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1. A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages

   https://doi.org/10.1128/spectrum.02897-23  

   Ipoutcha, Thomas; Racharaks, Ratanachat; Huttelmaier, Stefanie; Wilson, Cole J; Ozer, Egon A; Hartmann, Erica M (March 2024, Microbiology Spectrum)

   Andam, Cheryl P (Ed.)

   The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains ofP. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages. IMPORTANCE Pseudomonas aeruginosais a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications. 

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2. Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection

   https://doi.org/10.1128/spectrum.02833-23  

   Holtappels, D; Abelson, S A; Nouth, S C; Rickus, G_E J; Amare, S Z; Giller, J P; Jian, D Z; Koskella, B (March 2024, Microbiology Spectrum)

   Zeng, Quan (Ed.)

   ABSTRACT ThePseudomonas syringaespecies complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize twoP. syringaeisolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified theP. syringaeisolates as members of phylogroup 2, related to other strains previously isolated fromPyrusandPrunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related toPseudomonas syringaepv.actinidiaephage PHB09 and theFlaumdravirusgenus. Finally, usingin plantainfection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity ofP. syringaeand their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains ofPseudomonas syringaepv.syringaeisolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novelP. syringaepv.syringaeisolates that potentially pose a risk to local fruit production, or vice versa—but also provide us with novel associated phages, effective in disease mitigation. 

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3. Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens

   https://doi.org/10.1128/msystems.00801-24  

   Suttenfield, Laura C; Rapti, Zoi; Chandrashekhar, Jayadevi H; Steinlein, Amelia C; Vera, Juan Cristobal; Kim, Ted; Whitaker, Rachel J (September 2024, mSystems)

   Whiteson, Katrine (Ed.)

   ABSTRACT The opportunistic human pathogenPseudomonas aeruginosais naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity ofP. aeruginosaPA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat(CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogenPseudomonas aeruginosais persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers andcasgenes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations. 

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4. A Transducing Bacteriophage Infecting Staphylococcus epidermidis Contributes to the Expansion of a Novel Siphovirus Genus and Implies the Genus Is Inappropriate for Phage Therapy

   https://doi.org/10.1128/msphere.00524-22  

   Andrews, Taylor; Hoyer, J. Steen; Ficken, Karolyn; Fey, Paul D.; Duffy, Siobain; Boyd, Jeffrey M. (April 2023, mSphere)

   Imperiale, Michael J. (Ed.)

   ABSTRACT The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis -infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages’ putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy. 

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5. Lytic and temperate phage naturally coexist in a dynamic population model

   https://doi.org/10.1093/ismejo/wrae093  

   Kimchi, Ofer; Meir, Yigal; Wingreen, Ned_S (May 2024, The ISME Journal)

   Abstract When phage infect their bacterial hosts, they may either lyse the cell and generate a burst of new phage, or lysogenize the bacterium, incorporating the phage genome into it. Phage lysis/lysogeny strategies are assumed to be highly optimized, with the optimal tradeoff depending on environmental conditions. However, in nature, phage of radically different lysis/lysogeny strategies coexist in the same environment, preying on the same bacteria. How can phage preying on the same bacteria coexist if one is more optimal than the other? Here, we address this conundrum within a modeling framework, simulating the population dynamics of communities of phage and their lysogens. We find that coexistence between phage of different lysis/lysogeny strategies is a natural outcome of chaotic population dynamics that arise within sufficiently diverse communities, which ensure no phage is able to absolutely dominate its competitors. Our results further suggest a bet-hedging mechanism at the level of the phage pan-genome, wherein obligate lytic (virulent) strains typically outcompete temperate strains, but also more readily fluctuate to extinction within a local community. 

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