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JOURNAL OF BACTERIOLOGY, Jan. 2003, p. 325–331 0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.1.325–331.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 1

Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti Melanie M. Marketon,1 Sarah A. Glenn,1 Anatol Eberhard,2 and Juan E. Gonza´lez1* Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688,1 and Department of Chemistry, Ithaca College, Ithaca, New York 148502 Received 22 July 2002/Accepted 25 September 2002

Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis. Sinorhizobium meliloti is a soil bacterium capable of establishing a symbiotic relationship with the alfalfa plant, Medicago sativa. This process requires a series of molecular signaling events leading up to the formation of nitrogen-fixing nodules (for reviews, see references 21, 29, and 47). It has been shown that bacterially produced exopolysaccharides are essential for nodule invasion; however, their exact role in symbiosis is unknown (19, 26, 39). S. meliloti is capable of producing two distinct exopolysaccharides, succinoglycan and EPS II, either of which can function in symbiosis (8, 13, 16, 19, 27). Succinoglycan synthesis has been extensively characterized (18, 42). The genes encoding its production, the exo genes, reside in a 25-kb region of the pSymB megaplasmid (14, 15). Succinoglycan is composed of repeating octasaccharide subunits consisting of one galactose, seven glucoses, and one each of succinyl, acetyl, and pyruvyl modifications (1, 41). EPS II, on the other hand, is composed of repeating disaccharide subunits. In this case, each subunit contains one galactose and one glucose, along with acetyl and pyruvyl modifications (20, 28). The genes responsible for EPS II synthesis belong to a 32-kb gene cluster (the exp genes) also located on pSymB (5, 13). The exp gene cluster is composed of five putative operons designated as expA (nine genes), expC (one gene), expD (two genes), expE (eight genes), and expG (one gene) (5, 13). Both exopolysaccharides are synthesized in a range of sizes; however, it has been found that the low-molecular-weight fractions are sufficient to allow nodule invasion (4, 17, 39, 46). For succinoglycan, the active low-molecular-weight fraction is a trimer of the octasaccharide subunits, while for EPS II, a range of 15 to 20 of the disaccharide subunits allowed rescue of an exopolysaccharide-deficient mutant (4, 17, 39, 47).

Regulation of exopolysaccharide synthesis seems to be controlled, at least in part, by environmental signals, such as phosphate and nitrogen concentrations (3, 9, 26, 32, 45, 49). It was shown that EPS II synthesis is stimulated by low-phosphate conditions but repressed in high phosphate (2, 32, 44, 45, 49). In contrast, succinoglycan synthesis is increased under highphosphate conditions (32). Interestingly, the EPS II made by the commonly used wild-type strain Rm1021 in low phosphate is of high molecular weight and therefore is not active in invasion (17, 32). In addition to the exp gene cluster, which is located on pSymB, two chromosomal loci (expR101 and mucR) have been identified which are capable of regulating EPS II synthesis (13, 25, 50). MucR was shown to repress EPS II synthesis, and mutations in mucR lead to the production of high-molecular-weight EPS II, which is inactive in nodule invasion (17). A mutation defined as expR101, however, resulted in the production of both high- and low-molecular-weight EPS II (17, 19, 32). Recently it was shown that strain Rm8530 (formerly known as expR101) has an intact expR gene, while Rm1021 has a disrupted copy of this gene. A functional copy of the expR gene is required for the synthesis of low-molecularweight EPS II (38). Since the predicted expR gene product has homology to luxR-type quorum-sensing regulators, it was suggested that EPS II synthesis might also be controlled by quorum sensing (38). Marketon et al. have previously shown that S. meliloti strain Rm1021 harbors at least two quorum-sensing systems (30). One of these, the sinRI locus, was shown to be responsible for the production of numerous long-chain N-acyl homoserine lactones (AHLs) ranging in size from N-dodecanoyl homoserine lactone (C12-HL) to N-octadecanoyl homoserine lactone (C18HL) (31). When these genes were disrupted there was a corresponding decrease in the number of nodules per plant, indicating a role for quorum sensing in symbiosis (31). We therefore sought to identify the downstream targets of the

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Texas at Dallas, Mail Station FO 3.1, Box 830688, Richardson, TX 75083-0688. Phone: (972) 883-2526. Fax: (630) 604-3093. E-mail: [email protected]. 325

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TABLE 1. Bacterial strains and plasmids used in this work Strain

Rm1021 Rm11511 Rm11512 Rm8530 Rm9020 Rm9028 Rm9030-2 Rm9031 Rm9032 Rm9033 Rm9034 Rm11521 Rm11522 Rm11523 Rm11524 Rm11525 Rm11526 Rm11527 Rm11528 Rm11529 Rm11530 Rm11531 Rm11532 Rm11533 Rm11534 Rm11535 Rm11536 Rm11537 Rm11538 Rm11539

