MarR Family Dickeya

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Systematic targeted mutagenesis of the MarR/SlyA familymembers of Dickeya dadantii3937 reveals a role for MfbR inthe modulation of virulence gene expression in response toacidic pHmmi_7388 1..20

Sylvie Reverchon,1 Frdrique Van Gijsegem,2

Graldine Effantin,1 Ouafa Zghidi-Abouzid1 and

William Nasser1*1Univ Lyon, F-69622 Lyon France; Universit Lyon 1

Villeurbanne; INSA-Lyon F-69621 Villeurbanne; CNRS

UMR5240 Microbiologie, Adaptation et Pathognie,

Lyon, France.2Laboratoire Interaction Plantes Pathognes LIPPUMR217 INRA/AgroParisTech/UPMC F-75005 Paris,

France.

Summary

Pathogenicity of Dickeya dadantii is a process

involving several factors, such as plant cell wall-

degrading enzymes and adaptation systems to

adverse conditions encountered in the apoplast.

Regulators of the MarR family control a variety of

biological processes, including adaptation to hostile

environments and virulence. Analysis of themembers of this family in D. dadantii led to the iden-

tification of a new regulator, MfbR, which controls

virulence. MfbR represses its own expression but

activates genes encoding plant cell wall-degrading

enzymes. Purified MfbR increases the binding of

RNA polymerase at the virulence gene promoters

and inhibits transcription initiation at the mfbR pro-

moter. MfbR activity appeared to be modulated by

acidic pH, a stress encountered by pathogens

during the early stages of infection. Expression of

mfbR and its targets, during infection, showed that

MfbR is unable to activate virulence genes in acidic

conditions at an early step of infection. In contrast,

alkalinization of the apoplast, during an advanced

stage of infection, led to the potentialization of MfbR

activity resulting in plant cell wall degrading enzyme

production. This report presents a new example of

how pathogens adjust virulence-associated factors

during the time-course of an infection.

Introduction

Dickeya dadantii [ex Erwinia chrysanthemi (Samson

et al., 2005)] is described as a necrotrophic, Gram-

negative plant pathogen that causes disease in a widerange of plant species, including many crops of economic

importance such as vegetables and ornamentals and also

the model plant Arabidopsis (Perombelon, 2002; Dellagi

et al., 2005). However, recently a biotrophic phase during

the infection process has been postulated (Toth and Birch,

2005; Lebeau et al., 2008). Soft rot, the visible symptom,

is mainly due to the production of secreted degradative

enzymes, mostly pectate lyases (Pels), proteases and the

cellulase CelZ, that can destroy the plant cell wall (Barras

et al., 1994). During the early steps of infection, D. dadan-

tii is confronted with hostile environments in the inter-

cellular apoplastic fluid, such as acidic and oxidativestresses, iron starvation and the presence of bactericidal

compounds (Grignon and Sentenac, 1991; Expert, 1999).

Following the recognition of a variety of signals within the

host, sequential synthesis of plant cell wall degrading

enzymes is induced in the bacteria (Sepulchre et al.,

2007; Charkowski, 2009). The activity of these enzymes

results in lysis of the plant host cells. Thus, the ability of D.

dadantii to survive and grow in adverse environments is

important for virulence (El Hassouni et al., 1999; Expert,

1999; Santos et al., 2001; Reverchon et al., 2002; Llama-

Palacios et al., 2005). However, an efficient colonization

of the plant requires many additional bacterial factors,

including early factors (adhesins, exopolysaccharide, fla-

gella) which allow adhesion of the bacteria to the plant

and their entry into the apoplast (Condemine et al., 1999;

Rojas et al., 2002; Antunez-Lamas et al., 2009). D.

dadantii pathogenicity is clearly a multifactorial process

and the success of infection depends on the tight regula-

tion and co-ordinated expression of the various virulence

factors involved (Nasser et al., 2001; Nasser and Rever-

chon, 2002; Lautier and Nasser, 2007; Sepulchre et al.,

Accepted 30 August, 2010. *For correspondence. E-mail [email protected]; Tel. (+33) 4 72 43 26 95; Fax (+33) 4 72 43 1584.

Molecular Microbiology (2010) doi:10.1111/j.1365-2958.2010.07388.x

2010 Blackwell Publishing Ltd


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2007; Lebeau et al., 2008; Haque et al., 2009). The recent

completion of D. dadantii 3937 genome sequencing

reveals that approximately 9% of the predicted proteins

(about 400 proteins) are thought to play a role in transcrip-

tional regulation and 293 of these proteins are predicted

to be DNA-binding transcriptional regulators (J. Glassner

et al., in preparation). These transcriptional regulators are

distributed within 28 distinct families, each containing

from 1 to 62 members. The MarR/SlyA and DUF24/HxlR

families are quite well represented in D. dadantii with 10

and 7 members respectively. There is increasing evidence

that regulators of the MarR and DUF24/HxlR families are

structurally and functionally related. For example, the

redox-sensing transcriptional regulator QorR, in Coryne-

bacterium glutamicum, belongs to the DUF24 family and

uses a single Cys residue for redox-responsive regulation

(Ehira et al., 2009). This behaviour is similar to that of the

two regulators of the MarR family, SarZ and MgrA in

Staphylococcus aureus (Chen et al., 2009). For the pur-

poses of this study, we group the 10 MarR members and

the 7 DUF24/HxlR members of D. dadantiiall together asthe MarR family.

The MarR/SlyA family of prokaryotic transcriptional

regulators includes proteins which are critical for the

control of virulence (Ellison and Miller, 2006), for the bac-

terial response to antibiotic and oxidative stresses, and for

the catabolism of environmental aromatic compounds

(Wilkinson and Grove, 2006). The members of this family

which are involved in virulence control have been studied

in particular. These include SlyA, in Salmonella typhimu-

rium (Libby et al., 1994); RovA, in Yersinia enterocolitica

and in Yersinia pestis (Cathelyn et al., 2007); AphA, in

Vibrio cholerae(Kovacikova et al., 2004); MgrA and SarZ,in Staphylococcus aureus (Chen et al., 2009); Hor, in

Erwinia carotovora(Thomson et al., 1997); and PecS and

SlyA in D. dadantii (Reverchon et al., 1994; Haque et al.,

2009). In view of the important role played by these regu-

lators in the virulence control of the corresponding bacte-

ria, we performed a systematic analysis of the MarR/SlyA

proteins from D. dadantii. For this, each MarR/SlyA family

regulator was inactivated and mutants were screened for

virulence. This resulted in the identification of MfbR as a

new regulator of virulence. Consistently, MfbR is required

for optimal production of secreted degradative enzymes in

its host plant during infection. Furthermore, we showed

that MfbR represses its own gene expression and exerts

both a negative and a positive action on gene expression

by directly modulating RNA polymerase activity.

Results

The regulators related to the MarR family in D. dadantii

Proteins encoded by the D. dadantii 3937 genome were

submitted to Interproscan for signature searches and

those displaying a PFAM signature PF01047, specific for

proteins of the MarR/SlyA family, and a PFAM signature

PF01638, specific for proteins of the DUF24/HxlR family,

were retained for further analysis. Ten proteins showed a

MarR/SlyA signature and seven showed a DUF24/HxlR

signature (Table S1). Homologues of these regulators

were identified by Blast searches and four regulators have

a known or predicted function: PecS in D. dadantii, which

controls plant cell wall degrading enzymes, antioxydants

(Reverchon et al., 1994; Hommais et al., 2008), flagella

(Rouanet et al., 2004), and harpin synthesis (Nasser

et al., 2005); EmrR, which controls the multidrug efflux

pump EmrAB in E. coliand D. dadantii(Xiong et al., 2000;

Ravirala et al., 2007); SlyA, which controls a type three

secretion system in Salmonella (Linehan et al., 2005),

haemolysin E in E. coli (Lithgow et al., 2007) and pectate

lyase production in D. dadantii (Haque et al., 2009); and

OhrR, which controls the ohr gene encoding a thiol per-

oxidase involved in resistance to lipid hydroperoxide in

Xanthomonas campestris (Klomsiri et al., 2005). We

hypothesized that the OhrR regulator has the same func-tion in D. dadantii since the redox sensing Cys residues

are conserved and the genetic organization of the ohrR-

ohr locus is similar to that found in Xanthomonas. There

remain 13 D. dadantiiregulators related to the MarR/SlyA

family for which no function has yet been proposed.

