Development of a markerless deletion system for the fish-pathogenic bacterium Flavobacterium psychrophilum.
Development of a markerless deletion system for the fish-pathogenic bacterium Flavobacterium psychrophilum.
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DOI：
10.1371/journal.pone.0117969
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Guijarro JA
中科院分区：
文献类型：
--
作者：
Gómez E;Álvarez B;Duchaud E;Guijarro JA
嗜冷黄杆菌（Flavobacteriumpsychrophilum）是一种革兰氏阴性的鱼类病原菌，在世界范围内引起水产养殖的重大经济损失。虽然这种细菌的基因组已经确定，但与毒力相关的基因的功能和相对重要性仍有待确定。为了研究它们各自对细菌致病的贡献，需要有效的基因失活工具。在本研究中，一个无标记的基因删除系统已成功地开发了第一次在该细菌。利用该方法，对F.嗜冷菌fcpB基因，编码与化脓性链球菌链蛋白酶同源的预测的半胱氨酸蛋白酶，被删除。所开发的系统涉及构建含有fcpB基因侧翼序列和I-SceI大范围核酸酶限制性位点的接合质粒。一旦该质粒通过同源重组整合到基因组中，通过引入在fpp 2 F的控制下表达I-SceI的质粒来解析部分二倍体。嗜冷菌诱导型启动子与亲本菌株相比，所得缺失的fcpB突变体呈现细胞外蛋白水解活性的降低。然而，在肌内攻击虹鳟鱼感染模型中，它们的LD 50之间没有显著差异。在这项工作中开发的诱变方法代表了对这种“挑剔”细菌迄今存在的基因失活工具的改进。与转座子诱变和基因破坏不同，基因无标记缺失具有较小的极性效应潜力，并且允许几乎任何非必需基因或基因簇的突变。
Flavobacterium psychrophilum is a Gram-negative fish pathogen that causes important economic losses in aquaculture worldwide. Although the genome of this bacterium has been determined, the function and relative importance of genes in relation to virulence remain to be established. To investigate their respective contribution to the bacterial pathogenesis, effective tools for gene inactivation are required. In the present study, a markerless gene deletion system has been successfully developed for the first time in this bacterium. Using this method, the F. psychrophilum fcpB gene, encoding a predicted cysteine protease homologous to Streptococcus pyogenes streptopain, was deleted. The developed system involved the construction of a conjugative plasmid that harbors the flanking sequences of the fcpB gene and an I-SceI meganuclease restriction site. Once this plasmid was integrated in the genome by homologous recombination, the merodiploid was resolved by the introduction of a plasmid expressing I-SceI under the control of the fpp2 F. psychrophilum inducible promoter. The resulting deleted fcpB mutant presented a decrease in extracellular proteolytic activity compared to the parental strain. However, there were not significant differences between their LD50 in an intramuscularly challenged rainbow trout infection model. The mutagenesis approach developed in this work represents an improvement over the gene inactivation tools existing hitherto for this “fastidious” bacterium. Unlike transposon mutagenesis and gene disruption, gene markerless deletion has less potential for polar effects and allows the mutation of virtually any non-essential gene or gene clusters.
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