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 alpha-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 alpha-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-1alpha 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.