Identification and cloning of genes from Porphyromonas gingivalis after mutagenesis with a modified Tn4400 transposon from Bacteroides fragilis
Identification and cloning of genes from Porphyromonas gingivalis after mutagenesis with a modified Tn4400 transposon from Bacteroides fragilis
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DOI：
10.1128/iai.68.1.420-423.2000
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发表时间：
2000-01-01
影响因子：
3.1
通讯作者：
Duncan, MJ
中科院分区：
文献类型：
--
作者：
Chen, TT;Dong, H;Duncan, MJ
牙龈卟啉单胞菌是一种革兰氏阴性，黑色色素，口腔厌氧菌与成人牙周炎密切相关。先前的转座子诱变研究是基于拟杆菌转座子Tn4351。通过克隆鉴定Tn4351破坏的基因并不是分析大量突变体的有效方法，并且含有Tn4351的自杀传递载体的高协整率进一步复杂化。在本研究中，我们用脆弱拟杆菌转座子Tn4400的修饰版本诱变牙龈假单胞杆菌。将携带转座子的质粒pYT646B从大肠杆菌中接合到牙龈卟啉卟啉杆菌ATCC 33277上。正反转位频率相似（3 × 10(-8)）。然而，逆转座子（Tn4400’）包含一个pBR322复制子和一个β -内酰胺酶基因；因此，通过连接基因组片段并转化到大肠杆菌中，很容易完成逆转位突变体中断裂基因组dna的克隆。单次杂交实验可获得数千个转位子，而南方杂交表明，逆转位是随机的。通过这种方法，可以快速克隆、测序和鉴定牙龈卟啉单胞菌多效突变体的断裂基因。
Porphyromonas gingivalis is a gram-negative, black-pigmented, oral anaerobe strongly associated with adult periodontitis. Previous transposon mutagenesis studies with this organism were based on the Bacteroides transposon Tn4351. characterization of Tn4351-disrupted genes by cloning has not been an efficient way to analyze large numbers of mutants and is further complicated by the high rate of cointegration of the suicide delivery vector containing Tn4351, In this study, we mutagenized P. gingivalis with a modified version of the Bacteroides fragilis transposon Tn4400. Plasmid pYT646B carrying the transposon was mobilized from Escherichia coli to P. gingivalis ATCC 33277 by conjugation. Both normal and inverse transposition frequencies were similar (3 x 10(-8)). However, the inverse transposon (Tn4400') contains a pBR322 replicon and a beta-lactamase gene; thus, the cloning of disrupted genomic DNAs from inverse transposition mutants was easily accomplished after ligation of genomic fragments and transformation into E. coli. Thousands of transconjugants could be obtained in a single mating experiment, and inverse transposition was random as demonstrated by Southern hybridization. By this procedure the disrupted genes from P. gingivalis pleiotropic mutants were quickly cloned, sequenced, and identified.