Environmental PCR survey to determine the distribution of a non-canonical genetic code in uncultivable oxymonads

The universal genetic code is conserved throughout most living systems, but a non-canonical code where TAA and TAG encode glutamine has evolved in several eukaryotes, including oxymonad protists. Most oxymonads are uncultivable, so environmental RT-PCR and PCR was used to examine the distribution of this rare character. A total of 253 unique isolates of four protein-coding genes were sampled from the hindgut community of the cockroach, Cryptocercus punctulatus , an environment rich in diversity from two of the five subgroups of oxymonad, saccinobaculids and polymastigids. Four α-tubulins were found with non-canonical glutamine codons. Environmental RACE confirmed that these and related genes used only TGA as stop codons, as expected for the non-canonical code, whereas other genes used TAA or TAG as stop codons, as expected for the universal code. We characterized α-tubulin from manually isolated Saccinobaculus ambloaxostylus , confirming it uses the universal code and suggesting, by elimination, that the non-canonical code is used by a polymastigid. HSP90 and EF-1α phylogenies also showed environmental sequences falling into two distinct groups, and are generally consistent with previous hypotheses that polymastigids and Streblomastix are closely related. Overall, we propose that the non-canonical genetic code arose once in a common ancestor of Streblomastix and a subgroup of polymastigids.