Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to the Levivirus group and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found in Levivirus and Allolevivirus predate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making the Levivirus-like genome organization ancestral and the Allolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.