Molecular Microbiology (2010) 䊏
Systematic targeted mutagenesis of the MarR/SlyA family
members of Dickeya dadantii 3937 reveals a role for MfbR in
the modulation of virulence gene expression in response to
acidic pH mmi_7388 1..20
Sylvie Reverchon,1 Frédérique Van Gijsegem,2
Géraldine Effantin,1 Ouafa Zghidi-Abouzid1 and
William Nasser1*
1
Univ Lyon, F-69622 Lyon France; Université Lyon 1
Villeurbanne; INSA-Lyon F-69621 Villeurbanne; CNRS
UMR5240 Microbiologie, Adaptation et Pathogénie,
Lyon, France.
2
Laboratoire Interaction Plantes Pathogènes LIPP
UMR217 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 the
members 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
Accepted 30 August, 2010. *For correspondence. E-mail william.
nasser@insa-lyon.fr; Tel. (+33) 4 72 43 26 95; Fax (+33) 4 72 43 15
84.
© 2010 Blackwell Publishing Ltd
doi:10.1111/j.1365-2958.2010.07388.
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 wide
range 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 oxidative
stresses, 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.,
2 S. Reverchon et al. 䊏
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. dadantii all together as
the 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. coli and 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. dadantii regulators 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
© 2010 Blackwell Publishing Ltd, Molecular Microbiology

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 symptoms
more rapidly than the wild-type strain (data not shown). By
contrast, the virulence of the slyA and mfbR mutants 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 mfbR virulence (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
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
D. dadantii MarR/SlyA family and virulence gene regulation 3
Fig. 1. Virulence of the regulator mutants.
The kinetics of soft rot progression was
scored for a week in Saintpaulia (A) and
Arabidopsis (B). Symptoms were scored using
a 4-step scale as follows: stage 0, no
symptoms; stage 1, maceration of the
infiltrated area (Saintpaulia) or around the
bacterial suspension droplet (Arabidopsis);
stage 2, spreading maceration; stage 3,
maceration of the whole leaf, including the
petiole. The significance of the differences in
maceration symptoms distribution between
wild-type and mutants has been checked with
a Fisher test, comparing either the number of
asymptomatic plants or the number of plants
presenting the highest score on each day. At
all time points, disease development was
significantly decreased with the different
mutants, compared with the WT parent
(P < 0.05). No symptoms were observed for
negative control plants treated with sterile
buffer.
days post infection (Fig. 1B). However, as seen in Saint-
paulia, the mfbR mutant 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 mfbR derivative, 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 mfbR background than in
the parental strain (Fig. 2B). This therefore indicates that
MfbR negatively regulates the mfb operon. Six bases
4 S. Reverchon et al. 䊏

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

 D. dadantii MarR/SlyA family and virulence gene regulation 5

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 of
oligonucleotides used for amplification of the different fragments (A, B and C) revealed on the gel (left part) are indicated on the scheme by
the thin arrows. The DNA segments A, B and C were amplified using the oligonucleotides mfbRf and mfbRr, mfbRf and mfbArc, and mfbRf
and mfbBrc, respectively (Table S3). RNA was isolated from exponential cultures of the D. dadantii parental strain 3937 and added to
reactions in the presence of reverse transcriptase (lanes A, B and C on the gel), lane T1 corresponds to the same amplification as in A but
without the addition of reverse transcriptase to the reaction mixture. Lane T2 corresponds to amplification using the primer up, localized
upstream from the +1 transcription start site, and the reverse primer localized at the end of mfbR. The sizes of the amplified fragments are
indicated.
B. Determination of the D. dadantii mfb promoter start site. Primer extension reactions were performed in the presence of 5 mg of RNA
extracted 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 and
the putative Shine and Dalgarno sequence regions are underlined; the putative DNA binding site of MfbR and the translation initiation codon
ATG 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 mfbR
transcripts in both the parental strain and its mfbR derivative. The ffh gene was used as a reference for normalization. The pH stress was
performed 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 is
regulated in a growth phase-dependent manner, sensitive to acidic pH stress, and is submitted to a negative retro-control. Total RNA was
isolated from the parental strain 3937 and its mfbR derivative; the OD600 and the growth conditions are indicated. The same RNA extracts
were used for experiments presented in Fig. 2 and in Fig. 4. Each value represents the mean of two experiments. Bars indicate the standard
deviation.
E. Quantification of the asr gene expression. The experiments were performed as in (D).

