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Tectiviruses infecting the Bacillus cereus group represent part of the bacterial “plasmid repertoire” as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the “gene regulation-genome replication” module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

Some strains of the Bacillus cereus group can be implicated in two types of foodborne intoxication syndromes, namely diarrheal and emetic. The emetic syndrome results from the ingestion of the cereulide toxin produced by particular strains of B. cereus and Bacillus weihenstephanensis. It represents a major concern since it can lead, in severe cases, to the death of the intoxicated patients. This study aimed to characterize the virulent phage Deep-Blue and investigate its biocontrol potential against psychrotolerant B. weihenstephanensis. This myovirus was isolated from an agricultural soil and is stable for at least 6 months at 4 °C. It is specific to members of the B. cereus group and shows a lytic activity against five of twelve B. weihenstephanensis. Its lytic cycle lasts less than 1 h with a final phage progeny of ca. 300 virions. Regarding its bactericidal potential, Deep-Blue was able to reduce B. weihenstephanensis contamination under laboratory conditions (LB broth) and in milk, and to control bacterial growth in food matrices such as milk rice. Finally, the application of Deep-Blue as preventive treatment was shown to efficiently control the development of B. weihenstephanensis biofilm.

Introduction There is a renewed interest in phages and their derived proteins for various applications, including in the agro-food sector. Endolysins are peptidoglycan degrading enzymes synthetized at the end of the lytic cycle. In combination with holins, they are responsible for the lysis of the host cell allowing the release of newly synthetized virions. Endolysins from phages infecting Gram-positive bacteria are organized into two domains: a C-terminal Cell Wall Binding domain (CBD) and an N-terminal Enzymatically Active Domain (EAD) that cleaves the peptidoglycan. This study focuses on endolysins encoded by Deep-Blue [1] and Deep-Purple [2], two phages infecting Bacillus weihenstephanensis and belonging to the Myoviridae and Siphoviridae families, respectively. Materials and Methods Endolysin candidates were selected by bioinformatics and whole genes were cloned into the expression vector pET30a. Similarly, the corresponding CBD were cloned into pET30a with a N-terminal GFP fusion. The constructions were transformed into the strain BL21(DE3) and expression of each recombinant protein was induced by addition of IPTG. The proteins were purified on a Ni-NTA column. The lytic activity of the putative endolysins was assess by CFU reduction assay and reduction in Optical Density. As for the CBD, the interaction between the GFP fused domain and the bacteria was detected by fluorescent microscopy. Results and Discussion In silico analyses revealed that Deep-Blue gp221 and Deep-purple gp032 are good endolysin candidates. Gp221 has a Peptidase_M15_4 conserved domain from the VanY superfamily as EAD, which is also present in the endolysins of phages B4 [3] (LysB4) and Phrodo [4] (PlyP56) infecting members of the Bacillus cereus group. Gp032 possesses a GH25_PlyB-like domain belonging to the GH25_muramidase superfamily. PlyB is an endolysin from phage BcpI that displays activity against Bacillus anthracis [5]. As for the CBD, two SH3 domains were detected at the C terminus

Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60 °C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.

Bacteriophages are an important repository of genetic diversity and source of unexplored genes. In the particular case of tectiviruses infecting the Bacillus cereus group, these bacteriophages represent part of the bacterial mobilome and they are, so far, the only ones able to behave as linear plasmids during their lysogenic cycle. Recently, several novel tectiviruses have been found infecting diverse strains belonging to this bacterial lineage (Jalasvuori et al., 2013; Gillis and Mahillon 2014). Among them, partial DNA sequencing of tectiviruses Sole and Sato, isolated from B. cereus sensu lato and emetic B. cereus strains, respectively, uncovered a highly variable region potentially involved in lysogeny maintenance (Jalasvuori et al., 2013). Here, we analyze the complete genome sequences of bacteriophages Sole and Sato. The genome of Sole spans 14,445 bp with 28 putative CDSs, whereas the one of Sato has 14,852 bp comprising 31 putative CDSs. DNA sequence identity comparisons of Sato and Sole against Bacillus virus Bam35 (Betatectivirus type species) indicated 86 and 89% sequence identity, respectively. Additionally, phylogenetic analyses and genome alignments suggested that both isolates represent novel tectivirus species. Hence, tectiviruses in Gram-positive bacteria have shown to be more diverse than those infecting Gram-negative bacteria, as the latter ones display a very high level of sequence identity. In an effort to assess the host range of Sole and Sato against emetic B. cereus and Bacillus cytotoxicus, two species involved in food poisoning outbreaks, 50 strains of each species devoid of tectiviral-like elements were tested. The results showed that Sole and Sato have a narrow host range, only infecting particular strains. Overall, genome sequencing and comparative analysis of tectiviruses Sole and Sato have expanded the view of the genomic diversity occurring in plasmidial prophages found in members of the B. cereus group.

Bacteriophages are an important repository of genetic diversity and source of unexplored genes. In the particular case of tectiviruses infecting the Bacillus cereus group, these bacteriophages represent part of the bacterial mobilome and they are, so far, the only ones able to behave as linear plasmids during their lysogenic cycle. Recently, several novel tectiviruses have been found infecting diverse strains belonging to this bacterial lineage (Jalasvuori et al., 2013; Gillis and Mahillon 2014). Among them, partial DNA sequencing of tectiviruses Sole and Sato, isolated from B. cereus sensu lato and emetic B. cereus strains, respectively, uncovered a highly variable region potentially involved in lysogeny maintenance (Jalasvuori et al., 2013). Here, we analyze the complete genome sequences of bacteriophages Sole and Sato. The genome of Sole spans 14,445 bp with 28 putative CDSs, whereas the one of Sato has 14,852 bp comprising 31 putative CDSs. DNA sequence identity comparisons of Sato and Sole against Bacillus virus Bam35 (reference tectivirus) indicated 86 and 89% sequence identity, respectively. Additionally, phylogenetic analyses and genome alignments suggested that both isolates represent novel tectivirus species. Hence, tectiviruses in Gram-positive bacteria have shown to be more diverse than those infecting Gram-negative bacteria, as the latter ones display a very high level of sequence identity. In an effort to assess the host range of Sole and Sato against emetic B. cereus and Bacillus cytotoxicus, two species involved in food poisoning outbreaks, 50 strains of each species devoid of tectiviral-like elements were tested. The results showed that Sole and Sato have a narrow host range, only infecting particular strains. Overall, genome sequencing and comparative analysis of tectiviruses Sole and Sato have expanded the view of the genomic diversity occurring in plasmidial prophages found in members of the B. cereus group.

The Bacillus cereus emetic pathotype is responsible for important food-borne intoxications. Here, we describe the complete genome sequence of bacteriophage Deep-Blue, which is able to infect emetic strains of B. cereus. Deep-Blue is a 159-kb myophage of the Bastille-like group within the Spounavirinae.

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