Signaling by serine/threonine phosphorylation controls diverse processes in bacteria, and identification of the stimuli that activate protein kinases is an outstanding question in the field. Recently, we showed that nutrients stimulate phosphorylation of the protein kinase G substrate GarA in Mycobacterium smegmatis and Mycobacterium tuberculosis and that the action of GarA in regulating central metabolism depends upon whether it is phosphorylated. Here we present an investigation into the mechanism by which nutrients activate PknG. Two unknown genes were identified as co-conserved and co-expressed with PknG: their products were a putative lipoprotein, GlnH, and putative transmembrane protein, GlnX. Using a genetic approach, we showed that the membrane protein GlnX is functionally linked to PknG. Furthermore, we determined that the ligand specificity of GlnH matches the amino acids that stimulate GarA phosphorylation. We determined the structure of GlnH in complex with different amino acid ligands (aspartate, glutamate, and asparagine), revealing the structural basis of ligand specificity. We propose that the amino acid concentration in the periplasm is sensed by GlnH and that protein-protein interaction allows transmission of this information across the membrane via GlnX to activate PknG. This sensory system would allow regulation of nutrient utilization in response to changes in nutrient availability. The sensor, signaling, and effector proteins are conserved throughout the Actinobacteria, including the important human pathogen Mycobacterium tuberculosis, industrial amino acid producer Corynebacterium glutamicum, and antibiotic-producing Streptomyces species.