Abstract
Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of Escherichia coli where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. 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 can adapt to limited supplies of deoxyribonucleosides, as might occur in the 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 reveals that several replicate 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 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 life without ribonucleotide reduction.