Structural and functional characterization of MrpR, the master repressor of the Bacillus subtilis prophage SPß.

Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPß prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPß c2 mutant, we demonstrate that the lytic cycle of SPß c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPß genome. MrpR functions as a master repressor of SPß that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPß c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPß.

CRISPR-Cas9 Shaped Viral Metagenomes Associated with Bacillus subtilis.

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.

The Bacillus phage SPß and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components.

The Bacillus phage SPß 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 SPß-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPß-like phages. We extracted 55 SPß-like 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 SPß-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 SPß 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.

Chances and limitations when uncovering essential and non-essential genes of Bacillus subtilis phages with CRISPR-Cas9.

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.

The life cycle of SPß and related phages.

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. SPß 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, SPß 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, SPß presumably follows a two-step strategy, adsorbing primarily to teichoic acid and secondarily to a yet unknown factor. Once in the host, SPß-related phages pass through complex lysis-lysogeny decisions and either enter a lytic cycle or integrate as a dormant prophage. As prophages, SPß-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.