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.

The first phytoplasma RNase P RNA provides new insights into the sequence requirements of this ribozyme.

A high variability of RNase P RNA structures is seen among members of the Mycoplasma group. To gain further insight into the structure-function relations of this ribozyme, we have searched for the RNase P RNA gene from more distant relatives, the phytoplasmas. These mycoplasma-like organisms are the aetiological agents of many severe plant diseases. We report the sequence and catalytic properties of RNase P RNA from the phytoplasma causing apple proliferation disease. The primary and postulated secondary structure of this 443 nt long RNA are most similar to those of Acholeplasma, supporting the phylogenetic position of this pathogen. Remarkably, the extremely AT-rich (73.6%) phytoplasma RNA differs from the known bacterial consensus sequence by a single base pair, which is positioned close to the substrate cleavage site in current three-dimensional models. Phytoplasma RNase P RNA functions as an efficient ribozyme in vitro. Conversion of its sequence to the full consensus and kinetic analysis of the resulting mutant RNAs suggests that neither the sequence alone, nor the type of pairing at this position is crucial for substrate binding or catalysis by the RNase P ribozyme. These results refine the bacterial consensus structure close to the catalytic core and thus improve our understanding of RNase P RNA function.