Occurrence, genomic organization and relatedness of the pupylation gene locus. (a) Phylogenetic analysis of the combined Arc, Dop, Pup and PafA amino acid sequences reveals tight clustering of proteasome-harboring members (clusters I and II), whereas members without proteasomal genes in the pupylation locus exhibit much greater sequence variation (cluster III). The pupylation enzymes of Leptospirillum ferrooxidans, a Nitrospirae exponent, likely originate in a member of the acidimicrobiales, a subclass of the actinobacteria. (b) Genomic context of the pupylation-relevant enzymes. The genomes are listed counter clockwise as they appear in (a). The enzymatic order in the genome is rigidly conserved through all pupylation-competent organisms, although some species exist (as Saccharopolyspora erythraea or Salinispora tropica) with duplicated parts of the system. L. ferrooxidans contains two copies of the entire system (L1, L2), which are identical in terms of genetic context, but very different in their sequence (Pup in L2 even lacks the terminal GGE). The phylogenetic tree was calculated using PhyML  and displayed with iTOL  from MUSCLE-aligned  and GBLOCKS-refined  sequences.