Steven D. Bowden

Bacteriophages are increasingly explored and applied to combat antibiotic-resistant bacteria, yet rapid resistance development and narrow host range can limit their broader application. Engineering phage receptor-binding proteins (RBPs) to expand phage host ranges offers a promising strategy to overcome some of these constraints. However, current approaches remain labor-intensive and difficult to control. Here, we use a cell-free phage engineering workflow to rapidly modify and characterize the RBP of the Salmonella phage LPST153. Using the PHEIGES platform and engineering targeted RBP mutations, we identified residues 389 and 402 within the tail fiber as determinants of host specificity. The G389R substitution significantly increased phage adsorption (%), reaching 65% on E. coli BW25113 and 51% on the LPS-truncated E. coli BW25113 ΔwaaC, compared with 27% and 5.7% for the wild-type LPST153, respectively. Targeted engineering of tail fiber residue 389 generated phage variants with distinct host-range phenotypes, with a G389T variant that exhibited an alternative infection profile relative to G389R. Together, these findings demonstrate that minimal, site-specific modifications of RBPs can substantially modulate LPST153 host specificity. This work establishes a framework for rapid engineering of phages with tailored host ranges, advancing phage-based antimicrobial strategies.

Cell-free gene expression (CFE) systems provide a rapid and modular platform for synthesizing bacteriophages without the need for living host cells. However, the generalizability of CFE-based phage synthesis across diverse phages, as well as the functional comparability of in vitro-synthesized phages to host-derived counterparts, has not yet been fully explored. In this study, we evaluated the ability of an Escherichia coli-based CFE platform to synthesize diverse bacteriophages relevant to food safety and synthetic biology applications. Using an E. coli-based CFE system, we synthesized seven phages from four different families, achieving infectious titers ranging from 10⁶ to 10¹¹ plaque-forming units per milliliter (PFU/mL). Of these seven phages, five phages, vB_SalM-LPST153 (LPST153), vB_SenM-S16 (S16), SP6, vB_Sens_Jbel (Jbel), and vB_EcoM_Alf5 (Alf5), are reported here for the first time as successfully synthesized and characterized in a CFE system. CFE-synthesized phages remained capable of infecting host strains; however, significant differences in efficiency of plating (EOP) were observed across multiple Salmonella, E. coli, and Shigella hosts. Long-term storage tests for LPST153 showed that phage buffer (PB) stably maintained CFE titers above 10⁹ PFU/mL for 12 weeks, while purified CFE-synthesized and host-derived LPST153 lysates exhibited broadly comparable thermal stability profiles across the tested temperatures. Using the PHEIGES workflow, we also demonstrated complete in vitro assembly and expression of LPST153 from PCR fragments. In addition, phages were successfully synthesized using CFE lysates derived from the endotoxin-free E. coli strain ClearColi™, demonstrating the synthesis of infectious phages in an endotoxin-free system. These findings expand the diversity of bacteriophages compatible with cell-free synthesis (CFS) and demonstrate that CFE-synthesized phages remain infectious and retain several functional properties, although some differences in host-dependent infectivity were observed. The ability to synthesize infectious phages in endotoxin-free systems and assemble phage genomes entirely in vitro highlights the potential of CFE platforms for scalable, decentralized, and cold-chain-independent manufacturing of phage-based therapeutics and food safety interventions.

Phage Felix O1 (FO1) is one of the most widely used bacteriophages for targeting Salmonella enterica and is applied in both research and food safety settings. Despite its broad host range, we observed significantly reduced efficiency of plating (EOP) on a subset of Salmonella enterica strains, including serovar Typhimurium 4/74 ( S . 4/74). We found that O-antigen–mediated steric hindrance contributes to this resistance, which can be partially overcome by supplementation with the SP6 phage tailspike protein (TSP). However, full restoration of the EOP required deletion of the Gifsy-1 prophage, leading to the identification of a single prophage-encoded defense gene, gipd474 (Gifsy-encoded phage defense protein from strain S . 4/74). Heterologous expression of this gene in Escherichia coli increased resistance to multiple tailed phages. Bioinformatic analysis revealed that this gene is widely spread across the Enterobacteriaceae , suggesting a conserved role in phage resistance. Together, these findings uncover a dual barrier to FO1 infectivity in S . 4/74 and highlight gipd474 as a novel, prophage-encoded phage defense gene. IMPORTANCE: Phage therapy and biocontrol are increasingly explored as alternatives to antibiotics, particularly against drug-resistant pathogens such as Salmonella . FO1 is a widely used lytic phage with broad activity across Salmonella serotypes. However, variability in phage infectivity limits its reliability against certain strains. Here, we report that S . 4/74 restricts FO1 through a combination of O-antigen–associated interference and a novel prophage-encoded resistance gene. This gene also impairs the infectivity of other tailed phages and is conserved across multiple Enterobacteriaceae genera. Our findings highlight the importance of prophage-encoded defense systems in shaping phage susceptibility and highlight the need to account for such elements when developing phage-based applications.

