Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, ␥-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate ␥-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a ␥-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.Glutathione (␥-glutamyl-cysteinyl-glycine) (GSH) is the predominant low-molecular-weight peptide thiol present in living organisms. In bacteria it plays a pivotal role in many metabolic processes, chief among which are thiol redox homeostasis, protection against reactive oxygen species, protein folding, and provision of electrons via NADPH to reductive enzymes, such as ribonucleotide reductase. Low-molecular-weight nonribosomal peptides are assembled by the action of versatile multimodular enzymes termed nonribosomal peptide synthetases (NRPS) (22, 37) or through the consecutive actions of individual enzymes. GSH synthesis is a prime example of the latter, and GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (21). The ␥-carboxyl group of L-glutamate and the amino group of L-cysteine are ligated by the enzyme ␥-glutamylcysteine ligase (encoded by gshA) to give ␥-glutamylcysteine, which is then condensed with glycine in a reaction catalyzed by glutathione synthetase (encoded by gshB) to form GSH. Most grampositive bacteria do not contain GSH (9). However, a broad survey of the distribution of thiols in microorganisms revealed that several species of gram-positive bacteria, including Listeria, streptococci, and enterococci, produce significant amounts of GSH (23). The source of GSH in these bacteria has remained a puzzle, since their genomes do not contain a canonical gshB gene. The recent paper of Copley and Dhillon provides a clue to the origin of this GSH (7). These authors identified in the genomes of Listeria monocytogenes, Listeria innocua, Clostridium perfringens, and Pasteurella multocida an open reading frame (ORF) that is predicted to co...