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 mfbR was not
modified by the presence of pectin or by 100 mM H2O2
treatment (data not shown). In contrast, mfbR expression
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
rstA–rstB (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, mfb operon 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 mfbR transcripts
was about 65-fold higher in the mfbR mutant, compared
Table 1. Fold change in mfbR, pel, celZ, prtC, hrpN and asr gene
expression induced in response to pH stress, in the early exponential
phase, in the wild-type strain.
Gene name Fold change induced by pH stressa
mfbR 7.0 ⫾ 0.5
pelD -2.9 ⫾ 0.3
pelE -5.4 ⫾ 0.4
pelI -2.7 ⫾ 0.3
celZ -6.4 ⫾ 0.2
prtC -1.9 ⫾ 0.2
hrpN -1.2 ⫾ 0.3
asr 2800 ⫾ 200
a. Fold changes are expressed as the ratio of the specific gene
expression level during exposure to pH 4.3, compared with pH 7.0,
normalized to the level of expression of the ffh gene. Positive values
represent genes induced by acidic pH whereas negative values rep-
resent genes downregulated under acidic pH. The presented ratios
were obtained by quantitative RT-PCR as shown for Figs 2 and 4.
Results are means from two independent experiments ⫾ SD. The
results obtained at pH 4.3 are different from those obtained at pH 7.0,
with P-values of < 0.05 except for hrpN.

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

6 S. Reverchon et al. 䊏

Table 2. Differential expression of mfbR, pel, celZ, prtC and hrpN
genes under growth in minimal M63 medium, supplemented with
sucrose and polygalacturonate.
Fold changes compared with the wild-type straina
Early exponential Late exponential
Gene name phase phase
pH 7.0 pH 4.3 pH 7.0
mfbR 64 ⫾ 5 8.2 ⫾ 0.3 107 ⫾ 12
pelD -2.0 ⫾ 0.2 1.0 ⫾ 0.05 -49 ⫾ 7
pelE -1.4 ⫾ 0.1 -1.1 ⫾ 0.1 -32 ⫾ 5
pelI -1.5 ⫾ 0.1 1 ⫾ 0.05 -10 ⫾ 0.8
celZ -1.9 ⫾ 0.1 -1.2 ⫾ 0.1 -1.8 ⫾ 0.2
prtC -1.2 ⫾ 0.1 -1.3 ⫾ 0.1 -6.0 ⫾ 0.5
hrpN 1.0 ⫾ 0.1 1.0 ⫾ 0.1 1.0 ⫾ 0.1
a. Fold changes are expressed as the ratio of the specific gene
expression 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 mutant
whereas negative values represent genes downregulated in the mfbR
mutant, compared with the wild-type strain. The presented ratios were
obtained by quantitative RT-PCR as shown for Figs 2 and 4. Results
are means from two independent experiments ⫾ SD. At pH 7.0,
results obtained with the mfbR mutant are different from the wild-type
results, with P-values of < 0.05 except for hrpN.
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 mfbRAB operon, we
constructed mfbA and mfbB mutants to analyse their
involvement in D. dadantii virulence on Saintpaulia plants.
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 these
experiments 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 mfbR mutant in
late exponential growth phase, which corresponds to the
phase 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

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

 D. dadantii MarR/SlyA family and virulence gene regulation 7

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 on
polygalacturonate-containing plates and staining with copper (II) acetate solution. Cellulase (Cel) activity was detected after growth on
CMC-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 is
expressed as mmol of unsaturated product liberated per min per mg of bacterial dry weight. Each value represents the mean of three
experiments. Bars indicate the standard deviation.

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 pelB transcripts 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 mfbR mutant, a delayed and reduced
accumulation of virulence gene transcripts was observed
while a high amount of mfbR transcript accumulation was
seen throughout the infection period. In the mfbR mutant,
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

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

8 S. Reverchon et al. 䊏
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 MfbR–His and DNA fragments containing the
Fig. 4. Transcription of the D. dadantii
virulence genes. The data show qRT-PCR
results for the D. dadantii virulence genes.
The experiments were performed with the
same cDNA samples used previously for the
studies on the mfb operon described in Fig. 2.
Each value represents the mean of two
independent experiments. Bars indicate the
standard deviation.
regulatory regions of the mfbR, pelE and celZ genes.
Typical results are shown in Fig. S4. MfbR–His bound to
all the DNA fragments tested. However, there were differ-
ences between the MfbR–His-binding profiles obtained
with the different DNA probes. In particular, MfbR–His
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 MfbR–His 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 MfbR–His 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 MfbR–His. The mutual
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
 D. dadantii MarR/SlyA family and virulence gene regulation 9

