Heat shock increases conjugation efficiency in Clostridium difficile.
Heat shock increases conjugation efficiency in Clostridium difficile.
复制标题
热休克提高了艰难梭菌的共轭效率。
DOI：
10.1016/j.anaerobe.2016.06.009
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发表时间：
2016-12
期刊：
影响因子：
2.3
通讯作者：
Fagan, Robert P.
中科院分区：
文献类型：
--
作者：
Kirk, Joseph A.;Fagan, Robert P.
在过去的十年中，艰难梭菌感染的发病率和严重程度增加，并对人类健康构成独特的威胁。然而，C.艰难梭菌仍处于其幼年期，并且该细菌的特征仍相对较差。低效率的接合是目前唯一可用的将质粒DNA转移到C.很难这实际上是限制性的，并且减缓了对这种重要病原体的了解。菌株之间的缀合效率变化很大，重要的临床相关菌株如R20291对质粒转移特别难治。在这里，我们提出了一种优化的缀合方法，其中受体C。艰难梭菌在接合之前进行热处理。这显著提高了所有C中的缀合效率。检测的艰难梭菌菌株包括R20291。缀合效率也受进行缀合的培养基的选择的影响，其中标准BHI培养基提供大多数的转移缀合物回收。使用我们优化的方法大大增加了R20291的染色体可以通过同源重组精确操作的容易性。我们的方法改进了目前的接合协议，并将有助于加快基因操作的菌株，否则难以工作。艰难梭菌的结合效率可以通过受体C的热处理来增加。很难使用优化的热处理缀合方案的缀合效率受培养基选择的影响。当包括热处理步骤时，使用具有pBP 1复制子的质粒提高了缀合效率。热处理提高了所有C.测试的艰难菌株。
Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with. Conjugation efficiency in Clostridium difficile can be increased through heat treatment of recipient C. difficile. Conjugation efficiency using an optimised heat treatment conjugation protocol is affected by media choice. Conjugation efficiency is improved when a heat treatment step is included, using a plasmid with a pBP1 replicon. Heat treatment improves conjugation efficiency in all C. difficile strains tested.
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