Fig. 5: Evolution of Sgls in closely related ssRNA phages.

a Cladogram of ssRNA phages based on the corresponding Rep primary structures. Nodes with bootstrap values >80 (resampling n = 1000) are shown and nodes with at least two levels are grouped as clades and are highlighted in eight different colors. The genomes with sgls exhibiting function are colored blue, the sgls from the previously characterized ssRNA phages are colored black, and candidates that tested as non-functional are colored red. Closely related phages are indicated by brackets. b Nucleotide dot plots of full length contigs of the closely related ssRNA phage genomes (n = 11 genome pairs). Each dot plot compares two genomes and the dot plots are categorized as S (similar) and D (different) based on the relative genomic context of sgls. c An alignment of Hubei7_1 and Hubei8_1 wild-type and gain-of-function mutants. The amino acids in the alignment are highlighted according to clustalX and the missense changes found in gain-of-function mutants are highlighted as white letters on black background. The respective predicted membrane topologies are shown below the alignment as bar diagrams with different colors representing different predicted subcellular localizations. Blue (cytoplasmic, C), red (transmembrane domain, TMD), and purple (periplasmic, P). d The lysis profiles of gain-of-function Hubei7 mutants; pBAD24 (red open circle), Hubei7_1 (blue open squares), H7_mut5 (green open diamond), H7_mut6 (black cross), H7_mut10 (pink plus), H7_mut15 (light blue open triangle). H7_mut18 (black-filled circle), H7_mut20 (orange-filled square), H7_mut23 (blue-filled diamond), H7_mut24 (light gray-filled triangle). The mutants H7_mut6, mut18, and mut20 have the same change (W15L). e The lysis profiles of gain-of-function Hubei8 mutants; pBAD24 (red open circle), Hubei8_1 (blue open squares), H8_mut1 (green open diamond), H8_mut2 (black cross), H8_mut3 (pink plus), H8_mut4 (light blue open triangle). H8_mut5 (blue-filled circle), H8_mut6 (orange-filled square), H8_mut7 (blue-filled diamond), H8_mut8 (light gray-filled triangle), H8_mut9 (light pink-filled inverted triangle), H8_mut10 (pink circled dot). H8_mut1 and mut2 are siblings and H8_mut6 (S53P and S58P) is non-functional. The lysis profiles shown in this figure are representative of three biological replicates.