A CRISPR‐Cas9 tool to explore the genetics of
            <i>Bacillus subtilis</i>
            phages

Otte, Kolja L; Kühne, N.M.; Furrer, Alexandra Dominique; Lozada, Lina Paola Baena; Lutz, Veronika Theresa; Schilling, Tobias; Hertel, Robert

doi:10.1111/lam.13349

Cited by 10 publications

(14 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The AimX ncRNA may modulate the function of YopR and make it more slippery or tight. However, the arbitrium system is not essential for lysogeny establishment or resolvement but rather fine-tunes the system (Erez et al, 2017;Otte et al, 2020;Aframian et al, 2021;Brady et al, 2021). The question not asked before is what happens with the ncRNA AimX itself.…”

Section: Spβ Core Genes and Its Lysogeny Management Systemmentioning

confidence: 99%

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

Kohm

Floccari

Lutz

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Spβ Core Genes and Its Lysogeny Management Systemmentioning

confidence: 99%

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

Kohm

Floccari

Lutz

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cells of E. coli DH10B were prepared and transformed according to Sambrook and Russell (2001). The vector pRH030 (Otte et al, 2020) was digested with FastDigest™ BsaI (Eco31I; Thermo Fisher Scientific, USA) for 30 min at 37 C and subsequently purified on 1% agarose gel with QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Plaque assay and analysis were performed as described previously by Schilling et al (2018a) and Otte et al (2020). Briefly, overnight cultures of B. subtilis Δ6 strains holding a pRH030 derivate with a ready set gRNA was inoculated to OD 600 = 0.1 and cultivated at 30 C with vigorous shaking until an OD 600~1 .…”

Section: Plaque Assaymentioning

confidence: 99%

“…For B. subtilis phage modification, a CRISPR-Cas9 toolkit was designed based on a CRISPR-Cas9 plasmid (pJOE8999), itself made initially for B. subtilis modification (Altenbuchner, 2016). With the plasmid pRH030, this system was further adapted and optimized for the needs of B. subtilis phage modification (Otte et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…With the plasmid pRH030, this system was further adapted and optimized for the needs of B . subtilis phage modification (Otte et al ., 2020).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Kohm

Basu

Nawaz

et al. 2021

Environ Microbiol Rep

Self Cite

View full text Add to dashboard Cite

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.

show abstract