Phage vB_BsuP-Goe1: the smallest identified lytic phage of Bacillus subtilis

Hertel

2018

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We present the recently isolated virus vB_BthP-Goe4 infecting Bacillus thuringiensis HD1. Morphological investigation via transmission electron microscopy revealed key characteristics of the genus Phi29virus, but with an elongated head resulting in larger virion particles of approximately 50 nm width and 120 nm height. Genome sequencing and analysis resulted in a linear phage chromosome of approximately 26 kb, harbouring 40 protein-encoding genes and a packaging RNA. Sequence comparison confirmed the relation to the Phi29virus genus and genomes of other related strains. A global average nucleotide identity analysis of all identified φ29-like viruses revealed the formation of several new groups previously not observed. The largest group includes Goe4 and may significantly expand the genus Phi29virus (Salasvirus) or the Picovirinae subfamily.

Section: Introductionmentioning

confidence: 99%

Genomic Analysis of the Recent Viral Isolate vB_BthP-Goe4 Reveals Increased Diversity of φ29-Like Phages

Schilling

Hertel

2018

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“…To verify whether the pJOE8999 vector system [ 23 ] is suitable for in vivo genetic engineering of virulent phages, a proof of concept experiment with the smallest known B. subtilis phage Goe1 [ 24 ] was performed. The gene Goe1_c00180 coding for the head fiber protein was selected as target.…”

Section: Resultsmentioning

confidence: 99%

“…The virulent phage vB_BsuP-Goe1 (Goe1) [ 24 ] serves as model phage for this investigation. It reveals Podoviridae morphology and is a member of the genus Phi29virus [ 24 ]. It has a genome of 18,379 bp and is closely related to the well-understood phage phi29 (63.7% global nucleotide similarity).…”

Section: Introductionmentioning

confidence: 99%

A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages

Schilling

Dietrich

et al. 2018

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Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus. This toolkit comprises the application CutSPR, a bioinformatic tool for rapid primer design, and facilitates the cloning of specific CRISPR-Cas9-based mutagenesis plasmids. The employment of the prophage-free and super-competent B. subtilis TS01 strain enables an easy and fast introduction of specific constructs for in vivo phage mutagenesis. Clean gene deletions and a functional clean gene insertion into the genome of the model phage vB_BsuP-Goe1 served as proof of concept and demonstrate reliability and high efficiency. The here presented toolkit allows comprehensive investigation of the diverse phage genetic pool, a better understanding of phage biology, and safe phage applications.

“…Raw sewage from a municipal sewage treatment plant in Göttingen (Germany, 51°33′15.4″ N 9°55′06.4″ E) was used as inoculum. The isolation and propagation procedure were performed as described previously [18]. Plaque assays were performed by mixing 100 µL of the overnight host culture with 100 µL of virus suspension and incubating for 5 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Bacillus subtilis Viruses vB_BsuM-Goe2 and vB_BsuM-Goe3

Willms

Hertel

2017

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The Spounavirinae viruses are ubiquitous in nature and have an obligatory virulent lifestyle. They infect Firmicutes, a bacterial phylum containing an array of environmental non-pathogenic and pathogenic organisms. To expand the knowledge of this viral subfamily, new strains were isolated and investigated in this study. Here we present two new viruses, vB_BsuM-Goe2 and vB_BsuM-Goe3, isolated from raw sewage and infecting Bacillus species. Both were morphologically classified via transmission electron microscopy (TEM) as members of the Spounavirinae subfamily belonging to the Myoviridae family. Genomic sequencing and analyses allowed further affiliation of vB_BsuM-Goe2 to the SPO1-like virus group and vB_BsuM-Goe3 to the Bastille-like virus group. Experimentally determined adsorption constant, latency period, burst size and host range for both viruses revealed different survival strategies. Thus vB_BsuM-Goe2 seemed to rely on fewer host species compared to vB_BsuM-Goe3, but efficiently recruits those. Stability tests pointed out that both viruses are best preserved in LB-medium or TMK-buffer at 4 or 21 °C, whereas cryopreservation strongly reduced viability.