+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 MfbR–His 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-PCR
experiments were performed to analyse the transcript levels of
pelB, pelD, pelE, prtC, celZ and mfbR during plant infection.
Bacterial cDNA levels were equilibrated using the transcript level of
the constitutive rpoB gene. This gene was retained because
previous studies have revealed its stable expression during
infection (Lebeau et al., 2008).
B. Stability of MfbR in chloramphenicol chase experiment; the times
post-addition of chloramphenicol are indicated. The chase
experiment was performed, during early exponential phase, on
bacteria grown in M63 minimal medium containing sucrose.
Samples, corresponding to a constant bacterial number, were
removed between 0 and 9 h post chloramphenicol addition.
Samples were then submitted to Western blot analysis using a
polyclonal anti-MfbR as the primary antibody, and an anti-rabbit
peroxidase-conjugated antibody (SIGMA) as the secondary
antibody. A representative Western blot experiment is shown.

influence of MfbR–His 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
mfbR operator, addition of the two proteins together gave
a pattern similar to that obtained with MfbR–His alone
(Fig. 6). On the other hand, for the pelE and celZ opera-
tors, the addition of RNAP and MfbR–His together
showed an over-shift, corresponding to the binding of both
MfbR–His and RNAP. Moreover, more probes were
shifted in the presence of RNAP and MfbR–His together
than by adding the number of those shifted by the proteins
separately. Thus, it appears that MfbR–His and RNAP
bind synergistically at the pelE and celZ operators 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 Fig. 6. Binding of MfbR–His and RNA polymerase at the mfbR,
respect to the transcription start site (Fig. 7A). Increasing pelE and celZ promoter regions. The concentration of MfbR–His
(MfbR) and RNA polymerase (RNAP) used is indicated on the top.
the MfbR–His concentration up to 100 nM resulted in an The position of free DNA (F) and of the main MfbR–His/DNA
extension of the protected area from nucleotides -50 to complexes (C) is indicated.

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

10 S. Reverchon et al. 䊏
Fig. 7. DNase I footprinting of MfbR–His and RNAP binding at the
mfbR and pelE promoters.
A. Digestions at the mfbR promoter. Lane 1, no protein; lanes 2 to
5, incubation with 20, 30, 50 and 100 nM MfbR–His respectively;
lane 6, incubation with 150 nM RNAP; lane 7, incubation with
150 nM RNAP and 30 nM MfbR–His; lane 8, incubation with
150 nM RNAP and 100 nM MfbR–His. The arrowheads indicate
hypersensitivities 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 and
3, incubation with 200 and 800 nM MfbR–His respectively; lane 4,
incubation with 150 nM RNAP; lane 5, incubation with 150 nM
RNAP and 200 nM MfbR–His; lane 6, incubation with 150 nM
RNAP and 800 nM MfbR–His. The arrowheads and stars indicate
hypersensitivities induced by binding of RNAP and MfbR–His
respectively. The open circles indicate hypersensitivities induced by
binding of MfbR–His and RNAP. The areas protected by the
proteins are indicated on the right.
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 MfbR–His 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 MfbR–His 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, MfbR–His 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
MfbR–His 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 increasing
concentrations of MfbR–His 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 MfbR–His 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 vitro transcription to follow precisely the
effect of MfbR–His on RNAP activity. For this purpose, we
monitored mfb operon and pelE transcription using
pWN4100 and pTL5 DNA, respectively, with RNAP and
MfbR–His added either alone or in combination. As
expected, the addition of increasing MfbR–His 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 MfbR–His
specifically inhibits the mfb promoter and activates pelE
transcription in vitro.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
 D. dadantii MarR/SlyA family and virulence gene regulation 11

Fig. 8. MfbR–His 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 MfbR–His: lanes 2 and 7, no MfbR–His protein; lanes 3 and 8, 10 nM
MfbR–His; lanes 4 and 9, 20 nM MfbR–His; lanes 5 and 10, 30 nM MfbR–His; lanes 6 and 11, 50 nM MfbR–His. 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 MfbR–His
concentrations: lanes 2 and 5, no MfbR–His protein; lanes 3 and 6, 25 nM MfbR–His; lanes 4 and 7, 50 nM MfbR–His. Lane 1 corresponds to
the KMnO4 reaction without protein. The numbers -4, +3 and -4, -2 indicate the positions of KMnO4 sensitive nucleotides for mfbR and pelE
respectively. Asterisks indicate the transcription start sites of mfbR and pelE genes. DNA sequencing ladders (lanes A, C, G and T) were
introduced to indicate nucleotide positions. Dotted nucleotides in the DNA sequences indicate the KMnO4 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 obtained
in the absence of MfbR; 100% pelE relative expression corresponds to the expression obtained in the presence of 50 nM MfbR). Each value
represents the mean of two independent experiments. Bars indicate the standard deviation.