Viral infection of living cells, exemplified by bacteriophage interaction with bacteria, is fundamental to biology and universal across living systems. Here, we establish an all-cell-free viral cycle where T7 phages infect synthetic cells, equipped with lipopolysaccharides on the outer leaflet of the lipid membrane while encapsulating a cell-free gene expression system. We track each cycle step to demonstrate T7 phage-specific adsorption onto the liposomes, genome ejection, replication, expression, and assembly of new infectious virions within the synthetic cells. We quantify key characteristics of the cycle, including the multiplicity of infection, replication efficiency, liposome size constraints, and phage rebinding dynamics. This work establishes a versatile, fully defined in vitro platform for reconstructing and investigating viral infections from individual molecular components.

We isolated and characterized the novel polyvalent T-even type bacteriophage vB_SenS_Jbel from wastewater using an enrichment of three different Salmonella strains. The vB_SenS_Jbel virions have prolate icosahedral capsids approximately 100 nm long and 80 nm wide. The genome consists of linear, double-stranded DNA that is 165,566 bp long. Analysis of the genome and structure of vB_SenS_Jbel indicates that it belongs to the genus Tequatrovirus of the family Straboviridae. This novel polyvalent phage can infect Escherichia coli and multiple Salmonella and Shigella species through its unique tail fiber structure.

Five lytic bacteriophages specific for Salmonella enterica and Escherichia coli were isolated from wastewater in Minnesota. These phages, designated vB_Sal_EH1, vB_Sal_EH2, vB_Sal_EH3, vB_Sal_EH4, and vB_Sal_EH7, were characterized, and their genomes were sequenced. Phylogenetic analysis showed that they grouped within the genus Epseptimavirus, with genome sizes ranging from 108,554 to 115,218 bp. All five phages exhibited lytic activity against both S. enterica and Shiga-toxin-producing E. coli O157:H7. Transposon mutagenesis of the host genome identified the outer membrane protein BtuB as essential for phage infection, suggesting that it is a putative receptor. Genome sequence comparisons revealed genetic loci that are variable among the isolated phages and potentially influence their host specificity and virulence.

Predators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains of Escherichia coli and Salmonella enterica by competing a generalist T5-like phage able to infect both prey against P22 vir , an S. enterica -specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that the in vitro cost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest. IMPORTANCE: There is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and an in vitro system consisting of Escherichia coli , Salmonella enterica , a T5-like generalist phage, and the specialist phage P22 vir to highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections.

Cell-free transcription-translation (TXTL) enables achieving an ever-growing number of applications, ranging from the rapid characterization of DNA parts to the production of biologics. As TXTL systems gain in versatility and efficacy, larger DNAs can be expressed in vitro extending the scope of cell-free biomanufacturing to new territories. The demonstration that complex entities such as infectious bacteriophages can be synthesized from their genomes in TXTL reactions opens new opportunities, especially for biomedical applications. Over the last century, phages have been instrumental in the discovery of many ground-breaking biotechnologies including CRISPR. The primary function of phages is to infect bacteria. In that capacity, phages are considered an alternative approach to tackling current societal problems such as the rise of antibiotic-resistant microbes. TXTL provides alternative means to produce phages and with several advantages over in vivo synthesis methods. In this chapter, we describe the basic procedures to purify phage genomes, cell-free synthesize phages, and quantitate them using an all-E. coli TXTL system. Key words TXTL (cell-free transcription-translation) Bacteriophages E. coli Spotting assay Kinetic infection assay

To determine if the bacteriophage abortive infection system ToxIN is present in foodborne Salmonella and if it protects against infection by bacteriophages specific to enteric bacteria. A set of foodborne Salmonella Enteritidis isolates from a 2010 eggshell outbreak was identified via BLASTN queries as harboring a close homolog of ToxIN, carried on a plasmid with putative mobilization proteins. This homolog was cloned into a plasmid vector and transformed into the laboratory strain Salmonella Typhimurium LT2 and tested against a set of Salmonella-specific phages (FelixO1, S16, Sp6, LPST153, and P22 HT105/1 int-201). ToxIN reduced infection by FelixO1, S16, and LPST153 by approximately 1–4 log PFU mL⁻¹ while reducing the plaque size of Sp6. When present in LT2 and E. coli MG1655, ToxIN conferred cross-genus protection against phage isolates which infect both bacteria. Finally, the putative ToxIN plasmid was found in whole-genome sequence contigs of several Salmonella serovars, pathogenic E. coli, and other pathogenic enterobacteria. Salmonella and E. coli can resist infection by several phages via ToxIN under laboratory conditions; ToxIN is present in foodborne pathogens including Salmonella and Shiga-toxigenic E. coli.