CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease

Abstract

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called “CRISPRs.” Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

Viruses in corals: hidden drivers of coral bleaching and disease?

“…These findings suggest the cyanobacteria are regularly infected by bacteriophages and that phages may play a role in the disease development 24 .…”

“…The mechanisms by which lysogenic and lytic bacteriophages interfere with or contribute to coral disease pathogenesis are Roseofilum reptotaenium and Geitlerinema sp., two species dominating the black band disease mat in terms of biomass 24 .…”

Compositional homogeneity in the pathobiome of a new, slow-spreading coral disease

“…Further, both diseases show similarities in the taxonomy of disease-associated cyanobacteria. Roseofilum (the dominant cyanobacterium associated with BBD [18]) was recently shown to be closely related to Rivularia [30]. Likewise, seven of the 14 cyanobacteria that appeared to proliferate in GPD were classified as Rivulariaceae.…”

Novel T4 bacteriophages associated with black band disease in corals

“…In addition, the gp23 sequences were compared with a CRISPR-Cas spacer data set of the BBD associated cyanobacteria Geitlerinema sp. and R. reptotaenium (Buerger et al, 2016b). This step may reveal bacteriophages that previously infected the cyanobacteria, as spacers potentially contribute to a resistance against bacteriophage infections.…”

“…This virally-mediated pathogencity inducing mechanism has been suggested for the coral pathogen Vibrio coralliilyticus that causes some types of white syndromes (Weynberg et al, 2015). Relevance of phages in BBD pathogenesis is further suggested by the genomes of BBD-associated cyanobacteria harbouring CRISPR-Cas systems, which defend against bacteriophage infections (Buerger et al, 2016b). However, the phage community of BBD has not been described in detail and thus their possible role in BBD aetiology has not been identified.…”