Autoinducer-2 (AI-2) a signal produced by a range of phylogenetically distant microorganisms, enables inter-species cell-cell communication and regulates many bacterial phenotypes. Certain bacteria can interfere with AI-2-regulated behaviours of neighbouring species by internalizing AI-2 using the Lsr transport system (encoded by the lsr operon). AI-2 imported by the Lsr is phosphorylated by the LsrK kinase and AI-2-phosphate is the inducer of the lsr operon. Here we show that in Escherichia coli the phosphoenolpyruvate phosphotransferase system (PTS) is required for Lsr activation and is essential for AI-2 internalization. Although the phosphorylation state of Enzyme I of PTS is important for this regulation, LsrK is necessary for the phosphorylation of AI-2, indicating that AI-2 is not phosphorylated by PTS. Our results suggest that AI-2 internalization is initiated by a PTS-dependent mechanism, which provides sufficient intracellular AI-2 to relieve repression of the lsr operon and, thus induce depletion of AI-2 from the extracellular environment. The fact that AI-2 internalization is not only controlled by the community-dependent accumulation of AI-2, but also depends on the phosphorylation state of PTS suggests that E. coli can integrate information on the availability of substrates with external communal information to control quorum sensing and its interference.