The phosphoenolpyruvate phosphotransferase system (PTS) is a phosphotransfer cascade that regulates bacterial metabolism, behavior, and the transport of many sugars. Glucose-specific enzyme IIA (EllAGlc) is component of the PTS that carries out phosphotransfer and regulates both membrane-associated and cytoplasmic partners through direct protein-protein interactions. Here Vijayakumar et al identify an amphipathic helix at the N-terminus of Vibrio cholerae EIIAGlc that enables this protein to associate with the inner membrane. This amphipathic helix is essential for EIIAGlc regulation of membrane-associated partners, and Dr. Vijayakumar shows that unrelated amphipathic helices can rescue function. Removal of this amphipathic helix uncouples phosphotransfer through the PTS from sugar transport. Finally, Dr. Vijayakumar shows that EIIAGlc regulates opposing cytoplasmic and membrane-associated regulators of biofilm formation and bacterial metabolism in the mammalian intestine whose activities may be studied in isolation by removal of the amphipathic helix.