Synthetic Reconstruction and Characterization of Endogenous Bacteriophage Genomes from Desulfovibrio vulgaris

Microbiologically influenced corrosion (MIC) caused by sulfate-reducing bacteria (SRB) is a major challenge in industrial infrastructure, yet the limited availability of well-characterized bacteriophages has hindered the development of phage-based strategies for controlling these organisms. Here, we present an integrated genome-guided workflow for the discovery, validation, and synthetic reconstruction of endogenous prophages from the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Bioinformatic analysis using PHASTEST identified seven prophage-like regions distributed throughout the bacterial chromosome, including five classified as intact prophages. Ultraviolet (UV)-mediated induction followed by whole-genome sequencing of purified phage DNA experimentally validated four inducible prophage loci (Regions 1, 5, 6 and 7) detected in all three biological replicates, together with a fifth locus (Region 2) detected less consistently, demonstrating strong agreement between computational prediction and biological activity. Comparative analysis further showed that prophage functionality was not always correlated with computational completeness, as one computationally “questionable” region exhibited robust induction whereas another “intact” region remained inactive under the conditions tested. Based on these results, one highly inducible prophage was selected for synthetic engineering. Three progressively truncated synthetic prophage genomes (approximately 36, 33, and 29 kb) were assembled using long-range PCR and scarless isothermal DNA assembly and verified by Nanopore sequencing, which confirmed accurate reconstruction without detectable rearrangements. Following electroporation into D. vulgaris and UV induction, the engineered genomes produced reproducible localized turbid phenotypes rather than classical lytic plaques, indicating that the synthetic constructs remained biologically active but did not undergo efficient lytic propagation under the experimental conditions examined. Quantitative PCR analysis of representative structural and lytic genes in wild-type cells revealed coordinated transcriptional activation by 21 h after UV induction, defining a practical harvest time for subsequent characterization. Collectively, this work establishes an integrated computational and synthetic biology framework for engineering endogenous prophages from anaerobic SRB. The approach provides a foundation for investigating temperate phage biology in D. vulgaris and represents an important step toward the future development of engineered phage platforms for MIC control.