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.

Over 100 species of Proteobacteria use acyl-homoserine lactone (HSL) quorum-sensing signals to control expression of specific subsets of genes in a cell density-dependent manner (1–3). Signals are produced by members of the LuxI protein family and detected by transcription factors of the LuxR family. The cognate luxI and luxR genes are often adjacent to each other (4). Because acyl-HSLs can diffuse in and out of cells, the environmental signal concentration must reach a critical level to trigger a response.

All but one of the acyl-HSL signals described to date are fatty acyl-HSLs. A variety of different fatty acyl-HSLs have been described, and the acyl side chain confers signal specificity (1, 3, 5–7). The substrates for fatty acyl-HSL synthesis by LuxI homologs are S-adenosylmethionine (SAM) and the appropriate fatty acyl-acyl carrier protein (ACP) (8–10). The exception is RpaI from the photosynthetic bacterium Rhodopseudomonas palustris, which produces p-coumaroyl-HSL (pC-HSL) (11). Synthesis of pC-HSL by R. palustris requires an exogenous source of p-coumarate. As is true for many fatty acyl-HSL quorum-sensing systems, transcription of rpaI is positively autoregulated by pC-HSL and the transcription factor RpaR (11). Also similar to other acyl-HSL quorum-sensing systems, pC-HSL is found in fully grown cultures of R. palustris at a concentration on the order of 1–10 μM, and R. palustris shows a response to pC-HSL at concentrations as low as a few nanomolar, with saturation of the response at 20–50 nM pC-HSL (11).

The report describing pC-HSL production by R. palustris included a screen for other bacteria that, when grown in the presence of p-coumarate, produced a signal that could substitute for pC-HSL in bioassays for this material. Other than R. palustris itself, the bacterium that produced the most biological activity was Bradyrhizobium BTAi1, and the activity was about 1% of the activity found in R. palustris cultures (11). Members of the genus Bradyrhizobium are closely related to R. palustris, and the Bradyrhizobium BTAi1 LuxI homolog shows a high level of identity (58%) with the R. palustris pC-HSL synthase, RpaI. Bradyrhizobium BTAi1 is a curious member of the genus in that it is photosynthetic and that it can form nitrogen-fixing nodules on the stems of host plants, whereas with other bradyrhizobia, the nodules are restricted to plant roots (12, 13).

It is not clear whether p-coumarate is required for production of the R. palustris signaling molecule by Bradyrhizobium BTAi1, and it is not clear if the low level of activity was the result of low levels of pC-HSL synthesis or whether BTAi1 might be producing a different molecule that could trigger a relatively weak response in R. palustris. Thus, we sought to identify the signal produced by stem-nodulating bradyrhizobia. We report here that the molecule is cinnamoyl-HSL, that stem-nodulating bradyrhizobia do not require added cinnamic acid to synthesize cinnamoyl-HSL, and that the signal-dependent transcription factor responds to extraordinarily low cinnamoyl-HSL concentrations.

Results

Stem-Nodulating Photosynthetic Bradyrhizobia Produce a pC-HSL Substitute.

We previously showed that an R. palustris reporter sensitive to pC-HSL responded to something produced by Bradyrhizobium strain BTAi1 grown with p-coumarate. Because the response was quite low compared with responses to material produced by R. palustris, we hypothesized that Bradyrhizobium BTAi1 either makes very low levels of pC-HSL or makes a different but related aryl-HSL with a limited ability to serve as a substitute for pC-HSL (11). To begin to discriminate between these two possibilities, we separated the active material extracted with ethyl acetate from cultures of Bradyrhizobium BTAi1 and a second phototrophic stem-nodulating Bradyrhizobium strain, ORS278, by HPLC and assessed individual fractions for activity in the R. palustris pC-HSL reporter system (Fig. 1A). We found active fractions in extracts of culture fluid from both strains of Bradyrhizobium, but the majority of activity was found in fractions 34 and 35, which eluted later than the fraction containing pC-HSL (fraction 25). This indicated that the active material was less polar than pC-HSL. Furthermore, similar levels of active material were obtained from cultures grown with or without added p-coumarate.

