Chapter 50

Role of Quorum Sensing in theSinorhizobium meliloti –AlfalfaSymbiosis

Luciana V. Rinaudi-Marron and Juan E. GonzalezDepartment of Molecular and Cell Biology, University of Texas at Dallas, USA

50.1 INTRODUCTION

The ability to detect a change in local population, termedquorum sensing, is a widespread phenomenon in bacte-ria (Whitehead et al., 2001; see Chapters 70–77). Thiscapacity to perceive the presence of other related bacteriain the environment and accordingly adjust the expres-sion of specific genes relies on the production of uniquesignal molecules called autoinducers. The best charac-terized of these signal molecules are N-acyl homoserinelactones (AHLs), encoded by the LuxI family of AHLsynthases and produced by many gram-negative bacteria,such as Sinorhizobium meliloti, Photobacterium fischeri,Pseudomonas aeruginosa, and Agrobacterium tumefaciens(Whitehead et al., 2001). AHLs accumulate with a risein cell population density and, once they reach a par-ticular threshold level, bind to their cognate transcrip-tional regulators belonging to the LuxR family of AHL-binding proteins. These complexes interact with specificDNA sequences to facilitate the activation or repressionof target genes (Fig. 50.1). A variety of bacterial functionssuch as exopolysaccharide synthesis, motility, biofilm for-mation, and virulence are tightly controlled by quorumsensing in an array of symbiotic and pathogenic organisms(Whitehead et al., 2001).

Quorum sensing in S. meliloti has been studiedin some detail in two different strains, Rm41 andRm1021/Rm8530 (Marketon and Gonzalez, 2002; Mar-keton et al., 2002; Pellock et al., 2002; Chen et al., 2003;

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

Marketon et al., 2003; Teplitski et al., 2003; Hoang et al.,2004; Gao et al., 2005; Bartels et al., 2007; Glenn et al.,2007; Hoang et al., 2008; McIntosh et al., 2008; Gurichand Gonzalez, 2009; McIntosh et al., 2009; Mueller andGonzalez, 2011). At least two quorum-sensing systemshave been identified (Marketon and Gonzalez, 2002).One of them, the Tra system, is only present in anRm41-specific plasmid. The second quorum-sensingsystem, the ExpR/Sin system, is chromosomally locatedin all strains examined so far. This chapter summarizeswhat we and many others have learnt about the role ofthese two quorum systems.

50.2 S. meliloti QUORUM-SENSINGSYSTEMS

50.2.1 The Tra Quorum-SensingSystemThe S. meliloti Tra system, named for its homology to theTra system in A. tumefaciens, resides on a plasmid calledpRme41a (Marketon and Gonzalez, 2002). This plasmid isfound in the commonly used S. meliloti strain, Rm41, andit is also present in many natural Sinorhizobium isolates.Interestingly, the other commonly used S. meliloti strain,Rm1021, appears to have lost this plasmid, along withsome other traits. At least three regulatory genes (traR,traI, and traM), in addition to genes with homology to the

535

536 Chapter 50 Role of Quorum Sensing in the Sinorhizobium meliloti–Alfalfa Symbiosis

Figure 50.1 The basic quorum-sensing model.

trb operon, have been identified in pRme41a (Marketonand Gonzalez, 2002). TraI is an AHL synthase respon-sible for the production of at least three different signalmolecules, 3-O–C8 –HSL, 3-OH–C8 –HSL, and C8 –HSL(Marketon and Gonzalez, 2002). The presence of the tran-scriptional activator TraR is necessary for full synthaseactivity. In a manner analogous to the Tra system in A.tumefaciens, the product of the traM gene was shown tonegatively regulate TraR activity, ensuring that the Trasystem is active only at high population densities. The S.meliloti Rm41 Tra system also controls conjugal plasmidtransfer, as in other organisms, by mediating the transferof pRme41a (Marketon and Gonzalez, 2002). Disruptionof the traR, traI, or the trb operon abolishes plasmid trans-fer, while disruption of the traM gene results in a 100-foldincrease in transfer (Marketon and Gonzalez, 2002).

50.2.2 The ExpR/SinQuorum-Sensing SystemThe second quorum-sensing system in S. meliloti, theExpR/Sin system, is present in all the strains of Sinorhi-zobium studied so far. It consists of two transcriptionalregulators, SinR and ExpR, as well as the SinR-controlled autoinducer synthase SinI, responsible for the

biosynthesis of AHLs (Marketon and Gonzalez, 2002;Marketon et al., 2002) (Fig. 50.2). Interestingly, thewidely studied Rm1021 strain contains an insertionsequence in the expR gene, suggesting that the straincontaining an intact copy of this gene (Rm8530) shouldbe considered the true wild type (Pellock et al., 2002;Gurich and Gonzalez, 2009). SinI is responsible forthe production of several novel AHLs, ranging in sizefrom C12 –HSL to C18 –HSL (Marketon et al., 2002).Disruption of either sinI or sinR results in a delay as wellas an overall decrease in the appearance of nitrogen-fixingnodules, suggesting a role for the SinI-made AHLs inestablishing a successful symbiosis with Medicago sativa(Marketon et al., 2002). These AHLs, in conjunction withthe ExpR regulator, control a variety of downstream genes(Hoang et al., 2004; Gurich and Gonzalez, 2009). Morerecently, the Sin-quorum-sensing system, in conjunctionwith the ExpR regulator, has been shown to play a rolein various other important cellular processes (Muellerand Gonzalez, 2011; Fig. 50.2).

Whole-genome expression profiles between a strainwith intact expR and sinI genes and strains that lack eitherone of them during the early-, mid-, and late-log phasesof growth have provided a deeper understanding of thetiming and intensity of quorum-sensing control of manycell functions throughout the bacterial growth cycle in thefree-living state (Gurich and Gonzalez, 2009). This ledto the identification of groups of genes, including severalnovel genes that were dependent on one or more regu-latory components. These extensive microarray analysesallowed the identification of a group of genes that mightbe responsible for the invasion defect shown by the sinImutant. Furthermore, the role of quorum sensing after theestablishment of symbiosis was evaluated by measuringbacterial gene expression inside mature nodules (Gurichand Gonzalez, 2009). These studies have provided insightsinto the role of this regulatory system in free-living bacte-ria from low cell population density to high cell populationdensity, and throughout the course of the plant—bacterialinteractions.

Figure 50.2 The ExpR/Sin system regulates multiplephysiological phenomena in S. meliloti . Abbreviations: SinR,ExpR, Transcriptional Regulators; SinI, AutoinducerSynthase; LMW, Low Molecular Weight; HMW, HighMolecular Weight.

50.3 Role of S. meliloti Quorum-Sensing Systems 537

50.3 ROLE OF S. melilotiQUORUM-SENSING SYSTEMS

50.3.1 ExopolysaccharideSynthesisS. meliloti strain Rm1021 produces succinoglycan, a well-characterized exopolysaccharide polymer of repeatingoctasaccharide subunits (see Chapter 52). Each subunitconsists of one galactose and seven glucose residues andis decorated with a succinyl, an acetyl, and a pyruvyl mod-ification (Aman et al., 1981; Reuber and Walker, 1993a;Reinhold et al., 1994). Mutants of Rm1021 that fail to syn-thesize succinoglycan (exo mutants) form empty nodulesthat are devoid of bacteria and are unable to fix nitrogen(Glucksmann et al., 1993; Reuber and Walker, 1993b).A 25-kb cluster of exo genes has been identified on thesecond symbiotic megaplasmid (pSymB) that is requiredfor the production of succinoglycan, and biosyntheticroles have been assigned to most of the gene products(Leigh et al., 1987; Long et al., 1988; Buendia et al., 1991;Reed et al., 1991; Battisti et al., 1992; Becker et al., 1993a;Becker et al., 1993b; Muller et al., 1993; Reuberand Walker, 1993b; Reuber and Walker, 1993a;Gonzalez et al., 1996b; York and Walker, 1997;Gonzalez et al., 1998). A trimer fraction of the suc-cinoglycan octasaccharide subunit was shown to be theactive species in nodule invasion (Battisti et al., 1992;Gonzalez et al., 1998; Wang et al., 1999). Rm1021 alsohas the capacity to make a second distinct exopolysaccha-ride, designated EPS II (Glazebrook and Walker, 1989;Zhan et al., 1991), which is a polymer of repeatingdisaccharide subunits. Subunits of EPS II consist of anacetylated glucose connected to a pyruvylated galactoseresidue (Glazebrook and Walker, 1989; Her et al., 1989;Zhan et al., 1989). A 32-kb cluster of exp genes (distinctfrom the exo genes) in pSymB is responsible for thesynthesis of EPS II (Becker et al., 1997). Further analysisrevealed that a low molecular weight fraction, consistingof 15–20 disaccharide subunits of EPS II, efficientlyrescued nodule invasion in an exopolysaccharide-deficientstrain (Gonzalez et al., 1996a). EPS II production bystrain Rm1021 had been previously characterized ascryptic (Glazebrook and Walker, 1989). EPS II wasproduced at extremely low levels by this strain, butproduction was greatly increased in derivatives carryingthe expR101 mutation, which resulted in overexpressionof the exp genes. The expR101-mediated synthesis ofEPS II was shown to suppress the symbiotic defects ofsuccinoglycan-deficient strains on alfalfa (Glazebrook andWalker, 1989; Gonzalez et al., 1996a). Further analysisrevealed that the expR101 allele was not a mutation butactually a restoration of the expR gene responsible for

encoding the quorum-sensing regulator ExpR, which isdisrupted by an insertion sequence in the Rm1021 strain(Pellock et al., 2002).

The presence of, both, ExpR and the SinI-made AHLsare required for the synthesis of EPS II by S. meliloti(Pellock et al., 2002; Marketon et al., 2003). ExpR is apositive regulator of the exp genes, responsible for EPSII biosynthesis (Pellock et al., 2002). In a sinI mutant,expression of several of the exp genes is abolished, andthis deficiency can be fully rescued by the addition ofeither crude AHL extracts or by synthetic C16 : 1 –HSL(Marketon et al., 2003).

Recent work shows that the ExpR/Sin quorum-sensing system induces EPS II production by increasingthe expression of the expG–expC operon, encodingboth a transcriptional regulator (ExpG) and a glycosyltransferase (ExpC) (Mueller and Gonzalez, 2011). ExpGderepresses EPS II production at the transcriptional levelfrom MucR, while concurrently elevating expressionof expC, resulting in the synthesis of the symbioticallyimportant low molecular weight form (Mueller andGonzalez, 2011). While the ExpR/Sin system abolishesthe role of MucR in EPS II production, it preservesa multitude of other quorum-sensing-independent reg-ulatory functions, which promote the establishment ofsymbiosis. In free-living S. meliloti, MucR properlycoordinates a diverse set of bacterial behaviors byrepressing a variety of genes intended for expressionduring symbiosis and enhancing the bacterial ability toinduce root nodule formation. Quorum sensing preciselymodulates the functions of MucR to take advantage of,both, the production of symbiotically active EPS II aswell as the proper coordination of bacterial behaviorrequired to promote symbiosis.

The ExpR/Sin system also controls the productionof the second symbiotically important exopolysaccharidesuccinoglycan (Glenn et al., 2007) (see Chapter 52). Theabsence of one or more of the quorum-sensing regulatorycomponents results in a decrease in the production of suc-cinoglycan. Moreover, production of the symbiotically-active low molecular weight fraction of this polymer isstrongly controlled by the ExpR/Sin quorum-sensing sys-tem (Glenn et al., 2007).

50.3.2 Biofilm FormationQuorum sensing appears to also play a significant rolein biofilm formation by S. meliloti, a process medi-ated mostly through its regulation of EPS II and theproduction of the active low molecular weight fractionof this polymer. Biofilms are formed when one ormultiple bacterial species are enclosed in a self-producedpolymeric matrix attached to a surface and to eachother (Costerton et al., 1995; see Chapters 66, 68).

538 Chapter 50 Role of Quorum Sensing in the Sinorhizobium meliloti–Alfalfa Symbiosis

These polysaccharide-bound colonies often developinto complex three-dimensional microbial communitiespermeated by channels through which nutrients and waterflow (Stanley and Lazazzera, 2004). The compositionand structure of biofilms are highly dependent on thebacterially produced polysaccharides (Sutherland, 2001).Factors such as the effective polysaccharide concen-tration, degree of polymerization, type of carbohydratelinkages, ionic status, and the presence of other macro-molecules determine the stability of the bacterial-bindingmatrix (Mayer et al., 1999). It was recently shown thatthe presence of EPS II is essential for the formation ofbiofilms by S. meliloti in vitro and in vivo (Rinaudi andGonzalez, 2009). In the absence of succinoglycan, EPSII-producing strains form interconnected complex cellclusters or honeycomb-like biofilms. Bacteria in thesehighly organized biofilms are predominantly attachedto each other through lateral interactions, forming rowsof cells identically oriented. Moreover, the presence ofthe low molecular weight fraction of EPS II is vital forbiofilm formation, both in vitro and in vivo, providing forthe first time a role for the symbiotically active fractionof EPS II (Rinaudi and Gonzalez, 2009; see Chapter 66).

50.3.3 S. meliloti MotilityStudies from several laboratories, in addition to thecompleted S. meliloti genome, have substantially con-tributed to our understanding of the genetic componentsof this bacterium’s flagellar system. The S. melilotigenome contains at least 41 motility- and chemotaxis-related genes that are chromosomally located in a 45-kbregion commonly referred to as the flagellar regulon(Sourjik et al., 1998). These genes are categorized intothree main classes and are expressed in a hierarchicalmanner. Class I genes (visN and visR) lie at the apexand consequently control the expression of the genesin class II and class III. Sourjik et al. showed that amutation in either visN or visR is sufficient to dramaticallydecrease the expression of representative flagellar andchemotaxis genes in class II and class III, which, inturn, abolishes motility in the S. meliloti strain RU11(Sourjik et al., 2000). More recently, work by Rotter et al.demonstrated a regulator, which the authors named Remthat controls the expression of several classes of motilitygenes in the S. meliloti strain RU11 (Rotter et al., 2006).

Genome-wide expression analysis showed a largesubset of ExpR/Sin-regulated genes, which appeared to bealso dependent on the bacterial growth phase. The major-ity of these genes were related to motility and chemotaxis.In the quorum-sensing proficient strain, the accumulationof AHLs causes repression of the motility and chemotaxisgenes (Hoang et al., 2008). We also showed that ExpRis required for activation of these genes at a low cell

population density (i.e., no AHLs or low levels of AHLs).It seems that free ExpR and the ExpR–AHL complexcompete for the control of the motility genes. At alow cell population density, free ExpR is available forinduction of visN and visR. With a rise in cell-population-density and AHL concentration, the ExpR–AHL complexblocks the expression of visN and visR.

Expression analyses provided an explanation to theparticular observation that the sinI mutant does not invadeplants efficiently, while the expR mutant invades as well asthe quorum-sensing proficient strain. These data suggestedthat the sinI mutant’s inability to repress the synthesis offlagella at a high cell population density interfered withthe establishment of symbiosis, leading to a reduction inthe total number of nodules produced by the plant and adecrease in the ratio of nitrogen versus nonnitrogen-fixingnodules compared to plants inoculated with the quorum-sensing proficient strain or the expR mutant. Indeed, asinI flaAB double mutant (incapable of producing flagella)regained the full capacity to establish symbiosis (Gurichand Gonzalez, 2009). Therefore, the ExpR/Sin quorum-sensing system plays a critical role in the timely control ofexpression of the motility and chemotaxis genes. In nature,activation of these genes at low cell population densitiesis essential for the ability of bacteria to find nutrients or toreach their host. At high cell population densities, such asthose found in close proximity to the host root, repressionof flagellar production appears to be necessary for biofilmformation and maximal efficiency in plant invasion.

50.4 OTHER LUXR HOMOLOGS

In addition to ExpR, the S. meliloti genome containsvarious LuxR homologs that appear to play a role in geneexpression. S. meliloti Rm1021 contains four additionalputative orphan LuxR homologs (SMc04032, SMc00658,SMc00877, and SMc00878), the regulatory roles of whichremain to be fully determined (Galibert et al., 2001). Theseresponse regulators contain the characteristic domains ofthe LuxR family of proteins that include an N-terminalautoinducer/response regulatory domain and a C-terminalhelix-turn-helix domain (Galibert et al., 2001). Recently,the regulatory role of one of the orphan LuxR-typeresponse regulators, NesR (SMc04032), was elucidated(Patankar and Gonzalez, 2009). Through expression andphenotypic analyses, NesR was determined to affect theactive methyl cycle of S. meliloti. Disruption of nesR wasshown to influence the nutritional and stress–responseactivities in S. meliloti. The nesR mutant was deficient incompeting with the quorum-sensing proficient strain forplant nodulation. Taken together, these results suggestthat NesR potentially contributes to the adaptability of S.

References 539

meliloti when it encounters challenges such as high osmo-larity, nutrient starvation, and/or competition for nodula-tion, thus increasing its chances for survival in the stressfulrhizosphere. Interestingly, the function of NesR appearsto be independent of the SinI-encoded AHLs, suggestingthat there are potentially additional signal moleculesthat remain to be identified. Preliminary evidence showsthat the gene products of SMc00658, SMc00877, andSMc00878 may play a role in nitrogen utilization, aminoacid transport, and membrane stability, among others(Patankar and Gonzalez, unpublished results).

50.5 QUORUM SENSING INSIDETHE NODULE

Clearly, the ExpR/Sin quorum-sensing system plays animportant regulatory role in free-living S. meliloti as wellas during the host invasion process. Its role once inside theplant host remained elusive. Given the fact that bacteriaare found within nitrogen-fixing nodules in high numbers,allowing for the possible accumulation of AHLs, it wasinvestigated whether the ExpR–AHL complex continuesto regulate gene expression in the nodule. Microarrayand detailed gene expression analyses show that all keyquorum-sensing genes and their regulatory effects arerepressed within the nodule (Gurich and Gonzalez, 2009).All ExpR/Sin quorum-sensing components (sinR, sinI,and expR) were dramatically down-regulated inside thenodule. Equally, all quorum-sensing-induced genes, suchas exopolysaccharide and calcium-binding protein geneswere repressed as well. On the basis of these studies, itwas concluded that the ExpR/Sin quorum-sensing systemplays an essential role in gene regulation before andduring plant invasion but is inactivated once symbiosis isestablished (Gurich and Gonzalez, 2009).

50.6 CONCLUSION

Quorum sensing has been shown to play an important rolein many bacterial–host symbiotic and pathogenic interac-tions. In the case of S. meliloti, there is increasing evi-dence that these roles are multiple and quite varied. Recentwork revealed that ExpR is a highly versatile regulatorwith a unique ability to show different regulatory capa-bilities in the presence or absence of autoinducer. Onecentral role appears to be the regulation of the productionof the symbiotically important exopolysaccharides. TheExpR/Sin system not only regulates the amount of suc-cinoglycan and EPS II produced by the bacterium, but isalso crucial for the production of the invasion-proficientlow molecular weight fractions of these polymers. In addi-tion, the ExpR/Sin system, mainly through the regulation

of EPS II biosynthesis, controls biofilm formation andattachment to plant surfaces. Bacterial motility is also neg-atively regulated by high population density via ExpR andSinI. In the presence of these two components, flagellarand chemotaxis genes are repressed when cultures achievestationary phase. Once symbiosis is established, quorumsensing is repressed by a yet undetermined mechanism,and all metabolic functions of the bacteroids are directedtoward nitrogen fixation. Therefore, quorum sensing playsa crucial role in the regulation of many cell functions thatensure successful invasion of the host, and is inactivatedonce symbiosis is established.

ACKNOWLEDGMENTS

We thank the members of our laboratory for criticallyreading the manuscript. Our work was supported byNational Science Foundation grant MCB-9733532 andNational Institutes of Health grant 1R01GM069925 toJ.E.G.

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