Cooperation of two distinct ExpR regulators controls quorum sensing specificity and virulence in the...

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

Solveig Sjöblom, Günter Brader, Gudrun Koch and

E. Tapio Palva*

Viikki Biocenter, Faculty of Biosciences, Department of

Biological and Environmental Sciences, Division of

Genetics, University of Helsinki, P.O.B. 56, FIN-00014,

Helsinki, Finland.

Summary

Quorum sensing, the population density-dependent

regulation mediated by N-acylhomoserine lactones

(AHSL), is essential for the control of virulence in the

plant pathogen Erwinia carotovora ssp. carotovora

(Ecc). In Erwinia carotovora ssp. the AHSL signal with

an acyl chain of either 6 or 8 carbons is generated by

an AHSL synthase, the expI gene product. This work

demonstrates that the AHSL receptor, ExpR1, of Ecc

strain SCC3193 has strict specificity for the cognate

AHSL 3-oxo-C8-HSL. We have also identified a

second AHSL receptor (ExpR2) and demonstrate a

novel quorum sensing mechanism, where ExpR2 acts

synergistically with the previously described ExpR1

to repress virulence gene expression in Ecc. We show

that this repression is released by addition of AHSLs

and appears to be largely mediated via the negative

regulator RsmA. Additionally we show that ExpR2 has

the novel property to sense AHSLs with different acyl

chain lengths. The expI expR1 double mutant is able

to act in response to a number of different AHSLs,

while the expI expR2 double mutant can only respond

to the cognate signal of Ecc strain SCC3193. These

results suggest that Ecc is able to react both to the

cognate AHSL signal and the signals produced by

other bacterial species.

Introduction

Quorum sensing (QS) is a central cell-to-cell communica-

tion system that bacteria employ to monitor their population

density and coordinate functions for which high population

density is profitable (Fuqua et al., 2001; Waters and

Bassler, 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 secondary

metabolites and virulence in both plant and animal patho-

gens (Whitehead et al., 2001). QS is required for the

production of virulence determinants and biofilm formation

in the opportunistic human pathogen Pseudomonas

aeruginosa (Winson et al., 1995), exopolysaccharide pro-

duction in the plant pathogen Pantoea stewartii (Beck von

Bodman and Farrand, 1995) and production of plant cell

wall degrading enzymes (PCWDEs) and antibiotics in

another plant pathogen Erwinia carotovora (Jones et al.,

1993; Pirhonen et al., 1993).

The LuxR/LuxI system controlling bioluminescence in

Vibrio fischerii was the first to be characterized and has

become the paradigm of QS. The QS system is minimally

executed by an AHSL synthase, a LuxI-type protein, and a

QS regulator, a LuxR-type protein, controlling transcription

of downstream genes (Fuqua et al., 2001; Whitehead et al.,

2001). There is high degree of specificity in QS determined

by substrate specificity of the AHSL synthase and specific

recognition of the cognate AHSL by the LuxR-type protein,

modulating the expression of QS-regulated target genes.

Different bacteria produce AHSLs with diverse acyl side

chain lengths, ranging from 4 to 16 carbons, and with

alterations in the oxidative status of carbon 3 (Fuqua and

Greenberg, 2002; Pappas et al., 2004). The LuxR-type

proteins act as QS regulators, and they distinguish

between different AHSLs by showing binding of cognate,

but not non-cognate AHSLs suggesting that they are

mainly involved in intraspecies signaling (Lazdunski et al.,

2004). The LuxR-type proteins share a similar structure

with 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, their

operative mechanisms can be different. Many LuxR-type

activators, including CarR, LuxR and TraR, the only crys-

tallized LuxR-type protein so far (Qin et al., 2000; Vannini

et al., 2002; Zhang et al., 2002), form dimers or multimers

upon binding to AHSLs (Whitehead et al., 2001; PappasAccepted 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

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

et al., 2004). This stable complex then binds to a target

gene promoter, in many cases to a 20 bp palindromic lux

box, in order to activate target gene transcription (Fuqua

et al., 2001; Lazdunski et al., 2004). In contrast, EsaR, a

LuxR-type protein of the plant-pathogen Pantoea stewartii

(Beck von Bodman and Farrand 1995) binds the target

DNA in the absence of AHSL and represses transcription,

but after addition of the cognate AHSL EsaR is thought to

be 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 Erwinia

carotovora 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 have

shown that AHSL synthesis is required for the production

of PCWDEs, such as cellulases, polygalacturonases and

pectinases, the main virulence determinants of this patho-

gen and that QS is responsible for density-dependent and

coordinated production of these enzymes to establish a

successful infection (Pirhonen et al., 1991; 1993).

Mutants deficient in the AHSL synthase (the expI gene

product) are impaired in the production of PCWDEs and

are thus avirulent (Pirhonen et al., 1991; 1993; Jones

et al., 1993). Depending on the strain, the main

AHSLs 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 strain

SCC3193 (Brader et al., 2005). The so far best charac-

terized LuxR-type protein of Ecc is CarR, which positively

regulates the production of carbapenem antibiotics, in

response to its cognate autoinducer 3-oxo-C6-HSL in the

Ecc strain GS101 (Welch et al., 2000).

The QS regulators controlling PCWDE production have

so far remained more elusive. We originally identified

ExpR of the Ecc strain SCC3193 as a potential QS regu-

lator (Andersson et al., 2000). Although mutations in expR

did not show a clear phenotype, overexpression studies

suggested that ExpR might act as repressor of PCWDE

synthesis (Andersson et al., 2000). Interestingly, von

Bodman et al. (2003) have demonstrated that ExpRSCC3193

can bind to DNA in the absence of AHSL, but that this

binding is inhibited by AHSL addition. A recent study by

Cui et al. (2005) showed that a related LuxR-type protein

ExpREcc71 from another Ecc strain Ecc71 binds to the

promoter of a target gene rsmA, activating its transcription

in an AHSL free state. Addition of the cognate AHSL

released the ExpREcc71 from this promoter leading to

repression of the target gene that encodes the global

negative regulator RsmA (Chatterjee et al., 2005; Cui

et al., 2005). RsmA is an RNA-binding protein that

represses the production of PCWDEs (Chatterjee et al.,

1995). These results supported a close relation between

RsmA and the QS system already indicated in previous

studies (Chatterjee et al., 1995; Kõiv and Mae, 2001).

