All life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. A handful of obligate intracellular species are known to lack ribonucleotide reduction and are instead dependent on their host for deoxyribonucleotide synthesis. As ribonucleotide reduction has on occasion been lost in obligate intracellular parasites and endosymbionts, we reasoned that it should in principle be possible to knock this process out entirely under conditions where deoxyribonucleosides are present in the growth media. We report here the creation of a strain of E. coli where all three ribonucleotide reductase operons have been fully deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain is able to grow in the presence of deoxyribonucleosides and shows slowed but substantial growth. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines are able to adapt to limited supplies of deoxyribonucleosides, as might occur in the evolutionary switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis of replicate lines reveals that several lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than synthesis via this pathway complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to intracellular life without ribonucleotide reduction.
All genome data have been deposited to the Single Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accessions SRX17677859-SRX17677886. The following biosamples are associated with this project: SAMN30957267-SAMN30957273. The data have been deposited with links to BioProject accession number PRJNA882995 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).
Experimental evolution of an Escherichia coli line lacking ribonucleotide reduction.NCBI Bioproject, PRJNA882995.
- Jun Ogawa
- Anthony M Poole
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Amie K Boal, Pennsylvania State University, United States
© 2023, Arras et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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