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JOURNAL OF BACTERIOLOGY, Jan. 2011, p. 485–496 Vol. 193, No. 20021-9193/11/$12.00 doi:10.1128/JB.01129-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Complex Regulation of Symbiotic Functions Is Coordinated by MucRand Quorum Sensing in Sinorhizobium meliloti�†

Konrad Mueller and Juan E. Gonzalez*Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75080-0688

Received 22 September 2010/Accepted 27 October 2010

In Sinorhizobium meliloti, the production of exopolysaccharides such as succinoglycan and exopolysaccharideII (EPS II) enables the bacterium to invade root nodules on Medicago sativa and establish a nitrogen-fixingsymbiosis. While extensive research has focused on succinoglycan, less is known concerning the regulation ofEPS II or the mechanism by which it mediates entrance into the host plant. Previously, we reported that theExpR/Sin quorum-sensing system is required to produce the symbiotically active low-molecular-weight fractionof this exopolysaccharide. Here, we show that this system induces EPS II production by increasing expressionof the expG-expC operon, encoding both a transcriptional regulator (ExpG) and a glycosyl transferase (ExpC).ExpG derepresses EPS II production at the transcriptional level from MucR, a RosR homolog, while concur-rently elevating expression of expC, resulting in the synthesis of the low-molecular-weight form. While theExpR/Sin system abolishes the role of MucR on EPS II production, it preserves a multitude of other quorum-sensing-independent regulatory functions which promote the establishment of symbiosis. In planktonic S.meliloti, MucR properly coordinates a diverse set of bacterial behaviors by repressing a variety of genesintended for expression during symbiosis and enhancing the bacterial ability to induce root nodule formation.Quorum sensing precisely modulates the functions of MucR to take advantage of both the production ofsymbiotically active EPS II as well as the proper coordination of bacterial behavior required to promotesymbiosis.

Sinorhizobium meliloti is a Gram-negative soil bacteriumthat can establish a symbiotic relationship with Medicago sa-tiva, also known as alfalfa. As with most pathogenic and sym-biotic associations between prokaryotes and eukaryotes, thisprocess requires extensive coordination of bacterial gene ex-pression and function on a global scale for successful estab-lishment.

In the case of S. meliloti, the bacterium must first be recep-tive to the plant-produced chemoattractant luteolin. This phe-nolic compound is released by the alfalfa roots, drawing S.meliloti toward the rhizosphere while inducing the bacterialproduction of lipochitooligosaccharides (nod factors) (27, 32,44, 47). These, in turn, bind to receptors on the surface of rootcells, resulting in their rapid division and the formation ofnodules (39). S. meliloti subsequently invades these structuresby traveling through an infection thread, a plant-derived chan-nel extending along the length of a root hair, emptying into thenodule (19, 41). Invasion of the plant through the infectionthread is mediated by the production of symbiotically activeexopolysaccharides, such as succinoglycan and exopolysaccha-ride II (EPS II) (23, 25, 31, 42). In addition, recent work in ourlaboratory has shown that repression of motility is concertedwith this invasion process and is required for maximal effi-ciency in establishing symbiosis (26). Once inside, S. melilotidifferentiates into the bacteroid state, fixing free nitrogen for

the benefit of the plant while, in return, receiving a carbonsource in the form of dicarboxylic acids (33, 51).

S. meliloti regulates many of the functions required for as-sociating with M. sativa through the ExpR/Sin quorum-sensingsystem composed of the response regulator, ExpR, and theautoinducer synthase, SinI (21, 34). Constitutive sinI expres-sion results in the production of an exogenously released au-toinducer signal in the form of an N-acyl homoserine lactone,or AHL (35). The concentration of this signal acts as an indi-cator of population density for the bacteria. After sufficientautoinducer accumulation, the AHLs bind to ExpR, forming atranscriptional regulator with distinct functions (6, 26, 28).

Among the multitude of effects of quorum sensing on geneexpression, one is the progressive termination of motility inresponse to an increasing population density. Recently, wehave shown that maximal invasion efficiency of M. sativa by S.meliloti requires the repression of flagellar synthesis and isaccomplished by ExpR in the presence of AHLs (26). In ad-dition, the production of symbiotically active EPS II is inducedby this complex through the increased transcription of the expgene family spanning 32 kb. This gene cluster is composed ofthe operons expE (also called wge), expA (wga), and expD(wgd), as well as the expG-expC operon (wggR-wgcA) (8, 23).Expression of expE, expA, and expD is required for the struc-tural biosynthesis of EPS II, while expG-expC encodes both atranscriptional regulator, ExpG, and a glycosyl transferase,ExpC (4, 48). In the absence of quorum sensing, MucR, ahomolog of the Agrobacterium tumefaciens RosR, repressesEPS II biosynthesis through direct interactions with the pro-moter regions of expE, expA, and expD via its zinc finger DNA-binding domain (10, 36, 56). However, an intact ExpR/Sinquorum-sensing system significantly increases expression of the

* Corresponding author. Mailing address: RL11, Department ofMolecular and Cell Biology, University of Texas at Dallas, Richardson,TX 75080-0688. Phone: (972) 883-2526. Fax: (972) 883-2409. E-mail:[email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 5 November 2010.

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entire exp gene family, allowing for the production of EPS II inboth high-molecular-weight (HMW) and low-molecular-weight (LMW) fractions (24, 34). In S. meliloti, the EPS II-mediated capacity for biofilm formation, attachment, andinvasion of the host plant is provided specifically by the low-molecular-weight form (24, 46). Thus, this is considered thesymbiotically active fraction of this exopolysaccharide. While ithad been shown that HMW EPS II could be produced instrains lacking an intact quorum-sensing system under the con-dition of a disrupted mucR, it is clear that quorum sensing isrequired for the synthesis of symbiotically essential LMW EPSII (24, 34, 43, 56). Prior to this investigation, the mechanism bywhich the ExpR/Sin system resulted in the presence of thisfraction was unknown.

Historically, studies of S. meliloti have focused on strainRm1021, which lacks an intact quorum-sensing system due tothe presence of a disruption in expR (20). However, in additionto the ability to produce LMW EPS II, work with quorum-sensing-capable strains, such as Rm8530, continues to uncovera multitude of additional bacterial functions due to the abilityto detect quorum (23, 24, 26, 36, 46). As a result, Rm8530,carrying an intact quorum-sensing system, is commonly re-ferred to as the wild type, as it is in this article.

In order to ensure a successful symbiotic association, a va-riety of bacterial behaviors, from exopolysaccharide produc-tion and biofilm formation to motility and nitrogen fixation,are consistently and tightly regulated. Failure to adhere to anyof these complex regulatory patterns results in deficiencies ininvasion or the establishment of symbiosis (13, 23, 26, 31, 34,43, 45, 46, 53, 54). In this study, we originally intended toelucidate the mechanism by which the ExpR/Sin quorum-sens-ing system allows for symbiotically active LMW EPS II pro-duction. However, through our extended investigation into theinteractions of quorum sensing and MucR, an expansive reg-ulatory network connecting these bacterial functions is begin-ning to emerge.

