Abstract
Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.
Highlights
The family Tectiviridae is defined as a family of tailless, icosahedral viruses with a lipidic inner membrane and a linear, double-stranded DNA genome of approximately 15 kb, which is capped by the so-called terminal protein [1]
To obtain an extensive protein–protein interactome of Bam35 and B. thuringiensis by developing a novel and customizable approach, we established an experimental setup that combined traditional yeast two-hybrid with high-throughput sequencing methods
Others turned to fragment-based approaches for protein–protein interactions (PPIs) analysis, like those of Pseudomonas phage φKMV and that of mycobacteriophage Giles [35,39]
Summary
The family Tectiviridae is defined as a family of tailless, icosahedral viruses with a lipidic inner membrane and a linear, double-stranded DNA genome of approximately 15 kb, which is capped by the so-called terminal protein [1] These phages are currently divided into five genera, encompassing lytic and lysogenic bacteriophages that infect Gram-negative or Gram-positive bacteria, respectively. The early and best-known lytic viruses that prey on Gram-negative bacteria belong to the Alphatectivirus genus, whereas the temperate phages infecting Gram-positive bacteria were grouped in the Betatectivirus genus, and the recently reported new members of the family were assigned to the Gamma-, Delta-, and Epsilontectivirus genera [2] This family spans a wide genetic diversity under a common morphology with a broad host range and a predicted significant ecological importance [3,4]. Some infect pathogenic bacteria, such as phages Wip and AP50, can infect Bacillus anthracis, which is the etiological agent of anthrax [8,9], or Sole and Simila, which infects the food pathogen B. cereus [10]
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