Acquisition of the PK–AMGT energy shuttle and buffering system by C. eustigma through HGT. (A) Genomic location of the two PK genes and one AMGT gene. PK1 and AMGT are encoded in the scaffold Ceu0008, and PK2 is encoded in the scaffold Ceu0033 in the C. eustigma genome. (B) Phylogenetic relationship of PK proteins. The tree was constructed by the ML method (RaxML 8.0.0). ML BP >50% obtained by RaxML, and BI >0.95 obtained by Bayesian analysis (MrBayes 3.2.6) are shown above the branches. The accession numbers of the sequences are shown along with the names of the species. The branch lengths reflect the evolutionary distances indicated by the scale bar. (C) Overview of the phosphagen kinase energy buffering system (32). Taurocyamine (Tac) is produced by amidinotransferase (AMGT) from l-arginine (Arg) and taurine (Tau). Tac is phosphorylated to produce phosphotaurocyamine (PTac) by mitochondrial taurocyamine kinase (mtTK) by consuming ATP produced by oxidative phosphorylation. PTac is dephosphorylated by cytosolic taurocyamine kinase (cytTK) to produce ATP. ATP is, for example, consumed by plasma membrane H+-ATPase (PMA) to pump protons from the cytosol to outside the cell. Orn, ornithine. (D) Amino acid sequence alignment of the guanidine specificity region in C. eustigma phosphagen kinase with those of other organisms. The guanidine specificity regions (33) are shaded red, and taurocyamine kinases are shaded green. D1 and D2 represent domain 1 and domain 2, respectively, of two-domain enzymes. AvGK, Alitta virens glycocyamine kinase; CePK, C. eustigma phosphagen kinase; EfLK, Eisenia fetida lombricine kinase; LpAK, Limulus polyphemus arginine kinase; PiTK, Phytophthora infestans taurocyamine kinase; TcCK, Tetronarce californica creatine kinase. (E) Fluorometric detection of amines in cellular extractions by HPLC/OPA. The control is a chromatogram of a standard mixture of amino acids and taurocyamine. Asp, aspartic acid; OH-Pro, hydroxyproline; P-ET-Amine, o-phosphoethanolamine; Pro, proline; P-Ser, o-phosphoserine; Ser, serine; Tac, taurocyamine; Tau, taurine; Thr, threonine; α-A-A-A, α-aminoadipic acid.