We show that MucR mediates the repression of EPS IIproduction at extremely low population densities until theExpR/Sin system abolishes this effect at levels of quorum suf-ficient for biofilm formation and invasion of the host plant.Only the role of MucR in EPS II production is known to bespecifically negated with an increase in population. A variety ofother functions of MucR advantageous for the establishmentof symbiosis are maintained, including the enhanced produc-tion of succinoglycan as well as the repression of motility, bothpreviously reported (5, 9). Additionally, we describe the newlyuncovered role of MucR in increasing nod factor productionfor the development of nodules on the host plant (26, 32, 47).Furthermore, prior to symbiosis, MucR actively represses theunnecessary expression of a multitude of energy-expensivegenes intended for expression within the nodule, includingseveral involved in nitrogen fixation and respiration.

These data suggest the presence of a sophisticated circuitsimultaneously encompassing a wide variety of these behaviors,with MucR as a central factor. By allowing the ExpR/Sin sys-tem to modify specific roles of this transcriptional regulator, S.meliloti exploits the advantages of both quorum-sensing-de-pendent and -independent functions of MucR in order to en-sure the proper synchronization of gene expression and max-imal efficiency in establishing symbiosis.

MATERIALS AND METHODS

Bacterial strains and growth conditions. S. meliloti strains (Table 1) weregrown in Luria-Bertani (LB) broth or agar supplemented with 2.5 mM MgSO4,2.5 mM CaCl2 (MC), and appropriate antibiotics. For RNA isolation, 2 ml ofTYC broth (5 g of tryptone, 3 g of yeast extract, and 0.4 g of CaCl2 liter�1) withstreptomycin (500 �g ml�1) was inoculated with colonies grown on LB-MC agarplates and incubated for 48 h at 30°C with constant shaking. The strains werethen subcultured (1:100) in 20 ml of minimal glutamate mannitol (MGM) low-phosphate medium (50 mM morpholineethanesulfonic acid [MOPS], 19 mMsodium glutamate, 55 mM mannitol, 0.1 mM K2HPO4 � KH2PO4, 1 mM MgSO4,0.25 mM CaCl2, 0.004 mM biotin, pH 7) and grown at 30°C with constantshaking. The addition of luteolin to 10 �M was included when effects on nodoperon gene expression were to be analyzed. When necessary, chloramphenicol(20 �g ml�1), gentamicin (100 �g ml�1 for S. meliloti and 10 �g ml�1 forEscherichia coli), hygromycin (100 �g ml�1), neomycin (200 �g ml�1), tetracy-cline (10 �g ml�1), or trimethoprim (500 �g ml�1 for S. meliloti and 30 �g ml�1

for E. coli) was added.Construction of S. meliloti strains and plasmids. Oligonucleotide sequences

used in this study are listed in Table S1 in the supplemental material. Allmutations for this work were introduced into Rm8530 using the transducingphage �M12 as described previously (22). Expression of expC under a constitu-tively expressing promoter was accomplished by amplification of a fragment fromRm8530 carrying the open reading frame expC with primers expC�no-linker Fand expC�PstI R. Both pJN105 with the stability region from pTR101 (52) andthis amplicon were then digested with PstI and ligated together to form pJNexpC.This protocol was also followed for the mucR gene, only using primers

TABLE 1. Bacterial strains and plasmids used in this work

Strain orplasmid Relevant characteristicsa Reference or source

StrainsS. meliloti

Rm8530 Rm1021 expR� 43Rm11527 Rm8530 sinI::Kmr 34Rm11002 Rm8530 expA::Nmr 24Rm9020 Rm8530 exoY::OTCr 24Rm11603 Rm8530 expC::Hyr This workRm11604 Rm8530 mucR::Gmr 24Rm11605 Rm8530 expG::Nmr This workRm11606 Rm11604 expG::Nmr This workRm11607 Rm9020 mucR::Gmr This workRm11608 Rm11607 expG::Nmr This workRm11609 Rm9020 expA::Nmr This workRm11610 Rm11609 mucR::Gmr This work

E. coliDH5� F� �80dlacZ�M15 �(lacZYA-

argF)U169 recA1 endA1hsdR17(rK

� mK�) supE44

thi-1 gyrA relA1

Life Technologies

PlasmidspRK600 pRK2013 (npt::Tn9); Cmr 16pJN105 araC-PBAD cassette cloned in

pBBR1MCS-540

pTR101 RK2 plasmid containing thestability region

52

pJN105str pJN105 containing the stabilityregion from pTR101

Gonzalezlaboratory

pJNexpC pJN105str containing the expCgene

This work

pJNmucR pJN105str containing themucR gene

This work

pJTpexpC pJNexpC containing theDHFR cassette; Tpr

This work

pJTpmucR pJNmucR containing theDHFR cassette; Tpr

This work

a Kmr, kanamycin resistance; Nmr, neomycin resistance; OTCr, oxytetracyclineresistance; Hyr, hygromycin resistance; Gmr, gentamicin resistance; Cmr, chlor-amphenicol resistance; Tpr, trimethoprim resistance; Tcr, tetracycline resistance.

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mucR�SmaI F and mucR�SmaI R for sequence amplification and the SmaIendonuclease for digestion of both insert and plasmid, resulting in pJNmucR.Due to the redundant presence of gentamicin resistance in the pJN105 plasmidas well as in several strains used in this work, pJNexpC and pJNmucR weremodified with an EZ-Tn5 �DHFR-1� Insertion Kit from Epicentre to providetrimethoprim resistance instead. All plasmids were introduced into S. meliloti bytriparental mating. A list of plasmids used in this work is provided in Table 1.

Bacterial RNA isolation. Cultures were grown at 30°C to optical densities at600 nm (OD600) of 0.02, 0.1, and 1.2 in either 500 ml or 20 ml of MGMlow-phosphate medium. Larger volumes were required for bacteria of low pop-ulation densities in order to obtain sufficient RNA for analysis. Bacteria werecollected by centrifugation (17,000 g for 10 min at 4°C for 500-ml liquidcultures or 14,000 g for 2 min at 4°C for 20-ml liquid cultures), and the pelletswere frozen in liquid nitrogen. Total RNA was purified by using an RNeasy MiniKit (Qiagen) as described in Gurich et al. (26). RNA integrity was determined inan Agilent 2100 Bioanalyzer, and the concentrations were measured with Nano-drop spectrophotometer ND-1000.

