Cooperation of two distinct ExpR regulators controlsquorum sensing specificity and virulence in the plantpathogen Erwinia carotovora

Solveig Sjöblom, Günter Brader, Gudrun Koch andE. Tapio Palva*Viikki Biocenter, Faculty of Biosciences, Department ofBiological and Environmental Sciences, Division ofGenetics, University of Helsinki, P.O.B. 56, FIN-00014,Helsinki, Finland.

Summary

Quorum sensing, the population density-dependentregulation mediated by N-acylhomoserine lactones(AHSL), is essential for the control of virulence in theplant pathogen Erwinia carotovora ssp. carotovora(Ecc). In Erwinia carotovora ssp. the AHSL signal withan acyl chain of either 6 or 8 carbons is generated byan AHSL synthase, the expI gene product. This workdemonstrates that the AHSL receptor, ExpR1, of Eccstrain SCC3193 has strict specificity for the cognateAHSL 3-oxo-C8-HSL. We have also identified asecond AHSL receptor (ExpR2) and demonstrate anovel quorum sensing mechanism, where ExpR2 actssynergistically with the previously described ExpR1to repress virulence gene expression in Ecc. We showthat this repression is released by addition of AHSLsand appears to be largely mediated via the negativeregulator RsmA. Additionally we show that ExpR2 hasthe novel property to sense AHSLs with different acylchain lengths. The expI expR1 double mutant is ableto act in response to a number of different AHSLs,while the expI expR2 double mutant can only respondto the cognate signal of Ecc strain SCC3193. Theseresults suggest that Ecc is able to react both to thecognate AHSL signal and the signals produced byother bacterial species.

Introduction

Quorum sensing (QS) is a central cell-to-cell communica-tion system that bacteria employ to monitor their populationdensity and coordinate functions for which high populationdensity is profitable (Fuqua et al., 2001; Waters andBassler, 2005). QS is achieved by production and sensing

of diffusible chemical signals that in gram-negative bacte-ria are usually N-acylhomoserine lactones (AHSLs).AHSLs control a number of diverse functions in bacteria,such as bioluminescence (Eberhard et al., 1981; Engebre-cht and Silverman, 1984), conjugal transfer (Zhang et al.,1993), production of antibiotics as well as secondarymetabolites and virulence in both plant and animal patho-gens (Whitehead et al., 2001). QS is required for theproduction of virulence determinants and biofilm formationin the opportunistic human pathogen Pseudomonasaeruginosa (Winson et al., 1995), exopolysaccharide pro-duction in the plant pathogen Pantoea stewartii (Beck vonBodman and Farrand, 1995) and production of plant cellwall degrading enzymes (PCWDEs) and antibiotics inanother plant pathogen Erwinia carotovora (Jones et al.,1993; Pirhonen et al., 1993).

The LuxR/LuxI system controlling bioluminescence inVibrio fischerii was the first to be characterized and hasbecome the paradigm of QS. The QS system is minimallyexecuted by an AHSL synthase, a LuxI-type protein, and aQS regulator, a LuxR-type protein, controlling transcriptionof downstream genes (Fuqua et al., 2001; Whitehead et al.,2001). There is high degree of specificity in QS determinedby substrate specificity of the AHSL synthase and specificrecognition of the cognate AHSL by the LuxR-type protein,modulating the expression of QS-regulated target genes.Different bacteria produce AHSLs with diverse acyl sidechain lengths, ranging from 4 to 16 carbons, and withalterations in the oxidative status of carbon 3 (Fuqua andGreenberg, 2002; Pappas et al., 2004). The LuxR-typeproteins act as QS regulators, and they distinguishbetween different AHSLs by showing binding of cognate,but not non-cognate AHSLs suggesting that they aremainly involved in intraspecies signaling (Lazdunski et al.,2004). The LuxR-type proteins share a similar structurewith a ligand (AHSL) recognizing domain at the amino-terminus (N-terminus) and usually a very conserved DNA-binding domain at the carboxy-terminus (C-terminus).Although LuxR-type proteins have similar structures, theiroperative mechanisms can be different. Many LuxR-typeactivators, including CarR, LuxR and TraR, the only crys-tallized LuxR-type protein so far (Qin et al., 2000; Vanniniet al., 2002; Zhang et al., 2002), form dimers or multimersupon binding to AHSLs (Whitehead et al., 2001; Pappas

Accepted 24 April, 2006. *For correspondence. E-mail tapio.palva@helsinki.fi; Tel. (+358) 9 19159600; Fax (+358) 9 19159076.

Molecular Microbiology (2006) 60(6), 1474–1489 doi:10.1111/j.1365-2958.2006.05210.xFirst published online 18 May 2006

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et al., 2004). This stable complex then binds to a targetgene promoter, in many cases to a 20 bp palindromic luxbox, in order to activate target gene transcription (Fuquaet al., 2001; Lazdunski et al., 2004). In contrast, EsaR, aLuxR-type protein of the plant-pathogen Pantoea stewartii(Beck von Bodman and Farrand 1995) binds the targetDNA in the absence of AHSL and represses transcription,but after addition of the cognate AHSL EsaR is thought tobe released from DNA and the target gene is derepressed(Minogue et al., 2002; 2005).

QS is central to regulation of virulence of the gram-negative, broad host range plant pathogen Erwiniacarotovora ssp. carotovora (Ecc), and it also controls pro-duction of carbapenem antibiotics in some strains of Ecc(Pirhonen et al., 1991; 1993; Jones et al., 1993). We haveshown that AHSL synthesis is required for the productionof PCWDEs, such as cellulases, polygalacturonases andpectinases, the main virulence determinants of this patho-gen and that QS is responsible for density-dependent andcoordinated production of these enzymes to establish asuccessful infection (Pirhonen et al., 1991; 1993).Mutants deficient in the AHSL synthase (the expI geneproduct) are impaired in the production of PCWDEs andare thus avirulent (Pirhonen et al., 1991; 1993; Joneset al., 1993). Depending on the strain, the mainAHSLs produced and recognized by Ecc are 3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL) or 3-oxo-octanoylhomoserine lactone (3-oxo-C8-HSL) with 3-oxo-C8-HSL being the cognate AHSL of the Ecc strainSCC3193 (Brader et al., 2005). The so far best charac-terized LuxR-type protein of Ecc is CarR, which positivelyregulates the production of carbapenem antibiotics, inresponse to its cognate autoinducer 3-oxo-C6-HSL in theEcc strain GS101 (Welch et al., 2000).

The QS regulators controlling PCWDE production haveso far remained more elusive. We originally identifiedExpR of the Ecc strain SCC3193 as a potential QS regu-lator (Andersson et al., 2000). Although mutations in expRdid not show a clear phenotype, overexpression studiessuggested that ExpR might act as repressor of PCWDEsynthesis (Andersson et al., 2000). Interestingly, vonBodman et al. (2003) have demonstrated that ExpRSCC3193

can bind to DNA in the absence of AHSL, but that thisbinding is inhibited by AHSL addition. A recent study byCui et al. (2005) showed that a related LuxR-type proteinExpREcc71 from another Ecc strain Ecc71 binds to thepromoter of a target gene rsmA, activating its transcriptionin an AHSL free state. Addition of the cognate AHSLreleased the ExpREcc71 from this promoter leading torepression of the target gene that encodes the globalnegative regulator RsmA (Chatterjee et al., 2005; Cuiet al., 2005). RsmA is an RNA-binding protein thatrepresses the production of PCWDEs (Chatterjee et al.,1995). These results supported a close relation between

RsmA and the QS system already indicated in previousstudies (Chatterjee et al., 1995; Kõiv and Mae, 2001).

