Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440

Thymidylate kinases (TMPKs) play an essential role in DNA biosynthesis across all domains of life by catalyzing dTMP phosphorylation to dTDP. In Pseudomonas putida KT2440, a model Gram-negative soil bacterium, tmk is disrupted by a 65-kb genomic island (GI), posing questions about the origin of the essential TMPK function. To solve this long-standing evolutionary riddle, we addressed three competing hypotheses: (i) assembly of two Tmk segments into a functional protein, (ii) complementation by a deoxynucleotide monophosphate kinase encoded within the GI, or (iii) fulfillment of the essential function by the product of PP_3363, yet another gene annotated as “thymidylate kinase.” Systematic genome engineering, quantitative physiology and targeted proteomics, complementation assays, phylogenetic analysis, and structure homology modeling were combined to investigate the role of genes within the GI. Our findings revealed that the GI-encoded dNMPK gene PP_1964 plays a critical role in complementing the disrupted TMPK function—exposing a non-essential character for the native PP_3363 gene and the tmk pseudogene. This dNMPK was found to be structurally related to that of bacteriophage T4, as part of a distinct evolutionary domain connected to mobile genetic elements and phages. The recursive genome reduction approach in this work deepens our understanding of the genetic architecture of a model bacterium while it provides evidence that the essential TMPK function has been acquired by horizontal gene transfer. Furthermore, the insights gained in the present study have broader implications for understanding the essentiality and functionality of dNMPK homologs in other bacteria.