Evidence of Tailocin Persistence and Resistance in Pseudomonas

Tailocins are bacterially-produced, phage tail-like bacteriocins that have been proposed as therapeutics for treatment of bacterial infections. However, we have a limited understanding of how target populations survive tailocin exposure. In this paper, we demonstrate that cells of a target population of Pseudomonas syringae are able to survive lethal doses of a tailocin through both physiological and genetic mechanisms with stationary phase cells predominantly surviving using a physiological mechanism. Regardless of growth phase, a significant fraction of cells that survived tailocin exposure did not exhibit any increased resistance to subsequent exposure, indicating that these cells survive by persistence rather than resistance mechanism. Of those cells that did gain a detectable increase in tailocin resistance, there was a range from insensitive (complete resistance) to partially sensitive. We also recovered a mutant exhibiting a high-persistence like phenotype that showed significantly increased survival to transient exposure but no detectable growth after prolonged tailocin treatment. By sequencing the genome of multiple types of mutants, we identified several genes linked to lipopolysaccharide (LPS) biogenesis and implicated in tailocin tolerance and resistance. In particular, we found a specific genomic region that, when mutated, gave rise to the various classes of resistance and the high persistence phenotypes. Furthermore, we showed that a hypothetical gene involved in the high persistence phenotype is transcriptionally fused with the LPS biosynthetic region and contains a signal peptide and several trans-membrane domains. While the complete resistant mutants had lost their LPS O-antigen, incomplete resistant mutants contained minor to significant changes in their O-antigen. This work demonstrates that Gram-negative bacteria can survive tailocin exposure through multiple strategies, including the first description of a persistence-like mechanism for tolerating tailocin exposure. Author Summary With the rise in antibiotic resistant infections, it has become a necessity to study and find alternative treatment strategies. Bacteriocins, which are bacterially produced protein toxins, have been proposed as one of such alternatives. However, a deeper understanding of how target bacteria respond to bacteriocin exposure is lacking. Here, using a phage-tail like bacteriocin known as a tailocin, we show that target cells of the important plant pathogen Pseudomonas syringae are able to survive tailocin exposure through both physiological persistence and genetic resistance mechanisms. We show that bacterial cells that are not growing rapidly rely primarily on persistence mechanism, whereas rapidly growing cells are more likely to survive through genetic resistance. By genomic sequencing, we identified various types of mutations in the genes involved in lipopolysaccharide (LPS) biogenesis that cause increased tailocin persistence and resistance. In particular, we identified a mutation in a LPS-related genomic region that encodes a hypothetical protein and causes tailocin high persistence. We conclude that tailocin persistence is a real phenomenon and effects of both persistence and resistance in long term disease management needs to be considered before designing tailocins into biocontrol agents.