Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles
Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles
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DOI：
10.1016/j.resmic.2010.07.004
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发表时间：
2010-10-01
影响因子：
2.6
通讯作者：
Yamaguchi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Matsuo, Junji;Oguri, Satoshi;Yamaguchi, Hiroyuki
研究了细菌通过原生动物结合的机制。以携带pRT733和TnphoA的耐卡那霉素大肠杆菌sm0 λ(+)为供菌，通过偶联法导入耐环丙沙星大肠杆菌临床分离受体菌。等量的供体和受体细菌在存在或不存在原生动物（纤毛虫，自由生活的阿米巴，粘阿米巴）的情况下混合在Page的阿米巴生理盐水中24小时。用含有卡那霉素和环丙沙星的Luria肉汁琼脂选择转偶联物。结合的频率估计为每个受体的转缀合物的数量。在有纤毛虫存在的情况下，偶联频率估计约为10(-6)，而在没有纤毛虫或有其他原生动物存在的情况下，偶联频率约为10（-8）。在培养至少2小时后，纤毛虫也发生了偶联。经聚合酶链反应证实偶联成功。环己亚胺或拉曲库林B的加入抑制了偶联。热杀灭纤毛虫或细菌对偶联频率没有影响。表达绿色荧光蛋白的大肠杆菌和pkh -67生命染色的大肠杆菌在同一纤毛囊泡中共定位，表明供体和受体细菌在同一囊泡中聚集。在本研究中，我们发现从纤毛虫中纯化的囊泡中细菌的结合频率明显高于培养悬浮液中细菌的结合频率。我们得出结论，纤毛虫通过在囊泡中的细菌积累迅速增强了大肠杆菌菌株的结合。(C) 2010 Elsevier Masson SAS。版权所有。
The mechanism underlying bacterial conjugation through protozoa was Investigated. Kanamycin-resistant Escherichia colt SMI0 lambda(+) carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. colt clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were Mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page's amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10(-6) but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10(-8). Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E colt and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles. (C) 2010 Elsevier Masson SAS. All rights reserved.