The element chlorine is not a usual constituent of Escherichia coli, but it can be used to replace the methyl group in thymine (i.e., 5-methylu-racil) to yield a synthetic nucleic acid precursor, 5-chlorouracil. Marliere et al. exploited metabolic selection in an automated, self-cleaning culture device to evolve strains of E. coli in which the canonical base thymine was nearly fully replaced with the synthetic analog within 150 days of culture. Thymidylate synthase and the trmA gene were excised from the starting strains and nucleoside deoxyribosyltransferase was inserted. After about 1000 generations, the evolved strains became dependent on chlo-rouracil for growth. Analysis of the chemical composition of the DNA of these isolates showed that thymidine content had dropped to less than 2% of that in the original strain, but DNA sequencing revealed several thousands of base substitutions and chromosomal rearrangements. These mutations may cause base mispairing, but the consequences of this on subsequent evolution of the heterodox bacterium, or indeed the value of the novel organism for the biosynthesis of non-natural fermentation products, remain unclear.
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