FIG 2.

Shortage of important downstream metabolites, but not the toxic accumulation of homocysteine, partially accounts for MetH essentiality. (A) Minimal medium containing 5 mM Met and 5 μg/ml Dox was supplemented with a variety of metabolites (potentially relevant for downstream processes [see Fig. 1A]) in single and multiple addition. Growth assays showed that purines (adenine A and guanine G, 3 mM) could slightly reconstitute growth of the H_OFF strain. Additional supplementation with amino acids improved growth, but not to the wild-type levels. Folic acid (5 mM) could also reconstitute growth, but only when amino acids were the only N source. (B) Overexpression of genes that could detoxify a potential accumulation of homocysteine did not reconstitute growth in the absence of MetH activity. Further addition of adenine did not improve growth. The phenotypic analyses were repeated in three independent experiments. Representative plates are shown.