Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogenAgrobacterium tumefaciens . Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains ofA. tumefaciens . The coinoculation ofA. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages.In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putativep hagep eptidoglycanh ydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression ofpph in the absence of additional phage genes causes a block in cell division and subsequent lysis ofA. tumefaciens cells. When the presumed active site of theN -acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic.IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such asAgrobacterium tumefaciens , may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infectA. tumefaciens may provide insights for designing preventative strategies against bacterial pathogens.