Transposon Tn5 mutagenesis of Actinobacillus actinomycetemcomitans via conjugation.
Transposon Tn5 mutagenesis of Actinobacillus actinomycetemcomitans via conjugation.
复制标题
通过接合对放线杆菌伴放线杆菌进行转座子 Tn5 诱变。
DOI：
10.1111/j.1399-302x.1994.tb00073.x
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发表时间：
1994
影响因子：
--
通讯作者：
Kraig,E
中科院分区：
文献类型：
--
作者：
Kolodrubetz,D;Kraig,E
为了便于对牙周致病菌伴放线放线杆菌的毒力进行分子遗传学研究，我们将Tn 5转座子诱变的应用扩展到了这种生物体。使用自杀质粒通过接合大肠杆菌A来递送壮观霉素抗性Tn 5衍生物[mini-Tn 5（Sp）]。发现大观霉素抗性接合子的频率约为1 × 10 - 7/受体细胞。Southern杂交分析表明转座子已进入A.放线菌共生体基因组中，每一个接合子都有一个转座子。重要的是，mini-Tn 5（Sp）似乎转座到相对随机的位点：通过Southern印迹分析检查的33个接合子中有32个不同的整合位点。最后，mini-Tn 5（Sp）稳定整合，因为所有接合子在非选择性培养基上传代7次后仍具有壮观霉素抗性。因此，mini-Tn 5（Sp）系统是诱变A的有用工具。放线菌共生放线菌的毒力因子及其调控的研究。
To facilitate molecular genetic studies on the virulence of the periodontopathogenActinobacillus actinomycetemcomitans, we have extended the use ofTn5transposon mutagenesis to this organism. A suicide plasmid was used to deliver a spectinomycin‐resistantTn5derivative [mini‐Tn5(Sp)] via conjugation fromEscherichia colitoA. actinomycetemcomitans.Spectinomycin‐resistant exconjugants were found at a frequency of about 1 × 10‐7per recipient cell. Southern blot analysis showed that the transposon had moved into theA. actinomycetemcomitansgenome, and each exconjugant had a transposon at a single location. Importantly, the mini‐Tn5(Sp)appeared to transpose into relatively random sites: there were 32 different sites of integration among the 33 exconjugants examined by Southern blot analysis. Finally, the mini‐Tn5(Sp)was stably integrated, since all of the exconjugants were still spectinomycin‐resistant after 7 passages on non‐selective media. Thus, the mini‐Tn5(Sp)system is a useful tool for the mutagenesis ofA. actinomycetemcomitansin studies of its virulence factors and their regulation.