Biomolecular monitoring in the gastrointestinal tract could offer rapid, precise disease detection and management but is impeded by access to the remote and complex environment. Here, we present an ingestible micro-bio-electronic device (IMBED) for in situ biomolecular detection based on environmentally resilient biosensor bacteria and miniaturized luminescence readout electronics that wirelessly communicate with an external device. As a proof of concept, we engineer heme-sensitive probiotic biosensors and demonstrate accurate diagnosis of gastrointestinal bleeding in swine. Additionally, we integrate alternative biosensors to demonstrate modularity and extensibility of the detection platform. IMBEDs enable new opportunities for gastrointestinal biomarker discovery and could transform the management and diagnosis of gastrointestinal disease.
A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.
Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication. Though lipopolysaccharide (LPS) has been shown to act as a receptor for tailocins, the breadth of factors involved in tailocin sensitivity, and the mechanisms behind resistance to self-intoxication, remain unclear. Here, we employed genome-wide screens in four non-model pseudomonads to identify mutants with altered fitness in the presence of tailocins produced by closely related pseudomonads. Our mutant screens identified O-antigen composition and display as most important in defining sensitivity to our tailocins. In addition, the screens suggest LPS thinning as a mechanism by which resistant strains can become more sensitive to tailocins. We validate many of these novel findings, and extend these observations of tailocin sensitivity to 130 genome-sequenced pseudomonads. This work offers insights into tailocin–bacteria interactions, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.
Tailocins are bactericidal protein complexes produced by a wide variety of bacteria to compete against closely related strains. Like tailed bacteriophages, with whom they share an evolutionary and morphological relationship, tailocins bind and kill a narrow spectrum of target cells. Thanks to their high specificity, tailocins have garnered recent attention for their potential as precision antibacterial agents. Nevertheless, the field currently lacks a systematic investigation of genetic determinants of tailocin sensitivity. Here, we employed barcoded transposon-insertion mutant libraries and comparative genomics to assess genetic contributions to tailocin sensitivity in pseudomonads. Our mutant screens identified O-specific antigen (OSA) composition and display as most important in defining sensitivity to our tailocins. Additionally, the screens suggest lipopolysaccharide (LPS) thinning as a mechanism by which resistant strains can become more sensitive to tailocins. Our comparative genomics analyses show a loose relationship between OSA biosynthetic genes and tailocin sensitivity, as well as sensitivity nuances that require further investigation. Overall, our data reinforces the model that LPS molecules can act as either a receptor for, or shield against, tailocin binding and killing. This work offers insight into the specificity of tailocins and tailocin-mediated competition, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.
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