Eukaryotic association module in phage WO genomes from Wolbachia

Abstract

Viruses are trifurcated into eukaryotic, archaeal and bacterial categories. This domain-specific ecology underscores why eukaryotic viruses typically co-opt eukaryotic genes and bacteriophages commonly harbour bacterial genes. However, the presence of bacteriophages in obligate intracellular bacteria of eukaryotes may promote DNA transfers between eukaryotes and bacteriophages. Here we report a metagenomic analysis of purified bacteriophage WO particles of Wolbachia and uncover a eukaryotic association module in the complete WO genome. It harbours predicted domains, such as the black widow latrotoxin C-terminal domain, that are uninterrupted in bacteriophage genomes, enriched with eukaryotic protease cleavage sites and combined with additional domains to forge one of the largest bacteriophage genes to date (14,256 bp). To the best of our knowledge, these eukaryotic-like domains have never before been reported in packaged bacteriophages and their phylogeny, distribution and sequence diversity imply lateral transfers between bacteriophage/prophage and animal genomes. Finally, the WO genome sequences and identification of attachment sites will potentially advance genetic manipulation of Wolbachia.

Figure 1. Phage WO genomes harbour an EAM.

The complete phage WO genome for (a) WOVitA1 was sequenced directly from purified viral particles using high throughput, metagenomic sequencing. The prophage (b) WOVitA1, (c) WOCauB3 and (d) WOCauB2 genomes were reannotated based on sequencing reads obtained from purified particles; complete genomes of WOCauB3 and WOCauB2 were not obtained. Each genome consists of a bacteriophage-like region (recombinase to patatin) and EAM highlighted in white. Grey slash marks indicate illustrative continuation of the genome. Dark blue dots indicate the discovery of the attL and attR sites of the prophage, which adjoin in the packaged WO genome to form attP. Numbers above the open reading frames indicate locus tags. Scale bar, 5,000 base pairs.

Figure 2. Eukaryotic-like EAM genes are enriched in prophage WO regions.

EAM genes with (a) eukaryotic homology are most likely to be associated with prophage WO while those with (b) bacterial homology are both phage-associated and found scattered throughout the Wolbachia chromosome. *The two chromosomal latrotoxin-CTD domains (wNo_10650 and wHa_05390) are located within phage-associated genes and transposases, indicating a potential genomic rearrangement. ^†SecA represents one ‘domain type' but is listed separately because phage WO contains two different homologues (that is, wHa_3920 and wHa_3930). Putative functional categories are as follows: anti-eukaryotic toxins (orange); host–microbe interactions (green); host cell suicide (blue); secretion of virulence factors (pink); and unknown (black). Octomom refers to WD0513 of the wMel genome.

Figure 3. Latrotoxin-CTD comparative analyses support lateral genetic transfers.

(a) Phylogeny of phage WO latrotoxin-CTD protein domains and their eukaryotic homologues was constructed by Bayesian analysis of 74 amino acids using the JTT model of evolution. Consensus support values are shown at the nodes. Comparative protein architecture shows that spider venom (b) vertebrate-specific alpha-latrotoxins and (c) invertebrate-specific alpha- and delta-latrotoxins are highly conserved, whereas (d) phage WO are not. WO denotes the specific phage haplotype while genome locus tags are listed in parentheses. Predicted furin cleavage sites, listed in Supplementary Table 3, are illustrated with grey triangles. *A second L. hesperus sequence represents a recently described downstream paralogue with unknown toxin activity. ^†wNo_10650 is located within phage-associated genes and transposases, indicating a potential genomic rearrangement of a phage region. ^‡Architecture is not shown for sequences on incomplete contigs (WOBol1-b, WOAlbB, WODi, WOPipMol and WOVitB) because complete peptide information and specific phage association are unknown. Scale bar, 1,000 amino acids.

Figure 4. Related TPR and ankyrin proteins support lateral genetic transfer.

(a) A BLASTP query of WOVitA1's gwv_1093 N terminus reveals homologues in mosquitoes, ants, beetles, a mealy bug, a solitary bee and one obligate intracellular gammaproteobacteria. Bayesian phylogenetic trees were constructed based on (b) a 137-aa alignment of all homologues with E-value <e⁻⁴⁰ using the LG+G model of evolution. (c) To resolve taxa closest to phage WO, trees were reconstructed based on a 627-aa alignment of all homologues with an E-value of 0 using the JTT+I+G model of evolution. Isoforms were removed from each alignment. Both trees are unrooted. Consensus support values are shown at the nodes. Chromosomal neighbourhood analyses of available animal genome sequences indicate that animal homologues to the phage WO protein are on contigs with other animal genes. Scale bar, 1,000 amino acids.

Figure 5. Phylogeny and protein architecture of the cell death domain, NACHT.

(a) A BLASTP query of prophage WO's NACHT region reveals homologues throughout arthropods and crustaceans. (b) Bayesian phylogenetic trees were constructed based on a 271-aa alignment of all homologues with E-value <e⁻¹⁵ and coverage >70% using the cpREV+G model of evolution. To resolve taxa closest to prophage WO, all Daphnia sequences were removed from the alignment and clusters of highly divergent residues (that is, five or more sequential residues with <15% pairwise identity) were trimmed. (c) Trees were reconstructed based on this 262-aa alignment using the LG+G model of evolution. Consensus support values are shown at the nodes. Both trees are unrooted. Chromosomal neighbourhood analyses of available animal genome sequences indicate that animal homologues to the prophage WO protein are on contigs with other animal genes. Scale bar, 1,000 amino acids.

Figure 6. Models of lateral DNA transfer between eukaryotes and bacteriophages.

(a) The eukaryotic cell can harbour multiple microbes capable of horizontal gene transfer. Genetic transfers between eukaryotes and bacteriophages can, in theory, occur (b) directly between eukaryotic chromosomes and phage genomes; (c) indirectly between eukaryotic and Wolbachia chromosomes; or (d) indirectly between eukaryotic chromosomes and intermediary entities, such as eukaryotic viruses and other intracellular bacteria.