Aryl-homoserine lactone quorum sensing in stem-nodulating photosynthetic bradyrhizobia

Abstract

Many Proteobacteria possess LuxI-LuxR-type quorum-sensing systems that produce and detect fatty acyl-homoserine lactone (HSL) signals. The photoheterotroph Rhodopseudomonas palustris is unusual in that it produces and detects an aryl-HSL, p-coumaroyl-HSL, and signal production requires an exogenous source of p-coumarate. A photosynthetic stem-nodulating member of the genus Bradyrhizobium produces a small molecule signal that elicits an R. palustris quorum-sensing response. Here, we show that this signal is cinnamoyl-HSL and that cinnamoyl-HSL is produced by the LuxI homolog BraI and detected by BraR. Cinnamoyl-HSL reaches concentrations on the order of 50 nM in cultures of stem-nodulating bradyrhizobia grown in the presence or absence of cinnamate. Acyl-HSLs often reach concentrations of 0.1-30 μM in bacterial cultures, and generally, LuxR-type receptors respond to signals in a concentration range from 5 to a few hundred nanomolar. Our stem-nodulating Bradyrhizobium strain responds to picomolar concentrations of cinnamoyl-HSL and thus, produces cinnamoyl-HSL in excess of the levels required for a signal response without an exogenous source of cinnamate. The ability of Bradyrhizobium to produce and respond to cinnamoyl-HSL shows that aryl-HSL production is not unique to R. palustris, that the aromatic acid substrate for aryl-HSL synthesis does not have to be supplied exogenously, and that some acyl-HSL quorum-sensing systems may function at very low signal production and response levels.

Fig. 1.

HPLC analyses of acyl-HSLs produced by stem-nodulating bradyrhizobia. (A) Bradyrhizobium ORS278 and BTAi1 were grown with 500 μM p-coumarate, and ethyl acetate extracts of cultures were fractionated by HPLC. HPLC fractions were screened with the R. palustris CGA814 pC-HSL–sensitive bioassay. Extracts of strain ORS278 (■), and strain BTAi1 (○) are shown. Background β-galactosidase without added extract was 23,000 and is subtracted from the data shown in the figure. Synthetic pC-HSL was eluted in fraction 25. (B) Radiotracer assay for acyl-HSL produced by Bradyrhizobium ORS278. Cells were grown with ¹⁴C-methionine and a mixture of 13 aromatic compounds (Materials and Methods). The cultures were extracted, and extracts were separated by HPLC as above. The dashed lines show the percent methanol in the gradient.

Fig. 2.

(A) Chemical identity of the Bradyrhizobium ORS278 acyl-HSL: cinnamoyl-HSL (C₁₃H₁₃NO₃). (B) Mass spectra of chemically synthesized cinnamoyl-HSL (Upper) and material purified from Bradyrhizobium ORS278 (Lower). (C) Induction of β-galactosidase in R. palustris CGA814 (rpaI-lacZ) by synthetic pC-HSL (◆) and synthetic cinnamoyl-HSL (□).

Fig. 3.

The cinnamoyl-HSL–specific bioassay. (A) The Bradyrhizobium ORS278 braI-braR region and flanking genes. Hypothetical ORFs are shaded in gray, and genes of putative function are filled in black. The gene upstream of braR (shaded black) is annotated as a putative enoyl-CoA hydratase, and the gene downstream of braI (lined) shows some sequence similarity to putative nitroreductases; the genes further downstream of braI (shaded black) are annotated as putative acyl-CoA dehydrogenases. (B) We used Bradyrhizobium NA1, which contains a braI::lacZ insertion, as a bioassay, and we measured responses to synthetic pC-HSL (□) and cinnamoyl-HSL (●) at the concentrations indicated.

Fig. 4.

Cinnamoyl-HSL production by WT and mutant strains of Bradyrhizobium ORS278 and the R. palustris rpaI::lacZ bioassay strain containing pBraI. Cultures were extracted at an OD of 0.1. Cinnamoyl-HSL in extracts was measured by using the Bradyrhizobium braI::lacZ reporter strain NA1. Cinnamate (cinn) or a mixture of aromatic salts (aromatics) was added to the growth medium as indicated. BraI⁻ and BraR⁻ indicate extracts from the braI and braR mutants. Extracts of the R. palustris rpaI mutant (R. pal) containing pBraI grown with 100 μM or without added cinnamate were also tested. The data are means of six biological replicates, with each replicate assayed in triplicate. The error bars are SDs from the means.

Fig. 5.

Cinnamoyl-HSL production by Bradyrhizobium ORS278 as a function of growth: CFU per mL (○) and cinnamoyl-HSL concentration (●). We used the cinnamoyl-HSL bioassay to measure signal concentrations in cultures extracted at the indicated times. A standard curve was constructed by using synthetic cinnamoyl-HSL.

Fig. 6.

Concentrations of various HSLs required for half-saturation responses in the cinnamoyl-HSL bioassay. Each acyl-HSL was tested over a range of concentrations, and the concentrations at which the response was half-saturated were determined from the response curves. Data are the means of duplicate experiments, and error bars show the ranges.