In this study we identify a novel QS regulation wheretwo LuxR-type proteins, ExpR1 and ExpR2, act synergis-tically as negative regulators of PCWDE productionin the Ecc strain SCC3193. This negative regulationreleased by accumulation of AHSLs appears to be largelymediated by the global negative regulator RsmA. Intrigu-ingly, we demonstrate that the two ExpR proteins havedistinct AHSL specificities: while ExpR1 is specific to thecognate AHSL, the newly identified ExpR2 protein showsbroad signal sensing capacity and responds also to non-cognate AHSL, allowing both intra- and interspeciescommunication.

Results

Inactivation of expR alters AHSL sensing specificity

We have demonstrated that the AHSL synthase encodedby expI of Ecc strain SCC3193 (Pirhonen et al., 1993)produces mainly 3-oxo-C8-HSL (Brader et al., 2005). AnexpI mutant (SCC3065) of SCC3193 is not able toproduce the PCWDEs (Pirhonen et al., 1993), but can bespecifically rescued by addition of the cognate 3-oxo-C8-HSL (Brader et al., 2005). A much higher concentration(200-fold) of the non-cognate AHSL 3-oxo-C6-HSL isrequired to rescue the PCWDE negative phenotype of thismutant (Brader et al., 2005). To address the role of theExpR protein of SCC3193 (Andersson et al., 2000) in thisrecognition, we characterized the ability of the addednon-cognate 3-oxo-C6-HSL or cognate 3-oxo-C8-HSL torestore the cellulase (Cel) activity in the expI mutant(SCC3065) and the expI expR double mutant (SCC6005)(Fig. 1) using a Carboxymethylcellulose (CMC) plate

A

B

Fig. 1. An expI expR mutant shows lack of sensing specificity.Sensitivity of Ecc expI mutant (SCC3065) and expI expR mutant(SCC6005) to cognate and non-cognate AHSLs.A. The expI mutant and the expI expR mutant were grown for 16 hin L medium added with either no AHSL, 3-oxo-C6-HSL or3-oxo-C8-HSL. Cel activity of 10 ml growth culture supernatant wasdetected with CMC plates as described in Experimentalprocedures.B. For complementation studies expR was expressed in trans inthe expI expR mutant and grown in L medium with either no AHSL,3-oxo-C6-HSL or 3-oxo-C8-HSL for 16 h. Cellulase activity wasdetected on CMC plates as in A.

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assay (Pirhonen et al., 1993). Interestingly, the presenceor absence of the expR gene had a profound effect onAHSL sensing specificity; while the Cel activity of the expImutant was restored only by the cognate 3-oxo-C8-HSL,in the expI expR double mutant the Cel production wasrestored both by the non-cognate 3-oxo-C6-HSL and thecognate 3-oxo-C8-HSL. To confirm that this altered speci-ficity was due to loss of the ExpR function the expR-SCC3193 gene was expressed in trans in the expI expRdouble mutant (SCC6005) background. This resulted inrestoration of the requirement for the cognate 3-oxo-C8-HSL in Cel production (Fig. 1). These results demonstratethat the expR mutant has broader substrate specificityand suggest that ExpR is involved in sensing of 3-oxo-C8-HSL.

Identification of a second expR homologue in Ecc strainSCC3193

The altered AHSL sensing specificity (Fig. 1) and the lackof clear PCWDE phenotype (Andersson et al., 2000) ofthe expR mutant could be explained by the existence ofan additional ExpR homologue or possibly another partlyredundant QS regulator responsible for sensing the non-cognate 3-oxo-C6-HSL. Interestingly, two expR homo-logues have been reported in the newly sequencedgenome of Erwinia carotovora ssp. atroseptica strainSCRI1043 (Eca); one (expR) is linked to the expI genesimilar to the organization of expI and expR in SCC3193(Andersson et al., 2000) and the other one (ECA1561)exists separately (Bell et al., 2004). To explore the possi-bility of a second expR homologue in Ecc strain SCC3193we used the sequence data of Eca strain SCRI1043 todesign primers for PCR identification of a potential secondexpR homologue. Subsequently the presence of a secondexpR gene in SCC3193 was demonstrated (designatedexpR2, with the previously identified/characterized expRrenamed as expR1). The genomic organization ofsequences flanking expR2SCC3193 is rather similar betweenthe two subspecies of Erwinia carotovora. Downstream ofthe expR2SCC3193 gene is an open reading frame (orf) of252 aa that is 83% identical to a CDP-diacylglycerol pyro-phosphatase of Eca strain SCRI1043, while in theupstream region an approximately five kb fragment,present in the Eca genome, is lacking between expR2 andthe next orf (partially sequenced) that is 88% identical toa chemotaxis signal transduction protein (ECA1568) ofthe Eca strain SCRI1043 (Fig. 2A).

The expR2 orf consists of 735 bp encoding a putativeExpR2 protein of 245 aa. Structural comparison ofExpR2 with other LuxR-type proteins suggested thatExpR2 is likely a QS regulator (Fig. 2B). The ExpR2protein shows 94% aa identity (97% similarity) to apotential QS regulator (ECA1561) of Eca SCRI1043 and

62% aa identity (81% similarity) to ExpR of EcaSCRI1043, 58% aa identity (81% similarity) to ExpR1 ofEcc SCC3193, 62% aa identity (80% similarity) to aputative ExpR of Ecc SCC1, 54% aa identity to EsaR ofPantoea stewartii ssp. stewartii and 22% aa identity(46% similarity) to TraR of Agrobacterium tumefaciens(Fig. 2B). The DNA binding domain is highly conservedbetween the Erwinia carotovora and the Pantoea strains,while the AHSL binding domains show more sequencevariety. Taken together, these results indicate that EccSCC3193 has indeed two LuxR-type proteins andsuggest that this redundancy could explain the lack of aclear PCWDE phenotype in expR1 mutants.

Inactivation of both expR1 and expR2 suppresses thecellulase-negative phenotype of an AHSL deficientstrain

To explore the functional roles of the two ExpR proteins inthe QS system of E. carotovora we generated both singleand double expR1 and expR2 insertion mutants in thewild-type and expI genetic backgrounds of SCC3193.Interestingly, neither the expR1 or expR2 single mutantsnor the expR1 expR2 double mutant showed any clearimpairment of PCWDE production or virulence in the wild-type SCC3193 background suggesting that the corre-sponding proteins do not act as positive regulators ofvirulence. As there was the distinct possibility that one orboth of the ExpR proteins of SCC3193 could function asnegative regulators of virulence and enzyme production assuggested previously for ExpR1, EsaR and for theExpREcc71 and VirR of other E. carotovora strains (vonBodman et al., 1998; Andersson et al., 2000; Cui et al.,2005; Burr et al., 2006) we assessed the phenotypic effectsof expR1 and expR2 mutations in the expI mutant back-ground. The results firstly demonstrate that the presence ofeither ExpR1 or ExpR2 is sufficient for the PCWDE-negative phenotype of expI mutants (Fig. 3A). In contrast,inactivation of both expR genes in the expI mutant back-ground restores PCWDE production to wild-type levels(Fig. 3A). This was initially shown for Cel production(Fig. 3A), but a similar restoration of other enzyme activi-ties including polygalacturonase (Peh) and protease (Prt)was also evident (data not shown). The effect was inde-pendent of growth conditions as the PCWDE phenotypeswere identical in both rich and minimal medium. These datastrongly indicate that both ExpR1 and ExpR2 act as nega-tive regulators of PCWDE production and hence of viru-lence in Ecc and suggest that the function of AHSLs couldbe in relieving this repression at high population density.

AHSL specificity of ExpR1 and ExpR2

The generation of expI expR1 and expI expR2 doublemutants allowed us to explore the AHSL specificity of

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each ExpR protein. Mutant strains were grown in thepresence of either the cognate autoinducer 3-oxo-C8-HSL or the non-cognate 3-oxo-C6-HSL and Cel activity

was characterized (Fig. 3A). The analysis showed that theexpI expR2 mutant (SCC908) responded specifically to3-oxo-C8-HSL, in contrast to the broader specificity

Fig. 2. A. Organization of a 3 kb DNA fragment of Ecc SCC3193. The three open reading frames are indicated with arrows pointing in thedirection of transcription, the striped arrow indicate a partially sequenced CDS. The dotted region implies sequence region with no homologyto Eca SCRI1043.B. Alignment of ExpR amino acid sequences. Protein sequences are from Ecc strain SCC3193 (ExpR1 and ExpR2), Ecc strain SCC1 (ExpR),Eca strain SCRI1043 (ExpR and ExpR2 (ECA1561)), Pantoea stewartii strain SS104 (EsaR) and Agrobacterium tumefaciens strain NTL4(TraR). The alignments were performed using the CLUSTALW program (Thompson et al., 1994) and shaded using the BOX shade programversion 3.21 (Hofman and Baron, http://www.ch.embnet.org). Autoinducer binding domain is underlined and DNA binding domain is doubleunderlined.

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shown by the expI expR1 double mutant (SCC6005).According to these results ExpR1 is a specific LuxR-typeprotein, activating Cel production only after addition of3-oxo-C8-HSL. At physiological AHSL concentrationsExpR1 was indeed only able to respond to the cognatesignal 3-oxo-C8-HSL, while the addition of 3-oxo-C6-HSLdid not suppress the Cel negative phenotype of the expIexpR2 double mutant. Addition of 3-oxo-C6-HSL to a con-centration of 10 mM or more was required to restore Celactivity to this mutant (data not shown).

To determine the specificity of ExpR1 and ExpR2 asAHSL receptors, expI expR1 and expI expR2 doublemutants were exposed to a series of AHSLs: C4-HSL,C6-HSL, 3-oxo-C6-HSL, C7-HSL, C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, C12-HSL or 3-oxo-C14-HSL, andassayed for Cel activity (Fig. 3B). As suggested by previ-ous results (Fig. 3A) ExpR1 appeared to have a narrowAHSL specificity responding best to the cognate 3-oxo-C8-HSL, although it also could respond to some extent to3-oxo-C10-HSL and to C8-HSL. On the other hand ExpR2appeared to be promiscuous and respond to all theAHSLs tested except for the shortest (C4-HSL) and thelongest ones (C12-HSL and 3-oxo-C14-HSL). ThusExpR2 had a much broader AHSL recognition capacitycompared to ExpR1, which clearly is the more specificLuxR-type protein mainly responding to the cognate3-oxo-C8-HSL.

Having established the specificity of AHSL sensing foreach ExpR protein, we wanted to explore the joint effect ofthe two ExpR proteins in PCWDE regulation. As a tool weemployed the triple mutant (SCC906) lacking the AHSLsynthase as well as the two ExpR proteins. As shown thistriple mutant exhibited wild-type levels of Cel activitywithout the addition of any AHSL (Fig. 3A and C). Comple-mentation studies were used to demonstrate the specificroles for each ExpR protein. Plasmids carrying eitherexpR1 or expR2 were introduced to the expI expR1expR2 triple mutant and Cel activities were assessed inthe presence and absence of 3-oxo-C6-HSL or 3-oxo-C8-HSL (Fig. 3C). These results support the hypothesis thatboth ExpR proteins have distinct roles in AHSLrecognition. Introduction of expR1 into the triple mutantmade the strain dependent of the cognate 3-oxo-C8-HSLin Cel production whereas introduction of expR2 gener-ated a dependency of a variety of AHSLs. These datademonstrate that the ExpR1 and ExpR2 have individualroles in AHSL recognition and indicate that either ExpR issufficient to repress PCWDE production.

ExpR1 and ExpR2 mediated repression of PCWDEactivity is population density-dependent

Since the expI expR1 expR2 triple mutant was constitu-tively Cel positive without the addition of AHSL, we

Fig. 3. An expI expR1 expR2 mutant strain is constitutivelycellulase positive. The two ExpR proteins show differentspecificities to AHSLs.A. Ecc wild-type (SCC3193), expI mutant (SCC3065), expI expR1mutant (SCC6005), expI expR2 mutant (SCC908), expR1 expR2mutant (SCC907) and expI expR1 expR2 mutant (SCC906) weregrown in L medium added with no AHSL, 3-oxo-C6-HSL or3-oxo-C8-HSL and Cel activity of 10 ml of growth culturesupernatants were detected with CMC plates.B. The expI mutant, expI expR1 mutant, expI expR2 mutantwere grown in L medium with no AHSL, C4-HSL, C6-HSL,3-oxo-C6-HSL, C7-HSL, C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL,C12-HSL or 3-oxo-C14-HSL and the Cel activity of 10 ml of growthculture supernatants was detected as in 3A.C. expI expR1 expR2 triple mutant (SCC906) carrying either expR1or expR2 on a plasmid. The specificity of ExpR proteins wasdetermined by monitoring the Cel activity using a plate assay as in3A and 3B.

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wanted to elucidate the role of the QS system inpopulation density-dependent regulation of PCWDEs.Nothern analysis was used to explore the role of ExpR1and ExpR2 in regulation of PCWDE genes in the pres-ence of endogenous AHSLs. We compared PCWDEgene expression in the wild-type (SCC3193), the expR1expR2 mutant (SCC907) and the expI expR1 expR2mutant (SCC906) strains during the logarithmic growthusing PCWDE gene specific probes (Fig. 4). Bothmutant strains exhibited substantially up-regulatedPCWDE transcript accumulation compared to the wild-type strain. Similar expression levels were only reachedby the wild-type at the stationary growth phase. In accor-dance with this also the production of one of theenzymes, Cel was clearly up-regulated in the expR1expR2 double mutant at the early phases of growth(Fig. 4). This analysis demonstrates that ExpR1 andExpR2 are essential for the growth phase dependentcontrol of PCWDE production.

ExpR1 and ExpR2 control expression of RsmA, anegative regulator of PCWDE

Our results (Figs 3 and 4) demonstrate that ExpR1 andExpR2 are negative regulators of PCWDE gene expres-sion and PCWDE production. However, they do not indi-cate weather this repression is direct or mediated throughsome other regulator. An involvement of the global nega-tive regulator RsmA was recently suggested by Cui et al.(2005) showing that in Ecc strain Ecc71 ExpR71 binds to

the rsmA promoter and activates its expression in theabsence of AHSL. The activation of rsmA transcriptionwas prevented by the addition of 3-oxo-C6-HSL (Chatter-jee et al., 2005; Cui et al., 2005). We explored the possi-bility of an RsmA-mediated mechanism of ExpR1 andExpR2 control in Ecc SCC3193 using an rsmA-gusA pro-moter fusion. The rsmA promoter from Ecc strainSCC3193 used in this construct contained a putativeExpR-box, similar to the ExpR-binding site defined inEcc71 (Chatterjee et al., 2005). Our results fromb-glucuronidase (GUS) assays in the following mutantbackgrounds expI, expI expR1, expI expR2 and expIexpR1 expR2 demonstrate that in the absence of AHSLExpR1 and ExpR2 were both, either together or sepa-rately, able to activate the expression of rsmA (Fig. 5A). Incontrast, in the expI expR1 expR2 triple mutant the rsmAexpression was significantly down-regulated with orwithout the addition of AHSLs suggesting that at least oneof the ExpRs is needed for the transcriptional activation ofrsmA.

To further elucidate the role of ExpR1 and ExpR2 in thetranscriptional activation of rsmA the effect of 3-oxo-C6-HSL and 3-oxo-C8-HSL was characterized (Fig. 5A).Addition of the cognate AHSL of SCC3193 3-oxo-C8-HSL,resulted in a substantially decreased GUS activity in bothexpR1 and expR2 mutants. This argues that both ExpR1and ExpR2 are able to bind the cognate autoinducer,which will subsequently prevent the activation of rsmA.The residual GUS activity was at the level of that found inthe expI expR1 expR2 triple mutant. In contrast addition ofthe non-cognate AHSL 3-oxo-C6-HSL led to substantiallydecreased GUS activity only in the expI expR1 mutant,likely due to release/inactivation of ExpR2. ExpR2 wasnot able to activate the transcription of rsmA in the pres-ence of 3-oxo-C6-HSL, while under the same growth con-ditions ExpR1 activates the transcription of rsmA in theexpI expR2 mutant. These data demonstrate that in theabsence of AHSLs either of the ExpRs is sufficient fortranscriptional activation of rsmA, which in turn down-regulates expression of PCWDE genes. These data alsosuggest that accumulation of the cognate AHSL (3-oxo-C8-HSL) will release both ExpRs from rsmA leading tocoordinate activation of PCWDE genes. Interestingly,also the non-cognate AHSL (3-oxo-C6-HSL) appears torelease/inactivate one of the ExpRs, ExpR2, suggestingthat signals from other bacterial species may modulatethis response.

The binding of AHSL by ExpR1 and ExpR2 correlateswith rsmA transcriptional activity

To provide additional evidence for the mode of action ofthe ExpR1 and ExpR2 proteins and to elucidate whetherthe transcription of rsmA is indeed controlled by AHSL

Fig. 4. Effect of expR1 and expR2 on populationdensity-dependent regulation of exoenzymes. Northern blotanalysis of total RNA from Ecc wild-type SCC3193, expR1 expR2double mutant (SCC907) and expI expR1 expR2 triple mutant(SCC906). Total RNAs were extracted at logarithmic, OD600 1.0 (1)and stationary, OD600 1.8 (2) phase of growth. RNA was hybridizedwith celV, pehA, pelB and 16S rRNA probes. At the bottom thesame strains, as used for Northern analysis, are grown on a CMCplate for 4 h and 7 h and detected as earlier described.

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binding to ExpRs, we measured AHSL binding using thesame strains that were characterized for rsmA transcrip-tional activation (Fig. 5B). As expected the triple mutant,lacking both ExpR proteins, was not able to bind either ofthe AHSLs used, indicating that the presence of ExpRproteins is indeed required for AHSL binding. In contrast,the expI mutant with both ExpR proteins present bindseffectively both the cognate 3-oxo-C8-HSL and the non-cognate 3-oxo-C6-HSL. Binding of the cognate AHSL inexpI mutant background was clearly more effective than in

strains lacking either one of the ExpR proteins indicatingthat ExpR1 and ExpR2 cooperate in this binding. TheAHSL binding studies also support the observed specific-ity of the AHSL action. The expI expR1 double mutant withonly ExpR2 present was able to bind 3-oxo-C6-HSLequally well as the expI mutant indicating that 3-oxo-C6-HSL binding can be explained by the binding capacity ofExpR2. On the other hand the expI expR2 mutant with thespecific ExpR1 protein present was as expected able tobind only the cognate 3-oxo-C8-HSL. These AHSLbinding results correlate well with the ExpR mediatedrsmA regulation shown with the rsmA promoter fusionassay. Taken together these results strongly indicate thatExpR1 and ExpR2 act synergistically in binding of AHSLsand suggest that this binding leads to down-regulation ofrsmA transcription.

Role of RsmA in QS regulation of PCWDEs

Our data show that QS controls rsmA expression andsuggest that QS regulation of PCWDEs could be medi-ated by RsmA. However, our data does not exclude thepresence of other RsmA-independent mechanisms bywhich ExpR proteins could regulate the PCWDEproduction. To explore this possibility we employed anexpI rsmA double mutant. The effect of rsmA on PCWDEproduction was assessed by comparing Cel activities ofan expI mutant and an expI rsmA double mutant in thepresence and absence of AHSLs (Fig. 6; Table 1). Inaccordance with earlier studies, using another Ecc strain(Chatterjee et al., 1995), the expI rsmA double mutant ofSCC3193 showed a Cel positive phenotype even in theabsence of AHSL supporting the role of RsmA as a majorQS regulator of PCWDE production in Ecc SCC3193.Interestingly, addition of the cognate, but not the non-cognate AHSL to the expI rsmA double mutant resulted insomewhat higher Cel activity. To quantify this effect weassayed Cel activity and could show a 30% increase in

Fig. 5. A. ExpR protein is needed for the full transcription of rsmA.b-glucuronidase assay of Ecc expI mutant (SCC3065), expI expR1mutant (SCC6005), expI expR2 mutant (SCC908) and expI expR1expR2 mutant (SCC906) carrying rsmA-gusA promoter fusion on aplasmid (pSMS18). Samples were taken from bacteria grown to thelogarithmic phase, OD600 1.5, in L medium with no AHSL or addedwith 1 mM 3-oxo-C6-HSL or 1 mM 3-oxo-C8-HSL. Similar resultswere gained when bacteria were grown in M9 medium (data notshown).B. Specific and non-specific binding by ExpR1 and ExpR2.Bioluminescence assay was used to determine the AHSL bindingcapacity of Ecc expI mutant (SCC3065), expI expR1 mutant(SCC6005), expI expR2 mutant (SCC908) and expI expR1 expR2mutant (SCC906) carrying rsmA-gusA promoter fusion on aplasmid (pSMS18). Same samples as in (A) were used. Similarresults were obtained when the AHSL binding capacity of ExpRproteins was determined with LC-MS (data not shown). However,as expected in these analyses no AHSL was detectable in thecontrols without added autoinducer.

Fig. 6. An expI rsmA mutant strain has slightly enhanced Celactivity with the cognate AHSL. Ecc expI mutant (SCC3065) andexpI rsmA mutant were grown in L medium added with no AHSL,3-oxo-C6-HSL or 3-oxo-C8-HSL and Cel activity of 10 ml of growthculture supernatants were detected with CMC plates. Same resultswere gained on PGA plates (data not shown).

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Cel activity with the cognate AHSL added (Fig. 6). Theseresults suggest that QS control of PCWDE production islargely mediated by RsmA but that in response to thecognate 3-oxo-C8-HSL there is an additional RsmA-independent pathway to fine tune PCWDEs production.

The AHSL specificity of Ecc strain SCC3193 can bealtered with an ExpR protein from Ecc strain SCC1

To further elucidate the specificity of the QS systemamong Erwinia strains, we tested whether the system iscompatible with the LuxR-type protein expRSCC1 fromSCC1, a 3-oxo-C6-HSL producing strain of Ecc (Braderet al., 2005). The expRSCC1 gene was expressed in transin different expR1 and expR2 mutant backgrounds of theexpI mutant of SCC3193, and the specificity for 3-oxo-C6-HSL and 3-oxo-C8-HSL was characterized by assessingthe Cel phenotype (Fig. 7). Introduction of the expRSCC1gene into the triple mutant (expI expR1 expR2) back-ground repressed the Cel-positive phenotype of thismutant demonstrating that ExpRSCC1 is functional in thisheterologous background. The repression could bereleased by the cognate AHSL of the SCC1 strain (Braderet al., 2005) 3-oxo-C6-HSL, but not by the cognate AHSL(3-oxo-C8-HSL) of SCC3193 strain (Fig. 7). Thus, intro-duction of expRSCC1 into the triple mutant (SCC906)changed the strain’s AHSL sensing specificity from 3-oxo-C8-HSL to 3-oxo-C6-HSL. The specificity of the ExpRSCC1

protein was also evident when expRSCC1 was expressedin expI expR1 mutant background (SCC6005). The expIexpR1 mutant strain with the broader AHSL ligand speci-ficity was altered to the specific 3-oxo-C6-HSL sensingwhen expressing expRSCC1 in trans. Expression ofexpRSCC1 in trans in the presence of ExpR1 as in theexpI mutant (SCC3065) or the expI expR2 double mutantstrain (SCC908) resulted in a Cel-negative phenotypeindependent of the addition of AHSL. This might beexplained by the simultaneous presence of two AHSLreceptors ExpR1SCC3193 and ExpRSCC1 with distinct AHSLspecificities. These data demonstrate that the AHSLsensing specificity of the QS system in SCC3193 is deter-mined by the ExpR1 protein of either Ecc strain SCC3193or SCC1.

Lack of a functional quorum sensing system enhancesplant maceration

The QS system is one of the most important virulenceregulators in Ecc and controls PCWDE productionrequired for plant tissue maceration during infection. Toelucidate the role of the ExpR proteins on macerationcapacity, we inoculated potato tubers (Van Gogh cultivar)and Arabidopsis wild-type (Col-0) plants with wild-typestrain SCC3193, and the different mutants and the extentof maceration was determined (Fig. 8). As expected theavirulent expI mutant showed almost no maceration, whilethe expR1 expR2 double mutant and the expI expR1expR2 triple mutant strains showed even slightlyenhanced maceration compared to the wild-type (Fig. 8Aand B). Wild-type Arabidopsis plants were inoculated withthe same Ecc strains as used for potato above. Similarresults as in the potato assay were obtained with Arabi-dopsis: while no maceration was observed in plantsinoculated with the expI mutant, clear maceration wasevident in plants inoculated with wild-type, the expR1expR2 double mutant or the expI expR1 expR2 triplemutant (Fig. 8A). These results are in agreement with ourresults from both the PCWDE assays and Northern analy-sis, showing enhanced production of PCWDEs, the mainvirulence factors of Ecc in strains lacking both ExpRproteins.

Discussion

QS is central in the control of virulence and the productionof main virulence determinants like PCWDEs in the plantpathogen Erwinia carotovora ssp. carotovora (Ecc). Pre-vious studies have established that the QS system of Eccstrain SCC3193 consists of AHSL synthase, encoded by

Table 1. The Cel activity was additionally measured with a quantita-tive Cel assay, measuring the amount of nmol reduced sugar/h/OD600

released.

Ecc strain

AHSL added

– 3-oxo-C6-HSL 3-oxo-C8-HSL

expI 20 ± 8 17 ± 13 60 ± 4expI rsmA 96 ± 9 97 ± 9 130 ± 1

Parallel samples to the experiment in Fig. 6 were used.

Fig. 7. The QS system of Ecc strain SCC3193 could be altered toa 3-oxo-C6-HSL sensing system by using expRSCC1. expRSCC1was expressed in trans in expI expR1 expR2 triple mutant(SCC906), expI expR2 mutant (SCC908), expI expR1 mutant(SCC6005) and the expI mutant (SCC3065). Strains were grown inL medium complemented with no AHSL, 3-oxo-C6-HSL or3-oxo-C8-HSL and analyzed for there Cel activity as earlierdescribed.

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the expI gene, producing 3-oxo-C8-HSL as cognateAHSL, and a convergently transcribed expR gene, encod-ing a QS regulator ExpR (renamed ExpR1 in this article),which we proposed to be a negative regulator of PCWDEsin the absence of AHSL (Pirhonen et al., 1993; Anderssonet al., 2000; Brader et al., 2005). In this study we presentseveral major results: First we demonstrate that two ExpRproteins, ExpR1 and ExpR2, exist in Ecc SCC3193 andthat ExpR1 and ExpR2 act synergistically to repress theproduction of PCWDEs and thus also virulence in theabsence of AHSL. Second, we show that the two ExpRproteins produced by the Ecc strain SCC3193, have dis-tinct AHSL recognition and binding specificities withExpR1 responsible for recognition of cognate AHSL andExpR2 responsible for recognition of both cognate and

non-cognate AHSLs. Third, we provide data suggestingthat the ExpR control of PCWDE gene expression is to alarge extent, but not solely, mediated by the negativeregulator RsmA (Fig. 9).

The lack of a clear PCWDE phenotype in the expR1mutant (Andersson et al., 2000) suggested a redundancyin the AHSL recognition system of Ecc SCC3193. Thatthis was indeed the case was demonstrated in this studyby identifying a second LuxR-type protein ExpR2 in EccSCC3193. Our studies clearly demonstrated that (i) bothExpR1 and ExpR2 function as AHSL receptors andindeed bind AHSLs and (ii) they act as repressors ofPCWDE production in the absence of AHSLs. By inacti-vating the QS system of SCC3193, including the expI,expR1 and expR2 genes, we could show that the triplemutant strain was able to produce wild-type levels ofPCWDEs without the addition of any AHSLs and macer-ated plant tissue as well as or even better than the wild-type. The presence of either ExpR1 or ExpR2 in the expImutant background essentially abolished the productionof PCWDEs in the absence of AHSLs. This repressionwas relieved by addition of the cognate 3-oxo-C8-HSL toboth expI expR1 and expI expR2 double mutants.

These results suggest a novel QS mechanism wheretwo partially redundant QS regulators act in concert tocontrol a single characteristic, i.e. PCWDE production.The repression by single ExpRs was not always absolute:an expI expR1 double mutant showed partial relief fromthe PCWDE repression, observed as haloes around bac-terial colonies grown on CMC indicator plates (data notshown). This was not seen from the culture supernatantsof liquid cultures probably due to still too low level ofPCWDE production. This result is in agreement withrecent findings that show partial restoration of PCWDEproduction in an expI expR double mutant of another Eccstrain (Cui et al., 2005) and the slight increase inPCWDEs in the expR1 single mutant strain (Anderssonet al., 2000). Also the recently sequenced Eca strainSCR1043 (Bell et al., 2004) harbors two expR genes in itsgenome that are also organized in a similar fashion as inEcc SCC3193 suggesting a similar type of QS regulationas observed here. During preparation of this manuscriptBurr et al. (2006) showed mutant analysis of the expR2homologue of Eca (virR) suggesting that this gene codesfor a repressor of PCWDE production. Interestingly, thevirR mutant of Eca showed partial restoration of PCWDEproduction suggesting that the Eca ExpR (Ecc ExpR1homologue) might not be as strong repressor of PCWDEsas ExpR1 in Ecc.

How is the control of PCWDE production by AHSLs andthe two ExpR proteins executed? We showed that thetranscript levels of the PCWDE genes are affected. North-ern analysis was used to this aim and demonstrated thatat early growth phase the transcript levels examined were

Fig. 8. A. Inactivation of expR1 and expR2 slightly enhancesvirulence. Potato tubers (Van Gogh) and Arabidopsis (Col-0) wereinoculated with following Ecc strains: wild-type SCC3193, expImutant (SCC3065), expR1 expR2 mutant (SCC907) and expIexpR1 expR2 triple mutant (SCC906) using a bacterial inoculum of105 cfu ml-1 72 h after inoculation potato tubers were cut in themiddle, to observe the maceration.B. The macerated tissue of the inoculated potatoes was weighed todetermine the size of virulence symptoms. Significantly differentvalues indicated by different letters (P � 0.05) were calculated byone way ANOVA followed by LSD test.

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indeed constitutive in the expR1 expR2 double mutant.Here we present for the first time evidence showing thatthe ExpR proteins are truly responsible for the delay inPCWDE gene expression and demonstrate that QS isessential for population density-dependent regulation ofthese genes. These data do not, however, demonstrateweather the ExpR proteins directly control PCWDE genesor weather this regulation is executed at some other level.Recent data from Cui et al. (2005) indicated that theglobal negative regulator RsmA is controlled by ExpR inanother Ecc strain. To explore this possibility in Ecc

SCC3193 we employed an rsmA-gusA promoter fusionand demonstrated that ExpR-dependent repression ofPCWDE genes was apparently due to transcriptionalcontrol of rsmA. In the absence of AHSL both ExpR1 andExpR2 were able to activate rsmA transcription, leading todown-regulation of PCWDE genes (Figs 5A and 9) whilethe addition of the cognate 3-oxo-C8-HSL abolished thisactivation leading to up-regulation of PCWDE genes. Tofurther characterize the mechanism of this regulation weexamined the AHSL binding capacities of the differentexpR mutant strains. These results clearly show that

Fig. 9. A schematic model for regulation of PCWDEs by ExpR1 and ExpR2 in a signal dependent way mediated largely via RsmA in Ecc. Inthe absence of AHSLs both ExpR1 and ExpR2 activate the expression of rsmA. An up-regulated rsmA results in decreased exoenzymeproduction. After addition of 3-oxo-C6-HSL the ExpR2 is released from the transcriptional activation of rsmA, but ExpR1 is still presentactivating rsmA. But, in the presence of 3-oxo-C8-HSL both ExpR1 and ExpR2 are released from activating rsmA, which results in theproduction of exoenzymes. Striped arrows indicate a possible RsmA-independent regulation of PCWDEs.

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ExpR1 and ExpR2 are able to bind either together orseparately the cognate 3-oxo-C8-HSL. These resultsstrongly support the results gained with the rsmA tran-scription activation assay. This model is in agreement withthe data by Cui et al. (2005) showing that in the Ecc strainEcc71 the ExpREcc71 protein binds to the promoter regionof rsmA and activates its transcription in the absence ofAHSLs and that the DNA binding of ExpREcc71 and henceactivation of rsmA transcription was inhibited by the pres-ence of the cognate AHSL (Chatterjee et al., 2005; Cuiet al., 2005).

Although rsmA-mediated repression appears to be themain control of PCWDE production we show that this isnot the only pathway by which the AHSLs exert theircontrol. Interestingly, an expI rsmA double mutant was stillable to respond to the cognate 3-oxo-C8-HSL andincrease its Cel activity accordingly. This result suggests afurther fine-tuning in the system. This could be accom-plished by a dual function of ExpR proteins (Fig. 9)whereby the AHSL-bound forms of ExpR could act asdirect or indirect regulators of PCWDE production. Alter-natively ExpR could control expression of another yetunknown negative regulator of PCWDEs similarly to itscontrol of rsmA.

In most known AHSL receptors the N-terminal ligandbinding domain has been shown to be very specific dis-tinguishing between cognate and non-cognate ligands(Luo et al., 2003; Chai and Winans, 2004). We proposethat (Fig. 9) in Ecc the two ExpR proteins have synergis-tic, but individual roles, with ExpR1 acting as a cognateligand specific regulator, while ExpR2, interestingly,responds to both the cognate and the non-cognatesignals. We demonstrate that ExpR2 has a broad AHSLsensing capacity, in contrast to the other LuxR-type pro-teins of Erwinia strains known today (Welch et al., 2000;Chatterjee et al., 2005). This conclusion is based on fourkey observations: First, recently we showed that thePCWDE activity of an expI mutant of Ecc strain SCC3193could only be restored with physiological levels of thecognate AHSL 3-oxo-C8-HSL (Brader et al., 2005). Herewe show that the expI expR1 double mutant of SCC3193had lost its specificity for the cognate AHSL and reactedalso to non-cognate AHSLs, such as 3-oxo-C6-HSL indi-cating the presence of another AHSL receptor. We sub-sequently identified a second ExpR protein (ExpR2) thatwas shown to be responsible for the sensing of the non-cognate AHSLs. Second, by monitoring Cel activity weshow that ExpR2 senses AHSLs with acyl chains rangingbetween C6 and C10. The inability to sense the AHSLswith even longer acyl chains (C12 and C14), mightdepend on the lack of a reasonable transport system forthese long chain AHSLs into the cell (Fuqua et al., 2001).Third, in the expI expR1 double mutant the transcription ofrsmA can be prevented with either 3-oxo-C6-HSL or

3-oxo-C8-HSL, and leading to subsequent production ofPCWDEs. In contrast expression of rsmA is only abol-ished by the addition of 3-oxo-C8-HSL in, the expI expR2mutant (Fig. 9). Fourth, we show that the expI mutant bindboth 3-oxo-C6-HSL and 3-oxo-C8-HSL, while the the expIexpR2 mutant is only able to bind 3-oxo-C8-HSL. This isa clear evidence that ExpR2 is responsible of the bindingof 3-oxo-C6-HSL.

The special feature of ExpR2 to respond to severaldifferent AHSLs raises questions of its role. Why does thisEcc strain have both a specific and an unspecific QSregulator? The existence of expR2 could be a conse-quence of horizontal gene transfer from other bacteria(Pappas et al., 2004). This is supported by the fact thatexpR2 is located separately from the expI-expR1cassette. A biological advantage of having a receptor forvarious non-cognate AHSLs could be the ability to identifyneighboring bacteria by sensing the accumulation of dif-ferent kinds of AHSLs. Thus eavesdropping on possiblecompetitors (Lazdunski et al., 2004; Waters and Bassler,2005) or establishing cooperation with other bacteria tooverwhelm the plant host could be beneficial for thesuccess of Ecc as a pathogen. The special feature ofExpR2 to recognize AHSLs produced by its own speciesin addition to AHSLs produced by neighboring bacteriacould further enhance survival in a crowded niche. It isalso possible that ExpR2 in addition possess uniquetarget sites not recognized by ExpR1 and involved ininteractions with other bacterial species.

We present in this article a novel mechanism for QS(Fig. 9): the simultaneous need for two individual, butcooperatively acting QS regulators in controlling expres-sion of the same target gene. In this study we have shownthat ExpR1 and ExpR2 can function as activators eitheralone or synergistically when both proteins are present.Our results suggest that the amount of bound AHSLscorrelate with the amount of ExpR proteins present andthat the AHSL binding of the ExpR proteins correspond tothe transcriptional activity of rsmA. We propose thatExpR1 and ExpR2 act in synergy integrating cognate andnon-cognate AHSL signals to control expression of acentral regulatory gene rsmA. However, we show thatthey can also act independently and therefore are notnecessarily physically interacting. LuxR-type proteins usedifferent modes of action and operate in various waysdepending on bacterial species and tasks to beaccomplished. A hierarchical organization model whereone LuxR-type protein regulates the transcription ofanother LuxR-type protein is described in, e.g. Yersiniapseudotuberculosis (Atkinson et al., 1999) andPseudomonas aeruginosa (Pesci et al., 1997). The pos-sibility that ExpR1 would control expression of expR2 orvice versa, was tested using expR-gusA promoterfusions, but no transcriptional regulation between ExpR1

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and ExpR2 was observed (data not shown). Thus weprefer a model where the two ExpR proteins directlycontrol expression of the rsmA gene and possible othertarget genes. LuxR-type proteins, such as CarR, LuxRand TraR dimerize/multimerize after binding AHSL leadingto subsequent stabilization of the protein and activation oftarget gene expression (Qin et al., 2000; Welch et al.,2000; Urbanowski et al., 2004). In contrast, the ExpRproteins appear to bind target DNA and act as activatorsin the absence of AHSL and then be inactivated/releasedby AHSLs. Whether these states involve dimerizationremains to be demonstrated. Alternatively it could be pos-sible that after AHSL binding, the two ExpR proteins stayat the target gene promoter just shifting in their conforma-tion to block the function of the RNA polymerase. E.g.RhlR, the AHSL receptor of Pseudomonas aeruginosahas such a dual role always binding to DNA, but acting asan activator or a repressor depending on the presence ofAHSL (Medina et al., 2003). An open question is alsowhether the ExpR1 and ExpR2 bind the same promotersite or are there possibly several ExpR binding sites in thersmA promoter? In the rsmA promoter of Ecc71 one ExpRbinding site was recently identified (Chatterjee et al.,2005). We could identify a similar DNA element (ExpRbox) in the rsmA promoter of Ecc strain SCC3193 and inthe sequenced Eca strain SCRI1043. Furthermore, wecannot rule out the possibility that the ExpR proteins ofEcc SCC3193 have a dual role, working both in an AHSLbound state and in a ligand-free state. This hypothesis ispartly supported by the results with the expI rsmA mutantstrain, showing enhanced Cel activity only with the addi-tion of the cognate 3-oxo-C8-HSL. Such a model has alsobeen proposed for EsaR of Pantoea stewartii that binds toits target promoter in the absence of AHSL and is releasedby the addition of AHSL (Minogue et al., 2005).

The ability of Ecc strains lacking the whole QS systemto grow and macerate plant tissues as well as the wild-type under laboratory conditions indicate that the biologi-cal relevance of the QS system is mainly in the naturalhabitat, where the densities and the composition of bac-terial populations fluctuate in response to environmentalcues. An ecological study would be essential to eluci-date the significance of QS in controlling the success ofEcc in the environment (Manefield and Turner, 2002;Redfield, 2002; Toth and Birch, 2005; Keller and Surette,2006).

Experimental procedures

Bacterial strains and media

Bacterial strains and plasmids used in this study are listed inTable 2. Escherichia coli strains were cultured in L medium(Miller, 1972) at 37°C and Erwinia carotovora ssp. carotovorastrains at 28°C. Ampicillin (Amp) was added to media at

150 mg ml-1, chloramphenicol (Cm) at 50 mg ml-1 and kana-mycin (Km) at 50 mg ml-1 when required and if not otherwisementioned. AHSLs were used at a concentration of 1 mM ifnot otherwise mentioned.

Library screening and construction of mutant strains

Recombinant DNA techniques were used according to stan-dard procedures (Sambrook and Russell, 2001). PCR ampli-fications were performed with proofreading Pfu polymerase(Stratagene) and Dynazyme II (Finnzymes). Based on thesequence of Eca strain SCRI1043 an expR2 specific probewas PCR amplified from wild-type SCC3193 genomic DNAusing primers ProbR2F (5�-TCTGTATTTTGCTCTGATAA-3�)and ProbR2R (5�-CAGATCGCCATACTGTTTTA-3�). TheexpR2 probe was used to screen Lambda DASH library(Stratagene) containing 17–22 kb BamHI fragments ofSCC3193. An expR2 positive clone was amplified for lambdaDNA isolation and the purified DNA containing an approxi-mately 20 kb insert was cut with BamHI and EcoRI andverified with Southern blot analysis to be expR2 positive.Primers cdhF (5�-TGATTGCTATAGGTCCTCAG-3�) andcheR (5�-GGTGAGGTTTGTTCTCTCATC-3�) were used toPCR amplify a 3 kb DNA fragment, for subsequent sequenc-ing and cloning procedures. To obtain a deletion mutant ofexpR2, the upstream region of the expR2 gene was am-plified by PCR from SCC3193 with primers expRBapaR(5�-CCGGGCCCCCTGCGGCTATTGTGATAACG-3�) andexpRBhindF (5�-CCAAGCTTTCTGGCTGCGTTATCGATTATG-3�). The resulting 1461 bp PCR product was digestedwith ApaI and HindIII resulting in a 889 bp DNA fragment (thereduction in DNA length was due to an unobserved HindIIIrestriction site) and inserted into these sites in pBluescript(pSMS100). The cat gene and the kanamycin resistance (km)gene was PCR amplified with following primers: CatSmaF(5�-CCCCCGGGTTCGACCGAATAAATACCTGT-3�) andCatHindR (5�-CCAAGCTTCTATCGTCAATTATTACCTCCA-3�); KmSmaF (5�-CCCCCGGGCAGCTACTGGGCTATCTGGA-3�) and KmHindR (5�-CCAAGCTTGCGTCAATACGGGATAATAGTG-5�). Each fragment containing an antibioticresistance marker gene was digested with SmaI and HindIIIand inserted to pSMS100 to confer one chloramphenicol andone kanamycin resistant plasmid pSMS103(Km) andpSMS103(Cm). The down-stream region of expR2 gene wasPCR amplified with primers expRBspeF (5�-GACTAGTGTGTAGCGTAGTCAGGCAAC-3�) and expRBsmaR(5�-CCCCCGGGCTACTGTTACCCCATGATATCAC-3�). Theproduct was digested with SpeI and SmaI and inserted intoplasmids pSMS103(Km) and pSMS103(Cm) digested withsame enzymes, resulting in the constructs pSMS104(Km)and pSMS104(Cm). The DNA fragment (expR2::Km;expR2::Cm) of pSMS104 (Km/Cm) was digested with ApaIand SpeI and inserted into suicide vector pGP704 digestedwith same enzymes. The resulting plasmids pSMS105/Kmand pSMS105/Cm were transformed into E. coli S17-1 lpir and further transformed by conjugation into Eccstrain SCC3193, SCC3065 (expI), SCC5003 (expR1) andSCC6005 (expI expR1) (de Lorenzo and Timmis, 1994).Transconjugants were plated on either M9 minimal mediumsupplemented with 0.2% sucrose and chloramphenicol10 mg ml-1 or kanamycin 10 mg ml-1 or on L medium

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plates supplemented with chloramphenicol 50 mg ml-1 andkanamycin 50 mg ml-1. The resulting strains SCC905(Cm) (expR2::Cm), SCC905 (Km) (expR2::Km), SCC906(expIexpR1::Cm expR2::Km), SCC907 (expR1::CmexpR2::Km) and SCC908 (expI::Km expR2::Cm) were veri-fied to be CmR AmpS, KmR AmpS or CmR KmR AmpS andconfirmed genotypically.

Construction of plasmids

The expR1 and expR2 genes were amplified by PCR fromwild-type SCC3193 genomic DNA using the primers:ExpR(3193)eco2 (5�-CGGAATTCGAGATGTCGCAGTTATTCTACA-3�) and ExpR(3193)bam (5�-CGGGATCCGCCTATGACTGAACCGGTCG-3�); ExpR2 SMS17 Rev (5�-CGGGATCCCTATAGTGGTTCTGGCTTGATG-3�) and ExpRB pOKF(5�-CGGAATTCATGTCTGTATTTTGCTCTGATAATG-3�).The 737 bp expR1 PCR product and the 738 bp expR2 PCRproduct was digested with BamHI and EcoRI and ligated intopQE30, digested with corresponding enzymes, resulting inpSMS20 and pSMS21, respectively. The expRSCC1 wasamplified by PCR from wild-type SCC1 genomic DNA usingprimers: expR(1)eco (5�-CGGAATTCGAGATGTCGCCATTATTCACTG-3�) and expR(1)bam (5�-CGGGATCCTACCTGCCGCTATTGCACAGG-3�). The 729 bp expRSCC1 PCRproduct was digested with BamHI and EcoRI and ligated intopQE30, digested with corresponding enzymes, resulting inpSMS22. For promoter fusion studies a plasmid (pSMS18)

was constructed containing the rsmA promoter region andpartial coding sequence amplified by PCR using primers:RsmAF prom (5�-GCGTCGACCTGTTGTTGTGATAACAAAAG-5�) and RsmAR prom (5�-CCAAGCTTACCGTTACCTCATCGCCGA-3�). The 189 bp PCR product was digestedwith SalI and HindIII and ligated into pGUS102 digested withcorresponding enzymes.

RNA isolation and Northern blot analysis

Erwinia cells from overnight cultures were diluted 1/100 in Lmedium and grown at 28°C. Samples for RNA isolation weretaken at indicated time points and the growth was monitoredby measuring the OD600. Total RNA was isolated as describedby Sambrook et al. (1989). Northern analysis was performedwith 10 mg of total RNA separated in 1.5% formaldehyde gel.Filters were probed with specific digoxigenin labeled DNAfragments for celV1, pehA, pelB and 16S rRNA (Hyytiäinenet al., 2003). The blotting, hybridization and digoxigenindetection was performed according to the instructions of themanufacturer (Roche).

Enzyme assays

Cellulase (Cel) activities were analysed from 10 ml of super-natant of overnight grown liquid cultures on CMC indicatorplates using Congo Red to determine the cellulose diges-

Table 2. Bacterial strains, plasmids used in this study.

Strain or plasmid Genotype or description Reference

StrainsE. coliDH5a endAI hsdR17 supE44 thi-1 gyrA96 relA1 DlacU169 (f80 dLacD�15) Hanahan (1983)S17-1 l pir TpR SmRrecA, thi, pro, hsdR–M+ RP4 : 2-Tc: Mu: Km Tn7, l pir Miller and Mekalanos (1988)Erwinia carotovorasubsp carotovoraSCC1 Wild-type Pirhonen et al. (1988)SCC3193 Wild-type Saarilahti and Palva (1986)SCC3065 expI::km, KmR in SCC3193 background Pirhonen et al. (1991)SCC6005 expI expR1::cm, CmR in SCC3193 background Andersson et al. (2000)SCC5003 expR1::cm, CmR in SCC3193 background Andersson et al. (2000)SCC905 expR2::cm, CmR; expR2::km, KmR in SCC3193 background This workSCC906 expR2::km, KmR in SCC6005 background This workSCC907 expR2::km, KmR in SCC5003 background This workSCC908 expR2::cm, CmR in SCC3065 background This workexpI rsmA mutant DrsmA in SCC3065 background Andersson (unpublished)

PlasmidspSB402 pBR322 with luxRI� and a promoteless luxCDABE cassette Guard-Petter (1998)pBluescript SK+ Cloning vector, AmpR StratagenepGP704 Suicide vector, AmpR Miller and Mekalanos (1988)pQE30 Expression vector, AmpR QiagenpGUS102 pBR322 with promoterless uidA from E. coli Andersson et al. (2000)pSMS20 expR1SCC3193 cloned into pQE30 EcoRI and BamHI sites This workpSMS21 expR2SCC3193 cloned into pQE30 EcoRI and BamHI sites This workpSMS22 expRSCC1 cloned into pQE30 EcoRI and BamHI sites This workpSMS18 rsmASCC3193 promoter (189 nt) and partial CDS (67 nt) cloned into pGUS102

SalI and HindIII sitesThis work

pSMS100 889 bp DNA fragment containing upstream region of expR2 This workpSMS103 cat gene and km gene cloned into pSMS100 This workpSMS104 expR2SCC3193::Km, expR2SCC3193::Cm cloned into pBluescript ApaI and SpeI sites This workpSMS105 expR2SCC3193::Km, expR2SCC3193::Cm cloned into pGP704 ApaI and SpeI sites This work

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tion (Pirhonen et al., 1993). Quantitative Cel assay wasperformed as described previously (Pirhonen et al., 1991).For b-Glucuronidase (GUS) activity assay Erwinia cellsfrom overnight cultures were diluted 1/100 in L medium andgrown at 28°C. Samples for GUS activity assay were takenat indicated time points and the growth was monitoredby measuring the OD600. GUS activity was measured byusing p-nitrophenyl b-D-glucuronide as substrate (Novelet al., 1974; Marits et al., 2002). r-nitrophenol (r-NP),was detected at an absorbance of 405 nm and the specificactivity of GUS was expressed as nmol r-NP liberatedmin-1.

Assay for AHSL Binding

The samples for determining the AHSL binding capacitywere prepared as follows: 15 ml of bacteria was grown in Lmedia complemented with no AHSL, 1 mM 3-oxo-C6-HSL or1 mM 3-oxo-C8-HSL. The overnight grown bacterial cellswere collected and washed twice with 0.9% NaCl to removeAHSL from the supernatant. Washed cells were then resus-pended in 1.5 ml of lysis buffer (50 mM NaH2PO4 pH 8.0,300 mM NaCl, 10 mM imidazole) and lysozyme was added.After 30 min incubation on ice, cells were sonicated and thecell debris was removed. The AHSLs were extracted twicewith equal amounts of ethylacetate and the extracts dried ina Speed-Vac, with subsequent resuspension into 30 mlacetonitrile:0.1% formic acid (1:1 v/v) for LC-MS analysis asdescribed (Brader et al., 2005) or 50 ml L-medium for biolu-minescence assays. Here, overnight grown E. coli carryingpSB402 (Guard-Pette, 1998) was diluted 1:100 and grownfor 5 h. After this 50 ml of E. coli and the 50 ml AHSL extractwas mixed and incubated for 2 h. The bioluminescence wasmeasured with the 1420 multilabel counter VICTOR2.

Synthesis and analysis of AHSLs

AHSL standards have been purchased from Sigma-Aldrich(C7-, C8-, 3-oxo-C6-HSL) or synthesized (C4-, C6-, C12-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C14-HSL) asdescribed (Zhang et al., 1993). AHSL standards and profilesof culture supernatants have been analyzed by LC-MS asdescribed earlier (Brader et al., 2005).

Assay of maceration capacity

Erwinia strains were grown overnight, diluted into 0.9% NaCland samples containing 105 bacterial cells ml-1 were used forinoculation of potato tubers (Solanum tuberosum cv. VanGogh). The inoculation site was bored with a sterile toothpick.Infected potatoes were incubated at 28°C for 72 h underhumid conditions with wet tissue paper in the incubation box.The amount of soft rot was measured by cutting the potatotubers in half and scraping and subsequently weighing therotted tissue. Arabidopsis thaliana Col-0 was infiltrated with asyringe without needle with 105 bacterial cells ml-1 preparedas described above. The development of disease symptomswas documented after 48 h, kept in 22°C, 16 h light and highhumidity.

Nucleotide sequence accession number

The DNA sequence data determined in this study has beensubmitted to the DDB/EMBL/GenBank databases underaccession numbers DQ333187 and DQ333188.

Acknowledgements

We thank Leila Miettinen for excellent technical assistance.We thank Hannu Saarilahti (University of Helsinki, Finland)for kindly providing us the SCC3193 Lambda Dash II library.We thank Robert Andersson for kindly providing us the expIrsmA mutant. This study was supported by the HelsinkiGraduate School in Biotechnology, Molecular Biology andAcademy of Finland (projects 388033, 44252 and 44883;Finnish Centre of Excellence Programme 2000-05), Biocen-trum Helsinki and a grant from Leonardo da Vinci II Pro-gramme (to G. K.).

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