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

Abstract

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.

Living with Genome Instability: the Adaptation of Phytoplasmas to Diverse Environments of Their Insect and Plant Hosts

“…Most PMUs also contain a tmk gene encoding thymidylate kinase that synthesizes dTDP from dTMP for DNA synthesis. Similarly to AY-WB, the OY-M phytoplasma genome contains at least two tmk homologs, tmk-a and tmk-b, with tmk-a being present as multiple copies (68). We revealed that the tmk-a genes are part of PMUs.…”

“…We revealed that the tmk-a genes are part of PMUs. However TMK-b but not TMK-a was shown to have thymidylate kinase activity (68). Hence, the function of TMK-a is not yet clear.…”

Comparative Genome Analysis of “CandidatusPhytoplasma australiense” (Subgrouptuf-Australia I;rp-A) and “Ca. Phytoplasma asteris” Strains OY-M and AY-WB

“…Phytoplasma australiense" PMUs contained the tmk gene that encodes thymidylate kinase. This enzyme catalyzes the transfer of a terminal phosphoryl group from ATP to dTMP and is crucial for de novo synthesis and salvage pathways for pyrimidine deoxyribonucleotides (50). "Ca.…”

“…Phytoplasma australiense" carries multiple copies of the tmk gene in two paralog families. The OY-M phytoplasma genome encodes two tmk genes, tmk-a and tmk-b (50), where multiple copies of tmk-a exist in PMUs (9) but only tmk-b has thymidylate kinase activity (50). One paralog family containing two ORFs (CAM11639 and CAM11700) was similar to the tmk-a gene from OY-M and AY-WB but also to orthopoxviruses (6), and this supports a link between viruses and phytoplasma extrachromosomal DNA (42,55,57,58,67,84).…”

Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii

“…However, pseudogenes in general are found in bacteria (Andersson and Andersson 2001;Eiglmeier et al 2001;Parkhill et al 2001), and some may limit a bacterium's host range ) and/or contribute to inability to grow in artificial media . Recently, pseudogenes were found in the plant pathogenic mollicute clover phyllody (CPh) phytoplasma ) and onion yellows phytoplasma (Miyata et al 2003). The presence of pseudogenes in phytoplasmas has been interpreted as part of the ongoing process of the genome reduction or degradation , which may also be the case in S. kunkelii.…”