Quorum Sensing Signal Selectivity and the Potential for Interspecies Cross Talk

Abstract

Many species of proteobacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). Most AHL receptors are thought to be specific for their cognate signal, ensuring that bacteria cooperate and share resources only with closely related kin cells. Although specificity is considered fundamental to QS, there are reports of "promiscuous" receptors that respond broadly to nonself signals. These promiscuous responses expand the function of QS systems to include interspecies interactions and have been implicated in both interspecies competition and cooperation. Because bacteria are frequently members of polymicrobial communities, AHL cross talk between species could have profound impacts. To better understand the prevalence of QS promiscuity, we measured the activity of seven QS receptors in their native host organisms. To facilitate comparison of our results to previous studies, we also measured receptor activity using heterologous expression in Escherichia coli We found that the standard E. coli methods consistently overestimate receptor promiscuity and sensitivity and that overexpression of the receptors is sufficient to account for the discrepancy between native and E. coli reporters. Additionally, receptor overexpression resulted in AHL-independent activity in Pseudomonas aeruginosa Using our activation data, we developed a quantitative score of receptor selectivity. We find that the receptors display a wide range of selectivity and that most receptors respond sensitively and strongly to at least one nonself signal, suggesting a broad potential for cross talk between QS systems.IMPORTANCE Specific recognition of cognate signals is considered fundamental to cell signaling circuits as it creates fidelity in the communication system. In bacterial quorum sensing (QS), receptor specificity ensures that bacteria cooperate only with kin. There are examples, however, of QS receptors that respond promiscuously to multiple signals. "Eavesdropping" by these promiscuous receptors can be beneficial in both interspecies competition and cooperation. Despite their potential significance, we know little about the prevalence of promiscuous QS receptors. Further, many studies rely on methods requiring receptor overexpression, which is known to increase apparent promiscuity. By systematically studying QS receptors in their natural parent strains, we find that the receptors display a wide range of selectivity and that there is potential for significant cross talk between QS systems. Our results provide a basis for hypotheses about the evolution and function of promiscuous signal receptors and for predictions about interspecies interactions in complex microbial communities.

Keywords: acyl-homoserine lactone; bacterial communication; gene regulation; transcription factors.

FIG 1

Diagram of a generic AHL QS circuit and structures of AHLs used in this study. (A) AHL QS systems generally contain a synthase (I) that produces an AHL signal, depicted here as a star. The signal acyl chains vary in length from 4 to 20 carbons, with potential hydroxy or oxo modification on the 3rd carbon, double bonds, branching, and/or terminal aryl moieties. At low cell densities, signals diffuse away from cells. At high cell densities, signals accumulate and can bind the QS receptor (R), which is a cytosolic transcription factor that regulates genes involved in group behaviors. (B) Chemical structures of AHLs used. Non-IUPAC descriptions of compounds are as follows: (1) C4-HSL; (2) 3OHC4-HSL; (3) C6-HSL; (4) 3OC6-HSL; (5) 3OHC6-HSL; (6) C8-HSL; (7) 3OC8-HSL; (8) 3OHC8-HSL; (9) C10-HSL; (10) 3OC10-HSL; (11) 3OHC10-HSL; (12) C12-HSL; (13) 3OC12-HSL; (14) 3OHC12-HSL; (15) C14-HSL; (16) 3OC14-HSL; (17) 3OHC14-HSL; (18) C16-HSL; (19) 3OC16-HSL.

FIG 2

Activation of receptors in native organisms. Synthase-null mutants (PAO-SC4, CV026, MJ215, and JBT112) harboring receptor activity reporters were treated with the indicated AHLs (100 µM), which are labeled by length of acyl chain and modification on the 3rd carbon. The RhlR reporter PAO-SC4 (pPROBE-P_rhlA) was pretreated with 3OC12-HSL (10 µM). For all reporters, activation is normalized to that of the receptor’s most potent signal (cognate AHL for all receptors except QscR, which is normalized to C10-HSL). N-3-oxobutyryl-L-homoserine lactone (3OC4-HSL) and N-3-hydroxyhexadecanoyl-L-homoserine lactone (3OHC16-HSL) were not included in the panel. Values are means plus SEM (error bars) from n ≥ 3 independent experiments.

FIG 3

Dose-response curves showing activation of AHL receptors in P. aeruginosa or in recombinant E. coli. Activation was measured via promoter-gfp reporter constructs (pPROBE-P_rhlA, -P_rsaL, and -P_PA1897, for RhlR, LasR, and QscR, respectively) in the synthase-null P. aeruginosa mutant (PAO-SC4) or in E. coli (DH5α) harboring the indicated receptor gene on a plasmid (pJNR, pJNL, and pJNQ for rhlR, lasR, and qscR, respectively). The P. aeruginosa RhlR reporter PAO-SC4 (pPROBE-P_rhlA) was pretreated with 3OC12-HSL (10 µM). Activation is normalized to that of the receptor’s most potent signal. Data show the means and ranges for two biological replicates and are representative of n ≥ 3 independent experiments. See Tables S3 and S4 in the supplemental material for EC₅₀ values.

FIG 4

Effect of overexpression on AHL receptor activity in P. aeruginosa. (A) RhlR activity in the P. aeruginosa RhlR reporter strain PAO-SC4 (pPROBE-P_rhlA) harboring plasmid-borne, arabinose-inducible rhlR (pJNR) (triangles and circles) or an empty vector control (pJN) (squares). Treatment with arabinose (ara) and/or 3OC12-HSL (10 µM) is indicated. Data are the means and ranges of two biological replicates and are representative of three independent experiments. (B) EC₅₀ of 3OC12-HSL for LasR and QscR and of C4-HSL for RhlR when the indicated receptor is overexpressed (via pJNL, pJNQ, or pJNR) in the synthase-null P. aeruginosa mutant (PAO-SC4). EC₅₀ was measured via the activity reporters pPROBE-P_rhlA, -P_rsaL, and -P_PA1897, for RhlR, LasR, and QscR, respectively. Values are means and SEM from n ≥ 3 independent experiments. (C) Relative fluorescence (in relative fluorescence units [RFU]) of the synthase-null P. aeruginosa mutant (PAO-SC4) harboring the RhlR activity reporter pPROBE-P_rhlA and plasmid-borne, arabinose-inducible rhlR (pJNR) or an empty vector control (pJN). Treatment with 3OC12-HSL (10 µM) and/or arabinose is indicated. (D) Relative fluorescence of the synthase-null P. aeruginosa mutant (PAO-SC4) harboring the LasR activity reporter pPROBE-P_rsaL or the QscR activity reporter pPROBE-P_PA1897 and plasmid-borne, arabinose-inducible lasR (pJNL) or qscR (pJNQ) or an empty vector (e) control (pJN). Treatment with arabinose is indicated. (E and F) Activation of QscR by a panel of 19 AHL signals (100 µM) measured via pPROBE-P_PA1897 in the synthase-null P. aeruginosa mutant (PAO-SC4) harboring plasmid-borne, arabinose-inducible qscR (pJNQ) (F) or an empty vector control (pJN) (E). Activation is normalized to C10-HSL. Arabinose was added in both conditions. In panels C to F, bars are means and SEM from three independent experiments.