Relevant characteristics

Su47 str-21 Rm1021 sinI::KM Rm1021 sinR::GM Rm1021 expR⫹ Rm8530 exoY::Tn5–132 Rm8530 expR103::lacZ-GM Rm8530 expA1::lacZ-GM Rm8530 expC::lacZ-GM Rm8530 expD1::lacZ-GM Rm8530 expE2::lacZ-GM Rm8530 expG::lacZ-GM Rm9028 sinI::KM Rm9030-2 sinI::KM Rm9031 sinI::KM Rm9032 sinI::KM Rm9033 sinI::KM Rm9034 sinI::KM Rm8530 sinI::KM Rm8530 sinR::GM Rm9020 sinI::KM Rm1021 expA1::lacZ-GM Rm1021 expC::lacZ-GM Rm1021 expD1::lacZ-GM Rm1021 expE2::lacZ-GM Rm1021 expG::lacZ-GM Rm11511 expA1::lacZ-GM Rm11511 expC::lacZ-GM Rm11511 expD1::lacZ-GM Rm11511 expE2::lacZ-GM Rm11511 expG::lacZ-GM

Reference or source

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sinRI quorum-sensing system to determine how the sinRI locus influences nodulation. We show here that the sinR and sinI genes are required for synthesis of EPS II by the expR⫹ strain (Rm8530). Furthermore, we show that expression of several exp genes is abolished in a sinI background and can be fully complemented by the addition of either crude AHL extracts from wild-type Rm1021 or synthetic palmitoleyl-HL [C16:1-HL or N-(9-cis-hexadecenoyl)-L-homoserine lactone]. Finally, we show that EPS II regulation by sinRI is important for nodule invasion, since a strain that exclusively produces EPS II combined with a sinI mutation is no longer capable of forming nitrogen-fixing nodules. MATERIALS AND METHODS Media and genetic techniques. Bacteria were grown in Luria-Bertani broth or agar supplemented with MgSO4 and CaCl2 (LB/MC) and the appropriate antibiotics as previously described (12). Growth in low-phosphate medium (0.1 mM K2HPO4) has been described previously (32). Strains (Table 1) were created by generalized transduction using phage ␾M12 (12). ␤-Galactosidase assays. Bacteria were grown to log phase in 2 ml of lowphosphate medium at 30°C and assayed as previously described (33) to determine Miller units of activity. For complementation experiments, crude AHL extracts were prepared by extracting 5-ml LB/MC cultures with ethyl acetate (31) and the resulting extracts (or AHL standards) were added to the medium at the time of inoculation. AHL standards were purchased from Sigma [N-(3oxo-hexanoyl)-DL-homoserine lactone (oxo-C6-HL)] and Fluka (N-butyryl-DLhomoserine lactone [C4-HL], N-hexanoyl-DL-homoserine lactone [C6-HL], Noctanoyl-DL-homoserine lactone [C8-HL], N-dodecanoyl-DL-homoserine lactone [C12-HL], and N-tetradecanoyl-DL-homoserine lactone [C14-HL]) or were synthesized (C16:1-HL and N-octadecanoyl-L-homoserine lactone [C18-HL]) as described below.

Synthesis of synthetic AHLs. C16:1-HL was prepared by coupling the sodium salt of palmitoleic acid with L-homoserine lactone hydrobromide by using 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride in 50% (vol/vol) acetonitrile–50% (vol/vol) water, followed by preparative high-performance liquid chromatography (HPLC) with 97% (vol/vol) acetonitrile–3% (vol/vol) 1-propanol as the solvent. The mass spectrum of the C16:1-HL has been described previously (31). C18-HL was synthesized in a similar manner, starting with sodium stearate. Carbohydrate composition analyses. To characterize the carbohydrate composition of the exopolysaccharides produced by S. meliloti, a culture was grown to saturation in minimal mannitol glutamate medium (4, 24). Cells were removed by centrifugation, and the supernatant was dialyzed using a 1,000-molecularweight-cutoff membrane (Spectrum) against water for 2 days. A fraction of the dialyzed exopolysaccharide solution was mixed with an equal volume of 4 M trifluoroacetic acid and hydrolyzed overnight at 100°C in a sealed glass ampule. The hydrolyzed polymer solution was dried in a Brinkman SpeedVac centrifuge, washed twice, and resuspended in 100 ␮l of water. Analyses by high-pressure anion-exchange chromatography were performed on a Dionex DX 500 HPLC system with a CarboPac MA1 column using a pulsed amperometric detector with a gold working electrode. The waveform used for the analysis was as follows: 0.1 V for 0.4 s, 0.7 V for 0.2 s, and ⫺0.1 V for 0.4 s. The samples were eluted isocratically with 500 mM NaOH. The carbohydrate composition of the exopolysaccharides was calculated as a glucose/galactose ratio based on the elution times of known glucose and galactose standards. Because there was no free glucose or galactose before hydrolysis, all calculated glucose/galactose ratios represent hydrolyzed exopolysaccharide. Plant nodulation assays. M. sativa was inoculated with the different S. meliloti strains and grown on standard Jensen’s medium as previously described (26). Plants were inspected on a weekly basis. After 30 days, plant height was measured and pink (nitrogen-fixing) nodules were counted.

RESULTS The sinRI locus controls exopolysaccharide production. Marketon et al. have previously characterized the sinRI quorum-sensing locus and showed that mutations in sinR and sinI in Rm1021 had an effect on its symbiosis with alfalfa (31). Recent work by Pellock et al. showed that the expR gene is homologous to luxR-type regulators and provided evidence suggesting that quorum sensing may control exopolysaccharide production in S. meliloti (38). In light of these findings, we decided to investigate the possible correlation between the sinRI locus and exopolysaccharide synthesis. When we transduced the sinR and sinI mutations into an expR⫹ background (Rm8530 [formerly expR101]), we noticed a dramatic change from a highly mucoid colony phenotype to a much less mucoid phenotype, similar to that of Rm1021 (Fig. 1). Since Rm8530 has been shown to produce mainly EPS II (17, 32, 38), these results suggested that EPS II synthesis was indeed regulated by the products of the sinRI locus. Synthesis of EPS II, but not of succinoglycan, is abolished in a sinI mutant. To confirm that it was EPS II synthesis and not succinoglycan synthesis that was affected by the sinR and sinI mutations, we analyzed the carbohydrate composition of the exopolysaccharide produced by the mutants (Table 2). The glucose/galactose ratio of the exopolysaccharide produced by the expR⫹ strain (Rm8530) indicated that EPS II was the main exopolysaccharide present. Likewise, the expR⫹ exoY strain also made EPS II, which is in agreement with previous observations (32). For an expR⫹ strain carrying either sinI or sinR disruptions, however, only succinoglycan was detected. When the sinI mutation was combined with an exoY mutation which has been shown to abolish succinoglycan production (26, 42), only trace levels of glucose were detected, confirming that neither succinoglycan nor EPS II was synthesized. These data

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FIG. 1. Mutations in sinI and sinR abolish EPS II synthesis. Strains were plated on LB/MC. (A) expR⫹ (Rm8530); (B) expR⫹ sinI (Rm11527); (C) expR⫹ sinR (Rm11528); (D) expR⫹ exoY (Rm9020).

confirm that EPS II production is regulated, either directly or indirectly, by the sinRI locus. exp gene expression is regulated by sinRI. Modest changes in the expression of multiple exp gene fusions in response to the addition of conditioned medium in an expR⫹ background were previously observed (38). This suggested that the exp genes were induced by a quorum-sensing mechanism. To demonstrate unequivocally that this was indeed the case, we transduced the sinI mutation from the Rm1021 sinI derivative (Rm11511) into strains carrying lacZ transcriptional fusions in the following exp genes: expA1 (Rm9030-2), expC (Rm9031), expD1 (Rm9032), expE2 (Rm9033), expG (Rm9034), and expR (Rm9028) (Table 1). Gene expression was then assayed by growing the cells to log phase in low-phosphate medium, which has been previously shown to induce the exp genes (32, 43, 45). In a sinI⫹ background, the expC and expE2 gene fusions were expressed at high levels (Fig. 2A), which agrees with the observations made by Pellock et al. (38). However, in a sinI mutant, a dramatic decrease in expression levels was observed for the expE2 (41.7-fold), expC (7.1-fold), and expD1 (6.7-fold) gene fusions. Only slight differences were noticed for the other

TABLE 2. Quorum sensing controls production of symbiotically active EPS II Strain phenotype

Glc/Gal ratioa or glucose content

Exopolysaccharide producedb

% of plants with nitrogen-fixing nodulesc

expR⫹ expR⫹ sinI expR⫹ sinR expR⫹ sinI exoY expR exoY

1.5 (0.2):1 9.1 (0.2):1 9 (1.1):1 Trace glucose 1.6 (0.6):1

EPS II Succinoglycan Succinoglycan None EPS II

100 100 Not tested 0d 100

a Numbers in parentheses represent standard deviations determined from three trials. b The exopolysaccharide produced was determined based on glucose/galactose ratios determined as described in Materials and Methods. c Each strain was tested on at least 15 plants. d All plants lacked pink nodules but did develop white nodules.

FIG. 2. Effect of sinI on exp gene expression. (A) ␤-Galactosidase levels were measured in strains carrying lacZ fusions to expR, expA1, expC, expD1, expE2, and expG in sinI⫹ or sinI and expR⫹ or expR backgrounds. (B) Activity of the expE2::lacZ fusion in an expR⫹ sinI strain (Rm11525) was measured after adding different amounts of crude AHL extracts from Rm1021 or Rm1021 sinI.

gene fusions in the sinI background. These results clearly show that a functional sinI gene is required for normal expression of several genes involved in EPS II synthesis. exp gene expression is mediated by sin AHLs. To determine whether the observed differences in gene expression in the sinI mutants were specifically due to the absence of the sinI-specified AHLs, we conducted complementation experiments by adding back crude AHL extracts. Because expE2 had the most dramatic phenotype, we chose the strain with the expE2::lacZ fusion in a sinI background (Rm11525) for further analysis. We have previously shown that at least two AHL synthases are present in S. meliloti Rm1021. The sinI synthase was shown to be responsible for the production of several long-chain AHLs ranging in size from C12-HL to C18-HL, while the putative melI synthase was responsible for several short-chain AHLs, including C8-HL. Disruption of sinI did not affect the synthesis of the mel AHLs (31). Therefore, to confirm the role of the sin AHLs in exp gene expression, we added different concentrations of crude AHL extracts from either wild-type Rm1021 or Rm1021 sinI to expR⫹ expE2::lacZ sinI (Rm11525) cultures and measured the resulting ␤-galactosidase activity (Fig. 2B). Extracts were prepared as 10⫻ stocks, so 200 ␮l of extract represents the AHLs found in 2 ml of culture. As expected, we found that only the Rm1021 extract was able to complement the sinI

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FIG. 3. C16:1-HL induces expE2 expression. (A) The ability of different AHLs to induce exp gene expression was tested by adding excess amounts of each AHL to expR⫹ sinI expE2::lacZ (strain Rm11525) cultures. (B) Activity of the expE2::lacZ fusion in Rm11525 was measured after adding different amounts of C16:1-HL.

mutation in Rm11525. No induction of the expE2::lacZ fusion was observed from the Rm1021 sinI extracts, even when 200 ␮l of extract was added to the Rm11525 cultures. C16:1-HL can induce exp gene expression. Since the sinI synthase directs the production of several AHLs (C12-HL, oxoC14-HL, oxo-C16:1-HL, C16:1-HL, and C18-HL) (30), we sought to determine which AHL(s) was responsible for the complementation by adding back individual synthetic AHLs. We added approximately 5 ␮g of the following synthetic AHLs to expE2::lacZ sinI (Rm11525) cultures: C4-HL (⬃14.5 ␮M), C6-HL (⬃13.5 ␮M), oxo-C6-HL (⬃12 ␮M), C8-HL (⬃11 ␮M), C12-HL (⬃9 ␮M), C14-HL (⬃8 ␮M), C16:1-HL (⬃7.5 ␮M), and C18-HL (⬃7 ␮M) (Fig. 3A). We found that only C16:1-HL was able to restore expression of expE2. We therefore repeated the experiment with different amounts of C16:1-HL to determine how much of the AHL was necessary to restore expression and found that as low a concentration as 150 nM was sufficient to induce the expE2::lacZ fusion (Fig. 3B). Maximal induction was achieved with 1.5 to 7.5 ␮M C16:1-HL. Although induction of expE2 by C16:1-HL seems to be specific, the possibility of other AHLs modulating exp gene expression by competitively binding to the regulatory protein remained. Therefore, we performed competition assays using C16:1-HL and various AHLs and found that the other AHLs, such as C18-HL, were unable to inhibit C16:1-HL-mediated activation of expE2::lacZ (data not shown). This suggests that the induction of the exp genes by C16:1-HL is specific and that the other sin AHLs do not play a role in the regulation of the exp gene cluster. sinRI-induced exp expression requires expR. Pellock et al. recently showed that the expR gene in Rm1021 was disrupted by an insertion sequence (ISRm2011-1) (38). To demonstrate

J. BACTERIOL.

that an intact expR gene is required for the induction of the exp genes in response to AHLs, we introduced the different exp::lacZ fusions into Rm1021 and Rm1021 sinI (Table 1). Figure 2A shows ␤-galactosidase levels for each of the exp fusions in the Rm1021 backgrounds. In all cases the levels of expression were very low, similar to the low levels obtained from the corresponding fusions in an expR⫹ sinI background. These results indicate that in Rm1021, which lacks a functional expR gene, exp gene expression is not influenced by the sinRI quorum-sensing system. The data are in agreement with the previous observations and indeed suggest that the quorumsensing induction of the exp genes is through the expR gene product. sinRI-mediated EPS II production is involved in symbiosis. To confirm further the role of the sinRI quorum-sensing system in symbiosis, we conducted nodulation assays with the sin mutants. We inoculated alfalfa seedlings with expR⫹ (Rm8530), expR⫹ sinI (Rm11527), expR⫹ exoY (Rm9020), and expR⫹ sinI exoY (Rm11529) strains (Table 2). As expected, the expR⫹ and expR⫹ sinI strains were still able to invade the plants. This agrees with the exopolysaccharide analysis, which indicated that succinoglycan was still made in the sinI mutant. Therefore, nodule invasion can still occur. However, in plants inoculated with the expR⫹ sinI exoY mutant (Rm11529) no pink nitrogen-fixing nodules were observed, indicating that the bacteria were not capable of nodule invasion. This also agrees with the exopolysaccharide analysis, which showed that neither succinoglycan nor EPS II was synthesized by this strain. Without the presence of either exopolysaccharide, nodule invasion cannot occur. These results confirm that the symbiotically active EPS II is regulated by the sinRI quorum-sensing system. DISCUSSION Quorum sensing is known to regulate numerous phenomena, including the production of exopolysaccharides in soil organisms such as Rhodobacter sphaeroides, Ralstonia solanacearum, Pantoea stewartii subsp. stewartii, and Pseudomonas aureofaciens (10, 40, 48, 51). Recent studies with S. meliloti have indicated that the synthesis of symbiotically active EPS II may also be regulated by a quorum-sensing system (38). Those studies showed that the expR gene product was required for EPS II production and that expR was homologous to other luxR-type regulators. Here, we show that EPS II synthesis is indeed regulated by quorum sensing in S. meliloti, specifically by the sinRI system. Marketon et al. have previously shown that the sinR and sinI genes comprise a quorum-sensing system that is responsible for the synthesis of several long-chain AHLs ranging in size from C12-HL to C18-HL and that disruption of this system results in the absence of those AHLs (31). In the present study, we found that disruption of sinR and sinI also results in a dramatic decrease in exopolysaccharide production (Fig. 1) in the expR⫹ strain, Rm8530 (formerly expR101). Furthermore, carbohydrate composition analyses confirmed that EPS II synthesis was abolished in the sinR and sinI mutants (Table 2). However, succinoglycan was still present, indicating that sinRI controls production of EPS II but not succinoglycan. To investigate the role of sinRI in EPS II regulation, we analyzed the effect of a sinI mutation on exp gene expression (Fig. 2A). We found that in an expR⫹ background, the expE2

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and expC fusions were expressed at high levels and disruption of sinI resulted in 42- and 7-fold decreases in expression levels, respectively. Fusions to expD1 and expG had lower expression levels but still showed a decrease in activity in a sinI mutant. These results are in agreement with the observations by Pellock et al. in a study in which addition of conditioned media to expR⫹ strains led to a two- to fourfold increase in expression of several exp genes (38). The role of the SinI-produced AHLs was further investigated by analyzing the effect of crude extracts from either wild-type Rm1021 or Rm1021 sinI on the expression of the expE2 gene in an expR⫹ sinI background (Fig. 2B). Only the wild-type extract was able to induce the expE2 fusion, suggesting that one or more of the sin-AHLs were necessary. We confirmed this idea by testing the effect of various synthetic AHLs on the expression of the expE2::lacZ fusion (Fig. 3A). Of the AHLs tested, only C16:1-HL was able to restore expression of the expE2 gene. Furthermore, competition assays between C16:1-HL and C18-HL suggested that induction by C16:1-HL is fairly specific, since the presence of C18-HL did not hinder its ability to activate the expE2 gene. Unfortunately, we did not have synthetic oxo-C14-HL and oxo-C16:1-HL available for testing, but the results obtained here indicate that the induction of the exp genes is specific to a particular AHL, or subset of AHLs, made by the sinI synthase. It is possible that both C16:1-HL and oxo-C16:1-HL are the biologically active AHLs and that the others are minor byproducts of the synthetic pathways. Alternatively, the less-abundant AHLs may be capable of interacting with other LuxR-type regulators. The S. meliloti genome contains at least four open reading frames (SMc00658, SMc00877, SMc00878, and SMc04032) that are predicted to encode LuxR homologs in addition to SinR and ExpR (7, 11). The role of these putative regulators and their ability to associate with AHLs remain to be investigated. Interestingly, we found that at least 150 nM C16:1-HL was required to induce the expE2::lacZ fusion (Fig. 3B). This level is significantly higher than the amount of AHLs required to induce other quorum-sensing systems such as the lux operon, where 10 nM was shown to be sufficient for minimal induction (23). This difference is not surprising since long-chain AHLs probably partition to the cell membrane, as was seen in Pseudomonas aeruginosa (37). It is possible that purified AHLs added in trans become trapped in the membrane, resulting in a lower effective concentration inside the cell of AHLs that can interact with SinR and/or ExpR. On the other hand, endogenous C16:1-HL is probably also trapped by the membrane, resulting in a higher intracellular concentration. This hypothesis could explain the previous observation that the exp genes are activated in response to conditioned media at lower cell densities compared to other characterized quorum-sensing systems (38). Marketon et al. have recently shown that S. meliloti produces the longest AHLs described to date, with acyl chain lengths of 16 to 18 carbons (30). Here we establish that one of those long-chain AHLs, C16:1-HL, plays an important role in the symbiont-plant interaction. Whether these long-chain AHL signals are actively transported across the S. meliloti cell membrane and, if so, how this transport occurs remain to be explored. It was previously assumed that all AHLs are freely diffusible, since radiolabeled Photobacterium fischeri autoin-

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ducer N-3-oxo-hexanoyl homoserine lactone was shown to diffuse unrestrainedly in and out of P. fischeri and Escherichia coli cells (23, 36). This notion was recently challenged when Pearson and colleagues demonstrated that P. aeruginosa cells are not freely permeable to oxo-C12-HL, one of the two AHLs produced by this bacterium (36). In a series of elegant experiments, they showed that cellular concentrations of radiolabeled oxo-C12-HL are higher than external levels (36). This higher cellular level was not due to association with the response regulators LasR or RhlR nor was it the result of active inward transport (36). Instead, it seems that the high cellular level of oxo-C12-HL was due to partitioning into the cell membranes; furthermore, they proposed that this AHL was subject to active export by a specific efflux pump encoded by the mexAB-oprM operon (36). The fact that export, and perhaps import, of long-chain AHLs requires some type of active transport is not surprising, since highly hydrophobic acyl chains are inclined to strongly associate with the lipid bilayer. The mexAB-oprM operon implicated in P. aeruginosa oxo-C12-HL export encodes an efflux pump that belongs to the resistance/nodulation/cell division (RND) family that includes a number of other multidrug resistance proteins (AcrB and AcrF from E. coli and MtrD from Neisseria gonorrhoeae) (34, 35). Interestingly, this family also includes the NolGHI system from S. meliloti, which may be involved in the export of nodulation signals (44). The recently sequenced S. meliloti genome (7, 11) shows the presence of at least five RND homologous regions (SMc02867, SMc01095, SMc03971, SMa1662 and SMb21498). It is possible that one or more of these putative efflux pumps may be involved in long-chain AHL transport in S. meliloti cells. ExpR is homologous to LuxR-type regulators; however, its exact role in quorum-sensing regulation of EPS II is unclear. We compared the AHLs made by Rm1021 (expR) and Rm8530 (expR⫹) and found no difference in the pattern of AHLs produced. However, the expR⫹ strain did produce slightly higher levels of the sinI-specified AHLs (data not shown). In addition, sinRI-mediated induction of the exp genes required the presence of ExpR, since strains carrying the expR disruption produced only low levels of exp gene expression regardless of the presence or absence of sinI (Fig. 2A). It should be noted that expression of expR itself does not seem to be induced by sinRI (Fig. 2A). Furthermore, our data show that EPS II production in an expR⫹ sinR strain (Rm11528) could be restored by crossstreaking this derivative next to a wild-type (AHL-producing) strain (data not shown). These results indicate that the SinR regulator is required only for the production of the sin-AHLs and not for the production of EPS II. Since an expR⫹ sinR strain can still respond to exogenous AHLs, and since an expR⫹ strain produces higher levels of sin-AHLs, it is possible that the ExpR regulator is able to recognize AHLs (specifically C16:1-HL) and induce the expression of the exp genes as well as the sinRI operon. Whether ExpR is acting directly or indirectly on the exp genes is not yet clear and should be further investigated. The regulation of EPS II production by sinRI is an important finding that connects quorum sensing with the symbiosis process between S. meliloti and alfalfa. This link was confirmed with the observation that Rm 11529 expR⫹ sinI exoY, a strain that does not produce EPS II or succinoglycan, is unable to

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induce the formation of pink nitrogen-fixing nodules on alfalfa plants (Table 2). This supports the idea that sinRI, through the action of ExpR, activates the production of the symbiotically active low-molecular-weight EPS II, which, in turn, is important in nodule invasion (38). It is noteworthy that the quorumsensing/ExpR-mediated transcriptional activation involves mostly the expE and expC operons. Perhaps genes in these two operons are specifically involved in the production of lowmolecular-weight EPS II. Further investigation is necessary to confirm the idea that ExpR can recognize AHLs and, in response, activate target genes such as sinRI and the exp operon. Since we have shown that the sin system does not control expR expression, it should be interesting to determine what factors are responsible for expR induction. Previous work in several laboratories has shown that the exp genes are subject to many levels of control, including environmental phosphate levels and the negative regulator MucR (25, 32, 50). Work in a previous study (38) suggests that the expR-mediated induction of EPS II synthesis is tuned to low cell densities, which may be due to the partitioning of the C16:1-HL to the cell membrane. These findings, along with the fact that EPS II synthesis is also induced by low-phosphate conditions (17, 32), suggest that a complex regulatory network is responsible for modulating EPS II synthesis in the soil, where phosphate concentration and bacterial cell density are both low (6, 22). A mucR mutation or low-phosphate conditions induce the synthesis of high-molecular-weight EPS II, which is inactive in nodule invasion, whereas the presence of an intact expR gene induces production of low-molecular-weight EPS II, which is symbiotically active. It is possible that in the low-phosphate conditions of soil, mostly high-molecular-weight EPS II is made. As the bacteria approach plant roots, where the phosphate concentration is higher (6, 22) and bacterial cell density increases, the production of low-molecular-weight EPS II is induced, allowing for nodule invasion. Further investigation into how these different modes of regulation work together to determine which exopolysaccharide is made in soil and in the nodule environments should shed light on the details of the complex process of symbiosis. ACKNOWLEDGMENTS We thank members of the Gonza´lez laboratory for their helpful discussions and insights on this project. We also thank Brett Pellock and Graham Walker for communicating results before publication. A.E. thanks Ithaca College for a sabbatical leave for 2001 to 2002, Matthew R. Gronquist for help with the ESI MS/MS, and Chelsea Morgan for help with the AHL synthesis. Jerrold Meinwald of Cornell University graciously provided access to the mass spectrometer. This work was supported by National Science Foundation grant MCB-9733532 to J.E.G. This material was also based in part on work supported by the Texas Advanced Research Program under grant 009741-0022-2001. REFERENCES 1. Aman, P., M. McNeil, L.-E. Franzen, A. G. Darvill, and P. Albersheim. 1981. Structural elucidation, using HPLC-MS and GLC-MS, of the acidic exopolysaccharide secreted by Rhizobium meliloti strain Rm1021. Carbohydr. Res. 95:263–282. 2. Astete, S. G., and J. A. Leigh. 1996. mucS, a gene involved in activation of galactoglucan (EPS II) synthesis gene expression in Rhizobium meliloti. Mol. Plant-Microbe Interact. 9:395–400. 3. Bardin, S. D., and T. M. Finan. 1998. Regulation of phosphate assimilation in Rhizobium (Sinorhizobium) meliloti. Genetics 148:1698–1700. 4. Battisti, L., J. C. Lara, and J. A. Leigh. 1992. Specific oligosaccharide form

J. BACTERIOL.

5.

6. 7.

8. 9. 10. 11.

12. 13. 14. 15.

16.

17. 18.

19. 20. 21. 22. 23. 24.

25.

of the Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa. Proc. Natl. Acad. Sci. USA 89:5625–5629. Becker, A., S. Ruberg, H. Kuster, A. A. Roxlau, M. Keller, T. Ivashina, H. P. Cheng, G. C. Walker, and A. Pu ¨hler. 1997. The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J. Bacteriol. 179:1375–1384. Bieleski, R. 1973. Phosphate pools, phosphate transport, and phosphate availability. Annu. Rev. Plant Physiol. 24:225–252. Capela, D., F. Barloy-Hubler, J. Gouzy, G. Bothe, F. Ampe, J. Batut, P. Boistard, A. Becker, M. Boutry, E. Cadieu, S. Dreano, S. Gloux, T. Godrie, A. Goffeau, D. Kahn, E. Kiss, V. Lelaure, D. Masuy, T. Pohl, D. Portetelle, A. Pu ¨hler, B. Purnelle, U. Ramsperger, C. Renard, P. Thebault, M. Vandenbol, S. Weidner, and F. Galibert. 2001. Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc. Natl. Acad. Sci. USA 98:9877–9882. Cheng, H. P., and G. C. Walker. 1998. Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti. J. Bacteriol. 180:5183–5191. Doherty, D., J. A. Leigh, J. Glazebrook, and G. C. Walker. 1988. Rhizobium meliloti mutants that overproduce the R. meliloti acidic calcofluor-binding exopolysaccharide. J. Bacteriol. 170:4249–4256. Flavier, A. B., S. J. Clough, M. A. Schell, and T. P. Denny. 1997. Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. Mol. Microbiol. 26:251–259. Galibert, F., T. M. Finan, S. R. Long, A. Pu ¨hler, P. Abola, F. Ampe, F. Barloy-Hubler, M. J. Barnett, A. Becker, P. Boistard, G. Bothe, M. Boutry, L. Bowser, J. Buhrmester, E. Cadieu, D. Capela, P. Chain, A. Cowie, R. W. Davis, S. Dreano, N. A. Federspiel, R. F. Fisher, S. Gloux, T. Godrie, A. Goffeau, B. Golding, J. Gouzy, M. Gurjal, I. Hernandez-Lucas, A. Hong, L. Huizar, R. W. Hyman, T. Jones, D. Kahn, M. L. Kahn, S. Kalman, D. H. Keating, E. Kiss, C. Komp, V. Lelaure, D. Masuy, C. Palm, M. C. Peck, T. M. Pohl, D. Portetelle, B. Purnelle, U. Ramsperger, R. Surzycki, P. Thebault, M. Vandenbol, F. J. Vorholter, S. Weidner, D. H. Wells, K. Wong, K. C. Yeh, and J. Batut. 2001. The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293:668–672. Glazebrook, J., and G. C. Walker. 1991. Genetic techniques in Rhizobium meliloti. Methods Enzymol. 204:398–418. Glazebrook, J., and G. C. Walker. 1989. A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti. Cell 56:661–672. Glucksmann, M. A., T. L. Reuber, and G. C. Walker. 1993. Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti. J. Bacteriol. 175:7033–7044. Glucksmann, M. A., T. L. Reuber, and G. C. Walker. 1993. Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis. J. Bacteriol. 175:7045–7055. Gonza ´lez, J. E., A. Glucksman, T. L. Reuber, and G. C. Walker. 1993. Exopolysaccharides and Rhizobium meliloti-alfalfa interactions, p. 203–206. In R. Palacios, J. Mora, and W. E. Newton (ed.), New horizons in nitrogen fixation. Kluwer Academic Publishers, Dordrecht, The Netherlands. Gonza ´lez, J. E., B. L. Reuhs, and G. C. Walker. 1996. Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa. Proc. Natl. Acad. Sci. USA 93:8636–8641. Gonza ´lez, J. E., C. E. Semino, L. X. Wang, L. E. Castellano-Torres, and G. C. Walker. 1998. Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 95: 13477–13482. Gonza ´lez, J. E., G. M. York, and G. C. Walker. 1996. Rhizobium meliloti exopolysaccharides: synthesis and symbiotic function. Gene 179:141–146. Her, G. R., J. Glazebrook, G. C. Walker, and V. N. Reinhold. 1990. Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021. Carbohydr. Res. 198:305–312. Hirsch, A. M., M. R. Lum, and J. A. Downie. 2001. What makes the rhizobialegume symbiosis so special? Plant Physiol. 127:1484–1492. Israel, D. 1987. Investigation of the role of phosphorus in symbiotic dinitrogen fixation. Plant Physiol. 84:835–840. Kaplan, H. B., and E. P. Greenberg. 1985. Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J. Bacteriol. 163:1210–1214. Keller, M., W. Arnold, D. Kapp, P. Mu ¨ller, K. Niehaus, M. Schmidt, J. Quandt, W. M. Weng, and A. Pu ¨hler. 1990. Rhizobium meliloti genes involved in exopolysaccharide production and infection of alfalfa nodules, p. 91–97. In S. Silver, A. M. Chakrabarty, B. Iglewski, and S. Kaplan (ed.), Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, D.C. Keller, M., A. Roxlau, W. M. Weng, M. Schmidt, J. Quandt, N. Karsten, D. Jording, W. Arnold, and A. Pu ¨hler. 1995. Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccha-

VOL. 185, 2003

26. 27. 28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

rides succinoglycan and galactoglucan. Mol. Plant-Microbe Interact. 8:267– 277. Leigh, J. A., E. R. Signer, and G. C. Walker. 1985. Exopolysaccharidedeficient mutants of Rhizobium meliloti that form ineffective nodules. Proc. Natl. Acad. Sci. USA 82:6231–6235. Leigh, J. A., and G. C. Walker. 1994. Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function. Trends Genet. 10:63–67. Levery, S. B., H. Zhan, C. C. Lee, J. A. Leigh, and S. Hakomori. 1991. Structural analysis of a second acidic exopolysaccharide of Rhizobium meliloti that can function in alfalfa root nodule invasion. Carbohydr. Res. 210: 339–347. Long, S. R. 2001. Genes and signals in the Rhizobium-legume symbiosis. Plant Physiol. 125:69–72. Marketon, M. M., and J. E. Gonza ´lez. 2002. Identification of two quorumsensing systems in Sinorhizobium meliloti. J. Bacteriol. 184:3466–3475. Marketon, M. M., M. R. Gronquist, A. Eberhard, and J. E. Gonza ´lez. 2002. Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones. J. Bacteriol. 184:5686–5695. Mendrygal, K. E., and J. E. Gonza ´lez. 2000. Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti. J. Bacteriol. 182: 599–606. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Nikaido, H. 1996. Multidrug efflux pumps of gram-negative bacteria. J. Bacteriol. 178:5853–5859. Paulsen, I., M. Brown, and R. Skurray. 1996. Proton-dependent multidrug efflux systems. Microbiol. Rev. 60:575–608. Pearson, J. P., E. C. Pesci, and B. H. Iglewski. 1997. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacteriol. 179:5756–5767. Pearson, J. P., C. Van Delden, and B. H. Iglewski. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181:1203–1210. Pellock, B. J., M. Teplitski, R. P. Boinay, W. D. Bauer, and G. C. Walker. 2002. A LuxR homolog controls production of symbiotically active extracellular polysaccharide II by Sinorhizobium meliloti. J. Bacteriol. 184:5067–5076. Pellock, B. J., H. P. Cheng, and G. C. Walker. 2000. Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. J. Bacteriol. 182:4310–4318.

EXOPOLYSACCHARIDE PRODUCTION IN S. MELILOTI

331

40. Puskas, A., E. P. Greenberg, S. Kaplan, and A. L. Schaefer. 1997. A quorumsensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides. J. Bacteriol. 179:7530–7537. 41. Reinhold, B. B., S. Y. Chan, T. L. Reuber, A. Marra, G. C. Walker, and V. N. Reinhold. 1994. Detailed structural characterization of succinoglycan, the major symbiotically important exopolysaccharide of Rhizobium meliloti Rm1021. J. Bacteriol. 176:1997–2002. 42. Reuber, T. L., and G. C. Walker. 1993. Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74: 269–280. 43. Ruberg, S., A. Pu ¨hler, and A. Becker. 1999. Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR. Microbiology 145:603–611. 44. Saier, M. H., Jr., R. Tam, A. Reizer, and J. Reizer. 1994. Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol. Microbiol. 11:841–847. 45. Summers, M. L., J. G. Elkins, B. A. Elliott, and T. R. McDermott. 1998. Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti. Mol. Plant-Microbe Interact. 11:1094–1101. 46. Urzainqui, A., and G. C. Walker. 1992. Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants. J. Bacteriol. 174:3403– 3406. 47. van Rhijn, P., and J. Vanderleyden. 1995. The Rhizobium-plant symbiosis. Microbiol. Rev. 59:124–142. 48. von Bodman, S. B., D. R. Majerczak, and D. L. Coplin. 1998. A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proc. Natl. Acad. Sci. USA 95:7687– 7692. 49. Zhan, H. J., C. C. Lee, and J. A. Leigh. 1991. Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations. J. Bacteriol. 173:7391–7394. 50. Zhan, H. J., S. B. Levery, C. C. Lee, and J. A. Leigh. 1989. A second exopolysaccharide of Rhizobium meliloti strain SU47 that can function in root nodule invasion. Proc. Natl. Acad. Sci. USA 86:3055–3059. 51. Zhang, Z., and L. S. Pierson III. 2001. A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens. Appl. Environ. Microbiol. 67:4305–4315.