol. 1995 Oct;177(20):5773-7. doi: 10.1128/jb.177.20.5773-5777.1995.
We designed a transposon insertion mutagenesis system for Methanococcus species and used it to make mutations in and around a nifH gene in Methanococcus maripaludis. The transposon Mudpur was constructed with a gene for puromycin resistance that is expressed and selectable in Methanococcus species. A 15.6-kb nifH region from M. maripaludis cloned in a lambda vector was used as a target for mutagenesis. A series of 19 independent Mudpur insertions spanning the cloned region were produced. Four mutagenized clones in and around nifH were introduced by transformation into M. maripaludis, where each was found to replace wild-type genomic DNA with the corresponding transposon-mutagenized DNA. Wild-type M. maripaludis and a transformant containing a Mudpur insertion upstream of nifH grew on N2 as a nitrogen source. Two transformants with insertions in nifH and one transformant with an insertion downstream of nifH did not grow on N2. The transposon insertion-gene replacement technique should be generally applicable in the methanococci for studying the effects of genetic manipulations in vivo.
我们设计了一种用于甲烷球菌属物种的转座子插入诱变系统，并利用该系统对马氏甲烷球菌中nifH基因及其周围区域进行诱变。转座子Mudpur构建有一个在甲烷球菌属物种中可表达并可用于筛选的嘌呤霉素抗性基因。将克隆在λ载体中的来自马氏甲烷球菌的15.6 kb nifH区域用作诱变靶点。产生了一系列跨越克隆区域的19个独立的Mudpur插入突变体。通过转化将4个在nifH基因及其周围诱变的克隆引入马氏甲烷球菌，发现每个克隆都用相应的转座子诱变DNA取代了野生型基因组DNA。野生型马氏甲烷球菌和一个在nifH上游含有Mudpur插入的转化体以N2作为氮源生长。两个在nifH中有插入的转化体和一个在nifH下游有插入的转化体在N2上不能生长。转座子插入-基因替换技术在甲烷球菌中一般可用于研究体内基因操作的效果。
