Prevalence, genetic diversity and host range of tectiviruses among members of the Bacillus cereus group

Gillis, Annika [UCL] Mahillon, Jacques [UCL]

Tectiviridae comprises non-enveloped tail-less phages with a double-layer capsid where the ~15 kb linear dsDNA is located within a lipid-containing membrane/vesicle covered by a rigid icosahedral protein capsid. Upon infection the lipid membrane becomes a tail-like structure used in genome delivery. GIL01/Bam35, GIL16, AP50 and Wip1 are tectiviruses preying on the Bacillus cereus group. These phages also exhibit a strong similarity to the B. cereus linear plasmid pBClin15. Tectiviruses infecting the B. cereus group are temperate phages able to replicate autonomously as linear plasmids inside the host cell. Despite the significant contributions of phages in different biological processes, little is known about the relations taking place between tectiviruses and their Gram-positive hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group, by assessing their occurrence, genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 worldwide isolates, comprising all seven recognized members of this bacterial group, were evaluated using primers designed on two variable regions detected in previously described elements. The first variable region is located within the module that contains replication and regulatory genes (coordinates 3,162 - 4,442 in GIL01), referred to herein as the “RR region”, and codes for the C-terminal end of the DNA polymerase B protein (DNA polB, CDS 5 in GIL01) and for the N-terminal part of the LexA-like protein (CDS 6). The second variable region is located within the module containing the host recognition and cell lysis genes (coordinates 11,096 - 11,838 in GIL01), herein referred to as the “PL region”, and targets the N-acetyl-muramidase coding gene (mur1, CDS 26) and partially CDS 27 for which the predicted function is related to the pentameric spike base involved in host recognition. PCR screenings targeting the RR and PL regions, along with propagation tests, revealed that tectiviral elements occurred in less than 3 % of the isolates. Regardless of this limited distribution, 47 novel putative tectiviruses were discovered in the present work, beside the 10 members already known (including Sole, Sand, Sato, Emet and Lima). Overall, these findings (i) expand the view of tectiviral prophages occurrence in members of the B. cereus group, providing evidence for (ii) a greater genetic diversity than previously observed within the Tectiviridae. In addition, analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains, which did not harbor tectiviral-elements, were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship was found between the infection patterns of the tectiviruses and their diversity. Moreover, the genomic data exposed that diversity and apparent relationships between tectiviruses will differ with the analyzed variable region and if genes are subjected to different evolutionary processes, like recombination. As a consequence, the PL region is proposed to be used in large tectiviral occurrence screenings, whereas the RR region could be used to analyze the bona fide diversity. From an evolutionary perspective, the results indicate that the DNA polB gene has undergone genetic recombination, although the actual contribution of this event remained to be further explored.