The Discovery of Twenty-Eight New Encapsulin Sequences, Including Three in Anammox Bacteria

Abstract

Many prokaryotes encode protein-based encapsulin nanocompartments, including anaerobic ammonium oxidizing (anammox) bacteria. This study expands the list of known anammox encapsulin systems from freshwater species to include the marine genus Scalindua. Two novel systems, identified in "Candidatus Scalindua rubra" and "Candidatus Scalindua sp. SCAELEC01 167" possess different architectures than previously studied freshwater anammox encapsulins. Characterization of the S. rubra encapsulin confirms that it can self-assemble to form compartments when heterologously expressed in Escherichia coli. BLASTp and HMMER searches of additional genomes and metagenomes spanning a range of environments returned 26 additional novel encapsulins, including a freshwater anammox encapsulin identified in "Candidatus Brocadia caroliniensis". Phylogenetic analysis comparing these 28 new encapsulin sequences and cargo to that of their closest known relatives shows that encapsulins cluster by cargo protein type and therefore likely evolved together. Lastly, prokaryotic encapsulins may be more common and diverse than previously thought. Through searching a small sample size of all public metagenomes and genomes, many new encapsulin systems were unearthed by this study. This suggests that many additional encapsulins likely remain to be discovered.

Figure 1

Comparison of newly discovered and previously known Planctomycete encapsulins. (a) Four newly discovered Planctomycete encapsulin systems. The B. caroliniensis and uncultured Planctomycete RIFCSPHIGHO2 systems contain an encapsulin followed by a nitrite reductase/hydroxylamine oxidoreductase (NiR/HAO) fusion protein. The S. rubra system consists of an encapsulin, a multi-copper oxidase (MCO), and a downstream NiRK. The Scalindua SCAELEC01 167 system consists of a NiR/HAO fusion protein, an encapsulin fused to a hypothetical protein also found immediately before the S. rubra encapsulin, and a nitrite reductase. (b) Previously known encapsulin systems discovered in freshwater anammox genera (figure reproduced with permission from ref. ). These systems consist of a cytochrome c/encapsulin fusion protein (cEnc) followed by a NiR/HAO fusion protein. The blue bars represent cupredoxin copper binding domains and the red bars represent iron rich heme moieties. DUF2325, Trx, c1, and c554 are possible secondary cargo proteins.

Figure 2

The S. rubra encapsulin monomer can self-assemble into a spherical polymer 25 nm in diameter. (a) Protein gel electrophoresis reveals that a protein with the predicted molecular mass (34 kDa) of the S. rubra encapsulin can be expressed in and isolated from E. coli lysate. Enc 1 refers to a protein beginning at the first of two possible translation start sites, Enc 2 refers to a protein that begins at the second site. (b) TEM imaging of a solution containing purified S. rubra encapsulin reveals that the S. rubra encapsulin monomer self-assembles into a spherical polymer with a diameter of 25 nm.

Figure 3

HMMER searches of OMZ metagenomes, as well as other environmental metagenomes, with an HMM profile of all 913 previously known encapsulins and the Scalindua rubra encapsulin as query returned twenty-four new encapsulins. Green points indicate locations where new encapsulins were discovered while white points indicate locations where no significant HMMER hits were observed. Black text indicates the number of novel non-viral encapsulins discovered at a location.

Figure 4

Newly discovered full-length encapsulin sequences cluster into clades with identical cargo protein types. Dark green rectangles indicate that a cargo protein of that type is encoded immediately next to the encapsulin in the tree, while light green rectangles show that the indicated cargo type is not present. Grey rectangles indicate encapsulins that are present on contigs that are too short to reveal other genes. All bootstrap values above 0.75 are displayed on the tree as blue circles. Newly discovered encapsulins are shown in green, while their known closest relatives are shown in black. Newly discovered anammox encapsulins are displayed in bold green text while the entire anammox clade is outlined in blue.