Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320.
Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320.
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DOI：
10.1128/spectrum.03142-22
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发表时间：
2022-12-21
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
有序转座子文库是许多细菌物种的宝贵资源，特别是那些难以产生靶向基因突变的细菌。在这里，我们提出了构建细菌尿路病原体奇异变形杆菌HI4320菌株的有序转座子文库。该文库将为未来对奇异疫霉生物学的研究提供便利。对于大型实验筛查，它可以用来克服瓶颈限制，避免因基因长度而导致的偏颇结果。对于规模较小的研究，该文库允许避免费力地构建单一靶标突变体。这个包含18,432个井的文库被浓缩成一个包含1,728个突变体的较小文库。每个选定的突变体都在一个开放阅读框中有一个转座子插入，覆盖了P.mirabilis HI4320编码的45%的预测基因。这一覆盖率低于预期，这是由于没有映射插入的文库井，以及令人惊讶的高比例混合克隆和多转座子插入事件。我们为今后的图书馆建设提出了建议，并对如何利用这一图书馆资源提出了建议。重要性有序文库通过用最少数量的突变保证高基因组覆盖率来促进大型遗传筛选，并且它们可以通过减少构建靶向突变的需要来节省时间和精力。这一资源现在可用于奇异肺炎杆菌，这是一种常见的导管相关性尿路感染的复杂因素。我们还介绍了在构建文库过程中遇到的障碍，目的是帮助其他希望在其他物种中构建有序转座子文库的人。
Ordered transposon libraries are a valuable resource for many bacterial species, especially those with difficult methods for generating targeted genetic mutations. Here, we present the construction of an ordered transposon library for the bacterial urinary tract pathogen Proteus mirabilis strain HI4320. This library will facilitate future studies into P. mirabilis biology. For large experimental screens, it may be used to overcome bottleneck constraints and avoid biased outcomes resulting from gene length. For smaller studies, the library allows sidestepping the laborious construction of single targeted mutants. This library, containing 18,432 wells, was condensed into a smaller library containing 1,728 mutants. Each selected mutant had a single transposon insertion in an open reading frame, covering 45% of predicted genes encoded by P. mirabilis HI4320. This coverage was lower than expected and was due both to library wells with no mapped insertions and a surprisingly high proportion of mixed clones and multiple transposon insertion events. We offer recommendations for improving future library construction and suggestions for how to use this P. mirabilis library resource. IMPORTANCE Ordered libraries facilitate large genetic screens by guaranteeing high genomic coverage with a minimal number of mutants, and they can save time and effort by reducing the need to construct targeted mutations. This resource is now available for P. mirabilis, a common and complicating agent of catheter-associated urinary tract infection. We also present obstacles encountered during library construction with the goal to aid others who would like to construct ordered transposon libraries in other species.
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影响因子：
6.7
作者：
Alteri CJ;Himpsl SD;Pickens SR;Lindner JR;Zora JS;Miller JE;Arno PD;Straight SW;Mobley HL
通讯作者：
Mobley HL
影响因子：
6.4
作者：
Dale JL;Beckman KB;Willett JLE;Nilson JL;Palani NP;Baller JA;Hauge A;Gohl DM;Erickson R;Manias DA;Sadowsky MJ;Dunny GM
通讯作者：
Dunny GM
影响因子：
9.9
作者：
de Berardinis, Veronique;Vallenet, David;Castelli, Vanina;Besnard, Marielle;Pinet, Agnes;Cruaud, Corinne;Samair, Sumitta;Lechaplais, Christophe;Gyapay, Gabor;Richez, Celine;Durot, Maxime;Kreimeyer, Annett;Le Fevre, Francois;Schaechter, Vincent;Pezo, Valerie;Doering, Volker;Scarpelli, Claude;Medigue, Claudine;Cohen, Georges N.;Marliere, Philippe;Salanoubat, Marcel;Weissenbach, Jean
通讯作者：
Weissenbach, Jean
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
3
作者：
Himpsl, Stephanie D.;Lockatell, C. Virginia;Mobley, Harry L. T.
通讯作者：
Mobley, Harry L. T.