Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad- Host- Range RP4 Conjugative Machinery
Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad- Host- Range RP4 Conjugative Machinery
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DOI：
10.1128/jb.00621-10
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发表时间：
2010-12-01
影响因子：
3.2
通讯作者：
Ghigo, Jean-Marc
中科院分区：
文献类型：
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作者：
Ferrieres, Lionel;Hemery, Gaelle;Ghigo, Jean-Marc
随机转座子诱变是将表型与其未知遗传决定簇相关联的策略选择。它通常通过使用供体菌株携带的宽宿主范围RP 4接合机制将条件复制载体递送转座子动员到受体细胞来进行。在本研究中，我们证明，噬菌体Mu，这是故意引入的广泛使用的供体菌株SM 10 λ pir和S17-1 λ pir的原始建设期间，沉默转移到大肠杆菌受体细胞在高频率，无论是通过hfr和释放的Mu颗粒的供体菌株。我们的研究结果表明，噬菌体Mu可能已经污染了许多随机诱变实验与这些流行的供体菌株Mu敏感的物种，导致潜在的误解转座子突变表型，因此干扰突变体筛选分析。为了解决这个问题，我们精确地映射了Mu插入在SM 10 λ pir和S17-1 λ pir中，并构建了一种新的无Mu供体菌株MFDpir，其具有稳定的hfr缺陷型RP 4接合功能并维持Pi依赖性自杀载体的复制。因此，该菌株可以与大多数可用的转座子递送质粒一起使用，并且应该能够在E.大肠杆菌和其他Mu敏感的RP 4宿主细菌。
Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 lambda pir and S17-1 lambda pir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 lambda pir and S17-1 lambda pir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Pi-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.