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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. 

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system. 

ABSTRACT 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 ofEscherichia coliandSalmonella entericaby competing a generalist T5-like phage able to infect both prey against P22vir, anS. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that thein vitrocost 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. IMPORTANCEThere 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 anin vitrosystem consisting ofEscherichia coli,Salmonella enterica, a T5-like generalist phage, and the specialist phage P22virto 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. 

Abstract Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 10¹¹PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.