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We previously found that the glycine cleavage system (GCS) is needed for intracellular growth. The GCS decarboxylates glycine for generation of 1C-tetrahydrofolates (1C-THF), folate-dependent one-carbon donors needed for biosynthesis of other metabolites. We continued our studies on the GCS and showed that a\n                    <jats:italic>L. monocytogenes<\/jats:italic>\n                    \u0394\n                    <jats:italic>gcvPAB<\/jats:italic>\n                    mutant, lacking the GCS glycine dehydrogenase, is attenuated without resembling the phenotype of classical virulence factor mutants. The \u0394\n                    <jats:italic>gcvPAB<\/jats:italic>\n                    mutant also grew poorly in synthetic medium, explained by the presence of glycine that was toxic for this strain. Selection of glycine-resistant suppressors yielded a survivor, in which the N- and C-terminal parts of the formate-tetrahydrofolate ligase (\n                    <jats:italic>fhs<\/jats:italic>\n                    ) gene, which is naturally separated into two parts by a premature stop codon in the\n                    <jats:italic>L. monocytogenes<\/jats:italic>\n                    reference strain EGD-e were reassembled into a full-length open-reading frame. Like the GCS, Fhs also feeds the 1C-THF pool, and its restoration cured the virulence defects of the \u0394\n                    <jats:italic>gcvPAB<\/jats:italic>\n                    mutant. Another suppressor had a mutated\n                    <jats:italic>glyA<\/jats:italic>\n                    gene, encoding serine hydroxymethyltransferase, and combinatorial deletions of\n                    <jats:italic>gcvPAB<\/jats:italic>\n                    and\n                    <jats:italic>glyA<\/jats:italic>\n                    in\n                    <jats:italic>fhs\u207b<\/jats:italic>\n                    and\n                    <jats:italic>fhs<\/jats:italic>\n                    <jats:sup>+<\/jats:sup>\n                    backgrounds demonstrated a role of GlyA in 1C-THF metabolism. Our results show that three pathways feed the 1C-THF pool to support growth and virulence of\n                    <jats:italic>L. monocytogenes<\/jats:italic>\n                    and represent the first example of the spontaneous reactivation of an\n                    <jats:italic>L. monocytogenes<\/jats:italic>\n                    gene that is inactivated by a premature stop codon.\n                  <\/jats:p>","DOI":"10.7554\/elife.109227","type":"journal-article","created":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T16:30:13Z","timestamp":1764088213000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.109227","source":"Crossref","is-referenced-by-count":0,"title":["Three metabolic pathways replenishing the one-carbon pool collectively support growth and virulence of Listeria 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