TheBacillusphage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

Abstract

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.

Structural and functional analysis of YopR and identification of an additional key component of the SPβ phage lysis-lysogeny management system

“…Two recent studies uncovered several novel players of the SPβ lysis-lysogeny decision system [Brady et al, 2021; Kohm et al, 2022]. For instance, a suppressor analysis with a SPβ aimR mutant, which generates more lysogenic cells, revealed that the spontaneous inactivation of the yopN gene, encoding a protein of unknown function, partially restores the control of lysis-lysogeny [Brady et al, 2021].…”

“…For instance, a suppressor analysis with a SPβ aimR mutant, which generates more lysogenic cells, revealed that the spontaneous inactivation of the yopN gene, encoding a protein of unknown function, partially restores the control of lysis-lysogeny [Brady et al, 2021]. The same studies identified YopR as the master repressor of the lytic cycle of SPβ [Brady et al, 2021; Kohm et al, 2022]. Although it is unclear how AimR, YopN and YopR interact with each other, it has been proposed that YopN stimulates the repressor activity of YopR [Brady et al, 2021].…”

“…Although it is unclear how AimR, YopN and YopR interact with each other, it has been proposed that YopN stimulates the repressor activity of YopR [Brady et al, 2021]. Furthermore, a recent comparative genome analysis identified the core genes of SPβ-like phages and the yopR , yopQ , yopP , aimR and yokI genes, which are most likely transcriptionally active during the lysogenic cycle [Kohm et al, 2022]. The inactivation of the yokI gene indeed verified its role in the lysis-lysogeny decision system of SPβ because the yokI mutant released about four times more virions into the supernatant as compared to the parental strain [Kohm et al, 2022].…”

“…Furthermore, a recent comparative genome analysis identified the core genes of SPβ-like phages and the yopR , yopQ , yopP , aimR and yokI genes, which are most likely transcriptionally active during the lysogenic cycle [Kohm et al, 2022]. The inactivation of the yokI gene indeed verified its role in the lysis-lysogeny decision system of SPβ because the yokI mutant released about four times more virions into the supernatant as compared to the parental strain [Kohm et al, 2022]. It has been hypothesized that YokI could act as an RNase that prevents the accumulation of the AimX ncRNA, thereby hindering the entry into the lytic cycle (Figure 1) [Kohm et al, 2022].…”

Prevalence, Diversity and UV-Light Inducibility Potential of Prophages in Bacillus subtilis and Their Possible Roles in Host Properties

“…It is also considered as a key industrial organism due to its natural and engineered use in industrial production application, such as in food production (through fermentation), hydrolytic enzymes ( amylases , protases ), and fine chemicals (e.g., riboflavin) [ 18 ]. Despite the importance of B. subtilis strains in the food industry and biotechnology, the temperate phages SPbeta and phi3T are the two main phages that have been involved in research the most in past decades, and the B. subtilis 168 strain is the most frequently preferred host [ 19 , 20 ]. However, no data are available regarding the prevalence, diversity, and inducibility potential of the prophages in large collections of B. subtilis strains.…”