Quantitative real-time PCR. In order to quantify the presence of specificmRNA transcripts of interest, 1 �g of RNA isolated from bacteria grown inculture was used per Ambion RETROscript reverse-transcription reaction. Onemicroliter of resultant cDNA was used as a template for quantitative real-timePCR analysis. Each reaction mixture was prepared as described previously (26)and included appropriate primers for amplification of the desired mRNA (seeTable S1 in the supplemental material). Expression of SMc00128 was used as aninternal control for normalization (30). Fold change in expression between strainA versus strain B was calculated using observed threshold cycle (CT) values ofeach (CTa and CTb, respectively) and the following equation: fold change 2CTb � CTa. Expression levels of expE2, expA1, and expD1 are presented in thiswork as representations of transcriptional levels of operons since expression ofopen reading frames within each operon provided similar values (data notshown).

Microarray experiments and data analyses. Ten micrograms of total RNAfrom wild-type and mucR-mutant strains of S. meliloti grown to an OD600 of 1.2was used for analysis by microarray to evaluate gene expression levels. Thesynthesis of cDNA, labeling, and hybridizations to the S. meliloti/Medicago trun-catula Affymetrix GeneChip (Santa Clara, CA) were performed by the CoreMicroarray Facility at UT Southwestern Medical Centre (Dallas, TX). Data wereprocessed using GeneSifter as described by Gurich et al. (26) and uploaded tothe NCBI Gene Expression Omnibus (GEO) database. Genes were consideredto be differentially expressed if the fold change in expression was �1.5 with a Pvalue � 0.05. In order to observe potential metabolic differences between thewild type and mucR, the complete list of genes differentially expressed greaterthan 1.5-fold was further analyzed by Kegg Array, version 1.2.3.

EPS II isolation. Succinoglycan mutants (exoY) of S. meliloti grown on LB-MCagar plates were used to inoculate 5 ml of TYC broth with 500 �g ml�1 strep-tomycin. These were incubated for 48 h with constant shaking at 30°C. TYCcultures were centrifuged and resuspended in 1 liter of mannitol glutamate salts(MGS) medium used for S. meliloti EPS II production (37). MGS cultures weregrown for 7 days at 30°C with constant shaking, and EPS II released into themedium was extracted and purified as previously described (24). Cultures werecentrifuged, and supernatants were lyophilized. Samples were resuspended in 10ml of H2O and precipitated with three volumes of ethanol. This precipitate wasresuspended in the smallest volume of H2O possible and dialyzed for 4 days at4°C using a membrane with a 500-Da molecular mass cutoff. Samples were thenlyophilized and resuspended in H2O prior to analysis by high-performance an-ion-exchange chromatography (HPAEC).

High-performance anion-exchange chromatography. Analysis of the EPS IIoligosaccharides was performed by HPAEC on a Dionex metal-free BioLC witha CarboPac PA10 (anion-exchange) column (4 by 3 by 250 mm) using a pulsedamperometric detector reporting in nanocolumns (nC) with a gold workingelectrode and a triple-step carbohydrate waveform (Dionex), as described pre-viously (24). Eluent A consisted of 100 mM NaOH while eluent B consisted of1 M sodium acetate in 100 mM NaOH. Consistent flow of eluent A was main-tained with a gradient of eluent B as follows: 0 min, 10% eluent B; 3 min, 10%eluent B; 10 min, 20% eluent B; 25 min, 100% eluent B; 50 min, 100% eluent B;55 min, 10% eluent B; and 60 min, 10% eluent B. The flow rate was 1 ml min�1

at room temperature. Reconstitution of the column by passing 100% 300 mMNaOH for 30 min between samples was critical for maintaining the ability of thecolumn to separate fractions of EPS II.

Biofilm formation assay-microtiter plate method. The ability to form biofilmwas determined by a modified protocol described in Rinaudi and Gonzalez (46).Cultures were grown in low-phosphate MGM medium as described above, di-luted to an OD600 of 1.0, and inoculated into microtiter wells in 100-�l aliquots

in at least triplicate. A sterile microporous film was then placed over the entiretyof the plate (AeraSeal catalogue number BS-25), and the cover was added toprevent evaporation. The plate was then inverted to prevent the settling ofbacteria from interfering with true attachment to the walls of the well due tobiofilm formation and then incubated with gentle rocking for 48 h at 30°C and65% relative humidity. Free liquid culture was gently removed from the wells,and each well was air dried and stained for 15 min with 150 �l of 0.1% crystalviolet dye. Each well was then rinsed three times with water, and crystal violetwas resuspended with 150 �l ethanol. The absorbance of the solubilized dye atOD560 was measured using a microplate reader (Infinite M200; Tecan TradingAG, Mannedorf, Switzerland) as a measure of biofilm formation.

Plant nodulation assays. Plant nodulation and invasion assays with the sym-biotic host M. sativa were carried out in triplicate sets of 15 plants per strain perexperiment. Five-milliliter cultures of S. meliloti were grown in LB-MC brothwith 500 �g ml�1 streptomycin at 30°C for 48 h. Cultures were washed four timeswith sterile water, and 1 ml of a 1:100 dilution was used to inoculate 3-day-oldplant seedlings as described previously (26). Plates were incubated at 22°C with60% relative humidity and a 16-h light cycle. Plants were examined daily orweekly, depending on the observation of nodule development or successfulinvasion, respectively. To determine the capacity of a strain to produce symbi-otically active LMW EPS II, inoculated alfalfa plants were examined after 4weeks for the presence of pink nodules, indicative of the successful establishmentof symbiosis.

RESULTS

The ExpR/Sin quorum-sensing system of S. meliloti is criticalfor the production of both HMW and symbiotically activeLMW EPS II through the induced expression of the exp genefamily, composed of the operons expE, expA, expD, and expG-expC (23, 25, 42). Previous work in our laboratory has shownthat the absence of an intact quorum-sensing system, either bya disruption of expR or sinI, results in the termination of EPSII production due to repression by MucR (4, 24, 34). Anintermediate level of EPS II biosynthesis can be restored inthese strains through a mutation of the mucR gene. However,no symbiotically active LMW EPS II is produced in this man-ner (24). This suggests the presence of an independent mech-anism by which quorum sensing permits formation of the low-molecular-weight fraction of EPS II outside the derepressionby MucR. It has been shown that expression of expG results inthe transcription of several genes involved in EPS II produc-tion (3, 4, 48). However, the mechanism by which this permitsthe production of LMW EPS II in the presence of quorumsensing has not been reported.