Among these 13 regulators, eight are more similar to

proteins from plant-associated bacteria and might be

implicated in responses to conditions specifically encoun-

tered in plant tissues. Interestingly, the majority of the

MarR/SlyA proteins have no close homologues in the

related bacteria Pectobacterium atrosepticum and P.

carotovorum (Table S1).We performed a systematic analysis of the 17 regula-

tors related to the MarR/SlyA family. To facilitate this

analysis we gave a name to each of these genes/

proteins (Table S1): EmrR (ID15973), PecS (ID16089),

SlyA (ID15312), OhrR (ID15242), and for the other MarR

family regulators we arbitrarily chose the names

MfaR (ID15788), MfbR (ID16402), MfcR (ID18907),

MfdR (ID16171), MfeR (ID19341), MffR (ID17487), MfgR

(ID16861), MfhR (ID16472), MfiR (ID16515), MfjR

(ID16234), MfkR (ID18024), MflR (ID14507) and

MfmR (ID16781). ID numbers are those used in the

ASAP database. A systematic mutagenesis was carried

out to determine the importance of these D. dadantii

regulators in the plant-bacteria interaction (Table S2).

Virulence of the regulator mutants

The pathogenicity of each mutant was tested on Saint-

paulia ionantha(African violet), the plant from which strain

3937 has been isolated. Inoculation was performed by

infiltration of about 2 106 bacteria in one leaf per plant. In

2 S. Reverchon et al.

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such conditions, more than one half of the plants inocu-

lated with the wild-type strain showed maceration symp-

toms from 24 h after inoculation and most of them

harboured systemic symptoms 6 days after infection

(Fig. 1A). Of the 17 mutants analysed, only pecS, mfbR

and slyA behaved significantly differently from the wild-

type strain. As previously reported (Reverchon et al.,

2002), the pecS mutant developed severe symptomsmore rapidly than the wild-type strain (data not shown). By

contrast, the virulence of the slyA and mfbRmutants was

strongly affected because, compared with the wild-type

strain, fewer inoculated plants exhibited symptoms and

maceration spread more slowly (Fig. 1A). Thus, the integ-

rity of MfbR and SlyA is important for the development of

soft-rot disease on plants. The data we have obtained on

slyA are generally in accordance with those recently

reported by Haque et al. (2009). Indeed, these authors

have shown, on potato tubers, that the slyA mutant dis-

plays a threefold reduced virulence in comparison with the

wild-type strain. Thus, we concentrated our investigation

on MfbR. Inoculation of Saint Paulia with a lower bacterial

density (2 105 bacteria in one leaf) confirmed the strong

attenuation in the mfbRvirulence (data not shown). Since

Arabidopsis, the model for plant biology, has been shown

to be a host for D. dadantii (Dellagi et al., 2005; Fagard

et al., 2007), virulence of the mfbR mutant was also

analysed in Arabidopsis as well as in potato tubers. In

Arabidopsis, the mfbR mutant exhibited only a weak

agressivity, with most plants harbouring no symptoms 7

days post infection (Fig. 1B). However, as seen in Saint-

paulia, the mfbRmutant retained the ability to spread and

macerate the whole infected leaf. In potato tubers, the

mfbR mutant exhibited a strongly reduced maceration

capacity (Fig. S1).

Organization and regulation of the mfb locus

Since the targets of a regulator are frequently found in the

vicinity of its gene, the genetic environment of mfbR was

examined. Two genes, named as mfbA and mfbB, which

encode a tripartite efflux pump of the MFS family, are

located downstream of mfbR. MfbB is the cytoplasmic

membrane permease and MfbA is the membrane fusion

component of the efflux pump, which is located in the

periplasmic space and interacts with an outer-membrane

protein channel, such as TolC. By using reverse transcrip-

tion, coupled to polymerase chain reaction (RT-PCR)

experiments, we established that mfbA and mfbB are

organized in an operon with mfbR(Fig. 2A). Primer exten-

sion analysis with RNA extracted from the D. dadantii

parental strain 3937, or its mfbRderivative, revealed that

the mfb operon transcription was initiated at the A nucle-

otide, at position -26 upstream of the ATG translation

initiation codon of the mfbR gene (Fig. 2B and C). More-

over, a more abundant accumulation of the mfb operon

transcripts was observed in the mfbRbackground than in

the parental strain (Fig. 2B). This therefore indicates that

MfbR negatively regulates the mfb operon. Six bases

Fig. 1. Virulence of the regulator mutants.The kinetics of soft rot progression wasscored for a week in Saintpaulia(A) andArabidopsis(B). Symptoms were scored usinga 4-step scale as follows: stage 0, nosymptoms; stage 1, maceration of theinfiltrated area (Saintpaulia) or around thebacterial suspension droplet (Arabidopsis);stage 2, spreading maceration; stage 3,maceration of the whole leaf, including the

petiole. The significance of the differences inmaceration symptoms distribution betweenwild-type and mutants has been checked witha Fisher test, comparing either the number of

asymptomatic plants or the number of plantspresenting the highest score on each day. Atall time points, disease development wassignificantly decreased with the differentmutants, compared with the WT parent(P< 0.05). No symptoms were observed fornegative control plants treated with sterilebuffer.

D. dadantii MarR/SlyA family and virulence gene regulation 3



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further upstream the transcriptional start, there is a poten-

tial -10 element (TTAACT), separated by 17 bases from a

potential -35 element (TTGACT) (Fig. 2C), so the mfb

transcription start point mapped here is located in an

appropriate context.

Quantitative reverse transcription polymerase chain

reaction (qRT-PCR) was used to analyse the mfbRAB

operon expression dependence on growth, on the pres-

ence of pectin or after acid or oxidative stresse, two con-

ditions encountered by pathogens during infection. For

the stress assays, we used exponentially growing cul-tures, which are the best conditions for evaluating the

effects of pH challenge and oxidative stress on bacterial

gene expression (Arnold et al., 2001). In addition, acidic

and oxidative conditions are mainly encountered at early

phases of infection and we assume that, during these

phases, bacteria would probably be in conditions closer to

exponential growth phase rather than to stationary growth

phase. In the wild-type strain, expression of mfbRwas not

modified by the presence of pectin or by 100 mM H2O2

treatment (data not shown). In contrast, mfbRexpression

was induced by acidic treatment in the early exponential

phase (sevenfold) (Fig. 2D, Table 1). Quantification of the

asr transcripts was used as a control to visualize the

response to acidic pH stress. In E. coli, the asr gene

encodes an acid shock protein whose production is con-

trolled by the pH-responsive, two-component system

rstArstB (Ogasawara et al., 2007). As expected, the

expression of the asr gene was strongly increased by the

acidic shock in D. dadantii (2500-fold, Fig. 2E, Table 1),

which therefore validates our experimental conditions.

Moreover, mfboperon expression was seen to be depen-

dent on the growth phase, with maximal expression

occurring during early exponential growth and with

expression reduced by 2.4-fold in the late exponential

phase (Fig. 2D). A similar expression pattern was

observed for mfbA and mfbB (data not shown). Further-

more, no expression of mfbA and mfbB genes was

detected in the mfbR mutant, demonstrating the polar

effect of the mfbR::Cm insertion and confirming the

operon organization and the absence of an additional

promoter downstream of the MfbR ATG translation initia-

tion codon (data not shown). The level of mfbRtranscriptswas about 65-fold higher in the mfbR mutant, compared

Fig. 2. The D. dadantii mfb operon.A. Co-transcription analysis of mfbR, mfbA and mfbB genes by RT-PCR (left panel) and a schematic representation of RT-PCRs (right panel).The thick arrow indicates the transcription initiation position +1 determined in the primer extension experiment; the positions ofoligonucleotides used for amplification of the different fragments (A, B and C) revealed on the gel (left part) are indicated on the scheme bythe thin arrows. The DNA segments A, B and C were amplified using the oligonucleotides mfbRf and mfbRr, mfbRf and mfbArc, and mfbRfand mfbBrc, respectively (Table S3). RNA was isolated from exponential cultures of the D. dadantii parental strain 3937 and added toreactions in the presence of reverse transcriptase (lanes A, B and C on the gel), lane T1 corresponds to the same amplification as in A butwithout the addition of reverse transcriptase to the reaction mixture. Lane T2 corresponds to amplification using the primer up, localizedupstream from the +1 transcription start site, and the reverse primer localized at the end of mfbR. The sizes of the amplified fragments areindicated.

B. Determination of the D. dadantii mfb promoter start site. Primer extension reactions were performed in the presence of 5 mg of RNAextracted from the parental strain 3937 (WT) and its mfbR derivative. DNA sequencing ladders were generated with the same primer (lanes A,C, G and T). The retained transcriptional start point is indicated by an arrow and by a bold A base in the sequence presented on the left.C. Sequence of the mfbR promoter. Arrow indicates the transcription initiation site +1. The binding site for MfbR, as determined by DNase I

footprinting (Fig. 7A), is indicated in brackets; the KMnO4-sensitive bases are indicated by closed circles (Fig. 8). The probable -10/-35 andthe putative Shine and Dalgarno sequence regions are underlined; the putative DNA binding site of MfbR and the translation initiation codonATG are indicated in bold characters.D. Quantification of the mfbR gene expression by quantitative RT-PCR (qRT-PCR). The oligonucleotides used for qRT-PCR matches,upstream of the Cm cartridge insertion, which gave rise to the mfbR mutation. These oligonucleotides allowed for quantification of mfbRtranscripts in both the parental strain and its mfbR derivative. The ffh gene was used as a reference for normalization. The pH stress wasperformed by shifting pH from 7.0 to 4.3 by addition of malic acid. RNA was extracted 15 min after the pH stress. The expression of mfbR isregulated in a growth phase-dependent manner, sensitive to acidic pH stress, and is submitted to a negative retro-control. Total RNA wasisolated from the parental strain 3937 and its mfbR derivative; the OD600 and the growth conditions are indicated. The same RNA extractswere used for experiments presented in Fig. 2 and in Fig. 4. Each value represents the mean of two experiments. Bars indicate the standarddeviation.E. Quantification of the asr gene expression. The experiments were performed as in (D).

Table 1. Fold change in mfbR, pel, celZ, prtC, hrpN and asr geneexpression induced in response to pH stress, in the early exponentialphase, in the wild-type strain.

Gene name Fold change induced by pH stressa

mfbR 7.0 0.5pelD -2.9 0.3pelE -5.4 0.4

pelI -2.7 0.3

celZ -6.4 0.2prtC -1.9 0.2hrpN -1.2 0.3

asr 2800 200

a. Fold changes are expressed as the ratio of the specific geneexpression level during exposure to pH 4.3, compared with pH 7.0,normalized to the level of expression of the ffhgene. Positive valuesrepresent genes induced by acidic pH whereas negative values rep-resent genes downregulated under acidic pH. The presented ratioswere obtained by quantitative RT-PCR as shown for Figs 2 and 4.Results are means from two independent experiments SD. Theresults obtained at pH 4.3 are different from those obtained at pH 7.0,with P-values of < 0.05 except for hrpN.




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with the parental strain (Fig. 2D, Table 2), demonstrating

that MfbR plays a strong negative autoregulatory role. In

addition, mfbR expression is no longer induced by acidic

pH stress in the mfbR background, suggesting that MfbR

activity is modulated by an acidic pH and that MfbR is the

main regulator responsible for acidic pH shock control at

the level of the mfb operon. On the other hand, asr tran-

script accumulation after acidic stress is independent of

the presence of a functional MfbR protein (Fig. 2E).

Since mfbR is the first gene of the mfbRABoperon, weconstructed mfbA and mfbB mutants to analyse their

involvement in D. dadantiivirulence on Saintpauliaplants.

Neither mfbA nor mfbB mutants were affected for viru-

lence (Fig. S2), demonstrating that the mfbR phenotype

is not due to a polar effect. This result indicates that

MfbR controls additional targets involved in D. dadantii

virulence.

Phenotypes of the D. dadantii mfbR mutant

With regard to extracellular enzyme production, plate

assays revealed that the synthesis of proteases, cellulase

and pectate lyases (Pels) was decreased in the mfbR

mutant, compared with the parental strain (Fig. 3A). In

addition, the D. dadantii mfbR mutant is slightly less

motile on semisolid medium than the parental strain (data

not shown), but there is no significant reduction in the

growth rate in M63 sucrose minimal medium (Fig. 3B

and C).

The decrease in enzyme production associated with the

mfbR mutation was successfully complemented by the

presence of a low-copy-number plasmid bearing mfbR,

pRK767-mfbR, demonstrating that these phenotypes are

linked to MfbR (Fig. S3). We investigated the time-course

of Pel production during growth, in minimal medium, in the

absence (Fig. 3B) or presence of polygalacturonate

(PGA) (Fig. 3C). In the parental strain, Pels were prefer-

entially produced at the end of the exponential phase and

were induced in the presence of PGA. In the mfbR

mutant, the growth phase-dependence and induction by

PGA were retained but the production of Pels was

reduced in both conditions, at all sampling times (Fig. 3B

and C). Thus, in synthetic growth conditions, MfbR acti-

vates the production of Pels both in the absence or pres-

ence of pectic compounds.

MfbR controls transcription of genes encoding plant cell

wall degrading enzymes

To determine the mechanism of action of MfbR, we inves-

tigated the transcription of individual virulence genes

using qRT-PCR. The virulence genes selected for theseexperiments were prtC (prtC encodes one of the four

extracellular proteases), celZ (celZ encodes the major

cellulase), pelE, pelD, pelI, pelL (pelE and pelD encode

two of the five major Pels whereas pelI and pelL encode

two secondary Pels) and hrcC, hrpN (hrcC encodes a

component of the Hrp type III secretion system and hrpN

encodes an harpin secreted by the Hrp secretion system).

Except pelL whose transcription was not significantly

modified in the mfbR mutant (data not shown), transcrip-

tion of all other genes encoding plant cell wall degrading

enzymes was significantly reduced in the mfbRmutant in

late exponential growth phase, which corresponds to thephase of maximal expression of virulence genes in the

wild-type strain (Fig. 4). The pel genes were the most

severely affected by the mfbR mutation (32-, 49- and

10-fold decrease for pelE, pelD and pelI respectively),

followed by prtC (sixfold decrease), and then celZ (1.8-

fold decrease) (Table 2). This finding indicates an activa-

tor function of MfbR on prtC, celZ, pelE, pelD and pelI

gene expression. In contrast, transcription of hrcC and

hrpN genes is not significantly affected by the mfbR

mutation. MfbR then is not involved in the Hrp system

regulation.

Since mfbR expression is itself induced after an acidic

pH stress (Fig. 2D), expression of its downstream targets

would normally be modified by acidic pH as well. To

confirm this, we quantified the impact of acidic pH stress

on the pelD, pelE, pelI, celZ and prtC transcript

accumulation. Transcription of the virulence genes pelD,

pelE, pelI, prtC and celZ decreased in acidic pH, during

the early exponential phase, by 2.9, 5.4, 2.7, 1.9 and

6.4-fold respectively (Fig. 4, Table 1). These results indi-

cate an inactivation of MfbR by the acidic pH stress. This

Table 2. Differential expression of mfbR, pel, celZ, prtC and hrpNgenes under growth in minimal M63 medium, supplemented withsucrose and polygalacturonate.

Gene name

Fold changes compared with the wild-type straina

Early exponentialphase

Late exponentialphase

pH 7.0 pH 4.3 pH 7.0

mfbR 64 5 8.2 0.3 107 12pelD -2.0 0.2 1.0 0.05 -49 7pelE -1.4 0.1 -1.1 0.1 -32 5pelI -1.5 0.1 1 0.05 -10 0.8celZ -1.9 0.1 -1.2 0.1 -1.8 0.2prtC -1.2 0.1 -1.3 0.1 -6.0 0.5hrpN 1.0 0.1 1.0 0.1 1.0 0.1

a. Fold changes are expressed as the ratio of the specific geneexpression level in the mfbR mutant compared with that in the wild-type strain, normalized to the level of expression of the ffh gene.Positive values represent genes upregulated in the mfbR mutantwhereas negative values represent genes downregulated in the mfbRmutant, compared with the wild-type strain. The presented ratios wereobtained by quantitative RT-PCR as shown for Figs 2 and 4. Resultsare means from two independent experiments SD. At pH 7.0,

results obtained with the mfbR mutant are different from the wild-typeresults, with P-values of < 0.05 except for hrpN.




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assertion is reinforced by the lack of any difference in the

expression of the MfbR target genes between the wild-

type strain and its mfbR derivative in acidic conditions

(Fig. 4, Table 2).

Expression of virulence genes during infection

The effect of the mfbR mutation on the expression of

virulence genes and mfbR was also followed in planta

during the infection process of Arabidopsis plants

(Fig. 5A). In the parental strain, the accumulation of pelE,

pelD, prtC and celZ transcripts was already observed

between 6 and 9 h post inoculation, while the accumulation

of the pelBtranscripts was observed later, 24 h post inocu-

lation. The expression of these virulence genes remained

at a relatively high level until the end of the experiment

(48 h post inoculation). On the other hand, the number of

mfbR transcripts increased rapidly following inoculation.

They were detectable between 3 and 9 h post inoculation

but decreased thereafter, being non-detectable after 12 h

post inoculation (Fig. 5A). Thus, the expression of mfbR

precedes those of the virulence genes in planta. After

inoculation with the mfbRmutant, a delayed and reduced

accumulation of virulence gene transcripts was observed

while a high amount of mfbRtranscript accumulation was

seen throughout the infection period. In the mfbRmutant,

we measure a truncated mfbR transcript that is not auto-

regulated. The mfbR transcript accumulation was more

abundant in the earlier time period (from 3 to 9 h post

inoculation). These findings support the idea that MfbR is

acting to activate the expression of the virulence gene

tested and to repress its own gene transcription in the

middle/advanced stages of the infection. The assay of

MfbR stability corroborates this hypothesis since at least

90% of the MfbR protein was detected 9 h after the

chloramphenicol chase experiment (Fig. 5B). This shows

Fig. 3. Phenotypes of the D. dadantii mfbR mutant.A. Production of plant cell wall-degrading enzymes in the mfbR mutant. Pectinase activity was observed after growth onpolygalacturonate-containing plates and staining with copper (II) acetate solution. Cellulase (Cel) activity was detected after growth onCMC-containing plates, followed by staining with Congo red. Protease (Prt) activity was revealed, after growth on skim milk-containing plates,by a translucid halo surrounding the bacteria growth area.B and C. Pel activity in D. dadantii 3937 (WT) and its mfbR derivative. Bacteria were grown in liquid minimal M63 medium containing either

sucrose (2 g l-1

) (B), or sucrose (2 g l-1

) and polygalacturonate (4 g l-1

) (C), and samples were taken every hour. Pel-specific activity isexpressed as mmol of unsaturated product liberated per min per mg of bacterial dry weight. Each value represents the mean of threeexperiments. Bars indicate the standard deviation.




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that MfbR is very stable and would be available to quickly

ramp up plant cell wall-degrading enzyme production after

conditions become alkaline later in infection.

Binding of MfbR and RNA polymerase at the mfbR, celZ

and pelE promoters

Migration of purified MfbR through a gel filtration column

revealed that the native protein has a molecular mass of

35 kDa and exists as a dimer in solution (data not shown).

This result is consistent with those previously reported for

various members of the MarR/SlyA family (Praillet et al.,

1996; Stapleton et al., 2002; Ellison and Miller, 2006;

Wilkinson and Grove, 2006).

In vitro gel retardation assays were performed in the

presence of MfbRHis and DNA fragments containing the

regulatory regions of the mfbR, pelE and celZ genes.

Typical results are shown in Fig. S4. MfbRHis bound to

all the DNA fragments tested. However, there were differ-

ences between the MfbRHis-binding profiles obtained

with the different DNA probes. In particular, MfbRHis

showed a higher affinity for mfbR, followed by pelE and

then celZ. Up to a 50-fold molar excess of unlabelled

specific probe was needed to completely prevent binding

of MfbRHis on the labelled probes whereas no signifi-

cant difference in MfbR-binding was observed in the pres-

ence of similar amount of a non-specific probe (Fig. S4).

These findings suggest that MfbRHis regulates the

expression of mfbR, pelE and celZ genes by binding

specifically to the promoter regions.

Binding assays were next conducted with both RNA

polymerase (RNAP) and purified MfbRHis. The mutual

Fig. 4. Transcription of the D. dadantiivirulence genes. The data show qRT-PCRresults for the D. dadantii virulence genes.The experiments were performed with thesame cDNA samples used previously for thestudies on the mfb operon described in Fig. 2.Each value represents the mean of twoindependent experiments. Bars indicate thestandard deviation.




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influence of MfbRHis and RNA polymerase on their

binding ability was estimated using control reactions con-

taining only one of these two proteins. In the case of the

mfbRoperator, addition of the two proteins together gave

a pattern similar to that obtained with MfbRHis alone

(Fig. 6). On the other hand, for the pelE and celZ opera-

tors, the addition of RNAP and MfbRHis together

showed an over-shift, corresponding to the binding of both

MfbRHis and RNAP. Moreover, more probes were

shifted in the presence of RNAP and MfbRHis together

than by adding the number of those shifted by the proteins

separately. Thus, it appears that MfbRHis and RNAP

bind synergistically at the pelEand celZoperators while a

competitive binding is observed at the mfbR operator.

In the case of the mfbR operator, at a low MfbR con-

centration (20 nM), a DNase I footprinting assay revealed

a single protected region from nucleotides -50 to -2, with

respect to the transcription start site (Fig. 7A). Increasing

the MfbRHis concentration up to 100 nM resulted in an

extension of the protected area from nucleotides -50 to

+10 (Fig. 7A, compare lanes 2 and 5). In the presence of

RNAP alone, a clearly protected region spanning from

-67 to +14 was observed. This footprinting area, which

encompasses that of MfbR, is in the appropriate position

for initiating transcription at the mfbR promoter (Figs 7A

and 2C). The addition of MfbRHis prevents the RNA

polymerase binding at the mfbR promoter. Furthermore,

inspection of the MfbR high-affinity site revealed the pres-

ence of an inverted repeat, consisting of two decamers

separated by two T bases (ATTAGTTGACTTGTTAAC-

TAAT), located between positions -25 to -4, which might

Fig. 5. A. Effect of the mfbR mutation on transcript levels of

pathogenicity genes throughout plant infection. RT-PCRexperiments were performed to analyse the transcript levels ofpelB, pelD, pelE, prtC, celZ and mfbR during plant infection.Bacterial cDNA levels were equilibrated using the transcript level ofthe constitutive rpoB gene. This gene was retained becauseprevious studies have revealed its stable expression duringinfection (Lebeau et al., 2008).B. Stability of MfbR in chloramphenicol chase experiment; the timespost-addition of chloramphenicol are indicated. The chaseexperiment was performed, during early exponential phase, onbacteria grown in M63 minimal medium containing sucrose.Samples, corresponding to a constant bacterial number, wereremoved between 0 and 9 h post chloramphenicol addition.Samples were then submitted to Western blot analysis using apolyclonal anti-MfbR as the primary antibody, and an anti-rabbitperoxidase-conjugated antibody (SIGMA) as the secondaryantibody. A representative Western blot experiment is shown.

Fig. 6. Binding of MfbRHis and RNA polymerase at the mfbR,pelE and celZ promoter regions. The concentration of MfbRHis(MfbR) and RNA polymerase (RNAP) used is indicated on the top.The position of free DNA (F) and of the main MfbRHis/DNAcomplexes (C) is indicated.




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represent the MfbR DNA binding site (Fig. 2C). Scanning

of the E. chrysanthemi genome with this sequence, using

the Genome explorer program (Mironov et al., 2000), did

not reveal any other significant matches. This is probably

due to the fact that members of the MarR/SlyA family are

able to specifically interact with a degenerated consensus

(Rouanet et al., 2004).

Footprinting experiments performed with MfbRHis on

the pelE operator revealed modifications in the digestion

pattern, but with DNase I-hypersensitive sites (-125,

-118, -115, -85, -81, -77, -72, -63 and -40, noted by *)

and discrete protected areas (-52 to -44, -74 to -64 and

-95 to -90) rather than well defined strongly protected

region(s) (Fig. 7B). A weakly protected region, spanning

from -165 to -40, was observed in the presence of RNAP

alone. In addition to the protected area, typical DNase

I-hypersensitive sites induced by RNAP were observed

around positions -142, -130, -118 and -79 (noted by

arrows). In the presence of both MfbRHis and RNAP, the

regions protected by each of the two proteins became

more pronounced (Fig. 7B, compare lanes 2 and 4 with 5,

or lanes 3 and 4 with 6). Thus, MfbRHis and RNAP

synergistically bind at the pelE promoter. More notably,

new DNase I-hypersensitive sites were observed around

positions -176, -144, -133, -130, -118, -98, -94 and -88

(indicated by circles), whereas the hypersensitive sites

induced by RNA polymerase around positions -142, -130

and -118 disappeared. This therefore shows that MfbR

and RNAP form a nucleoprotein complex at the pelE

promoter.

MfbR acts at the transcription initiation step

We further characterized the MfbR regulatory mecha-nism at the mfbR and pelE promoters. The effect of

MfbRHis on RNAP activity was investigated, first, by

using potassium permanganate (KMnO4) footprinting on

supercoiled plasmids containing the mfbR regulatory

region (pWN4100) or the pelE regulatory region (pTL5).

KMnO4 preferentially targets the pyrimidine residues in

the untwisted regions of DNA and, thus, allows the

extent of the promoter opening to be measured. Follow-

ing the addition of RNAP, we observed that two bases

located around the transcription initiation position of both

genes are sensitive to KMnO4 (Fig. 8A, -4 and +3 for

mfbR, -4 and -2 for pelE). The addition of increasingconcentrations of MfbRHis substantially decreased the

KMnO4 reactivity at the mfbR promoter while an

increase in the KMnO4 reactivity was observed at the

pelE promoter. From these data, we can infer that

binding of MfbRHis in the mfb operon regulatory region

inhibits open complex formation by RNAP and supports

a direct negative retro-control mechanism by MfbR. By

contrast, MfbR promotes open complex formation by

RNAP at the pelE promoter.

We next used in vitrotranscription to follow precisely the

effect of MfbRHis on RNAP activity. For this purpose, we

monitored mfb operon and pelE transcription using

pWN4100 and pTL5 DNA, respectively, with RNAP and

MfbRHis added either alone or in combination. As

expected, the addition of increasing MfbRHis concentra-

tions decreased the accumulation of transcripts from the

mfb operon promoter (Fig. 8A and B) whereas the tran-

scription of the pelE promoter was noticeably increased

(Fig. 8A and B). These results demonstrate that MfbRHis

specifically inhibits the mfb promoter and activates pelE

transcription in vitro.

Fig. 7. DNase I footprinting of MfbRHis and RNAP binding at themfbR and pelE promoters.A. Digestions at the mfbR promoter. Lane 1, no protein; lanes 2 to5, incubation with 20, 30, 50 and 100 nM MfbRHis respectively;lane 6, incubation with 150 nM RNAP; lane 7, incubation with150 nM RNAP and 30 nM MfbRHis; lane 8, incubation with150 nM RNAP and 100 nM MfbRHis. The arrowheads indicatehypersensitivities induced by binding of RNAP. The areas protected

by the proteins are indicated on the left.B. Digestions at the pelE promoter. Lane 1, no protein; lanes 2 and3, incubation with 200 and 800 nM MfbRHis respectively; lane 4,incubation with 150 nM RNAP; lane 5, incubation with 150 nMRNAP and 200 nM MfbRHis; lane 6, incubation with 150 nMRNAP and 800 nM MfbRHis. The arrowheads and stars indicatehypersensitivities induced by binding of RNAP and MfbRHisrespectively. The open circles indicate hypersensitivities induced bybinding of MfbRHis and RNAP. The areas protected by theproteins are indicated on the right.




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The affinity of MfbR for its operators is pH sensitive

To assess the possibility that DNA binding may be compro-

mised at a lower pH, we first used the Chromatin Immuno-

Precipation (ChIP)technique to compare the in vivoMfbR

DNA complex formation at pH 7 and at pH 4.3. As control,

we used KdgR protein, a regulator that specifically regu-

lates pelgenes, such as pelEin response to pectin degra-

dation products (Reverchon et al., 1991). Figure 9 shows

the PCR analysis of the immunoprecipitated DNA using

primers that are specific to the promoter regions of mfbR,

pelEand pelDand, as a negative control, primers specific

to the rpoA coding region. Specific amplifications of the

mfbR, pelE, pelD regulatory regions were observed with

immunoprecipitated DNA using anti-MfbR in the parental

strain, grown at pH 7, while no amplification of the rpoA

negative control was observed in the same conditions.

Furthermore, no amplified mfbR, pelEand pelDfragments

were observed with immunoprecipitated chromatin using

the anti-MfbR antibodies from the mfbR mutant grown at

pH 7. These results confirmed the specific binding of MfbR

to its target operators. After an acidic stress, the binding of

MfbR to its target operators was strongly reduced whereas

no significant difference was observed with the control

KdgR protein. In addition, Western blot experiments, per-

formed with the same culture extracts and the anti-MfbR

Fig. 8. MfbRHis prevents transcription initiation at the mfbR promoter and activates pelE transcription.A. The KMnO4 and in vitro transcription experiments were performed on supercoiled templates. For mfbR, the reactions were carried out in the

presence of 100 nM RNAP and the following concentrations of MfbRHis: lanes 2 and 7, no MfbRHis protein; lanes 3 and 8, 10 nMMfbRHis; lanes 4 and 9, 20 nM MfbRHis; lanes 5 and 10, 30 nM MfbRHis; lanes 6 and 11, 50 nM MfbRHis. Lane 1 corresponds to the

KMnO4 reaction without protein. For pelE, the reactions were performed in the presence of 100 nM RNAP and the following MfbRHisconcentrations: lanes 2 and 5, no MfbRHis protein; lanes 3 and 6, 25 nM MfbRHis; lanes 4 and 7, 50 nM MfbRHis. Lane 1 corresponds tothe KMnO4 reaction without protein. The numbers -4, +3 and -4, -2 indicate the positions of KMnO4 sensitive nucleotides for mfbR and pelErespectively. Asterisks indicate the transcription start sites of mfbR and pelE genes. DNA sequencing ladders (lanes A, C, G and T) wereintroduced to indicate nucleotide positions. Dotted nucleotides in the DNA sequences indicate the KMnO 4 sensitive nucleotides.B. Quantitative analysis of the expression of mfbR and pelE promoters. The data were normalized to the value obtained for the bla promoter(an internal control, see Fig. 8A) and are expressed as a percentage (100% mfbR relative expression corresponds to the expression obtainedin the absence of MfbR; 100% pelE relative expression corresponds to the expression obtained in the presence of 50 nM MfbR). Each valuerepresents the mean of two independent experiments. Bars indicate the standard deviation.




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antibodies, revealed no significant variation in the MfbR

cellular content following an acidic shock. These results

demonstrated that MfbR activity is pH-sensitive. These in

vivo experiments were further confirmed in vitro by using

band-shift assays at various pH levels. The MfbR DNA

binding activity on mfbR and pelE promoters was signifi-

cantly reduced in vitro at pH 5.0 (Fig. S5). As a control,

KdgR DNA binding activity on the pelE promoter was not

affected by acidic pH.

Discussion

Pathogenic bacteria face a variety of adverse conditions

in the host environment, such as nutrient limitation, oxi-

dative and acidic stresses. At the same time, these

signals provide the necessary information for bacteria to

adjust the expression of their virulence factors. Most

pathogenic bacteria, including D. dadantii, have evolved

sophisticated systems to sense hostile environments and

trigger compensatory gene expression in order to survive

within the host (Llama-Palacios et al., 2005; Hommais

et al., 2008). Transcriptional regulators of the MarR/SlyA

family constitute an important part of this system and

mediate adaptative responses to a number of external

stimuli, such as temperature for RovA, a protein that

serves as a thermosensor to control virulence gene

expression in Yersinia (Tran et al., 2005; Cathelyn et al.,

2007; Herbst et al., 2009), or oxidative conditions for SarZ

Fig. 9. ChIP analysis of the in vivo sensitivity of MfbR to pH.The top panel depicts a gel on which PCR products, generated with primers designed to detect either mfbR, pelE and pelD promoter DNA, orthe control rpoA coding region in each immunoprecipitated sample, were analysed. ChIP analysis performed with anti-MfbR antibodies (A) orin the presence of the control anti-KdgR antibodies (B).C. ChIP fold enrichment values were measured by quantitative PCR and calculated relative to the non-target rpoA and the correspondingsignals obtained in the mutants. The oligonucleotides used are: pelD ChIPf and pelD ChIPr for pelD, pelE ChIPf and pelE ChIPr for pelE,mfbRup and PextmfbR2L for mfbR. Each value represents the mean of two independent experiments. Bars indicate the standard deviation.D. The MfbR cellular content measured in Western blot experiments.




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and MgrA, which use a single Cys residue to sense per-

oxide stress and control virulence in Staphylococcus

aureus (Chen et al., 2009).

Systematic analyses of the D. dadantii MarR/SlyA

members, to establish their involvement in virulence, led

to the identification of MfbR as a new regulator of viru-

lence genes in D. dadantii. mfbR is co-transcribed with

two genes, mfbA and mfbB, which encode an efflux pump

of the MFS family. This operon is driven by one unique

promoter and is submitted to growth phase-regulation,

acidic pH induction and, also, negative retro-control, an

autoregulation previously reported for several other

members of the MarR/SlyA family (Ellison and Miller,

2006; Herbst et al., 2009). Induction of the mfbRAB

operon by acidic pH is lost in the mfbR mutant indicating

that, in the wild-type strain, this pH modulation is medi-

ated by MfbR itself.

MfbR modulates RNA polymerase activity, both

positively or negatively, at different targetsAn important characteristic feature of the mfbR mutant is

its reduced production of plant cell wall-degrading

enzymes, as well as a reduced expression of the corre-

sponding virulence genes, which therefore suggests an

activator function for MfbR on these genes. Furthermore,

in vitro assays demonstrated that purified MfbRHis spe-

cifically binds to the regulatory region of the mfbR, pelE

and celZ genes. A competitive binding between RNA

polymerase and MfbRHis was observed at the mfbR

promoter region while a synergistic binding between the

two proteins was shown at the promoter of pelE and

celZ genes. KMnO4 and in vitro transcription experi-ments further demonstrate that MfbR specifically inhibits

mfb promoter expression and activates pelE transcrip-

tion. To the best of our knowledge these data represent

one of the first elucidations of the direct action of a

member of the MarR/SlyA family on RNA polymerase

activity, for both negative and positive regulation exerted

on different gene expressions. Although a study on the

mechanism of SlyA activity was initiated a while ago,

only its repressive function was elucidated at its own

gene promoter (Stapleton et al., 2002). Several anti-

repression mechanisms were proposed to explain the

positive action of SlyA on various target genes but the

direct impact of such a mechanism on gene expression

has not yet been reported (Ellison and Miller, 2006). By

contrast, the positive control of RovA, from Yersinia, has

been clearly established on inv genes (Tran et al.,

2005), but the negative function of this regulator has not

yet been clearly demonstrated in this bacteria. Further-

more, a dual role for OhrR as a repressor and as an

activator was recently reported in Streptomyces coeli-

color (Oh et al., 2007). However, this concerns two

divergently transcribed genes and the action of OhrR on

RNA polymerase activity was not described.

The DNA binding activity of MfbR is pH-sensitive

Another outcome of this work concerns the relationship

between the MfbR regulator and acidic stress. Our study

highlights the impact of stress induced by acidic shock

on mfbR and virulence gene expression. In the wild-type

strain, expression of genes encoding pectate lyases, cel-

lulase and proteases is decreased under acidic pH

stress and is similar to the expression observed in the

mfbR mutant under the same conditions. This result

reinforces the idea that MfbR is unable to activate viru-

lence genes in acidic conditions and that acidic shock

modulates the activity of MfbR. This finding is particu-

larly relevant because acidic shock stress is presumed

to mimic the environment bacteria encounter in plant

tissues, particularly during the initial/early stages of

infection. Both in vivo ChIP experiments and in vitro

DNA-binding assays confirm that the affinity of MfbR forits own promoter and for its target gene promoters is

significantly reduced at acidic pH. To date, the activity of

only one other regulator of the MarR/SlyA family, HucR

in Deinococcus radiodurans, has been reported to be

pH-sensitive (Bordelon et al., 2006). This regulator con-

trols the expression of an uricase gene and urate is its

natural ligand. Recently, a mechanism for the attenua-

tion of the DNA binding capacity of the HucR regulator

by urate was proposed (Perera et al., 2009). This

mechanism is based on a conformational change initi-

ated by charge repulsion, due to a bound ligand that

propagates to the DNA recognition helix of the regulator.The ligand interaction was assumed to change the ori-

entation of the recognition helix, resulting in attenuated

binding to DNA. The affinity of HucR for DNA is also

significantly reduced at acidic pH. Based on the crystal

structure of HucR, it was proposed that the a2/a2

helices that provide the dimer interface could serve as

pH-sensors, via the imidazole rings of His51 and His51 .

At pH 7.0, the imidazole moieties are involved in a

p-stacking interaction, but this interaction is disfavoured

following protonation of both the imidazole rings at acidic

pH (His pKa = 6.04). Protonation of His51/His51 leads

to a conformational change, induced by the repulsion of

like charges, that provokes a pH-dependent reorienta-

tion of the DNA-binding domain that, in turn, compro-

mises the formation of a complex (Bordelon et al.,

2006). Alignment of MfbR with HucR revealed that the

His residues of the a2/a2 helices are not conserved in

MfbR, which therefore suggests that the mechanism of

pH-sensing is different in MfbR. However, it is tempting

to speculate that acidic pH might modify the charge of

some critical amino acids in MfbR and this would result




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in an attenuation of the DNA-binding capacity of MfbR.

Further investigations should reveal the critical amino

acid residues of MfbR involved in pH-sensing.

MfbR is produced during the early phase of infection

before the expression of the virulence target genes

Although various members of the MarR family have beenshown to be important in the virulence of animal and plant

pathogens, it is rare that the effects of the regulators were

analysed in the natural host of the pathogen. We exam-

ined the impact of the mfbR mutation on D. dadantii viru-

lence, and on the expression of its virulence genes, during

the natural interaction with the plant host. It is noteworthy

that, overall, the results obtained on synthetic media are

in accordance with those obtained in planta. Transcripts

quantification during infection tests performed with the

parental strain showed expression of mfbR during the

initial-early stages of infection, followed by a decrease

in the middle-advanced stages of infection, while the

increase in the accumulation of the virulence gene tran-

scripts was more pronounced during the advanced stages

of infection. Thus, it is tempting to speculate that the

increase in mfbRexpression during the initial early stages

of infection is due to the adverse conditions encountered

by the bacteria in the apoplast, which is normally acidic.

Furthermore, similar experiments performed with the

mfbR mutant revealed a strong reduction in the induction

of virulence gene transcripts, concomitant with a strong

expression of the mfb operon throughout the experiment.

This therefore led us to suppose that MfbR activates the

expression of the genes encoding the extracellular

enzymes and represses its own gene expression in the

middle-advanced stages of infection. By integrating the

different results presented in this work, we propose a

model in which MfbR activity is modulated upon the alka-

linization of the infected tissue. This pH change is postu-lated to allow MfbR binding, which then leads to

repression of the mfbRABoperon and to activation of pel

virulence genes in the middle-advanced stages of infec-

tion (Fig. 10).

Acidic pH, via MfbR, rationalizes temporal virulence

expression in planta

When they enter a host plant, D. dadantii cells colonize

the intercellular spaces of the cortical parenchyma and

migrate within the cell walls, without causing any severe

injury to the cellular structure (Fagard et al., 2007; Lebeau

et al., 2008). During this colonization phase there is no

production of plant cell wall-degrading enzymes (Lebeau

et al., 2008), but bacteria have to adapt to the apoplast

environment, which is an acidic, low-nutrient medium. The

pH of apoplastic fluids is between 4 and 6.5, depending

on the plant species (Grignon and Sentenac, 1991). After

the colonization phase, the bacteria may either reside

latently in the plant intercellular spaces, without provoking

any symptoms, or they may start the disease process.

Fig. 10. Schematic representation of theregulation of virulence factors in D. dadantii atdifferent stages of infection. The lanes with abar and arrows represent a repression andactivation mechanism respectively; wide, thinand dotted arrows under the genes indicatehigh, intermediate and low transcriptionrespectively; i and a represent inactive andactive forms of the regulator MfbR underacidic or neutral pH respectively. MfbR activity

is modulated upon the alkalinization of theinfected tissue. This pH change is postulatedto allow MfbR binding which then leads torepression of the mfbRAB operon and to

activation of pel virulence genes in themiddle-advanced stages of infection.




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Thus, disease caused by D. dadantii is an intricate

process with two successive phases, an asymptomatic

phase and a symptomatic phase, that require the tempo-

ral expression of different groups of genes. MfbR might

help in this temporal regulation of synthesis of the factors

needed for pathogenesis. Indeed, the acidic conditions of

the apoplast could lead to a significant reduction of MfbR

activity. This would result in an increase in the expression

of the mfb operon due to a decrease in the auto-

repression mechanism. Furthermore, under acidic condi-

tions, MfbR would be unable to activate the expression of

the genes encoding for the plant cell wall-degrading

enzymes. This could prevent an early detection by the

host of plant breakdown products that signal the presence

of the pathogen before it reaches a population density

appropriate for successful infection (DOvidio et al.,

2004). Later during infection, alkalinization of the apoplast

may be a plant response to bacterial infection. Indeed,

oligogalacturonides or effectors secreted by type III secre-

tion systems of plant pathogenic bacteria, such as

harpins, have been shown to induce medium alkaliniza-tion of plant cell cultures (Popham et al., 1995; Mathieu

et al., 1998; Spiro et al., 1998). Whichever of these

mechanisms is operating in the early steps of D. dadantii

infection, an alkalinization of infected plant tissue has

been detected even before the occurrence of maceration

symptoms (Nachin and Barras, 2000). These changes in

the in planta conditions result in an activation of MfbR.

Thus, the large amount of MfbR accumulated up to this

point would stimulate increased production of the plant

cell wall-degrading enzymes which would, in turn, result in

a transition from biotrophic to necrogenic lifestyle. The

rapid production of the plant cell wall-degrading enzymeswould serve to macerate host cells and counter host

defences.

In conclusion, this work presents the characterization of

a new member of the MarR/SlyA family involved in viru-

lence control in a phytopathogenic bacterium. Our data

reveal important novel features, such as a considerable

reduction of plant virulence in an MfbR-deficient strain

and the use of a direct dual regulatory mechanism, involv-

ing repression and activation, by a member of the MarR

family to modulate gene expression in response to

changes in the environmental conditions. Further investi-

gations should focus on the identification of the MfbR

regulon using global approaches, such as transcriptome

analysis or in vivo expression technology, and on the

elucidation of the mechanism by which acidic pH stress

modulates the MfbR activity. It would also be interesting to

integrate this global regulator in the networks controlling

the synthesis of virulence factors, particularly that of

pectate lyases. Such approaches are a prerequisite for

elucidating the mechanisms used by D. dadantii to cause

disease.

Experimental procedures

Bacterial strains, plasmids, culture conditions and DNA

manipulation techniques

Bacterial strains, phages and plasmids are described in Table

S3. D. dadantii and E. coli were grown at 30C and 37C

respectively. The rich medium used was LuriaBertani broth

(LB); we used M63 minimal salts medium (Miller, 1972)

supplemented with a carbon source (polygalacturonate

(PGA) at 0.4% (w/v) and sucrose at 0.2% (w/v)). When

required, the antibiotics were as follows: ampicillin (Ap),

100 mg ml-1; kanamycin (Km), 50 mg ml-1; chloramphenicol

(Cm), 25 mg ml-1; and tetracycline (Tet), 20 mg ml-1. Liquid

cultures were grown in a shaking incubator (220 r.p.m.).

Media were solidified by the addition of 1.5% agar (w/v). To

test motility, equal quantities of bacteria, contained in 5 ml,

were loaded into holes in the middle of 0.4% LB agar plates.

Plates were incubated at 30C and checked between 12 and

24 h after inoculation. Motility was determined by measuring

the diameter of the colony. The pH stress was performed by

shifting pH from 7.0 to 4.3 by addition of malic acid, since

malate is naturally present in the plant apoplast (Lohaus

et al., 2001). DNA manipulations were performed using stan-dard methods (Sambrook and Russell, 2001).

Genetic techniques

Construction of mutants in each regulator of the MarR family

is described in supplementary material. To construct the mfbA

and mfbB mutants, the corresponding genes were specifi-

cally amplified using the primers mfbAf and mfbAr for mfbA,

and mfBf and mfBr for mfbB. The resulting PCR fragments

were cloned into the pGEMT plasmid using the TA cloning kit

from Promega. Inactivation of the mfbA and mfbBgenes was

carried out by ligation of a uidA-KmR cassette (Bardonnet and

Blanco, 1992) in the unique SmaI and EcoRI sites, located in

mfbA and mfbBrespectively. Insertion of a uidA-KmR

cassettein a gene, in the correct orientation, generates a transcrip-

tional fusion. These insertions were introduced into the D.

dadantii chromosome by marker exchange recombination

between the chromosomal allele and the plasmid-borne

mutated allele. The recombinants were selected after suc-

cessive cultures in low phosphate medium in the presence of

chloramphenicol or kanamycin, conditions in which pBR322

derivatives are very unstable (Roeder and Collmer, 1985).

Correct recombination was confirmed by PCR.

Transduction of the mutation from one strain to another

was performed using phage phiEC2 (Resibois et al., 1984).

Plate and enzyme assays

Detection of protease activity was performed on medium

containing Skim Milk (12.5 g l-1) and detection of cellulase

activity was performed using the Congo red procedure

(Teather and Wood, 1982). Detection of pectinase activity

was performed on medium containing PGA using the copper

acetate procedure, as previously described by Reverchon

et al. (1994).

Assay of pectate lyase was performed on toluenized cell

extracts. Pectate lyase activity was determined by the deg-

radation of PGA to unsaturated products that absorb at




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235 nm (Moran et al., 1968). Specific activity is expressed as

mmol of unsaturated products liberated min-1 mg-1 (dry

weight) bacteria. Bacterial concentration was estimated by

measuring turbidity at 600 nm, given that an optical density

(OD) of 1.0 at 600 nm corresponds to 109 bacteria per millili-

tre and to 0.47 mg of bacteria (dry weight) per millilitre.

MfbRHis purification

The coding region of mfbR was amplified by PCR using

primers mfbR tag D and mfbR rev tag His (Table S3), con-

taining unique restriction sites, so that the resulting fragment

contained an NdeI site at the ATG initiation codon and an

XhoI site before the stop codon of MfbR. The resulting 458 bp

NdeIXhoI restriction fragment PCR product was cloned into

the pET20b(+) vector (Novagen) to generate pSR3426. In the

resulting plasmid pSR3426, the mfbRgene was placed under

the control of the T7 RNA promoter and fused to a His6tag on

its C-terminus. Overproduction of the MfbRHis was carried

out in E. coli BL21(DE3)/pLysS. Purification of the recombi-

nant MfbR protein was achieved from cells grown at 30C in

LB medium containing ampicillin and chloramphenicol to

maintain both pSR3426 and pLysS. When the optical densityat 600 nm reached 0.6, IPTG was added to a final concen-

tration of 200 mM to induce T7 RNA polymerase synthesis.

Then the cells were grown for an additional 2 h at 30C. Cells

were collected by centrifugation at 5000 g for 10 min and

resuspended in an appropriate volume of lysis buffer

(Qiagen). The bacteria were disrupted at 138 00 kPa in a

French press (Aminco) and crude extracts were subse-

quently centrifuged at 20 000 g, for 15 min, to remove the

subcellular fractions. The supernatants obtained were used

for purification. Protein purification was performed under

native conditions at 4C, according to the QIA expressionist

handbook. Fractions containing the MfbR protein with more

than 95% purity, as measured by SDS-PAGE, were pooled

and dialysed twice for 2 h against 2 l of desalting buffer(20 mM Tris-HCl pH 7.9, 1 mM EDTA, 1 mM DTT, 10% glyc-

erol), then overnight against storage buffer (20 mM Tris-HCl

pH 7.5, 500 mM NaCl, 0.1 mM EDTA, 0.2 mM DTT, 50%

glycerol). The final preparation was stored at -20C.

To determine the native molecular mass of MfbR, approxi-

mately 100 mg of purified MfbRHis protein, suspended in

200 ml of buffer (50 mM Tris-HCl, pH 7.9, 0.4 M NaCl), was

loaded onto a standardized Superose 12 HR (Pharmacia)

previously washed with the same buffer. Elution was per-

formed at a 0.2 ml min-1 flow rate.

Degradation rate of MfbR measured by antibiotic chase

Overnight cultures were inoculated into fresh M63 minimal

medium containing sucrose. At the early exponential phase

(OD600 = 0.25), chloramphenicol was added, to a final con-

centration of 200 mg ml-1, from a freshly prepared stock

solution. Samples corresponding to a constant bacterial

number were removed between 0 and 9 h, centrifuged,

resuspended in an adequate volume of Laemmli sample

buffer, and boiled for 23 min. Samples were separated by

SDS-PAGE (15% polyacrylamide) and transferred, for

20 min, onto a nitrocellulose membrane, using a semi-dry

blotter. Western blots of the separated proteins were incu-

bated with a polyclonal anti-MfbR, as the primary antibody,

and an anti-rabbit peroxidase-conjugated antibody (SIGMA)

as the secondary antibody. The signals obtained in Western

experiments were detected by autoradiography on Amer-

sham MP film and quantified using ImageMaster TotalLab

version 2.01 software (GE Healthcare). Poly clonal MfbR

antibodies were obtained by collecting sera from rabbits fol-

lowing a course of four injections at intervals of 2 weeks using

200 mg of purified MfbRHis for the first injection and 100 mg

of protein for the subsequent booster injections.

RNA isolation, primer extension and quantitative reverse

transcription polymerase chain reaction analysis

Total RNA was extracted from D. dadantii by the frozen-

phenol method (Maes and Messens, 1992). Primer extension

experiments were essentially performed as described previ-

ously (Rouanet et al., 2004). The primer pmfbpextL2 (Table

S3), used for specific detection of mfbR mRNA, was 5 end-

labelled and annealed to mfbRmRNA molecules at positions

+118 to +137.

For RT-PCR analysis, cDNA was synthesized, usingrandom hexamers and Fermentas reverse DNA polymerase,

and qPCR was performed using the LightCyclerR faststart

DNA masterplus SYBR Green I kit from Roche (Roche Applied

Science), as previously described (Lautier et al., 2007).

Target gene expression is defined by the method described

by Pfaffl (2001) (Pfaffl, 2001). The statistical program used to

analyse the data was the Relative Expression Software Tool

(REST) (Pfaffl et al., 2002). The ffhgene was selected as the

reference gene for real-time RT-PCR to provide an accurate

normalization, based on studies performed in the related

plant pathogen Pectobacterium atrosepticum (Takle et al.,

2007). We confirmed that a similar ffh expression was

observed in the D. dadantii parental strain and in its mfbR

derivative in the different growth conditions used in this work.

ChIP

In ChIP experiments, a cells nucleoprotein is cross-linked

with formaldehyde, extracted and then fragmented by soni-

cation so that the average DNA fragment is 500 bp. Antibod-

ies, directed against the protein of interest, are then used to

select protein cross-linked DNA fragments that are analysed

by PCR after reversing the cross-links. Hence, in our experi-

ments, the parental strain and its mfbR or kdgR derivatives

were grown until the early exponential phase (OD600 = 0.25)

and then the pH of the cultures was shifted from 7 to 4.3.

Samples were taken 5 and 15 min after the pH stress and

submitted to Chlp, using anti-MfbR and anti-KdgR antibodies

to immunoprecipitate the DNA fragments attached to MfbR

and KdgR respectively. In vivo cross-linking of bacterial

nucleoprotein was initiated by the addition of formaldehyde

(at a final concentration of 1 %) to the bacterial cultures. After

a 30 min incubation at room temperature, cross-linking was

quenched by the addition of glycine (final concentration of

250 mM) followed by an additional 15 min incubation at room

temperature. Typically, cells were then harvested from 12 ml

of cultures by centrifugation, washed three times with Tris-




17/20

buffered saline (pH 7.5), and resuspended in 1 ml of lysis

buffer [20 mM HEPES-KOH pH 7.9, 50 mM KCl, 0.5 mM

DTT, 10% (v/v) glycerol] supplemented with a protease inhibi-

tor cocktail (Roche). Cellular DNA was then sheared, by

sonication, to an average size of 500 bp. The lysates were

adjusted with 10 mM Tris-HCl (pH 8), 150 mM NaCl and 0.1%

NP40, then the cell debris were removed by centrifugation

and the supernatant was retained for use as the input sample

in immunoprecipitation experiments.

An 800 ml aliquot of the input sample was used for each

immunoprecipitation experiment. The sample was incubated

with 5 ml of MfbR-antibodies or KdgR-antibodies, for 4 h at

4C, on a rotating wheel, then 50 ml of ProteinA-Sepharose

beads (Amersham) was added. The mixture was then incu-

bated overnight, at 4C, on a rotating wheel. The beads were

collected from each sample by a 2 min centrifugation, at

12 000 r.p.m., and washed five times with 1 ml of washing

buffer (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% NP40),

then twice with Tris-EDTA (Tris-HCl 10 mM pH 8, EDTA

1 mM). Immunoprecipitated complexes were next removed

from the beads by treatment with elution buffer (50 mM Tris-

HCl pH 8, 10 mM EDTA, 1% SDS) at 65C for 15 min. Immu-

noprecipitated samples were uncross-linked by incubation

overnight at 65C. Prior to analysis, DNA was purified using a

PCR purification Kit (QIAGEN) and resuspended in 100 ml of

0.5 elution buffer.

Following purification, PCR was used to analyse immuno-

precipitated DNA: 5 ml DNA samples were used in a 50 ml

reaction mix containing a 0.5 mM concentration of each oli-

gonucleotide primer. DNA amplification was catalysed, by Taq

DNA polymerase (Fermentas), and the PCR was allowed to

proceed for 2225 cycles before 10 ml of the reaction mixture

was analysed by electrophoresis on a 2 % agarose gel.

Alternatively, qPCR was performed in the presence of a 1 ml

DNA sample, as described above.

Virulence assays

Pathogenicity assays were performed as described in

Lebeau et al. (2008). For Saintpaulia assays, 1-month-old

potted S. ionanthacv. Blue Rhapsody cuttings were infected.

Bacteria cells grown on LB agar medium for 16 h at 30C

were suspended in a 100 mM KCl solution to an OD600 of

0.1, corresponding to a concentration of 10 8 cfu ml-1. About

20 ml of the resulting suspension (i.e. around 2 106 bacteria)

was inoculated into one leaf per plant by needle-free syringe

infiltration, after wounding the lower side of the leaf. Plants

were incubated in tropical conditions (day/night temperature

of 28C/26C; 16H light; relative humidity of 100%).

Twenty-four plants were tested for each bacterial strain.

Infections were also performed with a 10-fold lower inoculum

(i.e. around 2 105 bacteria). For Arabidopsis assays, bacte-

ria were suspended to a concentration of 10 4 bacteria ml-1 in

a 50 mM KH2PO4 pH 7 buffer and inoculation was performed

by wounding one leaf of 6-week-old Col-0 plants with a

needle and then depositing a 5 ml droplet of this bacterial

suspension (i.e. around 50 bacteria). Plants were incubated

at 24C/19C (day/night) in short day conditions (8 h light)

and in small containers with abundant watering to give 100%

humidity. Twenty-four plants were tested for each assay. Pro-

gression of symptoms was scored daily for a week.

Infections of potato tubers were performed as described

(Lautier and Nasser, 2007) with 2.5 106 bacteria in 5 ml of

50 mM KH2PO4 pH 7 buffer. Assays were carried out at least

in triplicate. Whatever the plant host, negative controls were

performed using sterile buffer.

RT-PCR expression analysis of bacterial genes after

infection

For D. dadantii gene expression analysis in Arabidopsis

plants, total RNAs were purified as described in Lebeau et al.

(2008). Briefly,6-week-old Arabidopsisplantswereinfected by

rapid immersion in a bacterial suspension (5 107 cfu ml-1) in

50 mM KPO4 pH 7 buffer containing 0.01% (vol/vol) of the

Silwet L-77 surfactant (van Meeuwen Chemicals BV, Weesp,

the Netherlands). Aerial plant tissues were collected at differ-

ent time points post inoculation and ground, in liquid N2, to a

fine powder. Total RNAs were purified and RT-PCR analysis

was performed. RT-PCR products were loaded on a 1.2%

(w/v) agarose gel and visualized by ethidium bromide fluores-

cence. Experiments were carried out twice, independently.

In vitro DNA/protein interaction

Band-shift assay and DNase I footprinting were performed as

previously described (Nasser et al., 1997). The regulatory

region of the mfbR, celZ and pelE DNA fragments were

recovered from plasmids pSR2790, pWN2965 and pSR1175,

respectively, by a PstINarI digestion for mfbR, a SalISacII

digestion for celZ, and an EcoRIHindIII digestion for pelE.

The DNA fragments obtained were further end-labelled by

filling up the NarI, SalI and HindIII extremities in the presence

of (a-32P)dCTP (3000 Ci mmol-1, GE Healthcare) and the

Klenow fragment of DNA polymerase. The labelled DNA frag-

ments were purified, after electrophoresis, on agarose gel

using the Qiagen gel extraction kit. The pH sensitivity analy-sis was performed, as described by Bordelon et al. (2006),

except that the electrophoresis buffer for the reactions per-

formed at pH 6 and pH 5 was replaced by Tris-acetate buffer

(pH 6.6).

Potassium permanganate reactivity assay and in vitro

transcription

The reactions for the potassium permanganate (KMnO4)

reactivity assay and in vitro transcription experiments were

performed with supercoiled templates, as previously

described (Lautier et al., 2007). The reaction products were

solubilized in water, divided into equal parts and then sub-

mitted to primer extension with radioactively end-labelled

primers pmfbpextL2, for mfbR mRNA, uidAdeb for pelE

mRNA and bla3B4 for the bla transcript (Table S3). The

extension with primers pmfbpextL2, uidAdeb and bla3B4

yields 137 bp, 96 bp and 100 bp fragments respectively. The

amplification products were analysed on a 6% sequencing

gel. The signals obtained were detected by autoradiography

on Amersham MP film and quantified using ImageMaster

TotalLab version 2.01 software (GE Healthcare). E. coli s70

RNA polymerase was obtained from GE Healthcare and the




18/20

protein molarity was determined based on the concentration

of the batches (mg ml-1).

Acknowledgements

We are grateful to Valerie James for the English corrections,

to A. Buchet for critical reading of the manuscript, and to our

colleagues G. Condemine, F. Hommais and N. Hugouvieux-

Cotte-Pattat for their support and advice. We thank A. Grove,

S. Castang and M. Lemaire for their advice regarding the pH

sensitivity and Chlp experiments. We are grateful to J.

Wawrzyniak for technical support. This work was supported

by grants from the Centre National de la Recherche Scienti-

fique (CNRS), and from French ANR blanc Rgupath 2007

Program, NANR-07-BLAN-0212. The funders were not

involved in the study design, data collection and analysis,

decision to publish, or preparation of the manuscript.

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MarR Family Dickeya