Genomic analyses of Symbiomonas scintillans show no evidence for endosymbiotic bacteria but do reveal the presence of giant viruses

Symbiomonas scintillans Guillou et Chrétiennot-Dinet, 1999 is a tiny (1.4 μm) heterotrophic microbial eukaryote. The genus was named based on the presence of endosymbiotic bacteria in its endoplasmic reticulum, however, like most such endosymbionts neither the identity nor functional association with its host were known. We generated both amplification-free shotgun metagenomics and whole genome amplification sequencing data from S. scintillans strains RCC257 and RCC24, but were unable to detect any sequences from known lineages of endosymbiotic bacteria. The absence of endobacteria was further verified with FISH analyses. Instead, numerous contigs in assemblies from both RCC24 and RCC257 were closely related to prasinoviruses infecting the green algae Ostreococcus lucimarinus, Bathycoccus prasinos, and Micromonas pusilla (OlV, BpV, and MpV, respectively). Using the BpV genome as a reference, we assembled a near-complete 190 kbp draft genome encoding all hallmark prasinovirus genes, as well as two additional incomplete assemblies of closely related but distinct viruses from RCC247, and three similar draft viral genomes from RCC24, which we collectively call SsVs. A multi-gene tree showed the three SsV genome types branched within highly supported clades with each of BpV2, OlVs, and MpVs, respectively. Interestingly, transmission electron microscopy also revealed a 190 nm virus-like particle similar to the morphology and size of the endosymbiont originally reported in S. scintillans. Overall, we conclude that S. scintillans currently does not harbour an endosymbiotic bacterium, but is associated with giant viruses.

Methods
Two strains of Symbiomonas scintillans were used to generate short-read Illumina NextSeq whole-genome amplified (WGA) sequencing data. The cells are individually isolated (50-100) for this. Viral contigs were identified and draft viral metagenomically assembled genomes (vMAGs) were generated using RagTag and annotated with Prokka. 

Three vMAGs were obtained from each of the strains RCC24 and RCC257. Only the most complete vMAGs (i.e., BpV2-vMAGs) were annotated.

Additionally, 22-core genes of prasinoviruses were searched in the WGA data to generate a multi-gene phylogeny. The identified genes were aligned with a previously compiled 22-core gene dataset and trimmed. After verifying each of the genes using 22 single-gene trees, the alignments were concatenated and used to construct multi-gene phylogenetic trees.