Indole production by Escherichia coli, discovered in the early 20th century, has been used as a diagnostic marker for distinguishing E. coli from other enteric bacteria. By using transcriptional profiling and competition studies with defined mutants, we show that cyclic AMP (cAMP)-regulated indole formation is a major factor that enables E. coli growth in mixed biofilm and planktonic populations with Pseudomonas aeruginosa. Mutants deficient in cAMP production (cyaA) or the cAMP receptor gene (crp), as well as indole production (tnaA), were not competitive in coculture with P. aeruginosa but could be restored to wild-type competitiveness by supplementation with a physiologically relevant indole concentration. E. coli sdiA mutants, which lacked the receptor for both indole and N-acyl-homoserine lactones (AHLs), showed no change in competitive fitness, suggesting that indole acted directly on P. aeruginosa. An E. coli tnaA mutant strain regained wild-type competiveness if grown with P. aeruginosa AHL synthase (rhlI and rhlI lasI) mutants. In contrast to the wild type, P. aeruginosa AHL synthase mutants were unable to degrade indole. Indole produced during mixed-culture growth inhibited pyocyanin production and other AHL-regulated virulence factors in P. aeruginosa. Mixed-culture growth with P. aeruginosa stimulated indole formation in E. coli cpdA, which is unable to regulate cAMP levels, suggesting the potential for mixed-culture gene activation via cAMP. These findings illustrate how indole, an early described feature of E. coli central metabolism, can play a significant role in mixed-culture survival by inhibiting quorum-regulated competition factors in P. aeruginosa. In nature, bacteria normally occur in polymicrobial communities. Interactions between community members typically involve several mechanisms, including responses to antimicrobial compounds, nutritional interactions, and signaling (9,12,42). Chemical signaling is widespread in bacteria, and in Gramnegative bacteria it involves several compounds, including N-acyl derivatives of homoserine lactone (AHLs), furans, small peptides, quinolones, and indole (37). In Pseudomonas aeruginosa, many genes involved with virulence and competition are regulated by AHL-and quinolone-based quorum signaling (35,50). Quorum signal disruption (quenching) has been shown to alter bacterial competition and reduce virulence (18). In one animal model study of P. aeruginosa lung infections, administration of a ginseng extract caused a reduction in AHL levels and AHL-regulated elastase without affecting bacterial growth (44). Microbial nutrition is also important in species' interactions. Central metabolism, long regarded as reflecting a number of routine housekeeping functions, is now being reexamined for its role in mixed-culture interactions within biofilm and planktonic populations (9).One component of central metabolism in Escherichia coli involves purine and pyrimidine nucleic acid synthesis (reviewed in reference 36). Pathways for de novo synthesis and salvage pathways...

The LuxI/R quorum-sensing system and its associated N-acylated homoserine lactone (AHL) signal is widespread among Gram-negative bacteria. Although inhibition by indole of AHL quorum signalling in Pseudomonas aeruginosa and Acinetobacter oleivorans has been reported previously, it has not been documented among other species. Here, we show that co-culture with wild-type Escherichia coli, but not with E. coli tnaA mutants that lack tryptophanase and as a result do not produce indole, inhibits AHL-regulated pigmentation in Chromobacterium violaceum (violacein), Pseudomonas chlororaphis (phenazine) and Serratia marcescens (prodigiosin). Loss of pigmentation also occurred during pure culture growth of Chro. violaceum, P. chlororaphis and S. marcescens in the presence of physiologically relevant indole concentrations (0.5-1.0 mM). Inhibition of violacein production by indole was counteracted by the addition of the Chro. violaceum cognate autoinducer, N-decanoyl homoserine lactone (C10-HSL), in a dosedependent manner. The addition of exogenous indole or co-culture with E. coli also affected Chro. violaceum transcription of vioA (violacein pigment production) and chiA (chitinase production), but had no effect on pykF (pyruvate kinase), which is not quorum regulated. Chro. violaceum AHL-regulated elastase and chitinase activity were inhibited by indole, as was motility. Growth of Chro. violaceum was not affected by indole or C10-HSL supplementation. Using a nematodefeeding virulence assay, we observed that survival of Caenorhabditis elegans exposed to Chro. violaceum, P. chlororaphis and S. marcescens was enhanced during indole supplementation. Overall, these studies suggest that indole represents a general inhibitor of AHL-based quorum signalling in Gram-negative bacteria.