Understanding the Encapsulins:Prediction and Characterization of Phage Capsid-like Nanocompartments in Prokaryotes

Encapsulins are a distinct family of prokaryotic compartments, comprised of an icosahedral shell encapsulating a variety of enzymes. The encapsulin shell protein is similar in sequence and structure to the capsid protein of dsDNA tailed bacteriophages. Although found across diverse phyla of Bacteria and Archaea, few encapsulins were previously characterized. My thesis work utilized a bioinformatic approach to identify and investigate the functions of all encapsulins found in bacteria and Archaea. I found 590 encapsulins showing definite sequence similarity to the previously described encapsulins, which I refer to as "classical encapsulins". I identified four new enzyme families strongly predicted to be classical encapsulin cargo enzymes: ferredoxins, rubrerythrin-like, Dps-bacterioferritin-like, and hemerythrin-like proteins. While most species encode encapsulin cargo proteins adjacent to the encapsulin gene, I discovered 113 species encoding cargo proteins in genomic regions far removed from the encapsulin gene. I also found 118 genomes encoding two or three different enzymes strongly predicted to be targeted to the same encapsulin. Genes encoding three protein families, radical SAM oxidoreductases, CutA1-like proteins and metalloproteases, are highly enriched near encapsulin genes, but without targeting motifs for direct encapsulation. In addition to classical encapsulins, I discovered 1060 novel putative encapsulins related to diverse families of phage capsids. The largest family of these novel encapsulins, with 986 proteins is larger than the classical encapsulin family, and conserves a sulphur metabolism operon encoding cysteine desulfurases, acetyltransferases, and rhodaneses. These activities are predicted to relieve sulphur restriction, and toxicity of cyanide and other oxidants. Overall, novel and classical encapsulins and their encapsulated enzymes are predicted to contribute to polyvalent cation dependent catalytic activities, and relieve heavy metal and cyanide toxicity. Phylogenies predicted capsids and encapsulins share intermingled ancestry. My work shows that encapsulins perform more diverse functions and are much more widely distributed than any other prokaryotic compartment.Ph.D