To examine the manner in which the ExpR/Sin system in-duces the biosynthesis of the low-molecular-weight fraction ofEPS II, the effects of disrupting mucR and expG on all mem-bers of the exp gene family (operons expE, expA, expD, andexpG-expC) were measured (Fig. 1). S. meliloti cells weregrown to stationary phase in minimal glutamate mannitol(MGM) low-phosphate medium in order to facilitate maximalgene expression related to EPS II production (37, 55). RNAwas extracted from each strain, and expression levels of the expgene family were measured by quantitative real-time PCR. Asexpected, compared to a quorum-sensing-deficient sinI mu-tant, wild-type S. meliloti showed significantly induced expres-sion of all exp genes. In the presence of an intact ExpR/Sinsystem, disruption of mucR resulted in negligible changes ingene expression. However, disruption of expG negated induc-tion of the exp gene family. In the mucR expG mutant, fullexpression of expE, expA, and expD was restored while tran-scription of expC remained hindered by a polar effect of theupstream transposon insertion in expG.

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Measurements in gene expression correlated with changes incolony morphology when grown on LB-MC agar plates (Fig.2). Wild-type S. meliloti appeared highly mucoid with auniquely watery consistency, the distinct visual indicators ofthe presence of LMW EPS II (24). As expected, the mucRmutation induced no noticeable change in appearance fromthe wild type while disruption of expG abolished EPS II pro-duction, resulting in a dry-colony phenotype. Introduction ofan additional disruption to mucR in the expG mutant restoredan intermediate level of EPS II production although the lowvolume and highly viscous consistency of the exopolysaccha-ride were characteristic of only HMW EPS II (24). Given thatthe only genetic differences between this and the mucR mutantwere the disruption of expG and the decrease in transcriptionof downstream expC, the cause of the apparent difference inmucoid phenotype was investigated through complementationof the expG mutation as well as supplementation of expressionlevels of expC in trans. Complementation of expG in the mucRexpG mutant resulted in no change (data not shown). However,introduction of pJTpexpC, a vector carrying expC under a con-stitutively expressing promoter, supplemented the low tran-scriptional levels of expC to approximately those measured inwild-type (Fig. 2) and restored the highly mucoid colony mor-phology associated with LMW EPS II production. Full resto-ration of expC expression increased overall EPS II productionroughly 20% compared to the mucR expG mutant according toanthrone assays (data not shown) (38). Because this increase intotal exopolysaccharide production may have contributed tothe highly mucoid phenotype in addition to the production ofthe watery LMW form, the molecular weight content of EPS IIproduced by these strains was verified by chromatographic andbiological assays.

Expression of expC is critical for the production of LMWEPS II. The molecular-weight content of the EPS II producedby the various mutants of S. meliloti was confirmed by threeindependent means of detecting the low-molecular-weightform specifically, regardless of overall EPS II production: high-

performance anion-exchange chromatography (HPAEC), thecapacity of the mutants to form biofilm, and the ability of eachto invade M. sativa in the absence of succinoglycan. Work inour laboratory has shown that EPS II, when separated byHPAEC and detected by pulsed amperometry, produces dis-cernible peaks indicative of each fraction. LMW EPS II isidentified by repeated peaks from 24 to 27 min of chromatog-raphy, while HMW EPS II produces a broad mound at 28 min(24). As expected, analysis of EPS II produced by wild-type S.meliloti grown in mannitol glutamate salts (MGS) indicated thepresence of both HMW and LMW fractions (Fig. 3A) (24).Evaluation of the exopolysaccharide produced by the mucRexpG mutant indicated the presence of HMW EPS II exclu-sively. Supplementation of expC expression levels with the in-troduction of pJTpexpC into this strain restored the productionof LMW EPS II. The mucR mutant produced a pattern iden-tical to that of the wild-type strain, as observed previously (24),while the expG mutant synthesized no EPS II for analysis.

Recently, studies have shown that the production of LMWEPS II results in biofilm formation and attachment at levels ashigh as 10-fold greater than those produced by strains of S.meliloti incapable of synthesizing this exopolysaccharide (18,46). Thus, the ability of the bacteria to establish levels ofbiofilm comparable to wild-type levels was utilized as a sec-ondary indicator of the presence or absence of the low-molec-ular-weight form. S. meliloti cells were inoculated in MGMlow-phosphate medium and grown in 96-well microtiter dishes.Attachment to the walls of the well due to biofilm formationwas measured by crystal violet staining. The deduced presenceof LMW EPS II, as observed by the capacity of each strain toform biofilm, coincided with the fractions detected by HPAEC(Fig. 3B). Wild-type levels of attachment were abolished in themucR expG mutant while supplementation of expC expressionlevels restored full biofilm formation.

Finally, invasion of M. sativa by S. meliloti is mediated eitherby succinoglycan (encoded by the exo gene family) or the LMWfraction of EPS II (24, 42). The ability of a strain to establish

FIG. 1. Expression of expC is not restored by disruption of mucR. Extracted RNA from various strains of S. meliloti grown in MGMlow-phosphate medium to an OD600 of 1.2 was analyzed by quantitative real-time PCR and compared to expression observed in a sinIquorum-sensing-deficient mutant. Disruption of expG resulted in expression levels of the exp gene family similar to those observed in an sinI mutantincapable of quorum sensing. Introduction of a disrupted mucR in the expG mutant restored expression of all members of the exp gene family withthe exception of expG and the downstream expC. WT, wild type.



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symbiosis after its capacity to produce succinoglycan has beendisrupted is a clear indicator of the production of the low-molecular-weight form, regardless of overall levels of EPS IIproduced (24). Thus, an exoY mutation was introduced into

each strain of S. meliloti, and the invasive capacities of thestrains were examined. Disruption of exoY in the wild typeabolished succinoglycan production but did not prevent thestrain from establishing a successful symbiosis with the hostplant due to the continued biosynthesis of symbiotically activeLMW EPS II. The mucR expG mutant lost its invasive abilityupon mutation of exoY (Fig. 3C) as it failed to produce thesymbiotically active fraction of EPS II. The introduction ofpJTpexpC into this mucR expG exoY mutant restored the abilityto invade the plant through the reestablished production ofLMW EPS II. These data confirm that expression levels ofexpC equivalent to those induced by quorum sensing are crit-ical for the production of symbiotically active EPS II.

MucR represses exp gene expression prior to the establish-ment of quorum. Although disruption of mucR in the absenceof an intact quorum-sensing system had been shown to dra-matically derepress several exp operons (48), in wild-type S.meliloti, no significant effect on EPS II-related gene expressionhad been previously reported at any stage of growth. Even atwhat had been considered to be “low population density” priorto the establishment of quorum (OD600 of 0.2), the ExpR/Sinsystem appeared to completely abolish transcriptional repres-sion by MucR (26). The purpose of the capacity of MucR torepress specific exp genes in S. meliloti was unclear, given thatits utilization had never been reported without additional mu-tations in quorum sensing. However, changes in exp gene ex-pression as a result of a disrupted mucR had not been previ-ously observed below the described low population density.

In order to examine the role of MucR at these earlier pointsin growth, exp transcriptional activity was measured by collect-ing RNA from MGM low-phosphate cultures grown to OD600

of 0.02, 0.1, and 1.2, followed by quantitative real-time PCR.At the lowest stage of growth (OD600 of 0.02), overall expres-sion of the exp gene family in wild-type S. meliloti was similarto that of a sinI mutant incapable of quorum sensing (Fig. 4A).To confirm that these transcriptional levels were specific to theexp operons and not artifacts of overall low gene expression,transcript levels of rem, previously reported to be high at lowpopulation density (26), were measured (Fig. 4B). By an OD600

of 0.1, full expression of the exp family similar to that observedat an OD600 of 1.2 had been restored, suggesting that thequorum required for EPS II production was achieved at somepoint between OD600 of 0.02 and 0.1. Disruption of mucR inthe population grown to an OD600 of 0.02 resulted in thederepression of operons expE, expA, and expD, responsible forthe biosynthesis of EPS II, 4.5-, 2.5-, and 2.4-fold, respectively(Fig. 4C). By an OD600 of 0.1, this mutation had no significanteffect on expression of these genes. These data suggest that therepressive effects of MucR on EPS II biosynthesis are activeonly at extremely low population densities previously unexam-ined. The quorum required for EPS II production is achievedat a particularly low stage of growth between OD600 of 0.02and 0.1, at which point repression by MucR is abolished.

MucR induces bacterial sensitivity to plant flavonoids, re-sulting in increased plant nodulation. At sufficient populationdensities, quorum sensing in S. meliloti abrogates transcrip-tional regulation by MucR on 22 open reading frames acrossexpE, expA, and expD operons without repressing the expres-sion of mucR (26). The complex manner by which S. melilotiutilizes the ExpR/Sin system to negate the repressive effects of

FIG. 2. Expression of expC is critical for the wild-type (WT) mu-coid phenotype. Mutant strains of S. meliloti streaked on LB-MC agarplates produced phenotypes correlating with gene expression of the expgene family measured by real-time PCR analysis. Disruption of expGprevented the production of EPS II, resulting in dry colonies. ThemucR expG mutant appeared mucoid although the viscosity and vol-ume of the exopolysaccharide were indicative of the absence of thelow-molecular-weight fraction. Restoration of expC expression (viapJTpexpC) to wild-type levels restored the fully mucoid phenotypecharacteristic of strains producing HMW and LMW EPS II.



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MucR at each specific binding site upstream of the exp operons(4, 34), rather than simply repress mucR at the transcriptionallevel, suggested the possibility that the maintained expressionof mucR provided the bacterium with significant advantages. Inaddition, the ability of MucR to induce the production ofsuccinoglycan (9) and repress motility (5) demonstrated itsdiverse regulatory potentials. Through microarray analysis de-scribed later in this article, a variety of alternative effects ofMucR were discovered, including a 1.7-fold increase in expres-sion of nodD, encoding the transcriptional activator of thedownstream nod genes required for the synthesis of bacterialnod factors and development of root nodules (17, 32) (seeTable S2 in the supplemental material). To determine if thismodest increase in expression of nodD had a measurable effecton the biosynthesis of nod factor, expression of the nodABCoperon responsible for its production was measured usingquantitative real-time PCR in wild-type S. meliloti as well as amucR mutant (Fig. 5). In the absence of the plant flavonoid(luteolin) required for full induction of the nod operon byNodD (32), only a slight decrease in expression of nodABC wasobserved with the disruption of mucR. However, in the pres-ence of 10 �M luteolin, the handicap in the transcriptionalinduction of nodABC appeared to be roughly 10-fold in theabsence of mucR. Complementation of mucR with thepJTpmucR vector restored this expression.

To confirm whether these changes in nod gene expressionresulted in measurable differences in the ability to induce nod-ule development, 3-day-old alfalfa seedlings were inoculatedwith S. meliloti carrying either an intact or disrupted mucRgene. The resulting numbers of developing nodules were re-corded (Fig. 6A). The prevention of successful invasion wascritical in order to avert the potential for established symbiosesinterfering with continued nodule organogenesis. Further-more, this isolated any potential effects of the mucR disruptionon invasion from nod factor production. In order to accom-

plish this, mutations in expA and exoY were introduced into allstrains to abolish the production of EPS II and succinoglycan,respectively. This permitted the observation of differences innodule development independent of plant invasion. Beginningwith the first observation of nodules 7 days after inoculation,the disruption of mucR resulted in a delay in nodule develop-ment persisting throughout the course of the experiment.Complementation of mucR recovered this deficiency. A com-parison of the number of nodules produced by each strain tothat of the mucR mutant indicates an initial 7-fold advantageprovided by carrying an intact copy of mucR, attenuating toroughly 1.3-fold after 30 days (Fig. 6B).

Disruption of mucR affects the transcription of a multitudeof genes necessary for symbiosis. The diverse effects of mucRin S. meliloti observed throughout this work warranted a morecomplete examination of any possible as yet undiscovered ad-ditional roles. In order to identify these potential functions,RNA was extracted from wild-type and mucR mutant strains ofS. meliloti and analyzed by microarray utilizing AffymetrixGeneChips (see Table S2 in the supplemental material). Intotal, 802 genes were differentially expressed by at least 2-fold.Of the 154 genes which showed dramatic changes in transcrip-tion (10-fold or greater), 138 of these were both induced in theabsence of mucR and located on symbiotic megaplasmid,pSymA.

The disruption of mucR resulted in the overexpression of 19nitrogen fixation and respiration genes by 8- to 1,017-fold, allof which had been previously shown to be induced in bacte-roids within the nodule after the establishment of symbiosis(Table 2) (7, 11, 14, 15, 26). Additionally, 14 motility andchemotaxis-related genes increased in expression 1.8- to 11-fold, an effect similarly observed in a quorum-sensing-deficientstrain of S. meliloti by Bahlawane et al. (5). In order for bac-teria to organize into sedentary biofilm communities, repres-sion of motility is crucial. While Gurich et al. have shown that

FIG. 3. Restored expression of expC results in the biosynthesis of LMW EPS II. (A) Purified EPS II produced from wild-type (WT) and mutantstrains was analyzed by HPAEC for the presence of LMW EPS II. Analysis of exopolysaccharide produced by the wild-type S. meliloti confirmedthe presence of LMW EPS II based upon distinctive peaks between 24 and 27 min. The mucR expG mutant failed to produce LMW EPS II.Supplementation (by introduction of pJTpexpC) of transcriptional levels of expC in this mutant strain restored the synthesis of the low-molecular-weight fraction. Results were overlaid for comparison. (B) The presence of LMW EPS II produced expected levels of biofilm formation andattachment. The mucR expG mutant strain failed to form biofilm while the strain carrying pJTpexpC attached at equivalent levels to the level ofthe wild type. (C) In the absence of succinoglycan production, the result of a disruption in exoY, the ability of S. meliloti to invade the host plantand establish a nitrogen-fixing symbiosis, visually discernible by the development of large pink nodules, is a clear indicator of the production ofLMW EPS II. The disruption of exoY in the mucR expG mutant abolished the capacity of the strain to invade the plant. Supplementation ofexpression levels of expC in this strain through the introduction of pJTpexpC resulted in an invasive capacity comparable to that of the exoY strain,confirming the synthesis of LMW EPS II as a result of restored expression of expC.



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this is predominantly accomplished in S. meliloti through re-pression by AHL-bound ExpR at high population density (26),these data suggest that MucR may also contribute to the mit-igation of motility.

In addition, expression of 11 open reading frames of the virBoperon involved in type IV secretion systems increased 15- to605-fold, and four genes encoding transport-related proteinswere induced 6- to 55-fold (see Table S2 in the supplementalmaterial). Seven of these open reading frames related to trans-port or secretion were also observed to have increased expres-sion within the nodule (Table 2). Differentially expressed ni-trogen fixation, motility, type IV secretion, and transport genesdescribed here were confirmed by quantitative real-time PCR(data not shown). These data show a multitude of downstreameffects from a disrupted mucR across a variety of bacterialfunctions. Furthermore, the derepression of numerous genesthat are also known to be induced during symbiosis indicates a

possible broad role of mucR as an inhibitor of bacteroid geneexpression in planktonic S. meliloti.

For a more global understanding of the extensive microarraydata, the entire list of genes differentially expressed more than1.5-fold was analyzed using a Kegg Array, presenting all theknown metabolic pathways affected. Compared to the wildtype, the mucR mutation impacted a diverse set of genes in-volved in numerous metabolic processes (see Fig. S1 in thesupplemental material). While the consequences of each areunclear, similar results were obtained when S. meliloti wasanalyzed in the bacteroid state during symbiosis to the inde-pendent planktonic form (26) (see Fig. S2). Few of the path-ways affected by the mucR disruption were not also observed asdifferentially expressed in the same direction after the estab-lishment of symbiosis (see Fig. S3). This, in addition to theobserved repression of gene expression related to nitrogenfixation, suggests that a significant portion of the role of MucR

FIG. 4. MucR represses the exp gene family prior to quorum. (A) Wild-type S. meliloti was grown in MGM low-phosphate medium to OD600of 0.02, 0.1, and 1.2. Analysis by real-time PCR and comparison to the sinI mutant indicated that expression levels of the exp gene family, inparticular, expE, expA, and expD directly responsible for EPS II biosynthesis, were first induced by quorum at a population density between anOD600 of 0.02 and an OD600 of 1.2. (B) Expression of rem confirmed that low expression levels observed at an OD600 of 0.02 were particular tothe exp gene family, not a by-product of low global gene expression at this stage of growth. (C) Disruption of mucR resulted in derepression ofexpE, expA, and expD operons at an OD600 of 0.02. However, the repressive effect of an intact mucR attenuated to negligible levels by an OD600of 0.1, suggesting that MucR facilitates the inhibition of EPS II production at a low population density until quorum is reached. At this point, theExpR/Sin system derepresses the exp gene family. Expression levels of expG and expC are not shown as this operon is not under the transcriptionalregulation of MucR.



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may involve repression of genes in planktonic S. meliloti thatare intended for maximal transcription only after symbiosis.

DISCUSSION

In order to establish a symbiotic association with M. sativa, S.meliloti simultaneously coordinates a multitude of activities,from nod factor and exopolysaccharide production prior to

invasion to nitrogen fixation and respiration during symbiosis.This study began as an investigation of the production of sym-biotically active LMW EPS II but evolved into a global exam-ination of networks connecting and governing this wide arrayof bacterial behaviors sharing quorum sensing and mucR ascentral factors.

EPS II production in response to population density. Wild-type S. meliloti produces two exopolysaccharides which allowfor host-plant invasion: succinoglycan and EPS II. While suc-cinoglycan appears to act as a signal, inducing appropriateresponses in the host plant (29), the method by which EPS IIfunctions is more elusive. Recent work by Rinaudi and Gonza-lez shows that EPS II production, specifically the synthesis ofthe low-molecular-weight fraction, results in organized biofilmformation and attachment at dramatically greater levels thanthose observed in the absence of this exopolysaccharide (46).In bacteria, biofilms permit the utilization of high populationdensities for the development of complex social organizations,providing various advantages, from antibiotic resistance to thechanneling of nutrients (1, 2, 12). The development of thesestructures in S. meliloti is coincident with the invasive abilitymediated by LMW EPS II. In wild-type S. meliloti, productionof all fractions of EPS II requires an active ExpR/Sin quorum-sensing system, paralleling the population-dependent nature ofbiofilms. In accordance with our real-time PCR expressiondata as well as previous observations (36) (Fig. 1), induction ofexpE, expA, and expD required for EPS II production is down-stream of increased expression of expG-expC by the ExpR/Sinquorum-sensing system. The transcriptional regulator encodedby expG derepresses these three operons from MucR (4), whileevidence presented in the present study shows that expressionof expC encodes a glycosyl transferase required for the pro-duction of the symbiotically active low-molecular-weight form

FIG. 5. MucR increases nod operon gene expression in response toluteolin. In the absence of induction by luteolin, a disruption in mucRresulted in a negligible change in nodA expression. Addition of luteolinresulted in an approximately 90-fold induction of the nod operon whiledisruption of mucR attenuated this effect to roughly 10-fold. Comple-mentation of mucR with pJTpmucR restored this expression. Expres-sion of the first open reading frame (nodA) of the nod operon is shownhere, as similar transcriptional levels were observed for downstreamnodB and nodC (data not shown).

FIG. 6. The presence of an intact mucR provides an advantage for nodule induction in M. sativa. (A) The ability of wild-type and mutant strainsof S. meliloti to develop nodules was examined daily after inoculation of bacteria onto seedlings. Disruption of mucR in strains incapable of invadingresulted in a decrease in the ability to induce the formation of nodules on the host plant. Complementation of mucR restored this ability towild-type levels. All strains were disrupted for expA and exoY in order to prevent invasion of the plant and establishment of symbiosis frominterfering with nodule development as an indicator of nod factor production. (B) Direct comparison of the ability of strains to produce nodules(panel A) between those carrying either an intact or disrupted mucR indicates an initial 7-fold advantage in nodule induction associated with thepresence of an intact mucR gene sequence. This advantage attenuates to roughly 1.3-fold by day 30.