In this study we identify a novel QS regulation where

two LuxR-type proteins, ExpR1 and ExpR2, act synergis-

tically as negative regulators of PCWDE production

in the Ecc strain SCC3193. This negative regulation

released by accumulation of AHSLs appears to be largely

mediated by the global negative regulator RsmA. Intrigu-

ingly, we demonstrate that the two ExpR proteins have

distinct AHSL specificities: while ExpR1 is specific to the

cognate AHSL, the newly identified ExpR2 protein shows

broad signal sensing capacity and responds also to non-

cognate AHSL, allowing both intra- and interspecies

communication.

Results

Inactivation of expR alters AHSL sensing specificity

We have demonstrated that the AHSL synthase encoded

by expI of Ecc strain SCC3193 (Pirhonen et al., 1993)

produces mainly 3-oxo-C8-HSL (Brader et al., 2005). An

expI mutant (SCC3065) of SCC3193 is not able to

produce the PCWDEs (Pirhonen et al., 1993), but can be

specifically 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 is

required to rescue the PCWDE negative phenotype of this

mutant (Brader et al., 2005). To address the role of the

ExpR protein of SCC3193 (Andersson et al., 2000) in this

recognition, we characterized the ability of the added

non-cognate 3-oxo-C6-HSL or cognate 3-oxo-C8-HSL to

restore 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 h

in L medium added with either no AHSL, 3-oxo-C6-HSL or

3-oxo-C8-HSL. Cel activity of 10 ml growth culture supernatant was

detected with CMC plates as described in Experimental

procedures.

B. For complementation studies expR was expressed in trans in

the 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 was

detected on CMC plates as in A.

Specificity of E. carotovora QS regulators 1475

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 60, 1474–1489

assay (Pirhonen et al., 1993). Interestingly, the presence

or absence of the expR gene had a profound effect on

AHSL sensing specificity; while the Cel activity of the expI

mutant was restored only by the cognate 3-oxo-C8-HSL,

in the expI expR double mutant the Cel production was

restored both by the non-cognate 3-oxo-C6-HSL and the

cognate 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 expR

double mutant (SCC6005) background. This resulted in

restoration of the requirement for the cognate 3-oxo-C8-

HSL in Cel production (Fig. 1). These results demonstrate

that the expR mutant has broader substrate specificity

and suggest that ExpR is involved in sensing of 3-oxo-

C8-HSL.

Identification of a second expR homologue in Ecc strain

SCC3193

The altered AHSL sensing specificity (Fig. 1) and the lack

of clear PCWDE phenotype (Andersson et al., 2000) of

the expR mutant could be explained by the existence of

an additional ExpR homologue or possibly another partly

redundant QS regulator responsible for sensing the non-

cognate 3-oxo-C6-HSL. Interestingly, two expR homo-

logues have been reported in the newly sequenced

genome of Erwinia carotovora ssp. atroseptica strain

SCRI1043 (Eca); one (expR) is linked to the expI gene

similar 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 SCC3193

we used the sequence data of Eca strain SCRI1043 to

design primers for PCR identification of a potential second

expR homologue. Subsequently the presence of a second

expR gene in SCC3193 was demonstrated (designated

expR2, with the previously identified/characterized expR

renamed as expR1). The genomic organization of

sequences flanking expR2SCC3193 is rather similar between

the two subspecies of Erwinia carotovora. Downstream of

the expR2SCC3193 gene is an open reading frame (orf) of

252 aa that is 83% identical to a CDP-diacylglycerol pyro-

phosphatase of Eca strain SCRI1043, while in the

upstream region an approximately five kb fragment,

present in the Eca genome, is lacking between expR2 and

the next orf (partially sequenced) that is 88% identical to

a chemotaxis signal transduction protein (ECA1568) of

the Eca strain SCRI1043 (Fig. 2A).

The expR2 orf consists of 735 bp encoding a putative

ExpR2 protein of 245 aa. Structural comparison of

ExpR2 with other LuxR-type proteins suggested that

ExpR2 is likely a QS regulator (Fig. 2B). The ExpR2

protein shows 94% aa identity (97% similarity) to a

potential QS regulator (ECA1561) of Eca SCRI1043 and

62% aa identity (81% similarity) to ExpR of Eca

SCRI1043, 58% aa identity (81% similarity) to ExpR1 of

Ecc SCC3193, 62% aa identity (80% similarity) to a

putative ExpR of Ecc SCC1, 54% aa identity to EsaR of

Pantoea stewartii ssp. stewartii and 22% aa identity

(46% similarity) to TraR of Agrobacterium tumefaciens

(Fig. 2B). The DNA binding domain is highly conserved

between the Erwinia carotovora and the Pantoea strains,

while the AHSL binding domains show more sequence

variety. Taken together, these results indicate that Ecc

SCC3193 has indeed two LuxR-type proteins and

suggest that this redundancy could explain the lack of a

clear PCWDE phenotype in expR1 mutants.

Inactivation of both expR1 and expR2 suppresses the

cellulase-negative phenotype of an AHSL deficient

strain

To explore the functional roles of the two ExpR proteins in

the QS system of E. carotovora we generated both single

and double expR1 and expR2 insertion mutants in the

wild-type and expI genetic backgrounds of SCC3193.

Interestingly, neither the expR1 or expR2 single mutants

nor the expR1 expR2 double mutant showed any clear

impairment of PCWDE production or virulence in the wild-

type SCC3193 background suggesting that the corre-

sponding proteins do not act as positive regulators of

virulence. As there was the distinct possibility that one or

both of the ExpR proteins of SCC3193 could function as

negative regulators of virulence and enzyme production as

suggested previously for ExpR1, EsaR and for the

ExpREcc71 and VirR of other E. carotovora strains (von

Bodman et al., 1998; Andersson et al., 2000; Cui et al.,

2005; Burr et al., 2006) we assessed the phenotypic effects

of expR1 and expR2 mutations in the expI mutant back-

ground. The results firstly demonstrate that the presence of

either 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 phenotypes

were identical in both rich and minimal medium. These data

strongly 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 could

be in relieving this repression at high population density.

