N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs

<i>N</i>⁴-methylcytosine is a modified heterocyclic base present both in RNA and DNA. The biosynthesis and function of this derivative are widely investigated. However, how the demethylation of this base occurs is not known. Here, we have investigated the growth of an <i>Escherichia coli</i> uracil auxotroph strain in minimal M9 medium supplemented with <i>N</i>⁴-methylcytosine. We have found that this compound, but not the related <i>N</i>⁴,<i>N</i>⁴-dimethylcytosine, well supports growth with a generation time of the bacterium being 3 h compared to 1.5 h for media supplemented with cytosine or uracil. Using high-performance liquid chromatography (HPLC), we have demonstrated that the concentration of <i>N</i>⁴-methylcytosine in the growth medium decreases by 12% after 24 h of growth. We have shown that <i>N</i>⁴-methylcytosine is not directly converted into uracil by <i>E. coli</i> CodA cytosine deaminase. Instead, we propose the enzymatic pathway in which <i>N</i>⁴-methylcytosine is converted into cytosine by yet unknown demethylase, whereas CodA converts the resulting cytosine to uracil, thereby supporting the growth.