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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.

european journal of plant pathology

Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection