Indole production promotes Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signaling

Abstract

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.

Fig 1

Influence of mutations in E. coli cAMP production (adenylate cyclase [cyaA]), cAMP receptor gene ([crp]), and cAMP phosphodiesterase, encoded by cpdA on E. coli monoculture planktonic (A) and biofilm (C) cultures and E. coli populations in mixed-planktonic (B) and biofilm cultures (D) with P. aeruginosa. The growth decline in E. coli cyaA mixed cultures was reversed by the addition of 1 mM cAMP. The growth medium in all figures is LB. Error bars in all figures represent standard errors of the means (n ≥ 3).

Fig 2

Influence of mutations in E. coli indole production (tryptophanase [tnaA]) and receptor gene (sdiA) on growth in mixed culture with P. aeruginosa in planktonic (A) and biofilm (C) cultures. Addition of 1 mM indole restored competitiveness of E. coli tryptophanase mutants (tnaA) to wt levels (A and C) as well as strains lacking adenylate cyclase (cyaA) and the cAMP receptor gene (crp) (B and D). (E) Monoculture growth of E. coli and P. aeruginosa in the presence and absence of 1 mM indole. Supplemented indole was metabolized by P. aeruginosa over 48 h in pure or mixed culture with an E. coli strain unable to produce indole (such as E. coli cyaA [panel F]).

Fig 3

Influence of pure and mixed-culture growth on indole production in wt and cyaA and cpdA mutants. Indole concentrations were calculated per E. coli cell.

Fig 4

Effects of 1 mM cAMP and 1 mM indole supplementation on pyocyanin production by P. aeruginosa in pure culture (A) and mixed culture with E. coli cyaA (B) or E. coli crp (C). The two E. coli strains are unable to produce indole.

Fig 5

Interaction of E. coli tryptophanase (tnaA) and P. aeruginosa AHL synthesis (lasI and rhlI) on E. coli (A [planktonic] and C [biofilm]) and P. aeruginosa (B [planktonic] and D [biofilm]) growth in mixed culture. Symbols for panels A to D are shown in panel A. Data for growth of E. coli wt with P. aeruginosa wt and of E. coli tnaA with P. aeruginosa wt from Fig. 2 are shown again for comparison. (E) In contrast to the wild type, P. aeruginosa AHL synthesis mutants were unable to degrade indole.