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 phytopathogen Agrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens. The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ϳ42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-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. IMPORTANCEThe characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium 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 infect A. tumefaciens may provide insights for designing preventative strategies against bacterial pathogens.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Time-lapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.

Bacteriophages are relatively ubiquitous in the environment and are highly abundant in the human microbiome. Phages can be commonly transmitted between close contacts, but the impact that such transmissions may have on their bacteria counterparts in our microbiomes is unknown.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with 2 new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemmoragic E. coli O111. We also found unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and adds to the known similarities between colicins and filamentous phage.

In the rhizosphere, bacteria–phage interactions are likely to have important impacts on the ecology of microbial communities and microbe–plant interactions. To better understand the dynamics of Agrobacteria–phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacterium tumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04—a T4-like phage belonging to the Myoviridae family—was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.