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首页> 外文期刊>Journal of bacteriology >Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans
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Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans

机译：通过使用广泛的宿主范围的载体直接选择IS903转座子插入：放线菌放线菌的过氧化氢酶缺陷突变体的分离。

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Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kangene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkaninsertion mutants were readily selected in Escherichia coliby their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogenActinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
机译：细菌中的转座子诱变通常需要有效地递送转座子自杀载体以允许选择相对罕见的转座事件。我们已经开发出了一种基于IS 903 的转座子诱变系统，用于多种革兰氏阴性细菌，该系统不受转移效率的限制。转座子IS 903φkan带有一个神秘的 kan 基因，只有在成功转座后才能表达。这允许将转座子递送载体稳定地引入宿主。然后，插入突变体的产生仅受转座频率的限制。将IS 903φkan置于具有转座酶基因的IncQ质粒载体上，该转座酶基因位于转座子之外，并由异丙基-β-d-硫代半乳糖吡喃糖苷（IPTG）诱导型启动子表达。转座酶诱导后，IS 903φkan插入突变体因其对卡那霉素的抗性而容易在大肠杆菌中选择。我们使用IS 903φkan从随机插入文库中分离出三种牙周病原体 Actinobacillus actinomycetemcomitans 的过氧化氢酶缺陷型突变体。该突变体显示出对过氧化氢的增加的敏感性，并且全部在开放阅读框内具有IS 903φkan插入，其预测产物与其他细菌过氧化氢酶密切相关。过氧化氢酶基因（指定为 katA ）和侧翼基因间区域的核苷酸序列分析还显示，多次出现11 bp序列，该序列与的自然转化的核心DNA摄取信号序列密切相关流感嗜血杆菌。我们的结果证明了IS 903φkan诱变系统在研究 A中的实用性。放线菌。因为IS 903φkan携带在可移动的，广泛宿主范围的IncQ质粒上，所以该系统可能在多种细菌中有用。

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