Abstract
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.
Original language | English |
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Article number | e01081-23 |
Journal | mBio |
Volume | 14 |
Issue number | 5 |
Number of pages | 22 |
ISSN | 2161-2129 |
DOIs | |
Publication status | Published - 2023 |
Keywords
- Pseudomonas putida
- Thymidylate kinase
- Prophage
- Bacterial evolution
- Genome reduction
- Horizontal gene transfer
- Genome engineering