In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner-family transposable element Himar1.
In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner-family transposable element Himar1.
复制标题
使用昆虫水手家族转座元件 Himar1 的改良版本对产甲烷古菌 Methanosarcina acetivorans C2A 进行体内转座子诱变。
DOI：
10.1073/pnas.160272597
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发表时间：
2000
影响因子：
11.1
通讯作者：
Metcalf,WW
中科院分区：
文献类型：
--
作者：
Zhang,JK;Pritchett,MA;Lampe,DJ;Robertson,HM;Metcalf,WW
我们在这里提出了一种方法forin vivo转座子诱变的产甲烷古菌，Methanosarcina acetivoransC 2A，这是因为它的独立性，从主机特定的因素可能有广泛的应用在许多微生物。因为没有已知的甲烷转座子，我们修改了最初在昆虫刺激血蝇中发现的甲烷转座元件Himar 1，使其能够在这种生物中使用。选择该元件是因为，像其他海洋元件一样，它的转座不依赖于宿主因素，只需要它的同源转座酶。构建了修饰的mini-Himar 1元件，其携带在甲烷杆菌物种中有功能的选择标记，并从已知的甲烷杆菌启动子表达Himar 1转座酶。这些迷你marinerelements在M中以高频转置。乙酰化酶基因组中的随机位点。微海洋元件内存在大肠杆菌可选择标记和质粒复制起点，这使得可以容易地克隆这些转座子插入以鉴定突变基因。在初步的实验中，我们已经分离出许多mini-mariner-inducedM。这些突变体包括具有插入物的突变体，这些插入物赋予对有毒类似物的抗性，以及编码涉及热休克、固氮和细胞壁结构的蛋白质的基因。
We present here a method forin vivotransposon mutagenesis of a methanogenic archaeon,Methanosarcina acetivoransC2A, which because of its independence from host-specific factors may have broad application among many microorganisms. Because there are no knownMethanosarcinatransposons we modified themarinertransposable elementHimar1,originally found in the insectHematobia irritans, to allow its use in this organism. This element was chosen because, like othermarinerelements, its transposition is independent of host factors, requiring only its cognate transposase. Modified mini-Himar1elements were constructed that carry selectable markers that are functional inMethanosarcinaspecies and that express theHimar1transposase from knownMethanosarcinapromoters. These mini-marinerelements transpose at high frequency inM. acetivoransto random sites in the genome. The presence of anEscherichia coliselectable marker and plasmid origin of replication within the mini-marinerelements allows facile cloning of these transposon insertions to identify the mutated gene. In preliminary experiments, we have isolated numerous mini-mariner-inducedM. acetivoransmutants, including ones with insertions that confer resistance to toxic analogs and in genes that encode proteins involved in heat shock, nitrogen fixation, and cell-wall structures.