Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.