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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10653934/ https://www.ncbi.nlm.nih.gov/pubmed/37732760 http://dx.doi.org/10.1128/mbio.01081-23 |
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author | Wirth, Nicolas T. Rohr, Katja Danchin, Antoine Nikel, Pablo I. |
author_facet | Wirth, Nicolas T. Rohr, Katja Danchin, Antoine Nikel, Pablo I. |
author_sort | Wirth, Nicolas T. |
collection | PubMed |
description | 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. IMPORTANCE: Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which “landed” in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host—instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings—yielding genome-reduced cell factories with enhanced properties and genetic stability. |
format | Online Article Text |
id | pubmed-10653934 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-106539342023-09-21 Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 Wirth, Nicolas T. Rohr, Katja Danchin, Antoine Nikel, Pablo I. mBio Research Article 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. IMPORTANCE: Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which “landed” in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host—instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings—yielding genome-reduced cell factories with enhanced properties and genetic stability. American Society for Microbiology 2023-09-21 /pmc/articles/PMC10653934/ /pubmed/37732760 http://dx.doi.org/10.1128/mbio.01081-23 Text en Copyright © 2023 Wirth et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Wirth, Nicolas T. Rohr, Katja Danchin, Antoine Nikel, Pablo I. Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title | Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title_full | Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title_fullStr | Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title_full_unstemmed | Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title_short | Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440 |
title_sort | recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in pseudomonas putida kt2440 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10653934/ https://www.ncbi.nlm.nih.gov/pubmed/37732760 http://dx.doi.org/10.1128/mbio.01081-23 |
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