The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Because LsrR and LsrK directly regulate AI-2 uptake, we hypothesized that they might play a wider role in regulating other QS-related cellular functions. In this study, we characterized physiological changes due to the genomic deletion of lsrR and lsrK. We discovered that many genes were coregulated by lsrK and lsrR but in a distinctly different manner than that for the lsr operon (where LsrR serves as a repressor that is derepressed by the binding of phospho-AI-2 to the LsrR protein). An extended model for AI-2 signaling that is consistent with all current data on AI-2, LuxS, and the LuxS regulon is proposed. Additionally, we found that both the quantity and architecture of biofilms were regulated by this distinct mechanism, as lsrK and lsrR knockouts behaved identically. Similar biofilm architectures probably resulted from the concerted response of a set of genes including flu and wza, the expression of which is influenced by lsrRK. We also found for the first time that the generation of several small RNAs (including DsrA, which was previously linked to QS systems in Vibrio harveyi) was affected by LsrR. Our results suggest that AI-2 is indeed a QS signal in E. coli, especially when it acts through the transcriptional regulator LsrR.Bacteria communicate with each other through small "hormone-like" organic molecules referred to as autoinducers. Autoinducer-based bacterial cell-to-cell communication, enabling population-based multicellularity, has been termed quorum sensing (QS) (27). Cellular functions controlled by QS are varied and reflect the needs of a particular bacterial species for inhabiting a given niche (10,38,65).QS among Escherichia coli and Salmonella strains has been a topic of great interest, and different intercellular signaling systems have been identified: that mediated by the LuxR homolog SdiA; the LuxS/autoinducer 2 (AI-2) system, an AI-3 system, and a signaling system mediated by indole (2,19,36,57,61,68). Among these systems, the LuxS/AI-2 system possesses the unique feature of endowing cell population-dependent behavior while interacting with central metabolism through the intracellular activated methyl cycle (20,21,45,65,73). Therefore, it has the potential to influence both gene regulation and bacterial fitness.AI-2's function has been studied using luxS mutants and by adding either conditioned medium or in vitro-synthesized AI-2 to bacterial cultures. It is noteworthy that the luxS transcription profile is not synchronous with the accumulation profile of extracellular AI-2 in bacterial supernatants (5,31,75). In E.coli, extracellular AI-2 activity peaks during the mid-to lateexponential phase and rapidly decreases during entry into the stationary phase. A corresponding decrease in LuxS protein levels is not observed (31,75). The disap...