Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions

Abstract

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria.IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

Keywords: Baltic Sea; Siphoviridae; TA system; brackish environment; phage-encoded CRISPR-Cas.

FIG 1

Genome map of cyanophage vB_AphaS-CL131 (CL 131) with annotated ORFs and assigned gene functions as described in the text. The color code is as follows: yellow, DNA replication, recombination, repair, and packaging; brown, transcription, translation, and nucleotide metabolism; blue, structural proteins; purple, chaperones/assembly; pink, protein with predicted catabolic activity; gray, ORFs of unknown function; red, CL 131-specific ORFs that encode unique proteins with no reliable identity to database entries; black, tRNA.

FIG 2

Phylogenetic analysis of cyanophage terminase large (TerL) subunit (A) and nucleotide sequence-based whole-genome comparisons (B) as tree diagrams. The scale bars indicate the average number of amino acid or nucleotide substitutions per site. Bacteriophage λ (family Siphoviridae) sequences were used as an outgroup. Red squares and green circles refer to cyanophages infecting unicellular and filamentous cyanobacteria, respectively. Bacteriophage family (M, Myoviridae; P, Podoviridae; S, Siphoviridae) assignments (A) or host cellular arrangement types (red, unicellular; green, filamentous) and habitat (marine or freshwater) (B) are provided.

FIG 3

Multiple pairwise genome alignments of cyanophages infecting filamentous cyanobacteria. The scale bar indicates the genome length. Blue and gray bars correspond to normal and inverted BLAST matches, respectively. WMP, Pf-WMP3; WMP4, Pf-WMP3; 2AV2, vB_NpeS-2AV2; CL131, vB_AphaS-CL131.

FIG 4

Representation of CRISPR-Cas locus in cyanophage vB_AphaS-CL131 genome (A) and multiple alignments of direct repeats of the cyanophage (CL 131) and Aphanizomenon flos-aquae strain 2012/KM1/D3 (afakm1d3-1 to afakm1d3-6) (B). Alignments were created using the CL 131 direct repeat sequence as the reference sequence; identities are normalized by aligned length and colored by identity. R, direct repeat; S1 to S10, spacers.

FIG 5

(A) Box plot representing the percentage of amino acid identities shared between cyanophage vB_AphaS-CL131 translated protein sequences and reads of 25 Baltic Sea metagenomes. The box depicts the upper and lower quartiles; the horizontal line indicates the median; whiskers indicate minimum and maximum amino acid identity values; circles indicate outliers. Boxes are colored to represent different years. (B) Dot plot of the number of total and cyanophage vB_AphaS-CL131-specific reads of structural genes. The samples (from August 2012; see text for details) with the highest number of total and vB_AphaS-CL131-specific reads are boxed.