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.

It seemed likely that the substance that could substitute for pC-HSL was a related aryl-HSL. If true, it is also possible that the active material might represent a small percent of the total amount of aryl-HSL synthesized, with the rest being material that is not active in the pC-HSL bioassay. To test this possibility, we used a radiotracer assay for acyl-HSLs that does not discriminate between bioactive and inactive material (14, 15). We used Bradyrhizobium ORS278 for the radiotracer experiment and most other experiments. The genome of strain ORS278 contains a single set of luxR-luxI homologs. Cultures of Bradyrhizobium ORS278 were incubated with a mixture of 13 different aromatic acids (as potential aryl substrates for the LuxI homolog) and l-[1-¹⁴C] methionine. Any acyl-HSL produced by the bacteria will have radiolabel in the HSL ring. The radiolabel extracted in acidified ethyl acetate was found almost exclusively in fractions in which most of the biological activity was detected (fractions 34 and 35) (Fig. 1B). Thus, it seems likely that Bradyrhizobium ORS278 produces primarily a single aryl-HSL that is less polar than pC-HSL but can serve as a pC-HSL substitute in bioassays.

Identity of the Aryl-HSL Produced by Bradyrhizobium ORS278.

To determine the chemical composition of the putative Bradyrhizobium ORS278 acyl-HSL, we extracted culture fluid with ethyl acetate, purified the active material in the ethyl acetate extract by methanol gradient and isocratic HPLC, and analyzed the purified material by high-resolution MS (Materials and Methods). The primary parent ion peak of the purified material had a mass of 232.0981, and there was an ion corresponding to a mass of 254.0797. These masses are within 5 ppm of the predicted M+H and M+Na masses (232.0974 and 254.0793, respectively) of a compound with the chemical composition of C₁₃H₁₃NO₃. This is the chemical composition of cinnamoyl-HSL, which differs from pC-HSL only in that it lacks the para-hydroxy group on the aromatic ring (Fig. 2A). Lack of the hydroxyl reduces the polarity of cinnamoyl-HSL with respect to pC-HSL, and this is consistent with the HPLC analysis. To gain evidence in support of the idea that the molecule produced by Bradyrhizobium ORS278 is cinnamoyl-HSL, we synthesized this compound chemically. The synthetic compound showed an HPLC elution profile that was identical to the purified biological material, and the MS spectra were indistinguishable (Fig. 2B). Furthermore, as predicted, cinnamoyl-HSL was active in the bioassay for pC-HSL; however, the amount of cinnamoyl-HSL required to give a half-maximal response was about 100 times the amount of pC-HSL needed to give a half-maximal response (Fig. 2C).

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 (□).

Cinnamoyl-HSL–Specific Bioassay.

The genome of Bradyrhizobium ORS278 contains a luxI homolog and a luxR homolog that are adjacent to each other, braI (BRADO0941) and braR (BRADO0942) (Fig. 3A). Because many luxI homologs are positively autoregulated by the acyl-HSL that they produce and their cognate LuxR homolog (1, 16), we suspected that braI might be responsive to cinnamoyl-HSL. It is often but not always the case that a lux box-like sequence, an 18- to 20-bp inverted repeat, can be identified in the promoter region of an acyl-HSL–dependent gene. This is the case for the pC-HSL–responsive rpaI promoter in R. palustris, for example (11). Although we were unable to identify such a sequence in the braI promoter region, we nevertheless constructed a strain with a promoterless lacZ inserted in the braI ORF. Because this strain has an uninterrupted braR and an inactive acyl-HSL synthase gene, transcription of lacZ should be activated by the addition of the cognate signal if there is positive autoregulation. We monitored lacZ expression by measuring β-galactosidase and found that the braI promoter was activated by as little as 2–5 pM cinnamoyl-HSL, and the response showed saturation at <100 pM cinnamoyl-HSL. The braI promoter also responded to pC-HSL, but it was much less sensitive to pC-HSL (Fig. 3B). Although the braI promoter response required about 50 times more pC-HSL than cinnamoyl-HSL, it was roughly equal in sensitivity to pC-HSL as was R. palustris, which shows specificity for pC-HSL (11). We conclude that the braI gene is positively autoregulated by cinnamoyl-HSL. Furthermore, the lacZ insertion strain can serve as a very sensitive bioassay of 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.

Production of Cinnamoyl-HSL Does Not Require an Exogenous Source of Cinnamate.

We determined the concentration of cinnamoyl-HSL in late logarithmic phase cultures of Bradyrhizobium ORS278 by extracting the signal with ethyl acetate and using the braI-lacZ mutant as a reporter in a bioassay. Fluid from cultures grown with or without cinnamate (5 μM) or a mixture of aromatic compounds contained about 10 nM cinnamoyl-HSL (Fig. 4). This level of acyl-HSL production is at the low end of the spectrum compared with many other acyl-HSL–producing bacteria. For example, R. palustris produces about 1–10 μM pC-HSL, but because Bradyrhizobium ORS278 is so exquisitely sensitive to cinnamoyl-HSL, the level of production is actually in great excess of that required for a signal response.

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.

BraI and BraR Define a Cinnamoyl-HSL–Dependent Quorum-Sensing Circuit.

We tested our hypothesis that braI is required for cinnamoyl-HSL synthesis by monitoring cinnamoyl-HSL production by a braI mutant and by monitoring cinnamoyl-HSL production by a recombinant R. palustris pC-HSL mutant (RpaI⁻) containing a braI expression plasmid. As predicted, the braI mutant did not produce any detectable cinnamoyl-HSL as measured by using the bioassay described above. The braI-containing recombinant R. palustris produced levels of cinnamoyl-HSL comparable with those produced by Bradyrhizobium ORS278, and it produced much higher levels (over 100 nM) when grown in the presence of cinnamate (Fig. 4). Thus, braI codes for cinnamoyl-HSL production.

To determine whether BraR is involved in cinnamoyl-HSL–dependent activation of braI, we measured cinnamoyl-HSL production in a braR mutant and found that there was about a 90% reduction in the level of cinnamoyl-HSL produced by the mutant compared with the parent (Fig. 4). This finding supports the hypothesis that BraR is a cinnamoyl-HSL–dependent transcription factor.

One characteristic of acyl-HSL production in positively autoregulated LuxI-LuxR–type circuits is that, in early logarithmic phase cultures, the signal remains low and increases rapidly when the culture reaches a sufficiently high density. This is precisely the result that we obtained when we monitored cinnamoyl-HSL production by Bradyrhizobium ORS278 with our bioassay (Fig. 5). Of note, the maximal amount of cinnamoyl-HSL was accumulated in the late logarithmic phase, and levels decreased in stationary phase. The level of cinnamoyl-HSL, which accumulated in the late logarithmic growth phase, was similar in strain BTAi1.

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.

The braI Promoter Responds Relatively Poorly to Fatty Acyl-HSLs.

The rpaI promoter in R. palustris is activated by RpaR and pC-HSL. We show here that it also responds to relatively high concentrations of cinnamoyl-HSL (Fig. 2C). The rpaI promoter was not responsive to fatty acyl-HSLs (11). We screened 11 different fatty acyl-HSLs with fatty acyl groups ranging from 4 to 12 carbons for the ability to activate expression of the braI-lacZ fusion in the Bradyrhizobium reporter strain. We used a range of concentrations for each acyl-HSL and determined a maximum response as well as the acyl-HSL concentration required to give a half-maximal response. The maximum responses for all of the fatty acyl-HSLs were similar to that for cinnamoyl-HSL, but the concentrations of these acyl-HSLs required to give a half-maximal response were anywhere from 3 to 6 orders of magnitude higher than the cinnamoyl-HSL half-maximal response (Fig. 6). Although the Bradyrhizobium ORS278 quorum-sensing system shows specificity for the acyl-HSL that it produces, cinnamoyl-HSL, the sensitivity is so great that, although fatty acyl-HSLs function as very poor substitutes for cinnamoyl-HSL, they function in physiologically relevant ranges from 10 nM to 10 μM.

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.

Discussion

We previously reported that R. palustris produced the aryl-HSL quorum-sensing signal pC-HSL and that production of pC-HSL was dependent on exogenously supplied p-coumarate. The genus Bradyrhizobium and the genus Rhodopseudomonas are closely related α-Proteobacteria (13). Because p-coumarate is produced by plants but not animals, we and others have speculated that quorum sensing in R. palustris might involve an intimate relationship with plants (11, 17). Thus, it was of particular interest that stem-nodulating bradyrhizobia produced a molecule that substituted, albeit poorly, for pC-HSL as an R. palustris quorum-sensing signal. We have determined that this molecule differs from pC-HSL only in that there is not a hydroxyl group on the aromatic ring. The compound is cinnamoyl-HSL, and perhaps even more interesting is that Bradyrhizobium ORS278 produces roughly equal amounts of this acyl-HSL when grown with or without aromatic acids added to the culture medium (Fig. 4). Furthermore, when given a choice of 13 different aromatic acids in the growth medium, the predominant Bradyrhizobium ORS278 acyl-HSL remained cinnamoyl-HSL (Fig. 1B). Thus, it is not universally true that aryl-HSL quorum-sensing systems are coupled to the presence of an exogenous supply of an aromatic compound.

Although Bradyrhizobium strains ORS278 and BTAi1 produce cinnamoyl-HSL in the absence of added cinnamate, the amount produced, on the order of 20 nM (Figs. 4 and 5), is very low compared with the amount of pC-HSL produced by R. palustris (11). We have shown that cinnamoyl-HSL production is dependent on the Bradyrhizobium luxI homolog braI (Fig. 4). By analogy to other LuxI homologs (8–11), we presume that the braI-encoded protein catalyzes the synthesis of cinnamoyl-HSL from SAM and an activated form of cinnamic acid. Some bacteria are capable of producing cinnamic acid by using phenylalanine ammonia lyase (PAL) to deaminate phenylalanine. Bradyrhizobium ORS278 contains a gene (BRADO1604) coding for a polypeptide, which shows similarity to the Streptomyces maritimus PAL (18), but the polypeptide contains conserved amino acid residues characteristic of histidine ammonia lyases (19, 20). This putative amino acid lyase might be involved in cinnamate production by Bradyrhizobium ORS278, but we note that recombinant braI-containing R. palustris also produced nanomolar concentrations of cinnamoyl-HSL without added cinnamate and that the R. palustris genome does not possess any obvious PAL homologs.

With the fatty acyl-HSL synthases, it has not been possible to relate primary polypeptide sequence to the preferred acyl-HSL product. We have now defined a family of two aryl-HSL synthases, RpaI from R. palustris and BraI from Bradyrhizobium ORS278. These two aryl-HSL synthases are much more similar to each other (56% identity) than they are to any known fatty acyl-HSL synthases (<32% identity). In fact, Bradyrhizobium japonicum possesses a LuxI homolog that is related to RpaI and BraI (38% and 34% identity, respectively). We are interested to determine the chemical nature of the putative B. japonicum acyl-HSL. R. palustris and Bradyrhizobium ORS278 are very closely related at the genomic level. Thus, we cannot discern whether the LuxI homolog similarity is because of constraints on enzymes that produce aryl-HSLs or a consequence of the close evolutionary relationship between these species. We hope that it will be possible to identify an aryl-HSL–producing bacterium that is not so closely related to bradyrhizobia and R. palustris. This would aid in discriminating between constraints on enzyme activity and evolutionary relatedness.

We were able to develop an assay for cinnamoyl-HSL by inserting a lacZ reporter into the Bradyrhizobium ORS278 braI. By using this reporter, we showed that a BraR mutant produced very little cinnamoyl-HSL (Fig. 4). This supports the conclusion that the LuxR homolog BraR is the receptor for cinnamoyl-HSL. We also were able to measure the amounts of cinnamoyl-HSL that accumulated in the culture fluid of WT stem-nodulating bradyrhizobia, and we found that the two strains that we examined made on the order of 30–65 nM cinnamoyl-HSL (Fig. 5). This is at the low end of the range of acyl-HSL concentrations found in culture fluid of other bacteria. For example, R. palustris produces 1–10 μM pC-HSL (11), and Pseudomonas aeruginosa produces two fatty acyl-HSLs, both in the range of 10 μM (21, 22). There is considerable strain variability in Vibrio fischeri acyl-HSL levels, with some strains producing very little 3-oxo-hexanoyl-HSL. In V. fischeri, the strain variation in levels of the acyl-HSL signal correlates with levels of quorum-controlled luminescence (23, 24). Culture fluid from brightly luminescent strains contains micromolar levels of signal, and culture fluid from dim strains contains nanomolar levels of signal. Furthermore, in vivo responses of LuxR homologs generally require nanomolar signal concentrations. However, our Bradyrhizobium braI-lacZ reporter strain responds to picomolar levels of cinnamoyl-HSL (Fig. 3B). We are aware of one other example of a response to picomolar levels of an acyl-HSL signal. This example involves engineered strains of Agrobacterium tumefaciens. Strains of A. tumefaciens that express normal levels of the acyl-HSL receptor TraR respond to nanomolar concentrations of the cognate signal 3OC8-HSL, and at relatively high concentrations, certain other fatty acyl-HSLs function as antagonists of 3OC8-HSL (25). Strains engineered to overexpress functional TraR respond to picomolar levels of 3OC8-HSL, and signals that function as antagonists with normally expressed TraR function as activators in the TraR overexpression constructs (25, 26).

There are several possible explanations for the ultrasensitive response of our Bradyrhizobium quorum-sensing reporter strain to picomolar levels of cinnamoyl-HSL. For example, there could be an active uptake system for cinnamoyl-HSL. However, we favor a simpler hypothesis that derives from the sensitivity of TraR overexpression strains of A. tumefaciens. It is plausible that BraR is a high-affinity cinnamoyl-HSL receptor that is produced at particularly high levels compared with WT levels of TraR, for example. If this is true, it might provide an explanation as to why the basal level of braI-lacZ transcription is relatively high and why exogenous signal affects a relatively meager two- to threefold induction of the β-galactosidase reporter. There are data indicating that overexpression of the V. fischeri LuxR results in an increased expression of the quorum-sensing, controlled luminescence genes in an acyl-HSL–independent manner (27). Expression can be further stimulated by acyl-HSL signal addition. Furthermore, the Bradyrhizobium cinnamoyl-HSL reporter is activated by relatively high concentrations of a wide variety of aryl-HSLs and fatty acyl-HSLs. This is consistent with findings for TraR overexpression strains of A. tumefaciens, which show activation of quorum-controlled genes by a range of acyl-HSLs that interfere with 3OC8-HSL gene activation in strains expressing normal levels of TraR. Of course, our favored hypothesis awaits direct testing.

Although our data indicate that Bradyrhizobium ORS278 responds relatively poorly to acyl-HSLs other than cinnamoyl-HSL, the level of activation at sufficiently high acyl-HSL concentrations is comparable with the level of cinnamoyl-HSL activation. The BraR response to noncognate acyl-HSLs occurs in the range of nanomolar to micromolar. This is within the range to which the cognate LuxR homologs respond and well within the range of signals produced by cultures of other bacteria. This information raises the possibility that, in certain soil habitats, a Bradyrhizobium ORS278 cell might respond to signals produced by groups of other species. We do not have any idea about the advantage that might be conferred by the potential ability to detect and respond to quorums of other species. We have not yet identified the functions other than BraI that are regulated by cinnamoyl-HSL. Obviously, such knowledge might inform us about the role of quorum sensing and potentially, eavesdropping on other species in stem-nodulating bradyrhizobia. Also, quorum sensing has been shown to affect interactions of other members of the Rhizobiales with their plant hosts (28–32). It will be of interest to compare plant interactions of BraI and BraR mutants with the parent. Finally, we point out that it seems a real possibility that we have overlooked acyl-HSL signaling systems in other bacteria, because we have focused on systems that require nanomolar concentrations of signals for a response. That we have now identified one system that functions below the nanomolar range suggests to us that there are other such systems.

Materials and Methods

Bacterial Strains and Growth Conditions.

Bradyrhizobium ORS278 (13) and BTAi1 (12) were grown in Arabinose-Gluconate (AG) medium (33) at 30 °C with shaking. Strain NA1 (described below) was grown in AG medium plus kanamycin (50 μg/mL) unless otherwise specified. The BraR mutant of strain ORS278 contains a Tn5-Gm insertion at base pair 392 of braR (locus tag BRADO0942). The mutant was selected by PCR from a Tn5 mutant library (34) by using a Tn5 and a braR-specific primer. For growth of R. palustris CGA814, an rpaI::lacZ derivative of strain CGA009 (11), we used Photosynthesis Minimal (PM) medium in the light with 10 mM succinate as a carbon source (35). For growth of R. palustris CGA814 containing the braI expression vector pBraI, we added gentamicin (100 μg/mL) to the PM medium for plasmid maintenance. Individual aromatic salts were added at the indicated concentrations. Where indicated, we used a pool of 13 aromatic salts, including benzoate, p-coumarate, m-coumarate, o-coumarate, cinnamate, caffeate, ferulate, methoxycinnamate, sinapate, p-hydroxybenzoate, vanillate, phenylalanine, and tryptophan, each at a concentration of 100 μM.

Mutant and Plasmid Construction.

To construct the braI-lacZ mutant Bradyrhizobium NA1, we replaced all of the Bradyrhizobium ORS278 braI gene (locus tag BRADO0941), except the translation start and stop codons, with the promoterless lacZ-kanamycin resistance cassette from pHRP314 (36). First, we PCR-amplified the braI flanking regions with primers containing 5′ EcoRI and 3′ PstI restriction sites on the upstream flanking region of braI and 5′ PstI and 3′ XbaI sites on the downstream flanking region. These two fragments were ligated with EcoRI- and XbaI-digested pSUP202pol6k (Tet^r) (37) to form an intermediate cloning vehicle. The intermediate plasmid was digested with PstI, and this was ligated with an lacZ::Km^r cassette containing the PstI fragment of pHRP314. The resultant lacZ::Km^r suicide plasmid was introduced into Bradyrhizobium ORS278 by conjugation with Escherichia coli S17-1. Recombinants were selected on AG-kanamycin agar plates containing naladixic acid (35 μg/mL), and double cross-over recombinants were identified as those that did not grow when screened on AG tetracycline plates (50 μg/mL).

To construct pBraI, we PCR-amplified a braI DNA fragment from Bradyrhizobium ORS278 DNA. The PCR product extended from 21 bp upstream of the braI start codon through the braI stop codon. The primers were designed to yield a product with a 5′ HindIII restriction site and a 3′ XbaI restriction site. The PCR fragment was cloned into pCR-TOPO 5.1 (Invitrogen Corp.), subsequently excised with HindIII and XbaI, and inserted into HindIII- and XbaI-digested pBBR1MCS-5, a broad-host range expression vector (38), to yield a P_lac-braI fusion vector.

