Phages are viruses of bacteria and are the smallest and most common biological entities in the environment. They can reproduce immediately after infection or integrate as a prophage into their host genome. SPβ is a prophage of the Gram-positive model organism Bacillus subtilis 168, and it has been known for more than 50 years. It is sensitive to dsDNA damage and is induced through exposure to mitomycin C or UV radiation. When induced from the prophage, SPβ requires 90 min to produce and release about 30 virions. Genomes of sequenced related strains range between 128 and 140 kb, and particle-packed dsDNA exhibits terminal redundancy. Formed particles are of the Siphoviridae morphotype. Related isolates are known to infect other B.subtilis clade members. When infecting a new host, SPβ presumably follows a two-step strategy, adsorbing primarily to teichoic acid and secondarily to a yet unknown factor. Once in the host, SPβ-related phages pass through complex lysis–lysogeny decisions and either enter a lytic cycle or integrate as a dormant prophage. As prophages, SPβ-related phages integrate at the host chromosome's replication terminus, and frequently into the spsM or kamA gene. As a prophage, it imparts additional properties to its host via phage-encoded proteins. The most notable of these functional proteins is sublancin 168, which is used as a molecular weapon by the host and ensures prophage maintenance. In this review, we summarise the existing knowledge about the biology of the phage regarding its life cycle and discuss its potential as a research object.

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPβ-like phages. We extracted 55 SPβlike prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. We could also reveal the SPβ-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

Prophages need to tightly control their lifestyle to either be maintained within the host genome or enter the lytic cycle. The SPβ prophage present in the genome ofBacillus subtilis168 was recently shown to possess anarbitriumsystem defining its replication stage. Using an historicB. subtilisstrain harboring the heat-sensitive SPβ c2 mutant, we analyzed a key component of the lysis-lysogeny decision system called YopR, which is crucial for maintenance of lysogeny. Here, we demonstrate that the heat-sensitive SPβ c2 phenotype is due to a single nucleotide exchange in theyopRgene, rendering the encoded YopRG136Eprotein temperature sensitive. Structural characterization of YopR revealed that the protein is a DNA-binding protein with an overall fold like tyrosine recombinases. Biochemical and functional analyses indicate that YopR has lost the recombinase function and the G136E exchange impairs its higher order structure and DNA binding activity. We also show that the serine recombinase SprA and its accessory factor SprB are not required for the heat-dependent induction of the lytic cycle of the SPβ c2 prophage. Finally, an evolution experiment with aB. subtilisstrain carrying SPβ c2 identified YosL as a novel component of the lysis-lysogeny management system, as the presence ofyosLis crucial for the induction of the lytic cycle of SPβ.

Virulent bacterial viruses, also known as phages or bacteriophages, are considered as a potential option to fight antibiotic-resistant bacteria. However, their biology is still poorly understood, and only a fraction of phage genes is assigned with a function. To enable the first classification, we explored new options to test phage genes for their requirement on viral replication. As a model, we used the smallest known Bacillus subtilis phage Goe1, and the Cas9-based mutagenesis vector pRH030 as a genetic tool. All phage genes were specifically disrupted, and individual survival rates and mutant genotypes were investigated. Surviving phages relied on the genome integrity through host intrinsic non-homologues end joining system or a natural alteration of the Cas9 target sequence. Quantification of phage survivors and verifying the underlying genetic situation enables the classification of genes in essential or non-essential sets for viral replication. We also observed structural genes to hold more natural mutations than genes of the genome replication machinery.