Three metabolic pathways replenishing the one-carbon pool collectively support growth and virulence of Listeria monocytogenes

The bacterium Listeria monocytogenes can grow in the cytoplasm of infected human cells, but there it relies on specific biosynthetic pathways for intracellular nutrient supply. 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 L. monocytogenes Δ gcvPAB mutant, lacking the GCS glycine dehydrogenase, is attenuated without resembling the phenotype of classical virulence factor mutants. The Δ gcvPAB 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 ( fhs ) gene, which is naturally separated into two parts by a premature stop codon in the L. monocytogenes 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 Δ gcvPAB mutant. Another suppressor had a mutated glyA gene, encoding serine hydroxymethyltransferase, and combinatorial deletions of gcvPAB and glyA in fhs⁻ and fhs + 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 L. monocytogenes and represent the first example of the spontaneous reactivation of an L. monocytogenes gene that is inactivated by a premature stop codon.