Genomic analysis of a Bacillus subtilis prophage vB_Bsu-yong1

Abstract:

[Objective] Bacillus subtilis, a Gram-positive bacterium ubiquitous in the nature, has strong stress tolerance and environmental adaptability and can inhibit the propagation of a variety of harmful bacteria. It is a common and important enzyme-producing bacterium, with the yield of proteases and amylases accounting for 50% of the industrial enzyme yield. Prophages integrated in host genomes are capable of providing additional genes and endow hosts with biological properties, demonstrating great research significance. The available studies about B. subtilis prophages focus on the defective ones. In this study, we analyzed the genome of an active prophage to expand the knowledge about non-defective prophages. [Methods] We induced a phage fromB. subtilis by using mitomycin C and named the phage as Bacillus phage Bsu-yong1. We employed transmission electron microscopy (TEM) to observe the negatively stained Bsu-yong1, Illumina MiSeq to sequence the genome of Bsu-yong1, and bioinformatics tools to annotate the genome. Furthermore, a proteomic tree of Bsu-yong1 was built based on genome-wide sequence alignment. [Results] Bsu-yong1 showed the morphology similar to that of PBSX-like defective prophages of B. subtilis. Unlike defective prophages which package DNA segments from random portions of the host genome, Bsu-yong1 was capable of packaging its own genome. The full-length genome of Bsu-yong1 was 43 590 bp with the G+C content of 41%. A total of 62 open reading frames (ORFs) were predicted with modular arrangement in the Bsu-yong1 genome. Bsu-yong1 harbored the ORFs encoding T7SS effector LXG polymorphic toxin, IMMA/IrrE and SMI1/KNR4. The former two were bacterial toxins and the later was an antitoxin. Toxins and antitoxins are members of the bacterial immune system, participating in bacterial competition and environmental adaptation. The gene encoding LXG polymorphic toxin in bacteriophages had never been reported before. A proteomic tree was built based on the whole genome alignment, revealing a long evolution distance between Bsu-yong1 and other phages. In the proteomic tree, Bsu-yong1 clustered with phages sv105, rho14, and vB_BteM-A9Y to form a monophyletic clade. These four phages all had PBSX-like morphology and shared 29 core genes. Pairwise sequence comparison (PASC) illustrated that the highest nucleotide sequence similarity between Bsu-yong1 and known phages was only 46.72%, which was much lower than the threshold (70%) to define a genus.[Conclusion] Bsu-yong1 represented a new unknown genus. We suggest the establishment of a novel family composed of Bsu-yong1, sv105, rho14, and vB_BteM-A9Y, which share 29 core genes. Bsu-yong1 carries immunity-related genes, which may benefit the host in bacterial competition and environmental adaptation. The findings enrich the bacteriophage gene database and enrich the knowledge of active Bacillus prophages.