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of EPS II. Mutant strains incapable of LMW EPS II synthesiscan regain this capacity by transcriptional supplementation ofexpC expression in trans to wild-type levels (Fig. 3). The directmechanism by which increased levels of expC results in de-creased molecular weights of EPS II is unclear. However, di-rect disruption of expC results in the complete termination ofEPS II production (23) while basal levels permit only thehigh-molecular-weight form (Fig. 3). This suggests the possi-bility that ExpC is required for initiation of EPS II biosynthesis

while factors encoded by expE, expA, and expD are responsiblefor the actual polymerization. Increased levels of ExpC withmaintained expression of expE, expA, and expD, as demon-strated in this work, would result in a greater number of ex-opolysaccharide chains of lower molecular weight, distributedamong more points of initiation. By tethering expression ofboth expG and expC to the same transcript, S. meliloti ensuresthat the quorum-sensing activation of EPS II biosynthesisthrough ExpG occurs alongside induced expression levels ofthe ExpC glycosyl transferase, allowing for the production ofthe low-molecular-weight fraction. This linkage results in thesynchronization of overall EPS II production with the synthesisof its symbiotically active form. These data suggest that theprimary utility of this exopolysaccharide is achieved throughfunctions of the low-molecular-weight fraction. Furthermore,the production of LMW EPS II is concurrent with the popu-lation density-dependent abolishment of motility, a factorknown to conflict with biofilm formation and recently shown tointerfere with plant invasion (26). The fact that the synthesis ofthe necessary exopolysaccharides for biofilm formation is co-incident with the termination of interfering behaviors, such asmotility, suggests that bacterial aggregation and attachmentmay be primary functions of EPS II.

The ExpR/Sin quorum-sensing system in S. meliloti dere-presses EPS II production across a broad range of populationdensities (26). However, at early stages of growth previouslyunexamined (OD600 of 0.02), overall expression of the exp genefamily closely paralleled that observed in sinI mutants incapa-ble of detecting quorum (Fig. 4A). The disruption of mucR inthese cultures restored expression to roughly 50% of full in-duction, suggesting a role of MucR in tightening the repressionof EPS II production at inadequate population densities for itsutilization (Fig. 4C). The impact of the mucR mutation atten-uates significantly at high population densities due to the dom-inance of activation by the ExpR/Sin quorum-sensing system.The observation that MucR actively represses EPS II produc-tion at extremely low stages of growth supports the argumentthat the major functions of this exopolysaccharide, potentiallybacterial organization and attachment coincident with plantinvasion, require sufficient population densities. Furthermore,these data suggest that quorum may be achieved for certainregulatory circuits at earlier stages of growth than previouslythought. Bacterial functions, such as EPS II production, havebeen acknowledged as dependent on the presence of an intactquorum-sensing system but independent of population density(26). However, a reexamination of the definition of low pop-ulation density has shown this to be imprecise. At the OD600 of0.02 studied in this work, the quantification of viable bacteriaby serial dilutions and plating indicates roughly 107 bacteriaper ml of liquid culture, suggesting an average of approxi-mately 50 �m between each bacterium. While this populationdensity appears insufficient for quorum in relation to EPS IIproduction, it is not unfeasible that other functions may acti-vate at yet lower thresholds. Caution must be taken in definingregulation as population density independent as dilute culturesunder laboratory conditions may still be too populous.

Quorum-sensing-independent functions of MucR. Theglobal examination of potential roles of MucR completed inthis study suggests a variety of functions beneficially affectingthe establishment of symbiosis. One such example is the ob-

TABLE 2. Microarray of mucR mutant versus the wild type andbacteroid versus planktonic

Function and locus Gene ID Gene name

Relative expression(fold change)b

mucRvs WT

Bacteroidvs

planktonic

Nitrogen fixation/respiration

NT01SMA0591 SMa0760 fixT2 10.2 2.2NT01SMA0592 SMa0762 fixK2 23.3 74.8NT01SMA0593 SMa0763 fixM 33.1 115.6NT01SMA0594 SMa0765 fixN2 20.1 23.6NT01SMA0595 SMa0766 fixO2 21.0 16.6NT01SMA0596 SMa0767 fixQ2 36.4 59.3NT01SMA0598 SMa0769 fixP2 30.7 42.5NT01SMA0599 NAa fixI 12.9 26.7NT01SMA0768 SMa0956 hemL 1,017.1 26.6NT01SMA0945 SMa1186 nosL 8.2 3.8NT01SMA0958 SMa1208 fixS1 127.0 8.6NT01SMA0959 SMa1209 fixI1 60.3 4.8NT01SMA0960 SMa1210 fixH 29.1 2.4NT01SMA0962 SMa1213 fixP1 37.8 5.7NT01SMA0964 SMa1216 fixO1 18.5 3.8NT01SMA0965 SMa1220 fixN1 12.7 8.2NT01SMA0966 SMa1223 fixM 12.4 24.2NT01SMA0967 SMa1225 fixK1 16.7 14.2NT01SMA0968 SMa1226 fixT1 13.6 3.1

Transport/secretionNT01SM1492 SMc02661 ABC

transporter55.5 9.8

NT01SMA1019 SMa1302 virB11 33.6 1.4NT01SMA1021 SMa1306 virB9 605.3 26.8NT01SMA1022 SMa1308 virB8 67.2 1.4NT01SMA1023 SMa1310 virB7 366.7 7.9NT01SMA1024 SMa1311 virB6 80.2 2.3NT01SMA1032 SMa1321 virB1 82.4 3.5

Motility/chemotaxisNT01SM0878 SMc03004 icpA 3.8NT01SM0879 SMc03005 cheX 2.2NT01SM0881 SMc03007 cheA 1.8NT01SM0883 SMc03009 cheR 2.1NT01SM0890 SMc03015 visN 2.4NT01SM0891 SMc03016 visR 2.4NT01SM0908 SMc03028 flgC 11.2NT01SM0913 SMc03032 flgI 3.8NT01SM0917 SMc03035 fliL 2.1NT01SM0925 SMc03040 fljL 2.1NT01SM0928 SMc03043 motC 5.0NT01SM0929 SMc03044 fliK 3.4NT01SM0932 SMc03047 flgE 4.6NT01SM0934 SMc03048 flgK 3.3

a NA, not available.b All values are positive in either the mucR mutant or bacteroid compared to

planktonic wild type (WT).



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servation that the presence of an intact mucR gene sequenceresults in a modest decrease in the expression of 14 motility-related genes, as similarly observed in a quorum-sensing-defi-cient strain (5). Gurich and Gonzalez showed that the presenceof flagella interferes with the invasion of nodules and thatquorum sensing at a high population density abolishes motilityat the transcriptional level (26). These data suggest two mech-anisms by which S. meliloti regulates this behavior: mitigationby MucR independent of growth, followed by complete termi-nation by quorum sensing. In addition, transcription of the exooperon is increased, resulting in the previously reported induc-tion of succinoglycan production (see Table S2 in the supple-mental material) (9).

The potential for successful symbiosis is further enhanced byMucR through an increase in the biosynthesis of nod factor forthe induction of nodule formation (Fig. 6). Expression of thenodABC operon responsible for the production of bacterialnod factor requires induction by the NodD transcriptionalregulator in the presence of the plant signal, luteolin. Withonly a slight increase in expression of nodD (see Table S2 inthe supplemental material), MucR significantly enhances in-duction of nod factor biosynthesis by luteolin, as measured byexpression of the nodABC operon (Fig. 5). Although in mostcases MucR acts as a repressor, the capacity for this transcrip-tional regulator to increase expression of genes such as exoYhas been demonstrated (9). In addition, features typical ofMucR binding sites as described by Becker et al. (9, 10) havebeen observed upstream of nodD, suggesting the potential fora direct effect. This positive role of MucR on nod factor pro-duction is carefully regulated as MucR increases expression ofnodD, rather than the nodABC operon directly. This therebyallows for an increase in nod factor production only in re-sponse to the detection of plant-produced luteolin, preventingexcess biosynthesis in the absence of a host plant. During plantnodulation assays, a sharp advantage in nodule developmentcoincident with an intact mucR is initially observed althoughthis quickly attenuates. This is possibly a consequence of thegradual accumulation of nod factor from excess inoculated S.meliloti. Ultimately, 3 to 4 weeks after inoculation, the num-bers of nodules developed and invaded become essentiallyequivalent between the wild type and the mucR mutant underlaboratory conditions. However, in a more unfavorable envi-ronment, either due to harsh conditions or lower numbers ofbacteria, this difference in luteolin sensitivity may bear greaterconsequences.

Once symbiosis has been established, differentiation of S.meliloti into the bacteroid state leads to a multitude of changesin gene expression. Interestingly, a disruption of mucR inplanktonic S. meliloti results in many of these same effects,including increased expression of multiple operons requiredfor nitrogen fixation and respiration, as well as numerous typeIV secretion system and putative transport-related genes (Ta-ble 2). While the functions of nitrogen fixation and respirationwithin the nodule are clear, the purposes of secretion andtransport-related gene expression are more elusive. These maycontribute to the exchange of plant signals and peptides re-ported to regulate bacterial growth and other cellular pro-cesses during symbiosis (49, 50). A more complete analysis byKegg Array of all known cellular processes affected by both thetransition into the bacteroid state and the disruption of mucR

presents remarkable parallels in the impacted pathways (seeFig. S1, S2, and S3 in the supplemental material). Due to theexpansiveness of affected functions, a complete understandingof the purpose of each modulation in gene expression requiresexhaustive analysis. However, the prevalent similarities inglobal changes strongly suggest a role of MucR in planktonicbacteria as a suppressor of premature bacteroid behavior. Pre-vious data from our laboratory have shown that within thenodule, mucR expression is repressed roughly 3.3-fold (26).While this appears to be a fairly modest decrease in transcrip-tion, the effects of disrupting mucR are more significant inamplitude than those observed with differentiation into thebacteroid state. This suggests the likelihood that a similar de-crease in mucR transcription, as opposed to complete disrup-tion, may produce equivalent values.

The ability of the ExpR/Sin system to modulate specificregulatory effects produces both quorum-sensing-dependentand-independent functions of MucR (Fig. 7). By this mecha-nism, derepression of EPS II production, suppression of pre-mature bacteroid-gene expression, and maximal synthesis ofnodules are simultaneously achieved in planktonic S. meliloti.

Trends in the roles of symbiotic megaplasmids pSymA andpSymB. Wild-type S. meliloti carries three major genetic ele-ments: the chromosome and symbiotic megaplasmids pSymAand pSymB. Throughout this study, clear patterns emerged inthe spatial organization of the genes involved in each regula-tory network. Prior to symbiosis, at a low population density,expression of motility persists as S. meliloti seek nutrients orhost plants with which to establish symbiosis. Maintained ex-pression of expR and sinI allows for the constitutive ability ofthe bacteria to detect quorum and respond appropriately. Fur-thermore, transcription of mucR suppresses bacteroid geneexpression in this planktonic state. Each of these genes resideson the chromosome of S. meliloti. As the bacterial populationdensity increases, AHL-bound ExpR permits the induction ofthe exp gene family present on pSymB. Once symbiosis hasbeen established, MucR is suppressed, and nitrogen fixation,type IV secretion, specific respiration, and transport-relatedgene expression, all on pSymA, are induced. In addition, 90%of the 154 genes most significantly differentially expressed(greater than 10-fold) as a result of a mucR mutation reside onthis megaplasmid. These observations suggest key utilization ofpSymB as population densities increase and expression frompSymA during the maintenance of symbiosis.

Furthermore, the unique organization of each of these reg-ulatory circuits provides a broad understanding of the sophis-ticated developmental history of S. meliloti, as well as theevolutionary versatility of the MucR repressor. With the ac-quisition of pSymB, the ability to abolish motility and produceEPS II in response to population allowed for the developmentof biofilms and the advantages associated with these commu-nities. However, this required the precise adjustment of theroles of MucR: a repressive capacity on EPS II production atearly stages of growth, followed by the abolishment of thiseffect at sufficient population densities. Additionally, the adop-tion of pSymA expanded the functions of pSymB. Synthesis ofsymbiotically active exopolysaccharides and the ability to de-velop biofilm could now be utilized for plant invasion andsubsequent nitrogen fixation. With this new capacity, the ad-ditional responsibility of preventing premature symbiotic gene



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expression was included among the already diverse functionsof MucR. The complex roles of this single transcriptional reg-ulator provide for more speculation than insight, but extensiveanalyses in greater detail may result in a clearer understandingof the means and order of adoption of each megaplasmid, aswell as the evolutionary progression to the modern S. meliloti.

ACKNOWLEDGMENTS

We thank Anke Becker for generously providing the strains, as wellas the members of our laboratory for critically reading the manuscript.We thank particularly Luciana Rinaudi for help in assembling thefigures, as well as David Allen for help with the Kegg Array analysis.

This work was supported by National Science Foundation grantMCB-9733532 and National Institutes of Health grant1R01GM069925 to J.E.G.

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FIG. 7. Schematic model for the quorum-sensing (QS)-dependent and -independent roles of MucR. At a low population density (OD600 of�0.02), MucR prevents the unnecessary production of EPS II prior to the quorum required for attachment and biofilm formation. At a highpopulation density (OD600 of �0.1), AHL-bound ExpR induces expression of the expG-expC operon encoding both the ExpG transcriptionalregulator and the ExpC glycosyl transferase. ExpG derepresses operons expE, expA, and expD from MucR, allowing for the biosynthesis of EPSII (4). Concurrently, increased levels of ExpC result in the production of the symbiotically active low-molecular-weight fraction. Throughout allstages of growth prior to symbiosis, MucR increases production of nod factor as well as the symbiotically active exopolysaccharide succinoglycanwhile repressing premature expression of bacteroid genes and mitigating motility.



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complex regulation of symbiotic functions is coordinated by … · or ahl (35). the concentration...

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