Fast and reliable screening assay developed to preselect candidate Soft Rot Pectobacteriaceae Tn5 mutants showing resistance to bacteriophage infection

Abstract

We present a simple, fast and inexpensive screening assay to preselect candidate Pectobacterium spp. and Dickeya spp. Tn5 mutants, which carry transposon insertions in genes putatively encoding proteins used by lytic bacteriophages to interact with host cells, for the follow-up studies. The proposed method is fast and cost-effective and it does not need any specialized laboratory equipment and/or technical support. The Tn5 mutants are generated using random transposon mutagenesis with the mini-Tn5 transposon. The obtained bacterial mutants are incubated in the presence of viable lytic bacteriophage particles in liquid bacterial growth medium supplemented with resazurin for 12 h at 28 °C in a 96-well microtiter plate assay. During the cultivation, the Tn5 mutants that are susceptible to phage infection are lysed. The mutants that are resistant to a viral infection (not lysed after contact with bacteriophages) irreversibly reduce the resazurin violet dye to pink/yellowish-colored resorufin indicating active bacterial metabolism (a positive reaction). The change of the culture color can be observed by eye. The Tn5 mutants that are positive in the screen are selected for sequencing of the Tn5 insertion site directly from bacterial genome. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes that can be later selected for further examinations. As a proof-of-concept, we used this method to evaluate resistance to viral infection of Tn5 mutants of Dickeya solani strain IPO2222 and Pectobacterium parmentieri strain SCC3193 using lytic bacteriophages ɸD5 and ɸA38, respectively.