Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility
Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility
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DOI：
10.1128/jb.01288-12
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发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Melville, Stephen B.
中科院分区：
文献类型：
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作者：
Liu, Hualan;Bouillaut, Laurent;Melville, Stephen B.
产气荚膜梭菌是一种厌氧革兰氏阳性病原体，可引起许多人类和动物疾病，包括食物中毒和气性坏疽。C.产气荚膜杆菌缺乏鞭毛但具有IV型皮利（TFP）。我们以前已经证明，C。产气荚膜杆菌可以由端对端连接的单个细菌组成的长丝状物滑过琼脂表面，并且为此需要两种TGF-相关蛋白PilT和PilC。为了发现在滑行中发挥作用的其他基因产物，我们开发了一种基于质粒的水手转座子诱变系统，该系统在C。产气荚膜杆菌对超过10，000个克隆进行了筛选，寻找缺乏远离菌落边缘能力的突变体。24个突变体（0.24%）被确定为符合标准。包含影响滑行运动的插入的基因分为九个不同的类别。一个基因，CPE 0278，它编码的佐贺细胞壁依赖性内肽酶的同源物，获得不同的转座子插入在两个独立的突变体。佐贺突变体由于完全缺乏滑行运动所必需的端对端连接而不能形成细丝。互补的佐贺突变体与野生型拷贝的基因恢复滑行运动。我们在佐贺基因中构建了一个框内缺失突变，发现该突变体具有与转座子突变体相似的表型。我们假设佐贺突变株不能形成保持细胞端对端方向所需的分子复合物，导致子细胞分离，不能进行滑动运动。
Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.