One-carbon metabolism (OCM) pathways are responsible for several functions, producing a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl) that are required for the synthesis of various amino acids and other biomolecules such as purines, thymidylate, redox regulators, and, in most microbes, folate. As humans must acquire folate from the diet, folate production is a target for antimicrobials such as sulfonamides. OCM impacts the regulation of microbial virulence such that in a number of instances, limiting the availability of para-aminobenzoic acid (pABA), an essential OCM precursor, causes a reduction in pathogenicity. Porphyromonas gingivalis, however, displays increased pathogenicity in response to lower pABA levels, and exogenous pABA exerts a calming influence on heterotypic communities of P. gingivalis with pABA-producing partner species. Differential responses to pABA may reflect both the physiology of the organisms and their host microenvironment. OCM plays an integral role in regulating the global rate of protein translation, where the alarmones ZMP and ZTP sense insufficient stores of intracellular folate and coordinate adaptive responses to compensate and restore folate to sufficient levels. The emerging interconnections between OCM, protein synthesis, and context-dependent pathogenicity provide novel insights into the dynamic host-microbe interface.

Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry-that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N-formyl-tetrahydrofolate (N-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates.