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First published online December 11, 2003; 10.1104/pp.103.029918 Plant Physiology 134:137-146 (2004) © 2004 American Society of Plant Biologists Chlamydomonas reinhardtii Secretes Compounds That Mimic Bacterial Signals and Interfere with Quorum Sensing Regulation in Bacteria1ARC Centre of Excellence for Integrative Legume Research Genomics Interaction Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capitol Territory 2601, Australia (H.C., B.G.R.); Graduate Program in Biophysics (S.R., R.T.S.) and Departments of Plant Pathology (M.M.) and Plant Biology (R.T.S.), Ohio State University, Columbus, Ohio 43210 (M.T., M.G., W.D.B.); and Department of Biology, University of Dayton, Dayton Ohio 45469 (J.B.R.)
The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-L-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression. More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C. reinhardtii culture filtrates. Colonies of C. reinhardtii and Chlorella spp. stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp. Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C. reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's own AHL signals, providing evidence that the algal mimic affected quorum sensing-regulated functions in this wild-type bacterium. Peptide mass fingerprinting identified 32 proteins affected by the bacterium's AHLs or the purified algal mimic, including GroEL chaperonins, the nitrogen regulatory protein PII, and a GTP-binding protein. The algal mimic was able to cancel the stimulatory effects of bacterial AHLs on the accumulation of seven of these proteins, providing evidence that the secretion of AHL mimics by the alga could be effective in disruption of quorum sensing in naturally encountered bacteria.
The production and exchange of specific signal substances between individual cells enables many bacterial species to coordinate their gene expression in a population density-dependent manner (Miller and Bassler, 2001  ; von Bodman et al., 2003). This kind of regulation, known as quorum sensing, affects the expression of many genes and behaviors in various bacteria (Whitehead et al., 2001; Schuster et al., 2003; Wagner et al., 2003). Bacterial pathogens and symbionts of both animals and plants require quorum sensing to colonize and invade their eukaryotic hosts (Eberl et al., 1996; Tan et al., 1999; Visick and Ruby, 1999; de Kievit and Iglewski, 2000; Loh et al., 2002).
In view of the bacterial dependence on quorum sensing for infection of hosts, it makes good evolutionary sense that eukaryotes have acquired the ability to recognize and respond to bacterial quorum sensing signals (Telford et al., 1998
More recently, various higher plants were also shown to secrete AHL signal mimic substances (Teplitski et al., 2000
In this study, we have investigated the production of AHL mimic substances by the unicellular green alga Chlamydomonas reinhardtii. In contrast to D. pulchra, C. reinhardtii is well suited to genetic and molecular genetic analysis of AHL signal mimic production. The alga has been used as a model organism for the molecular genetic and biochemical analysis of photosynthesis and other processes (Harris, 1989
Production of Substances Affecting Quorum Sensing by Colonies of C. reinhardtii and Chlorella Spp.
Colonies of C. reinhardtii and Chlorella spp. growing on agar plates were overlaid with suspensions of quorum sensing reporter strains (Table I), and their luminescence responses were monitored with a CCD camera. As shown in Figure 1A, colonies of C. reinhardtii, C. mutablis, Chlorella vulgaris, and Chlorella fusca all stimulated luminescence responses over a period of 24 h in the Vibrio harveyi BB170 reporter strain, which selectively responds to the furanosyl borate diester AI-2 quorum sensing signal (Chen et al., 2002
Culture filtrates from C. reinhardtii cells grown phototrophically in mineral salts medium were extracted with ethyl acetate and the concentrated extracts were fractionated by reverse-phase C18 HPLC. As shown in Figure 2, the E. coli LasR AHL reporter strain, most responsive to 3-oxo-C12-HSL, detected a number of substances in the extracts that stimulated strong quorum sensing-regulated responses. Two major and perhaps two minor, partially separated peaks with LasR stimulatory activity were detected in extracts from 4-d-old cultures, whereas extracts from 12-d-old cultures had perhaps six major and two or three minor peaks of LasR stimulatory activity. The activity peaks were quite consistent in duplicate cultures, although some variation occurred in activity level for individual peaks. Peaks of LasR activity with similar retention times were detected in extracts from cultures grown on Tris-acetate phosphate (TAP) medium containing acetate as a carbon source, but the activity levels in these peaks were generally 5 to 10 times lower (data not shown).
Solvent-only injection controls to test for contaminating activities released from the column during fractionation were negative. Experiments to detect active compounds present in the HS medium and the ethyl acetate solvent were conducted with 1 L of HS medium extracted with 2 x 300 mL ethyl acetate. HPLC fractions obtained after injection of the dried solvent residue had no activity detectable with the LasR or CepR reporters.
As illustrated in Figure 3, when the HPLC fractions of Figure 2 were assayed with the Pseudomonas putida CepRI'::GFP reporter strain of Steidle et al. (2001
No substances that stimulated either the E. coli LuxR or AhyR reporters, which are most responsive to 3-oxo-C6-HSL and C4-HSL, respectively, were detected in any of the HPLC fractions of the ethyl acetate extracts (data not shown), nor was there any evidence of substances that affected violacein synthesis in the Chromobacterium violaceum CV026 reporter strain, most responsive to C6-HSL, or that inhibited the responses of the LasR, LuxR, or CepR reporters to partially inducing concentrations of their cognate AHLs (data not shown). Storage of the crude extracts for 6 months at -20 C in 50% (v/v) acetonitrile resulted in substantial loss of the original activity for most of the LasR and CepR stimulatory substances (data not shown). When freeze-dried culture filtrate from C. reinhardtii was extracted first with methanol and then 1:1 (v/v) methanol:water, HPLC fractions obtained after injection of the combined extracts contained no detectable LasR, LuxR, or CepR stimulatory substances when tested at the same levels used to test fractions in Figures 2 and 3 (data not shown). This is in contrast to pea (Pisum sativum) and Medicago truncatula, where most of the AHL mimic activities were recovered in the methanol and 50% (v/v) methanol extracts, not the ethyl acetate extract (Teplitski et al., 2000
To test whether physiological concentrations of AHL mimic substances from C. reinhardtii might be able to affect important functions in wild-type bacteria, we exposed low-density cell cultures of S. meliloti to a purified preparation of one of the LasR stimulatory substances from the alga and looked for mimic-induced changes in protein accumulation in the bacterial cells. Like C. reinhardtii, S. meliloti is a common inhabitant in soils. S. meliloti was selected as a suitable model for such testing because a previous study showed global proteome responses to added AHLs in low-density cultures of the wild type (Chen et al., 2003 To obtain a highly purified AHL mimic from the alga, substances from approximately 10 L of C. reinhardtii culture filtrate were extracted, fractionated, and assayed with the LasR reporter strain (Fig. 4A). Fractions 52 to 55, corresponding to the last, most clearly separated peak of LasR stimulatory activity in Figure 4A, were pooled and refractionated to obtain a highly purified preparation for testing of responses in bacteria (fractions 52-54, Fig. 4B).
Proteome responses in S. meliloti to this purified substance were determined by redissolving the putative signal mimic substance in 1 mL of TA medium and adding portions to duplicate, washed, early log phase (A600 = 0.03) cultures of S. meliloti 1021 at a concentration calculated to be approximately equivalent to its concentration in the original C. reinhardtii cultures. After 2 h, the bacteria were collected by centrifugation, freeze dried, and later extracted to recover proteins for two-dimensional gel separation. A second set of duplicate cultures of the bacterium was treated with a mixture of the AHLs extracted from filtrates of an early stationary phase, defined medium culture of S. meliloti 1021. These AHLs were added to the early log culture of the bacterium at approximately the same concentration present in the original early stationary phase bacterial cultures. A third set of duplicate cultures was treated with a mixture of the bacterial AHLs and the C. reinhardtii LasR stimulatory mimic substance. Duplicate control cultures were treated with the residue from LasR inactive HPLC fractions 48 to 50 (Fig. 4B).
Table II lists the S. meliloti proteins that accumulated to significantly different levels in response to either the purified C. reinhardtii LasR mimic or the bacterium's own AHLs or to the mixture of bacterial AHLs and C. reinhardtii AHL mimic. Thirty-two of the 34 differentially accumulated proteins were identified by peptide mass fingerprint comparison with peptides predicted from the genomic sequence (Galibert et al., 2001
Production of Substances by C. reinhardtii That Affect Bacterial Quorum Sensing
The present study provides evidence that C. reinhardtii CC2137 is capable of producing and secreting at least a dozen chromatographically separable substances capable of specifically stimulating LasR- and CepR-mediated quorum sensing functions in well-characterized reporters (Figs. 2 and 3). For comparison, the model legume M. truncatula was found to produce about 15 to 20 separable AHL mimics (Gao et al., 2003
Because the LasR and CepR active substances from C. reinhardtii did not elicit responses in several other AHL reporter strains (LuxR, AhyR, and CviR), it appears that the algal compounds are acting in a receptor-specific manner. We speculate that the algal compounds are most likely interacting directly with the LasR or CepR receptor polypeptides in a manner similar to the interactions reported between the D. pulchra furanone AHL mimics and the LuxR receptor (Manefield et al., 1999
The chemical identities of the C. reinhardtii compounds responsible for activation of quorum sensing-regulated responses in the LasR and CepR reporters are still unknown. Because the active substances all stimulated the reporters, they are unlikely to be halogenated furanones like those made by D. pulchra, which all have inhibitory effects on bacterial quorum sensing. Because they partition into ethyl acetate in the same manner as bacterial AHLs, it is possible that the active compounds from C. reinhardtii are identical or very similar to bacterial AHLs. However, an initial mass spectral analyses of purified LasR active fractions from the alga using the methods described by Marketon et al. (2002 Both culture age and growth conditions were important factors in production of AHL mimics by C. reinhardtii. The levels of the LasR and CepR mimic substances were considerably higher when the alga was cultured phototrophically than when cultured on medium containing acetate as a carbon source, despite the fact that cell number did not increase appreciably during phototrophic culture. The LasR mimic activities, but not the CepR mimic activities, changed markedly with length of culture (Figs. 2 and 3). The observed changes in LasR activities with culture age suggest that the LasR active mimic compounds are not simply accumulating in the culture medium but may be subject to biotic or abiotic inactivation and changes in regulation of production or secretion. No LasR inhibitory mimic compounds were detected in ethyl acetate or methanol extracts of C. reinhardtii culture filtrate. Nonetheless, the LasR reporter's responses to its cognate AHL were strongly inhibited by algal streaks or colonies in overlays (Fig. 1B), as were the LuxR and AhyR reporters (not shown). This inhibition does not appear to be due to nonspecific toxicity or to an inhibition of luminescence by algal substances because a constitutively luminescent derivative of E. coli was not inhibited in similar overlays. These results suggest that that the algal colonies are secreting specific inhibitors of AHL quorum sensing and that these inhibitory substances are probably more hydrophylic than the stimulatory mimics, not readily extracted in organic solvents. This possibility is supported by the observation that LasR stimulatory substances could be detected on polyvinylidene difluoride membranes after passing C. reinhardtii culture filtrate through the membrane but only after rinsing the membranes with water, presumably removing the more hydrophilic inhibitory compounds (data not shown). Further studies are clearly needed to isolate, purify, and characterize these putative inhibitory AHL mimics.
Both the V. harveyi wild-type and BB170 reporter strains were stimulated to luminesce in overlays of C. reinhardtii and Chlorella spp. colonies (Fig. 1A), providing evidence that these algal isolates secrete compounds that are identical to, or capable of mimicking, the quorum sensing signals of V. harveyi. Quorum sensing-regulated luminescence in V. harveyi is complex, stimulated both independently and synergistically by an AHL (3-OH-C4-HSL = AI-1) and by a furanosyl borate diester (AI-2), and is mediated by pairs of two-component phosphorelay proteins rather than typical AHL receptors (Mok et al., 2003
S. meliloti was used here as a representative soil bacterium to test whether its quorum sensing regulation could be disrupted by exposure to physiological levels of one of the AHL signal mimics from C. reinhardtii. The present study should be regarded as preliminary because it involved treatment with an algal mimic compound that is not chemically identified, only partially purified and added at undefined concentrations. Nonetheless, a comparison of the S. meliloti proteins affected by a mixture of its own AHL signals with those affected by the purified AHL mimic from the alga shows a high degree of overlap (16 of 25 proteins, Table II). This provides evidence that the purified AHL mimic from C. reinhardtii, identified initially through its specific stimulation of responses in the E. coli LasR reporter strain, can affect diverse aspects of quorum sensing regulation in a representative wild-type soil bacterium.
The furanone AHL mimics of D. pulchra rather specifically target quorum sensing-regulated functions in bacteria and do not appreciably disrupt other aspects of bacterial metabolism or behavior (Manefield et al., 1999
Many of the AHL/mimic responsive proteins correspond to open reading frames (ORFs) listed as hypothetical, with unknown function. However, several proteins with potentially interesting functions were identified based on the S. meliloti 1021 annotated genome database, available at http://sequence.toulouse.inra.fr/meliloti.html. The PII nitrogen regulatory protein encoded by glnB (Smc00947 was newly detected in response to the algal mimic. GlnB is similar to the central PII regulators of carbon and nitrogen balance in both prokaryotes and eukaryotes (Ninfa and Atkinson, 2000
When added by itself, the algal mimic had effects that were qualitatively and quantitatively similar to those of the bacterium's AHLs on the accumulation of the 16 coresponsive proteins (Table II). However, the proteins encoded by moaB (Smc0863) and ppiB (Smc01208 responded in opposite ways to the addition of bacterial AHLs and algal mimic. The MoaB homolog, which is involved in synthesis of the molybdopterin cofactor required for many oxidoreductases (McLuskey et al., 2003
When added together, the algal mimic frequently cancelled the effect of the bacterial AHLs. Proteins encoded by Sma0967, Smb20602 and 21566; chromosomal genes 00328, 00469, and 01876; and isoform II encoded by Smc02111all accumulated significantly in response to either the AHLs alone or the mimic alone. However, when the bacteria were exposed to the mixture of AHLs and mimic, there was no significant change in the level of these proteins. Because AHL receptors typically appear to be active as dimers (Qin et al., 2000
Organisms and Growth Conditions
Chlamydomonas reinhardtii wild-type strain 2137 (CC-1021) was maintained on TAP (mineral salts + acetate) agar under 12 h of 40 µmol photons m-2 s-1 of white light at 25°C, restreaking every 3 weeks. Individual colonies were inoculated into TAP liquid shake cultures and grown axenically in light for 5 to 6 d to A750 > 1.2. The TAP-grown starter cultures were centrifuged, and the cells were resuspended in 20 volumes of HS mineral salts medium lacking a carbon source. Cultures were incubated for 4 to 12 d on a shaker (175 rpm) under continuous white light (75 µmol photons m-2 s-1). TAP and HS media were prepared as described (Harris, 1989
Bacterial strains used as reporters for AHL and AI-2 quorum sensing signals are listed in Table I and were cultured as described (Teplitski et al., 2000
C. reinhardtii cultures maintained in HS medium for 4, 8, or 12 d were tested for bacterial contamination and uncontaminated batches were centrifuged, filtered through 0.8-µm nitrocellulose membranes, and partitioned twice with 0.3 volumes of ethyl acetate. The ethyl acetate extracts were rotary evaporated at 32°C, and the dried extracts were resuspended in 50% (v/v) acetonitrile:water in 1 µL mL-1 of ethyl acetate. This solution was fractionated on a reverse-phase C18 analytical HPLC column (5 µm, 250-4 LiChrospher100, Agilent Technologies, Palo Alto, CA) mounted with a guard column by injecting 200-µL samples, eluting for 5 min with water, then for 40 min with a linear gradient of increasing acetonitrile to 100% (v/v), and maintaining 100% (v/v) acetonitrile elution for an additional 15 min. A flow rate of 0.75 mL min-1 was maintained throughout the sample run.
Bioassays to detect algal compounds with the ability to affect bacterial AHL reporter strains were conducted as described previously (Teplitski et al., 2000
TAP-grown cultures of C. reinhardtii CC-2137 (A750 = 0.8-1.0) were subcultured into 3 volumes of fresh HS medium and incubated in light at 25°C for a week (A750 = 0.3-0.4). The algae were then removed by centrifugation, and the culture filtrates (pH 6.4-6.8) were collected. The cell-free culture filtrates were extracted twice with an equal volume of ethyl acetate, and the extract was rotary evaporated to dryness over a 40°C water bath and stored in glass vials at -20°C. For HPLC analysis, the residue from about 10 L of culture was brought up in 1 mL of acetonitrile, centrifuged, and the precipitate was extracted with 1 mL of acetonitrile:water (1:1 [v/v]). The supernatants were combined and injected onto a 40% (v/v) acetonitrile:60% (v/v) water-equilibrated semipreparatory C18 column (Whatman Partisil 10, ODS-3, Whatman, Clifton, NJ) fitted with a guard column. The column was eluted at 2 mL min-1 with a linear water:acetonitrile gradient starting at 40% (v/v) acetonitrile and increasing to 100% (v/v) acetonitrile over 70 min, followed by an additional 10 min with 100% (v/v) acetonitrile. One-minute fractions were assayed with the LasR AHL reporter.
S. meliloti 1021 cells were subcultured and washed several times to reduce the concentration of endogenous AHLs as much as possible as described previously (Chen et al., 2003
Proteins were extracted from freeze-dried cells as described (Chen et al., 2000a
Proteins were identified by tryptic digestion of the polypeptides isolated from the Coomassie-stained control gels followed by peptide mass fingerprinting with matrix-assisted laser-desorption ionization time of flight mass spectrometry performed on a Micromass TofSpec 2E Time of Flight Mass Spectrometer (Waters Corporation, Milford, MA) at the Australian Proteome Analysis Facility (Macquarie University, Sydney). Peptide mass fingerprints were identified by comparison with the S. meliloti 1021 proteomic database using Mascot software (Micromass; Waters Corp) as described previously (Weiller et al., 2001
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. However, no supplies of the purified LasR mimic or other active compounds are presently available, and their isolation requires considerable effort.
We thank Brian Ahmer for use of his CCD camera, James Metzger for generous help with HPLC; Simon Swift, Bonnie Bassler, and Leo Eberl for providing AHL reporter strains; and Anatol Eberhard for providing synthetic AHLs. Received July 14, 2003; returned for revision August 4, 2003; accepted September 15, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.029918.
1 This work was supported by the U.S. Department of Agriculture National Research Initiatives (grant no. 2002-3531911559 to B.G.R., J.B.R., and W.D.B.), by the Ohio Plant Biotechnology Consortium (grant no. to J.B.R. and W.D.B), by the Ohio State University Office of International Education Travel (grants to W.D.B and M.T.), by the Ohio Agricultural Research and Development Center (presidential fellowship to M.T.), by the Ohio State University (Research Enhancement grant and Extension Sustainable Agriculture grant to M.T.), and by state and federal funds appropriated to the Ohio Agricultural Research and Development Center in partial salary and research support to W.D.B. This is contribution no. 03-14 of Department of Horticulture and Crop Science.
2 Present address: Department of Microbiology, Ohio State University, 484 W. 12th Avenue, Columbus, OH 43210. * Corresponding author; e-mail bauer.7{at}osu.edu; fax 614-292-9035.
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ABSTRACT
RESULTS
2 was less than 0.05. 
