A simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neo^R. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

已经开发出一种简单而快速的方法来在编码噬菌体所使用的受体与其宿主相互作用的受体的基因中产生软腐菌菌（SRP：Pectobacterium spp。和Dickeya spp。）Tn5突变体，用于后续研究。该过程便宜，并且不需要任何专门的工具和/或专门的技术支持。抗新霉素的SRP Tn5突变体是通过与携带pFAJ1819-mini-Tn5- neo ^R的转座子供体大肠杆菌ST18菌株（需要5-氨基乙酰丙酸（5-ALA）存活）结合产生的。。在补充有5-ALA的固体培养基上进行缀合。结合后，收集细菌细胞，将其悬浮在液体细菌培养基中，添加到含有裂解性噬菌体的悬浮液中，并在摇动（120 rpm）下再孵育30分钟。在此阶段，裂解易受噬菌体感染的转座子受体（油杆菌属和/或迪卡氏菌属Tn5突变体）。同样，由于生长培养基中缺乏5-ALA，大肠杆菌ST18（转座子供体）细胞在此阶段死亡。最后，温育后，在补充有新霉素的固体生长培养基上选择具有对噬菌体感染抗性的Tn5插入的细菌突变体。对Tn5插入位点进行测序，以获取有关Tn5突变基因及其在噬菌体-宿主相互作用中推定功能的知识。拟议的测定法允许在短时间内（约48小时）生成表达噬菌体抗性表型的大量立即可用的Tn5突变体，这些突变体随后可用于表征其他各种表型特征。在本研究中，作为概念验证，已使用此方法来产生耐Dickeya solani  IPO2222 Tn5突变体，抵抗由溶菌性噬菌体ɸD5引起的感染。