Acyl-HSL Bioassays.

We used an R. palustris rpaI-lacZ reporter strain (CGA814), which responds best to pC-HSL, and a Bradyrhizobium ORS278 braI-lacZ derivative (NA1) as a reporter, which responds best to cinnamoyl-HSL in bioassays. Bioassays were either with extracts of whole bacterial cultures or with cell-free culture fluid extracted with two equal volumes of acidified ethyl acetate (0.1 mL glacial acetic acid per 1 L ethyl acetate). Separation of cells and culture fluid was by centrifugation followed by filtration through a 0.22-μm membrane. Ethyl acetate extracts were fractionated as described below. For bioassays with R. palustris CGA814, we used the method of Schaefer et al. (11). For bioassays with Bradyrhizobium NA1, ethyl acetate solutions containing acyl-HSLs were dried in 1.5-mL Eppendorf tubes or 2-mL wells in 96-well microtiter plates. We added 500 μL culture that had reached an OD of 0.01 at 600 nm to the dried samples, and after 16–20 h at 30 °C with shaking, we measured β-galactosidase activity as described elsewhere (39). Standard curves were generated by using commercially supplied pC-L-HSL and cinnamoyl-L-HSL (Syntech).

HPLC Separation and Purification of Acyl-HSLs from Spent Culture Fluid.

Fluid from cultures of stem-nodulating bradyrhizobia or recombinant R. palustris was extracted with two equal volumes of acidified ethyl acetate after removal of cells by centrifugation and filtration through a 0.22-μm filter. Ethyl acetate extracts of culture fluid were concentrated by rotary evaporation, and the concentrated extracts were applied to a C18 reverse-phase HPLC column. The flow rate was 1 mL/min, and 1-mL fractions were collected and tested for acyl-HSLs as described in the text. We used a 10–100% methanol in water gradient spanning 70 min or a 35% isocratic methanol separation as indicated.

To purify the active compound produced by Bradyrhizobium ORS278, we extracted fluid from a 1-L culture grown to an OD of 1. The ethyl acetate extract was concentrated by rotary evaporation, and acyl-HSLs were separated by HPLC in a 10–100% methanol in water gradient; 1-mL fractions were collected, and activity in each fraction was assessed by using the Bradyrhizobium NA1 bioassay. The three 1-mL fractions showing greatest activity were pooled, and the pooled fractions were concentrated and then separated by HPLC in 35% methanol. The Bradyrhizobium NA1 bioassay showed that activity was eluted in fractions 26–29. These 1-mL fractions were pooled and evaporated to dryness. We used a Waters Micromass High-Definition Time of Flight MS System (collision energy = 30 eV, cone = 35 V) to determine an accurate molecular mass for the dried material and to compare the mass spectrum of this material to synthetic cinnamoyl-HSL.

Radiotracer Analysis of Acyl-HSLs.

We used a ¹⁴C-tracer method (14) to detect acyl-HSLs produced by Bradyrhizobium ORS278 as follows. Cells were grown to mid-logarithmic phase (OD of 0.5) and diluted in 5 mL fresh medium to an OD of 0.1 in 15-mL plastic tubes. We then added l-[1-¹⁴C] methionine (American Radiolabeled Chemicals) as described elsewhere (14) and the aromatic acid mixture described above. Cultures were incubated for 20 h at 30 °C with shaking. Radioactive acyl-HSLs were extracted in acidified ethyl acetate, and the extract was separated by methanol-gradient HPLC. Fractions were mixed with 4 mL Complete Counting Mixture (Research Products International), and radioactivity was measured with a Beckman LS 1800 liquid scintillation counter.