AHSL specificity of ExpR1 and ExpR2

The generation of expI expR1 and expI expR2 double

mutants allowed us to explore the AHSL specificity of

1476 S. Sjöblom et al.


each ExpR protein. Mutant strains were grown in the

presence 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 the

expI expR2 mutant (SCC908) responded specifically to

3-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 the

direction of transcription, the striped arrow indicate a partially sequenced CDS. The dotted region implies sequence region with no homology

to 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 program

version 3.21 (Hofman and Baron, http://www.ch.embnet.org). Autoinducer binding domain is underlined and DNA binding domain is double

underlined.



http://www.ch.embnet.org

shown by the expI expR1 double mutant (SCC6005).

According to these results ExpR1 is a specific LuxR-type

protein, activating Cel production only after addition of

3-oxo-C8-HSL. At physiological AHSL concentrations

ExpR1 was indeed only able to respond to the cognate

signal 3-oxo-C8-HSL, while the addition of 3-oxo-C6-HSL

did not suppress the Cel negative phenotype of the expI

expR2 double mutant. Addition of 3-oxo-C6-HSL to a con-

centration of 10 mM or more was required to restore Cel

activity to this mutant (data not shown).

To determine the specificity of ExpR1 and ExpR2 as

AHSL receptors, expI expR1 and expI expR2 double

mutants 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, and

assayed for Cel activity (Fig. 3B). As suggested by previ-

ous results (Fig. 3A) ExpR1 appeared to have a narrow

AHSL specificity responding best to the cognate 3-oxo-

C8-HSL, although it also could respond to some extent to

3-oxo-C10-HSL and to C8-HSL. On the other hand ExpR2

appeared to be promiscuous and respond to all the

AHSLs tested except for the shortest (C4-HSL) and the

longest ones (C12-HSL and 3-oxo-C14-HSL). Thus

ExpR2 had a much broader AHSL recognition capacity

compared to ExpR1, which clearly is the more specific

LuxR-type protein mainly responding to the cognate

3-oxo-C8-HSL.

Having established the specificity of AHSL sensing for

each ExpR protein, we wanted to explore the joint effect of

the two ExpR proteins in PCWDE regulation. As a tool we

employed the triple mutant (SCC906) lacking the AHSL

synthase as well as the two ExpR proteins. As shown this

triple mutant exhibited wild-type levels of Cel activity

without the addition of any AHSL (Fig. 3A and C). Comple-

mentation studies were used to demonstrate the specific

roles for each ExpR protein. Plasmids carrying either

expR1 or expR2 were introduced to the expI expR1

expR2 triple mutant and Cel activities were assessed in

the presence and absence of 3-oxo-C6-HSL or 3-oxo-C8-

HSL (Fig. 3C). These results support the hypothesis that

both ExpR proteins have distinct roles in AHSL

recognition. Introduction of expR1 into the triple mutant

made the strain dependent of the cognate 3-oxo-C8-HSL

in Cel production whereas introduction of expR2 gener-

ated a dependency of a variety of AHSLs. These data

demonstrate that the ExpR1 and ExpR2 have individual

roles in AHSL recognition and indicate that either ExpR is

sufficient to repress PCWDE production.

ExpR1 and ExpR2 mediated repression of PCWDE

activity 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 constitutively

cellulase positive. The two ExpR proteins show different

specificities to AHSLs.

A. Ecc wild-type (SCC3193), expI mutant (SCC3065), expI expR1

mutant (SCC6005), expI expR2 mutant (SCC908), expR1 expR2

mutant (SCC907) and expI expR1 expR2 mutant (SCC906) 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 growth culture

supernatants were detected with CMC plates.

B. The expI mutant, expI expR1 mutant, expI expR2 mutant

were 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 growth

culture supernatants was detected as in 3A.

C. expI expR1 expR2 triple mutant (SCC906) carrying either expR1

or expR2 on a plasmid. The specificity of ExpR proteins was

determined by monitoring the Cel activity using a plate assay as in

3A and 3B.



wanted to elucidate the role of the QS system in

population density-dependent regulation of PCWDEs.

Nothern analysis was used to explore the role of ExpR1

and ExpR2 in regulation of PCWDE genes in the pres-

ence of endogenous AHSLs. We compared PCWDE

gene expression in the wild-type (SCC3193), the expR1

expR2 mutant (SCC907) and the expI expR1 expR2

mutant (SCC906) strains during the logarithmic growth

using PCWDE gene specific probes (Fig. 4). Both

mutant strains exhibited substantially up-regulated

PCWDE transcript accumulation compared to the wild-

type strain. Similar expression levels were only reached

by the wild-type at the stationary growth phase. In accor-

dance with this also the production of one of the

enzymes, Cel was clearly up-regulated in the expR1

expR2 double mutant at the early phases of growth

(Fig. 4). This analysis demonstrates that ExpR1 and

ExpR2 are essential for the growth phase dependent

control of PCWDE production.

ExpR1 and ExpR2 control expression of RsmA, a

negative regulator of PCWDE

Our results (Figs 3 and 4) demonstrate that ExpR1 and

ExpR2 are negative regulators of PCWDE gene expres-

sion and PCWDE production. However, they do not indi-

cate weather this repression is direct or mediated through

some 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 the

absence of AHSL. The activation of rsmA transcription

was 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 and

ExpR2 control in Ecc SCC3193 using an rsmA-gusA pro-

moter fusion. The rsmA promoter from Ecc strain

SCC3193 used in this construct contained a putative

ExpR-box, similar to the ExpR-binding site defined in

Ecc71 (Chatterjee et al., 2005). Our results from

b-glucuronidase (GUS) assays in the following mutant

backgrounds expI, expI expR1, expI expR2 and expI

expR1 expR2 demonstrate that in the absence of AHSL

ExpR1 and ExpR2 were both, either together or sepa-

rately, able to activate the expression of rsmA (Fig. 5A). In

contrast, in the expI expR1 expR2 triple mutant the rsmA

expression was significantly down-regulated with or

without the addition of AHSLs suggesting that at least one

of the ExpRs is needed for the transcriptional activation of

rsmA.

To further elucidate the role of ExpR1 and ExpR2 in the

transcriptional 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 both

expR1 and expR2 mutants. This argues that both ExpR1

and 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 in

the expI expR1 expR2 triple mutant. In contrast addition of

the non-cognate AHSL 3-oxo-C6-HSL led to substantially

decreased GUS activity only in the expI expR1 mutant,

likely due to release/inactivation of ExpR2. ExpR2 was

not 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 the

expI expR2 mutant. These data demonstrate that in the

absence of AHSLs either of the ExpRs is sufficient for

transcriptional activation of rsmA, which in turn down-

regulates expression of PCWDE genes. These data also

suggest that accumulation of the cognate AHSL (3-oxo-

C8-HSL) will release both ExpRs from rsmA leading to

coordinate activation of PCWDE genes. Interestingly,

also the non-cognate AHSL (3-oxo-C6-HSL) appears to

release/inactivate one of the ExpRs, ExpR2, suggesting

that signals from other bacterial species may modulate

this response.

The binding of AHSL by ExpR1 and ExpR2 correlates

with rsmA transcriptional activity

To provide additional evidence for the mode of action of

the ExpR1 and ExpR2 proteins and to elucidate whether

the transcription of rsmA is indeed controlled by AHSL

Fig. 4. Effect of expR1 and expR2 on population

density-dependent regulation of exoenzymes. Northern blot

analysis of total RNA from Ecc wild-type SCC3193, expR1 expR2

double 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 hybridized

with celV, pehA, pelB and 16S rRNA probes. At the bottom the

same strains, as used for Northern analysis, are grown on a CMC

plate for 4 h and 7 h and detected as earlier described.



binding to ExpRs, we measured AHSL binding using the

same 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 of

the AHSLs used, indicating that the presence of ExpR

proteins is indeed required for AHSL binding. In contrast,

the expI mutant with both ExpR proteins present binds

effectively both the cognate 3-oxo-C8-HSL and the non-

cognate 3-oxo-C6-HSL. Binding of the cognate AHSL in

expI mutant background was clearly more effective than in

strains lacking either one of the ExpR proteins indicating

that ExpR1 and ExpR2 cooperate in this binding. The

AHSL binding studies also support the observed specific-

ity of the AHSL action. The expI expR1 double mutant with

only ExpR2 present was able to bind 3-oxo-C6-HSL

equally well as the expI mutant indicating that 3-oxo-C6-

HSL binding can be explained by the binding capacity of

ExpR2. On the other hand the expI expR2 mutant with the

specific ExpR1 protein present was as expected able to

bind only the cognate 3-oxo-C8-HSL. These AHSL

binding results correlate well with the ExpR mediated

rsmA regulation shown with the rsmA promoter fusion

assay. Taken together these results strongly indicate that

ExpR1 and ExpR2 act synergistically in binding of AHSLs

and suggest that this binding leads to down-regulation of

rsmA transcription.

Role of RsmA in QS regulation of PCWDEs

Our data show that QS controls rsmA expression and

suggest that QS regulation of PCWDEs could be medi-

ated by RsmA. However, our data does not exclude the

presence of other RsmA-independent mechanisms by

which ExpR proteins could regulate the PCWDE

production. To explore this possibility we employed an

expI rsmA double mutant. The effect of rsmA on PCWDE

production was assessed by comparing Cel activities of

an expI mutant and an expI rsmA double mutant in the

presence and absence of AHSLs (Fig. 6; Table 1). In

accordance with earlier studies, using another Ecc strain

(Chatterjee et al., 1995), the expI rsmA double mutant of

SCC3193 showed a Cel positive phenotype even in the

absence of AHSL supporting the role of RsmA as a major

QS 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 in

somewhat higher Cel activity. To quantify this effect we

assayed 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 expR1

mutant (SCC6005), expI expR2 mutant (SCC908) and expI expR1

expR2 mutant (SCC906) carrying rsmA-gusA promoter fusion on a

plasmid (pSMS18). Samples were taken from bacteria grown to the

logarithmic phase, OD600 1.5, in L medium with no AHSL or added

with 1 mM 3-oxo-C6-HSL or 1 mM 3-oxo-C8-HSL. Similar results

were gained when bacteria were grown in M9 medium (data not

shown).

B. Specific and non-specific binding by ExpR1 and ExpR2.

Bioluminescence assay was used to determine the AHSL binding

capacity of Ecc expI mutant (SCC3065), expI expR1 mutant

(SCC6005), expI expR2 mutant (SCC908) and expI expR1 expR2

mutant (SCC906) carrying rsmA-gusA promoter fusion on a

plasmid (pSMS18). Same samples as in (A) were used. Similar

results were obtained when the AHSL binding capacity of ExpR

proteins was determined with LC-MS (data not shown). However,

as expected in these analyses no AHSL was detectable in the

controls without added autoinducer.

Fig. 6. An expI rsmA mutant strain has slightly enhanced Cel

activity with the cognate AHSL. Ecc expI mutant (SCC3065) and

expI 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 growth

culture supernatants were detected with CMC plates. Same results

were gained on PGA plates (data not shown).



Cel activity with the cognate AHSL added (Fig. 6). These

results suggest that QS control of PCWDE production is

largely mediated by RsmA but that in response to the

cognate 3-oxo-C8-HSL there is an additional RsmA-

independent pathway to fine tune PCWDEs production.

The AHSL specificity of Ecc strain SCC3193 can be

altered with an ExpR protein from Ecc strain SCC1

To further elucidate the specificity of the QS system

among Erwinia strains, we tested whether the system is

compatible with the LuxR-type protein expRSCC1 from

SCC1, a 3-oxo-C6-HSL producing strain of Ecc (Brader

et al., 2005). The expRSCC1 gene was expressed in trans

in different expR1 and expR2 mutant backgrounds of the

expI mutant of SCC3193, and the specificity for 3-oxo-C6-

HSL and 3-oxo-C8-HSL was characterized by assessing

the Cel phenotype (Fig. 7). Introduction of the expRSCC1

gene into the triple mutant (expI expR1 expR2) back-

ground repressed the Cel-positive phenotype of this

mutant demonstrating that ExpRSCC1 is functional in this

heterologous background. The repression could be

released by the cognate AHSL of the SCC1 strain (Brader

et 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 expressed

in expI expR1 mutant background (SCC6005). The expI

expR1 mutant strain with the broader AHSL ligand speci-

ficity was altered to the specific 3-oxo-C6-HSL sensing

when expressing expRSCC1 in trans. Expression of

expRSCC1 in trans in the presence of ExpR1 as in the

expI mutant (SCC3065) or the expI expR2 double mutant

strain (SCC908) resulted in a Cel-negative phenotype

independent of the addition of AHSL. This might be

explained by the simultaneous presence of two AHSL

receptors ExpR1SCC3193 and ExpRSCC1 with distinct AHSL

specificities. These data demonstrate that the AHSL

sensing specificity of the QS system in SCC3193 is deter-

mined by the ExpR1 protein of either Ecc strain SCC3193

or SCC1.

Lack of a functional quorum sensing system enhances

plant maceration

The QS system is one of the most important virulence

regulators in Ecc and controls PCWDE production

required for plant tissue maceration during infection. To

elucidate the role of the ExpR proteins on maceration

capacity, we inoculated potato tubers (Van Gogh cultivar)

and Arabidopsis wild-type (Col-0) plants with wild-type

strain SCC3193, and the different mutants and the extent

of maceration was determined (Fig. 8). As expected the

avirulent expI mutant showed almost no maceration, while

the expR1 expR2 double mutant and the expI expR1

expR2 triple mutant strains showed even slightly

enhanced maceration compared to the wild-type (Fig. 8A

and B). Wild-type Arabidopsis plants were inoculated with

the same Ecc strains as used for potato above. Similar

results as in the potato assay were obtained with Arabi-

dopsis: while no maceration was observed in plants

inoculated with the expI mutant, clear maceration was

evident in plants inoculated with wild-type, the expR1

expR2 double mutant or the expI expR1 expR2 triple

mutant (Fig. 8A). These results are in agreement with our

results from both the PCWDE assays and Northern analy-

sis, showing enhanced production of PCWDEs, the main

virulence factors of Ecc in strains lacking both ExpR

proteins.

Discussion

QS is central in the control of virulence and the production

of main virulence determinants like PCWDEs in the plant

pathogen Erwinia carotovora ssp. carotovora (Ecc). Pre-

vious studies have established that the QS system of Ecc

strain 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 ± 4

expI 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 to

a 3-oxo-C6-HSL sensing system by using expRSCC1. expRSCC1

was 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 in

L medium complemented with no AHSL, 3-oxo-C6-HSL or

3-oxo-C8-HSL and analyzed for there Cel activity as earlier

described.



the expI gene, producing 3-oxo-C8-HSL as cognate

AHSL, 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 PCWDEs

in the absence of AHSL (Pirhonen et al., 1993; Andersson

et al., 2000; Brader et al., 2005). In this study we present

several major results: First we demonstrate that two ExpR

proteins, ExpR1 and ExpR2, exist in Ecc SCC3193 and

that ExpR1 and ExpR2 act synergistically to repress the

production of PCWDEs and thus also virulence in the

absence of AHSL. Second, we show that the two ExpR

proteins produced by the Ecc strain SCC3193, have dis-

tinct AHSL recognition and binding specificities with

ExpR1 responsible for recognition of cognate AHSL and

ExpR2 responsible for recognition of both cognate and

non-cognate AHSLs. Third, we provide data suggesting

that the ExpR control of PCWDE gene expression is to a

large extent, but not solely, mediated by the negative

regulator RsmA (Fig. 9).

The lack of a clear PCWDE phenotype in the expR1

mutant (Andersson et al., 2000) suggested a redundancy

in the AHSL recognition system of Ecc SCC3193. That

this was indeed the case was demonstrated in this study

by identifying a second LuxR-type protein ExpR2 in Ecc

SCC3193. Our studies clearly demonstrated that (i) both

ExpR1 and ExpR2 function as AHSL receptors and

indeed bind AHSLs and (ii) they act as repressors of

PCWDE 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 triple

mutant strain was able to produce wild-type levels of

PCWDEs 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 expI

mutant background essentially abolished the production

of PCWDEs in the absence of AHSLs. This repression

was relieved by addition of the cognate 3-oxo-C8-HSL to

both expI expR1 and expI expR2 double mutants.

These results suggest a novel QS mechanism where

two partially redundant QS regulators act in concert to

control a single characteristic, i.e. PCWDE production.

The repression by single ExpRs was not always absolute:

an expI expR1 double mutant showed partial relief from

the PCWDE repression, observed as haloes around bac-

terial colonies grown on CMC indicator plates (data not

shown). This was not seen from the culture supernatants

of liquid cultures probably due to still too low level of

PCWDE production. This result is in agreement with

recent findings that show partial restoration of PCWDE

production in an expI expR double mutant of another Ecc

strain (Cui et al., 2005) and the slight increase in

PCWDEs in the expR1 single mutant strain (Andersson

et al., 2000). Also the recently sequenced Eca strain

SCR1043 (Bell et al., 2004) harbors two expR genes in its

genome that are also organized in a similar fashion as in

Ecc SCC3193 suggesting a similar type of QS regulation

as observed here. During preparation of this manuscript

Burr et al. (2006) showed mutant analysis of the expR2

homologue of Eca (virR) suggesting that this gene codes

for a repressor of PCWDE production. Interestingly, the

virR mutant of Eca showed partial restoration of PCWDE

production suggesting that the Eca ExpR (Ecc ExpR1

homologue) might not be as strong repressor of PCWDEs

as ExpR1 in Ecc.

How is the control of PCWDE production by AHSLs and

the two ExpR proteins executed? We showed that the

transcript levels of the PCWDE genes are affected. North-

ern analysis was used to this aim and demonstrated that

at early growth phase the transcript levels examined were

Fig. 8. A. Inactivation of expR1 and expR2 slightly enhances

virulence. Potato tubers (Van Gogh) and Arabidopsis (Col-0) were

inoculated with following Ecc strains: wild-type SCC3193, expI

mutant (SCC3065), expR1 expR2 mutant (SCC907) and expI

expR1 expR2 triple mutant (SCC906) using a bacterial inoculum of

105 cfu ml-1 72 h after inoculation potato tubers were cut in the

middle, to observe the maceration.

B. The macerated tissue of the inoculated potatoes was weighed to

determine the size of virulence symptoms. Significantly different

values indicated by different letters (P � 0.05) were calculated by

one way ANOVA followed by LSD test.



indeed constitutive in the expR1 expR2 double mutant.

Here we present for the first time evidence showing that

the ExpR proteins are truly responsible for the delay in

PCWDE gene expression and demonstrate that QS is

essential for population density-dependent regulation of

these genes. These data do not, however, demonstrate

weather the ExpR proteins directly control PCWDE genes

or weather this regulation is executed at some other level.

Recent data from Cui et al. (2005) indicated that the

global negative regulator RsmA is controlled by ExpR in

another Ecc strain. To explore this possibility in Ecc

SCC3193 we employed an rsmA-gusA promoter fusion

and demonstrated that ExpR-dependent repression of

PCWDE genes was apparently due to transcriptional

control of rsmA. In the absence of AHSL both ExpR1 and

ExpR2 were able to activate rsmA transcription, leading to

down-regulation of PCWDE genes (Figs 5A and 9) while

the addition of the cognate 3-oxo-C8-HSL abolished this

activation leading to up-regulation of PCWDE genes. To

further characterize the mechanism of this regulation we

examined the AHSL binding capacities of the different

expR 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. In

the absence of AHSLs both ExpR1 and ExpR2 activate the expression of rsmA. An up-regulated rsmA results in decreased exoenzyme

production. After addition of 3-oxo-C6-HSL the ExpR2 is released from the transcriptional activation of rsmA, but ExpR1 is still present

activating rsmA. But, in the presence of 3-oxo-C8-HSL both ExpR1 and ExpR2 are released from activating rsmA, which results in the

production of exoenzymes. Striped arrows indicate a possible RsmA-independent regulation of PCWDEs.



ExpR1 and ExpR2 are able to bind either together or

separately the cognate 3-oxo-C8-HSL. These results

strongly support the results gained with the rsmA tran-

scription activation assay. This model is in agreement with

the data by Cui et al. (2005) showing that in the Ecc strain

Ecc71 the ExpREcc71 protein binds to the promoter region

of rsmA and activates its transcription in the absence of

AHSLs and that the DNA binding of ExpREcc71 and hence

activation of rsmA transcription was inhibited by the pres-

ence of the cognate AHSL (Chatterjee et al., 2005; Cui

et al., 2005).

Although rsmA-mediated repression appears to be the

main control of PCWDE production we show that this is

not the only pathway by which the AHSLs exert their

control. Interestingly, an expI rsmA double mutant was still

able to respond to the cognate 3-oxo-C8-HSL and

increase its Cel activity accordingly. This result suggests a

further 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 as

direct or indirect regulators of PCWDE production. Alter-

natively ExpR could control expression of another yet

unknown negative regulator of PCWDEs similarly to its

control of rsmA.

In most known AHSL receptors the N-terminal ligand

binding 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 propose

that (Fig. 9) in Ecc the two ExpR proteins have synergis-

tic, but individual roles, with ExpR1 acting as a cognate

ligand specific regulator, while ExpR2, interestingly,

responds to both the cognate and the non-cognate

signals. We demonstrate that ExpR2 has a broad AHSL

sensing 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 four

key observations: First, recently we showed that the

PCWDE activity of an expI mutant of Ecc strain SCC3193

could only be restored with physiological levels of the

cognate AHSL 3-oxo-C8-HSL (Brader et al., 2005). Here

we show that the expI expR1 double mutant of SCC3193

had lost its specificity for the cognate AHSL and reacted

also 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) that

was shown to be responsible for the sensing of the non-

cognate AHSLs. Second, by monitoring Cel activity we

show that ExpR2 senses AHSLs with acyl chains ranging

between C6 and C10. The inability to sense the AHSLs

with even longer acyl chains (C12 and C14), might

depend on the lack of a reasonable transport system for

these long chain AHSLs into the cell (Fuqua et al., 2001).

Third, in the expI expR1 double mutant the transcription of

rsmA can be prevented with either 3-oxo-C6-HSL or

3-oxo-C8-HSL, and leading to subsequent production of

PCWDEs. In contrast expression of rsmA is only abol-

ished by the addition of 3-oxo-C8-HSL in, the expI expR2

mutant (Fig. 9). Fourth, we show that the expI mutant bind

both 3-oxo-C6-HSL and 3-oxo-C8-HSL, while the the expI

expR2 mutant is only able to bind 3-oxo-C8-HSL. This is

a clear evidence that ExpR2 is responsible of the binding

of 3-oxo-C6-HSL.

The special feature of ExpR2 to respond to several

different AHSLs raises questions of its role. Why does this

Ecc strain have both a specific and an unspecific QS

regulator? 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 that

expR2 is located separately from the expI-expR1

cassette. A biological advantage of having a receptor for

various non-cognate AHSLs could be the ability to identify

neighboring bacteria by sensing the accumulation of dif-

ferent kinds of AHSLs. Thus eavesdropping on possible

competitors (Lazdunski et al., 2004; Waters and Bassler,

2005) or establishing cooperation with other bacteria to

overwhelm the plant host could be beneficial for the

success of Ecc as a pathogen. The special feature of

ExpR2 to recognize AHSLs produced by its own species

in addition to AHSLs produced by neighboring bacteria

could further enhance survival in a crowded niche. It is

also possible that ExpR2 in addition possess unique

target sites not recognized by ExpR1 and involved in

interactions with other bacterial species.

We present in this article a novel mechanism for QS

(Fig. 9): the simultaneous need for two individual, but

cooperatively acting QS regulators in controlling expres-

sion of the same target gene. In this study we have shown

that ExpR1 and ExpR2 can function as activators either

alone or synergistically when both proteins are present.

Our results suggest that the amount of bound AHSLs

correlate with the amount of ExpR proteins present and

that the AHSL binding of the ExpR proteins correspond to

the transcriptional activity of rsmA. We propose that

ExpR1 and ExpR2 act in synergy integrating cognate and

non-cognate AHSL signals to control expression of a

central regulatory gene rsmA. However, we show that

they can also act independently and therefore are not

necessarily physically interacting. LuxR-type proteins use

different modes of action and operate in various ways

depending on bacterial species and tasks to be

accomplished. A hierarchical organization model where

one LuxR-type protein regulates the transcription of

another LuxR-type protein is described in, e.g. Yersinia

pseudotuberculosis (Atkinson et al., 1999) and

Pseudomonas aeruginosa (Pesci et al., 1997). The pos-

sibility that ExpR1 would control expression of expR2 or

vice versa, was tested using expR-gusA promoter

fusions, but no transcriptional regulation between ExpR1



and ExpR2 was observed (data not shown). Thus we

prefer a model where the two ExpR proteins directly

control expression of the rsmA gene and possible other

target genes. LuxR-type proteins, such as CarR, LuxR

and TraR dimerize/multimerize after binding AHSL leading

to subsequent stabilization of the protein and activation of

target gene expression (Qin et al., 2000; Welch et al.,

2000; Urbanowski et al., 2004). In contrast, the ExpR

proteins appear to bind target DNA and act as activators

in the absence of AHSL and then be inactivated/released

by AHSLs. Whether these states involve dimerization

remains to be demonstrated. Alternatively it could be pos-

sible that after AHSL binding, the two ExpR proteins stay

at 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 aeruginosa

has such a dual role always binding to DNA, but acting as

an activator or a repressor depending on the presence of

AHSL (Medina et al., 2003). An open question is also

whether the ExpR1 and ExpR2 bind the same promoter

site or are there possibly several ExpR binding sites in the

rsmA promoter? In the rsmA promoter of Ecc71 one ExpR

binding site was recently identified (Chatterjee et al.,

2005). We could identify a similar DNA element (ExpR

box) in the rsmA promoter of Ecc strain SCC3193 and in

the sequenced Eca strain SCRI1043. Furthermore, we

cannot rule out the possibility that the ExpR proteins of

Ecc SCC3193 have a dual role, working both in an AHSL

bound state and in a ligand-free state. This hypothesis is

partly supported by the results with the expI rsmA mutant

strain, showing enhanced Cel activity only with the addi-

tion of the cognate 3-oxo-C8-HSL. Such a model has also

been proposed for EsaR of Pantoea stewartii that binds to

its target promoter in the absence of AHSL and is released

by the addition of AHSL (Minogue et al., 2005).

The ability of Ecc strains lacking the whole QS system

to 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 natural

habitat, where the densities and the composition of bac-

terial populations fluctuate in response to environmental

cues. An ecological study would be essential to eluci-

date the significance of QS in controlling the success of

Ecc 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 in

Table 2. Escherichia coli strains were cultured in L medium

(Miller, 1972) at 37°C and Erwinia carotovora ssp. carotovora

strains 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 otherwise

mentioned. AHSLs were used at a concentration of 1 mM if

not 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 the

sequence of Eca strain SCRI1043 an expR2 specific probe

was PCR amplified from wild-type SCC3193 genomic DNA

using primers ProbR2F (5�-TCTGTATTTTGCTCTGATAA-3�)

and ProbR2R (5�-CAGATCGCCATACTGTTTTA-3�). The

expR2 probe was used to screen Lambda DASH library

(Stratagene) containing 17–22 kb BamHI fragments of

SCC3193. An expR2 positive clone was amplified for lambda

DNA isolation and the purified DNA containing an approxi-

mately 20 kb insert was cut with BamHI and EcoRI and

verified with Southern blot analysis to be expR2 positive.

Primers cdhF (5�-TGATTGCTATAGGTCCTCAG-3�) and

cheR (5�-GGTGAGGTTTGTTCTCTCATC-3�) were used to

PCR amplify a 3 kb DNA fragment, for subsequent sequenc-

ing and cloning procedures. To obtain a deletion mutant of

expR2, the upstream region of the expR2 gene was am-

plified by PCR from SCC3193 with primers expRBapaR

(5�-CCGGGCCCCCTGCGGCTATTGTGATAACG-3�) and

expRBhindF (5�-CCAAGCTTTCTGGCTGCGTTATCGATTA

TG-3�). The resulting 1461 bp PCR product was digested

with ApaI and HindIII resulting in a 889 bp DNA fragment (the

reduction in DNA length was due to an unobserved HindIII

restriction 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�) and

CatHindR (5�-CCAAGCTTCTATCGTCAATTATTACCTCCA-

3�); KmSmaF (5�-CCCCCGGGCAGCTACTGGGCTATCTG

GA-3�) and KmHindR (5�-CCAAGCTTGCGTCAATACGGG

ATAATAGTG-5�). Each fragment containing an antibiotic

resistance marker gene was digested with SmaI and HindIII

and inserted to pSMS100 to confer one chloramphenicol and

one kanamycin resistant plasmid pSMS103(Km) and

pSMS103(Cm). The down-stream region of expR2 gene was

PCR amplified with primers expRBspeF (5�-GACTA

GTGTGTAGCGTAGTCAGGCAAC-3�) and expRBsmaR

(5�-CCCCCGGGCTACTGTTACCCCATGATATCAC-3�). The

product was digested with SpeI and SmaI and inserted into

plasmids pSMS103(Km) and pSMS103(Cm) digested with

same enzymes, resulting in the constructs pSMS104(Km)

and pSMS104(Cm). The DNA fragment (expR2::Km;

expR2::Cm) of pSMS104 (Km/Cm) was digested with ApaI

and SpeI and inserted into suicide vector pGP704 digested

with same enzymes. The resulting plasmids pSMS105/Km

and pSMS105/Cm were transformed into E. coli S17-1 lpir and further transformed by conjugation into Ecc

strain SCC3193, SCC3065 (expI), SCC5003 (expR1) and

SCC6005 (expI expR1) (de Lorenzo and Timmis, 1994).

Transconjugants were plated on either M9 minimal medium

supplemented with 0.2% sucrose and chloramphenicol

10 mg ml-1 or kanamycin 10 mg ml-1 or on L medium



plates supplemented with chloramphenicol 50 mg ml-1 and

kanamycin 50 mg ml-1. The resulting strains SCC905

(Cm) (expR2::Cm), SCC905 (Km) (expR2::Km), SCC906

(expIexpR1::Cm expR2::Km), SCC907 (expR1::Cm

expR2::Km) and SCC908 (expI::Km expR2::Cm) were veri-

fied to be CmR AmpS, KmR AmpS or CmR KmR AmpS and

confirmed genotypically.

Construction of plasmids

The expR1 and expR2 genes were amplified by PCR from

wild-type SCC3193 genomic DNA using the primers:

ExpR(3193)eco2 (5�-CGGAATTCGAGATGTCGCAGTTATT

CTACA-3�) and ExpR(3193)bam (5�-CGGGATCCGCCTAT

GACTGAACCGGTCG-3�); ExpR2 SMS17 Rev (5�-CGGGA

TCCCTATAGTGGTTCTGGCTTGATG-3�) and ExpRB pOKF

(5�-CGGAATTCATGTCTGTATTTTGCTCTGATAATG-3�).

The 737 bp expR1 PCR product and the 738 bp expR2 PCR

product was digested with BamHI and EcoRI and ligated into

pQE30, digested with corresponding enzymes, resulting in

pSMS20 and pSMS21, respectively. The expRSCC1 was

amplified by PCR from wild-type SCC1 genomic DNA using

primers: expR(1)eco (5�-CGGAATTCGAGATGTCGCCATT

ATTCACTG-3�) and expR(1)bam (5�-CGGGATCCTACCT

GCCGCTATTGCACAGG-3�). The 729 bp expRSCC1 PCR

product was digested with BamHI and EcoRI and ligated into

pQE30, digested with corresponding enzymes, resulting in

pSMS22. For promoter fusion studies a plasmid (pSMS18)

was constructed containing the rsmA promoter region and

partial coding sequence amplified by PCR using primers:

RsmAF prom (5�-GCGTCGACCTGTTGTTGTGATAACAA

AAG-5�) and RsmAR prom (5�-CCAAGCTTACCGTTACCT

CATCGCCGA-3�). The 189 bp PCR product was digested

with SalI and HindIII and ligated into pGUS102 digested with

corresponding enzymes.

RNA isolation and Northern blot analysis

Erwinia cells from overnight cultures were diluted 1/100 in L

medium and grown at 28°C. Samples for RNA isolation were

taken at indicated time points and the growth was monitored

by measuring the OD600. Total RNA was isolated as described

by Sambrook et al. (1989). Northern analysis was performed

with 10 mg of total RNA separated in 1.5% formaldehyde gel.

Filters were probed with specific digoxigenin labeled DNA

fragments for celV1, pehA, pelB and 16S rRNA (Hyytiäinen

et al., 2003). The blotting, hybridization and digoxigenin

detection was performed according to the instructions of the

manufacturer (Roche).

Enzyme assays

Cellulase (Cel) activities were analysed from 10 ml of super-

natant of overnight grown liquid cultures on CMC indicator

plates using Congo Red to determine the cellulose diges-

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

Strain or plasmid Genotype or description Reference

Strains

E. coli

DH5a 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 carotovora

subsp carotovora

SCC1 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 work

SCC906 expR2::km, KmR in SCC6005 background This work

SCC907 expR2::km, KmR in SCC5003 background This work

SCC908 expR2::cm, CmR in SCC3065 background This work

expI rsmA mutant DrsmA in SCC3065 background Andersson (unpublished)

Plasmids

pSB402 pBR322 with luxRI� and a promoteless luxCDABE cassette Guard-Petter (1998)

pBluescript SK+ Cloning vector, AmpR Stratagene

pGP704 Suicide vector, AmpR Miller and Mekalanos (1988)

pQE30 Expression vector, AmpR Qiagen

pGUS102 pBR322 with promoterless uidA from E. coli Andersson et al. (2000)

pSMS20 expR1SCC3193 cloned into pQE30 EcoRI and BamHI sites This work

pSMS21 expR2SCC3193 cloned into pQE30 EcoRI and BamHI sites This work

pSMS22 expRSCC1 cloned into pQE30 EcoRI and BamHI sites This work

pSMS18 rsmASCC3193 promoter (189 nt) and partial CDS (67 nt) cloned into pGUS102

SalI and HindIII sites

This work

pSMS100 889 bp DNA fragment containing upstream region of expR2 This work

pSMS103 cat gene and km gene cloned into pSMS100 This work

pSMS104 expR2SCC3193::Km, expR2SCC3193::Cm cloned into pBluescript ApaI and SpeI sites This work

pSMS105 expR2SCC3193::Km, expR2SCC3193::Cm cloned into pGP704 ApaI and SpeI sites This work



tion (Pirhonen et al., 1993). Quantitative Cel assay was

performed as described previously (Pirhonen et al., 1991).

For b-Glucuronidase (GUS) activity assay Erwinia cells

from overnight cultures were diluted 1/100 in L medium and

grown at 28°C. Samples for GUS activity assay were taken

at indicated time points and the growth was monitored

by measuring the OD600. GUS activity was measured by

using p-nitrophenyl b-D-glucuronide as substrate (Novel

et al., 1974; Marits et al., 2002). r-nitrophenol (r-NP),

was detected at an absorbance of 405 nm and the specific

activity of GUS was expressed as nmol r-NP liberated

min-1.

Assay for AHSL Binding

The samples for determining the AHSL binding capacity

were prepared as follows: 15 ml of bacteria was grown in L

media complemented with no AHSL, 1 mM 3-oxo-C6-HSL or

1 mM 3-oxo-C8-HSL. The overnight grown bacterial cells

were collected and washed twice with 0.9% NaCl to remove

AHSL 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 the

cell debris was removed. The AHSLs were extracted twice

with equal amounts of ethylacetate and the extracts dried in

a Speed-Vac, with subsequent resuspension into 30 ml

acetonitrile:0.1% formic acid (1:1 v/v) for LC-MS analysis as

described (Brader et al., 2005) or 50 ml L-medium for biolu-

minescence assays. Here, overnight grown E. coli carrying

pSB402 (Guard-Pette, 1998) was diluted 1:100 and grown

for 5 h. After this 50 ml of E. coli and the 50 ml AHSL extract

was mixed and incubated for 2 h. The bioluminescence was

measured 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) as

described (Zhang et al., 1993). AHSL standards and profiles

of culture supernatants have been analyzed by LC-MS as

described earlier (Brader et al., 2005).

Assay of maceration capacity

Erwinia strains were grown overnight, diluted into 0.9% NaCl

and samples containing 105 bacterial cells ml-1 were used for

inoculation of potato tubers (Solanum tuberosum cv. Van

Gogh). The inoculation site was bored with a sterile toothpick.

Infected potatoes were incubated at 28°C for 72 h under

humid conditions with wet tissue paper in the incubation box.

The amount of soft rot was measured by cutting the potato

tubers in half and scraping and subsequently weighing the

rotted tissue. Arabidopsis thaliana Col-0 was infiltrated with a

syringe without needle with 105 bacterial cells ml-1 prepared

as described above. The development of disease symptoms

was documented after 48 h, kept in 22°C, 16 h light and high

humidity.

Nucleotide sequence accession number

The DNA sequence data determined in this study has been

submitted to the DDB/EMBL/GenBank databases under

accession 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 expI

rsmA mutant. This study was supported by the Helsinki

Graduate School in Biotechnology, Molecular Biology and

Academy 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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