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 vivo MfbR–
DNA complex formation at pH 7 and at pH 4.3. As control,
we used KdgR protein, a regulator that specifically regu-
lates pel genes, such as pelE in 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,
pelE and pelD and, 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, pelE and pelD fragments
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

© 2010 Blackwell Publishing Ltd, Molecular Microbiology

12 S. Reverchon et al. 䊏

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, or
the control rpoA coding region in each immunoprecipitated sample, were analysed. ChIP analysis performed with anti-MfbR antibodies (A) or
in 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 corresponding
signals 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.

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

© 2010 Blackwell Publishing Ltd, Molecular Microbiology


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 targets
An 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 MfbR–His spe-
cifically binds to the regulatory region of the mfbR, pelE
and celZ genes. A competitive binding between RNA
polymerase and MfbR–His 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
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
D. dadantii MarR/SlyA family and virulence gene regulation 13
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 for
its 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
14 S. Reverchon et al. 䊏
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 been
shown 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 mfbR expression 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
Fig. 10. Schematic representation of the
regulation of virulence factors in D. dadantii at
different stages of infection. The lanes with a
bar and arrows represent a repression and
activation mechanism respectively; wide, thin
and dotted arrows under the genes indicate
high, intermediate and low transcription
respectively; ‘i’ and ‘a’ represent inactive and
active forms of the regulator MfbR under
acidic or neutral pH respectively. MfbR activity
is modulated upon the alkalinization of the
infected tissue. This pH change is postulated
to allow MfbR binding which then leads to
repression of the mfbRAB operon and to
activation of pel virulence genes in the
middle-advanced stages of infection.
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 mfbRAB operon 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.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology

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 (D’Ovidio 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 enzymes
would 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.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
D. dadantii MarR/SlyA family and virulence gene regulation 15
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 30°C and 37°C
respectively. The rich medium used was Luria–Bertani 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 30°C 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 mfbB genes 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 mfbB respectively. Insertion of a uidA-KmR cassette
in 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
16 S. Reverchon et al. 䊏
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.
MfbR–His 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
NdeI–XhoI restriction fragment PCR product was cloned into
the pET20b(+) vector (Novagen) to generate pSR3426. In the
resulting plasmid pSR3426, the mfbR gene was placed under
the control of the T7 RNA promoter and fused to a His6tag on
its C-terminus. Overproduction of the MfbR–His was carried
out in E. coli BL21(DE3)/pLysS. Purification of the recombi-
nant MfbR protein was achieved from cells grown at 30°C in
LB medium containing ampicillin and chloramphenicol to
maintain both pSR3426 and pLysS. When the optical density
at 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 30°C. 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 4°C, 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 -20°C.
To determine the native molecular mass of MfbR, approxi-
mately 100 mg of purified MfbR–His 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 2–3 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 MfbR–His 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 mfbR mRNA molecules at positions
+118 to +137.
For RT-PCR analysis, cDNA was synthesized, using
random 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 ffh gene 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 cell’s 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-
© 2010 Blackwell Publishing Ltd, Molecular Microbiology

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
4°C, on a rotating wheel, then 50 ml of ProteinA-Sepharose
beads (Amersham) was added. The mixture was then incu-
bated overnight, at 4°C, 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 65°C for 15 min. Immu-
noprecipitated samples were uncross-linked by incubation
overnight at 65°C. 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 22–25 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. ionantha cv. Blue Rhapsody cuttings were infected.
Bacteria cells grown on LB agar medium for 16 h at 30°C
were suspended in a 100 mM KCl solution to an OD600 of
0.1, corresponding to a concentration of 108 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 28°C/26°C; 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 104 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 24°C/19°C (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.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology
D. dadantii MarR/SlyA family and virulence gene regulation 17
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 Arabidopsis plants were infected 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 PstI–NarI digestion for mfbR, a SalI–SacII
digestion for celZ, and an EcoRI–HindIII 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 S. Reverchon et al. 䊏
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 Régupath 2007
Program, N°ANR-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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