We have shown that the quorum-sensing signals acylhomoserine lactones, autoinducer-2 (AI-2) and indole influence the biofilm formation of Escherichia coli. Here, we investigate how the environment, that is, temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30 degrees C (186 genes) than at 37 degrees C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25 and 30 degrees C than at 37 degrees C and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30 degrees C compared to 37 degrees C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD, pyrF and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30 degrees C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37 degrees C, indole more significantly decreased flagella-related qseB, flhD and fliA promoter activity, enhanced antibiotic resistance and inhibited cell division at 30 degrees C. In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37 degrees C (63 genes) than at 30 degrees C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37 degrees C (but not at 30 degrees C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37 degrees C but not at 30 degrees C. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined.