A MarR-type regulator directly activates transcription from the

A MarR-type regulator directly activates transcription 1

from the Brucella abortus virB promoter by sharing a 2

redundant role with HutC 3

4 Rodrigo Sieira1*, Gastón M. Arocena 1, Angeles Zorreguieta 1,3, Diego J. Comerci 2, and 5

Rodolfo A. Ugalde2†. 6

7

Author affiliation: 1 Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias 8 Argentinas 435, Buenos Aires C1405BWE, Argentina. 2 Instituto de Investigaciones 9 Biotecnológicas, Universidad Nacional de General San Martín, IIB-INTECH-10 CONICET, San Martín 1650, Buenos Aires, Argentina. 3 Facultad de Ciencias Exactas 11 y Naturales-Departamento de Química Biológica, Universidad de Buenos Aires, 12 Argentina. 13 14

15

16

17

18

19

20

21

22

23

24

25

*Corresponding author: Fundación Instituto Leloir (IIBBA-CONICET), Av. Patricias 26

Argentinas 435, Buenos Aires C1405BWE, Argentina. Tel. (54 11) 5238-7500. E-mail: 27

[email protected] 28 †: Deceased 17 august 2009. 29

30

31

32

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01007-12 JB Accepts, published online ahead of print on 21 September 2012

on April 14, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

2

Abstract 33

Type-IV Secretion Systems (T4SS) are multiprotein structures that direct the 34

translocation of specific molecules across the bacterial cell envelope. As in other 35

bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the 36

T4SS-encoding virB operon, whose expression is regulated by multiple transcription 37

factors belonging to different families. Previously, we identified IHF and HutC, two 38

direct regulators of the virB genes that were isolated from total protein extracts of 39

Brucella. Here, we report the identification of MdrA, a third regulatory element that was 40

isolated using the same screening procedure. This transcription factor, which belongs to 41

the MarR-family of transcriptional regulators, binds at two different sites of the virB 42

promoter and regulates expression in a growth phase-dependent manner. Like other 43

members of the MarR family, specific ligands were able to dissociate MdrA from DNA 44

in vitro. Determination of the MdrA-binding sites by DNase I Footprinting and analyses 45

of protein-DNA complexes by electrophoresis mobility shift assays (EMSA) showed 46

that MdrA competes with IHF and HutC for the binding to the promoter because their 47

target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the 48

promoter without inducing bending of DNA. Moreover, these two latter transcription 49

factors activated virB expression to similar extents and in doing so, they are functionally 50

redundant. Taken together, our results show that MdrA is a regulatory element that 51

directly modulates activity of the virB promoter and is probably involved in 52

coordinating gene expression in response to specific environmental signals. 53

54

55

56


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

3

INTRODUCTION 57

Type-IV Secretion Systems (T4SS) are multiprotein structures that direct the 58

translocation of specific molecules across the bacterial cell envelope (3). During 59

evolution, these machineries have specialized to secrete different kind of substrates, 60

which led to the emergence of the different biological functions that T4SSs can exert. 61

Both the Escherichia coli Tra and the Agrobacterium tumefaciens VirB T4SSs are 62

responsible for secreting protein-DNA complexes during plasmid conjugation or 63

oncogenic T-DNA translocation, respectively (48, 54). Instead, other T4SSs such as the 64

Helicobacter pylori Cag system, the Legionella pneumophila Dot/Icm system, or the 65

Bordetella pertussis Ptl system, have evolved to deliver protein substrates towards the 66

eukaryotic host, where they subvert cellular processes and favor the development of the 67

bacterial infection (8, 44). As in all these pathogenic bacteria, virulence of Brucella is 68

also fully dependent on the integrity of a T4SS, since deletion of one or more T4SS-69

encoding virB genes completely abrogates the ability of this genus of facultative 70

intracellular bacteria to colonize its eukaryotic host (30, 47). 71

Brucella comprises many species which are the causative agent of a worldwide 72

distributed zoonotic disease known as brucellosis. The members of Brucella differ in 73

their host preference, affecting mammals that range from rodents to cetaceans (52). 74

Brucella abortus, the causal agent of bovine brucellosis, is one of the species that can be 75

transmitted from infected animals to humans through inhalation, contact with skin 76

wounds or mucosa, or by consumption of contaminated dairy products. In the natural 77

host, brucellosis produces sterility in males and abortion in pregnant females, whereas 78

in humans it causes debilitating symptoms and complications that can lead to death 79

(16). 80


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

4

The ability of Brucella to infect and persist within the mammalian host relies on 81

different mechanisms that allow these bacteria to overcome the innate and adaptive 82

immune host responses. Among all these strategies, bacterial survival and intracellular 83

replication into the host professional phagocytes is a key process underlying brucellosis. 84

After entering the host macrophages, the bacterium is located in the lumen of the so-85

called Brucella-containing vacuole (BCV) (32). During the first stages of the 86

intracellular infection, this compartment undergoes sustained interactions with 87

endoplasmic reticulum (ER) membranes. This process, which depends essentially on the 88

activity of the VirB system, is necessary for the biogenesis of an ER-derived bacterial 89

replication compartment, since abrogation of the early BCV-ER interactions prevents 90

Brucella from multiplying intracellularly and leads to bacterial degradation in 91

lysosomes (7). Recently, screening procedures based on different methods led to the 92

identification of proteins that are translocated into the host cell cytoplasm in a VirB-93

dependent manner (10, 22). Until now, the function of these substrates in the 94

establishment of the host cell infection have not yet been described except for RicA, a 95

protein that interacts with and recruits the small GTPase Rab2 into BCVs during the 96

intracellular trafficking of Brucella (9). 97

The first reported evidence of intracellular regulation of the VirB T4SS showed 98

that expression of the virB genes is induced after internalization of the bacterium in 99

macrophage cell lines (4). Such induction is dependent on the acidification of the BCV, 100

and reaches the maximal level of expression at 5 h post infection. Subsequently, 101

expression of the virB operon is rapidly turned off, which suggests that the system is 102

controlled by regulatory mechanisms that respond to specific signals sensed by the 103

bacterium within the host cell (46). 104


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

5

The main regulatory protein controlling expression of the Brucella virB genes is 105

VjbR, a Quorum Sensing (QS)-related regulator that binds the virB promoter (PvirB) at 106

position -94 relative to the transcription start site (2, 12). In addition, many other 107

regulators were shown to directly or indirectly influence virB expression, including the 108

stringent response mediator Rsh and transcription factors belonging to different families 109

(13, 18, 37). In addition, although their binding sites in the virB promoter have not yet 110

been reported, it was recently shown that both the Two-component regulator BvrR and 111

the second QS-related transcription factor BabR/BlxR participate in regulation of virB 112

expression by directly interacting with PvirB, as demonstrated by pull-down experiments 113

and electrophoresis mobility shift assays (EMSA), respectively (6, 25). 114

In previous works from our group, we identified two transcriptional regulators that 115

directly contribute to induction of virB expression: (i) IHF, a nucleoid associated protein 116

that binds to PvirB at position -162.5 and induces a DNA bend that is necessary for the 117

promoter activation (46); and (ii) HutC, a GntR-family regulator that binds at position -118

188 and links virB expression to histidine catabolism (45). Here, we report the 119

identification of MdrA, a third regulatory element that was isolated using the same 120

screening procedure. Our experiments showed that this transcription factor binds at two 121

different sites of PvirB and regulates virB expression in a growth phase-dependent 122

manner. Analysis of promoter activities under different mutant backgrounds revealed 123

that MdrA exerts its regulatory role in redundancy with the function of HutC. Besides, 124

consistently with both the architecture and localization of the MdrA-binding sites within 125

the promoter region, we also observed that this transcription factor competes with IHF 126

and HutC for the binding to PvirB. As is usually the case with MarR family members, our 127

experiments showed that the presence of a specific ligand dissociates MdrA from a 128


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

6

target DNA binding site. Such ligand-induced disruption of promoter binding indicates 129

that this process may allow MdrA to receive and respond to environmental signals. 130

. 131

132

133


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

7

MATERIALS AND METHODS 134

Media and culture conditions 135

Brucella strains were grown in triptic soy broth (TSB); or in minimal media Gerhardt-136

Wilson (15) or MM1 (45) at pH 4.5, 5.5, or 7.0. Bacteria were cultured at 37˚C in a 137

rotary shaker (250 rpm). Media were supplemented with kanamycin (50 μg ml-1) and/or 138

nalidixic acid (5 μg ml-1), as needed. Sodium Deoxycholate (Sigma) was eventually 139

added to the media at final concentration of 100, 250, 500, 750, or 1000 μM. 140

Escherichia coli strains were cultured in Luria-Bertani (LB) broth at 37°C in a rotary 141

shaker at 250 rpm. Media were supplemented with ampicilin (100 μg ml-1) or 142

kanamycin (25 μg ml-1), as needed. 143

144

Isolation and identification of MdrA 145

The protein detected by EMSA was isolated from exponential phase cultures (OD600 ~ 146

0.5) of the avirulent strain B. abortus Δpgm (51). Bacteria were harvested and disrupted 147

as described previously (46). After ultracentrifugation and filtration, the total protein 148

extract was fractionated by ammonium sulphate precipitation. The different fractions 149

were analyzed by EMSA using probe vir-up or the control probe B10. The positive 150

fraction (0-45% ammonium sulphate saturation) was suspended in 35 mM phosphate 151

buffer (pH 6.8), 3 mM β-mercaptoethanol and dialyzed against the same buffer O.N. at 152

4°C. The solution was loaded onto a Mono-S column and eluted with a linear gradient 153

of 35 mM phosphate buffer (pH 6.8), 3 mM β-mercaptoethanol, 1M NaCl. DNA-154

binding activity of the fractions was again analyzed by EMSA using the probes 155

described above. Positive fractions were subjected to affinity chromatography: the 156

biotinylated probe biot-vir-up, which corresponds to the -430 to -202 region of PvirB, 157

was constructed by PCR using Taq, the 5´-biotinylated primer pvd228, the primer pirup 158


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

8

and plasmid genomic DNA of B. abortus 2308 as template. The biotinylated control 159

probe was constructed as described previously (45). The biotinylated probes were 160

bound to streptavidin paramagnetic spheres (Promega) and a binding reaction was 161

performed using binding buffer and the positive fractions of the Mono-S column. After 162

two washes with binding buffer with 0.2 M NaCl, the DNA-bound proteins were eluted 163

with 0.85 M NaCl and analyzed by 12.5% SDS-PAGE. The gel was silver-stained with 164

a mass spectrometry compatible method for visualizing the protein bands. A band which 165

was observed with the probe biot-vir-up but was absent in samples from the control 166

probe was excised form the gel and analyzed by mass spectrometry in Vital probes Inc. 167

(PA, USA). 168

169

Construction of plasmids 170

pK18mob-sacB-ΔmdrA: two PCRs were carried out using Pfx (Invitrogen), genomic 171

DNA of B. abortus 2308 as template, and primers SpeMarA (5’- 172

GGACTAGTTGACGATATGTTTGCCGCAT-3’) and COMMarA (5’- 173

CTGCAAGAAGGAGAGTATCGAATGCAGGCGCTCGACAAGC-3’), or SpeMarB (5’- 174

GGACTAGTAACCGCTCTTGCGGCAATC-3’) and COMMarB (5’- 175

TCGATACTCTCCTTCTTGCAGTTTATGGCGATGAACAAGTCG-3’). Both PCR 176

products, corresponding to two 400-bp flanking regions of mdrA, were annealed and 177

used as templates for a PCR performed with primers SpeMarA and SpeMarB. The 178

product was digested with SpeI and cloned into the kanamycin-resistant plasmid 179

pK18mob-sacB (42). 180

Complementation plasmid pK18mob-sacB-Knock-in-mdrA: a PCR was carried out 181

using Pfx, genomic DNA of B. abortus 2308 as template, and primers SpeMarA (5’- 182

GGACTAGTTGACGATATGTTTGCCGCAT-3’) and SpeMarB (5’- 183


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

9

GGACTAGTAACCGCTCTTGCGGCAATC-3’). The PCR product, which contains the 184

wild type-sequence of the mdrA gene and two 400-bp flanking regions, was digested 185

with SpeI and cloned into the kanamycin-resistant plasmid pK18mob-sacB. 186

Expression vector pQE-31-mdrA: a PCR reaction was performed using Pfx, genomic 187

DNA of B. abortus 2308 as template, and primers BamMarII (5’-188

CGGGATCCGACCAATACCCAGCGCAAGAT-3’) and HindMar (5’- 189

GGTACCTCAGCCGCGAGATGGCGT-3’). The PCR product were digested with BamHI 190

and HindIII, and cloned into plasmid pQE-31 (Qiagen). 191

192

Construction of strain B. abortus ΔmdrA 193

Plasmid pK18mob-sacB-ΔmdrA was transferred to B. abortus 2308 by biparental 194

conjugation. Kanamycinr colonies were selected as single-homologous recombinants. 195

Selection with sucrose, excision of plasmids and generation of deletion mutants was 196

performed as described previously (46). PCR analyses of kanamycins colonies were 197

carried out with primers SpeMarA and SpeMarB to identify clones that contain the 198

deletion of mdrA. 199

200

Construction of the double mutant deletion strain B. abortus ΔhutC ΔmdrA 201

Plasmid pK18mob-sacB-ΔmdrA was transferred to the strain B. abortus ΔhutC (45) by 202

biparental conjugation. Kanamycinr colonies were selected as single-homologous 203

recombinants. After selection with sucrose and excision of the plasmid, a PCR analysis 204

of kanamycins colonies was carried out with primers SpeMarA and SpeMarB to identify 205

clones that contain the deletion of mdrA in the ΔhutC mutant background. 206

207

Construction of the Knock-in complemented strain B. abortus ΔhutC ΔmdrA-KI 208


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

10

Plasmid pK18mob-sacB-Knock-in-mdrA was transferred to the strain B. abortus 209

ΔhutC ΔmdrA by biparental conjugation. Kanamycinr colonies were selected as single-210

homologous recombinants. After selection with sucrose and excision of the plasmid, a 211

PCR analysis of kanamycins colonies was carried out with primers SpeMarA and 212

SpeMarB to identify clones that incorporated the wild type mdrA gene. 213

214

Construction of strains containing a single PvirB-lacZ chromosomal transcriptional 215

fusion. 216

Plasmid pK18mob-PvirB-lacZ was transferred by biparental conjugation into the wild 217

type strain B. abortus 2308 or into strain B. abortus ΔmdrA, B. abortus ΔhutC, B. 218

abortus ΔhutC ΔmdrA, or B. abortus ΔhutC ΔmdrA-KI. Kanamycinr colonies were 219

selected as single-homologous recombinants. 220

221

Electrophoresis Mobility Shift Assays (EMSA) 222

32P-labeling of probes, incubations and running conditions were performed as described 223

previously (46). The radiolabeled probe vir-up was constructed using Taq (Invitrogen), 224

primers pvirup (5’-ATGACAGGCATATTTCAAC-3’) and pvd228 (5’-225

GTGATTTTCATATTTTTGCGG-3’) and genomic DNA of B. abortus 2308 as 226

template. The radiolabeled probes vir-down and vir-down-ihf- were constructed by PCR 227

using primers pvd229 and pvirdownI, and genomic DNA of B. abortus 2308 or plasmid 228

pBluescript-PvirB-IHF-sacB/R as template, respectively, as described previously (46). The 229

radiolabeled control probe B10 was constructed with primers B10Qu and B10d2, as 230

described previously (45). The radiolabeled probe 286/59 was constructed using primers 231

pvu144 (5’-CGGGATCCGATTCTTGGTCGGGTTAC-3’) and pvirdownIII (5’-232


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

11

TGTTTAAGCCGATATATGGAC-3’), and genomic DNA of B. abortus 2308 as 233

template. 234

The unlabeled competitors were constructed by PCR using Taq, genomic DNA of B. 235

abortus 2308 as template, and primers pvirup and pvd228 for competitor up; primers 236

pvu77 (5’-CGGGATCCGATGCCGCCTAATGGAGC-3’) and pvd228 for competitor 237

77; primers pvu144 and pvd228 for competitor 144; and primers pvu229 and pvirdownI 238

for competitor down. The unlabeled competitors A1 and B1 were constructed as 239

described previously (46). Binding reactions were performed in binding buffer [15 mM 240

Tris-HCl (pH 8.0), 0.5 mM EDTA, 10 μg ml-1 bovine serum albumin (BSA), 1 mM 241

DTT, 30 mM KCl, 6% glycerol, 50 μg ml-1 salmon sperm DNA]. 242

243

DNase I Footprinting 244

Labelling reactions, purification of probes, binding reactions, DNase I digestion, 245

purification of digested fragments, and electrophoresis conditions were performed as 246

described previously (46). Probe vir-upII was constructed using Taq, primer pvd228, 247

the 32P-labeled primer pvirup, and genomic DNA of B. abortus 2308 as template. Probe 248

vir-downII was constructed using primer pvu144, the 32P-labeled primer pvirdownI, and 249

genomic DNA of B. abortus 2308 as template. Probe vir-upIII was constructed using 250

primer pvirup, the 32P-labeled primer pvirdownIII, and genomic DNA of B. abortus 251

2308 as template. 252

253

Expression and purification of recombinant proteins 254

Recombinant IHF and HutC were prepared as described previously (45-46). 255

Recombinant MdrA was prepared as follows. Plasmid pQE-31-mdrA was transferred 256

into E. coli M15 (pREP4) (Qiagen). Transformed bacteria were cultured in LB until 257


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

12

DO600 = 0.6 and induced with 1 mM IPTG. After 4 h, bacteria were harvested, 258

suspended in lysis buffer [20 mM Tris-HCl (pH 7.6), 1 mM PMSF] and disrupted by 259

sonication. After centrifugation NaCl was added to a final concentration of 0.5 M and 260

the sample was loaded into a Hi-Trap Nickel-chelating column (Amersham 261

Biosciences). After washing with buffer B [20 mM Tris-HCl (pH 7.6), 0.5 M NaCl, 50 262

mM imidazole] the column was equilibrated with buffer A [20 mM Tris-HCl (pH 7.6), 263

0.5 M NaCl] and eluted with a linear gradient of buffer C [20 mM Tris-HCl (pH 7.6), 264

0.5 M NaCl, 1 M Imidazole]. Eluates were analyzed in a 12.5% SDS-PAGE, and the 265

fractions containing the recombinant protein MdrA (purity near to 95%) were pooled 266

and dialized against buffer D [20 mM Tris-HCl (pH 7.6), 0.5 M NaCl, 3 mM β-267

mercaptoethanol]. 268

269

β-galactosidase activity determinations 270

β-Gal activity was determined in whole bacterial cells as described (46), and it was 271

expressed as Miller Units [A420 / volume × OD600] ×100. 272

273

274


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

13

RESULTS 275

Isolation and identification of MdrA 276

In previous studies, two transcription factors implicated in the control of 277

expression of the virB operon were isolated by affinity chromatography from B. abortus 278

total protein extracts (45-46). Such analyses were performed by using DNA-probes that 279

contained sequences corresponding to positions -201 to +24 relative to the transcription 280

start site of the virB promoter (PvirB). In order to identify possible additional regulatory 281

proteins interacting with further upstream regions, we performed EMSA analyses using 282

crude protein extracts and a radioactive-labeled probe corresponding to positions -430 283

to -202 of PvirB. As shown in Figure 1, a protein-DNA complex was detected using total 284

protein extracts of B. abortus grown in rich medium (TSB) until exponential phase, 285

whereas no signal was observed with stationary-phase extracts. To isolate the factor 286

responsible for the observed complex, proteins were fractionated by ammonium 287

sulphate precipitation and ion-exchange chromatography, followed by EMSA analysis 288

of each fraction during the purification process (see Materials and Methods). 289

Subsequently, the partially purified protein fractions were subjected to affinity 290

chromatography using a biotin-labeled probe vir-up or a non-related control probe 291

bound to streptavidin-coated paramagnetic spheres, and the corresponding eluates were 292

analyzed by SDS-PAGE. Using this procedure we observed a specific band, which was 293

excised from the gel and identified by mass spectrometry as YP_414216, a protein 294

belonging to the MarR family of transcriptional regulators (14). Because of its 295

biochemical properties and its role on regulation of virB expression (see below), we 296

called this protein MdrA (for MarR-like sodium deoxicholate-responsive activator). As 297

revealed by BLAST analyses, MdrA shares 29% identity and 44% similarity with the 298

Escherichia coli prototypical member MarR (24), whereas it displayed slightly higher 299


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

14

homology with other representative MarR members such as PecS, SlyA, and RovA 300

(33% identity and 54% similarity; 30% and 49%; 31% and 50%; respectively) (21, 27, 301

34). As shown in Figure 2, a multiple sequence alignment indicated that MdrA displays 302

a high degree of amino acid conservation at postions corresponding to the winged-helix 303

turn helix DNA-binding domain [β1-α3- α4(recognition helix)-β2-W1(wing)-β3], 304

which is characteristic of this family of transcriptional regulators (1). 305

306

MdrA interacts with PvirB at two different binding sites 307

To study the MdrA-PvirB interaction, a His-tagged recombinant protein was 308

expressed and assayed by EMSA using different radioactive-labeled probes (see Fig. 309

3A). As shown in Fig. 3B, recombinant MdrA interacted with probe vir-up showing an 310

electrophoretic mobility similar to that of the native nucleoprotein complex, whereas no 311

signal was observed with the control probe B10. Incubation of MdrA with probe vir-312

down also resulted in the formation of a protein-DNA complex, which indicated that 313

this regulator specifically recognizes two separated regions of the promoter (Fig. 3B). 314

On the other hand, incubation of MdrA with a probe that lacks the IHF-binding site 315

produced no signal by EMSA (Fig. 3B), suggesting that the MdrA-binding site overlaps 316

with that of IHF. Competition experiments performed in the presence of an excess of 317

different unlabeled fragments indicated that the sequences recognized by MdrA in PvirB 318

are located at two regions residing between positions -353 and -286, and between -201 319

and -130 (Fig. 3C-D). Hereafter, we will refer to the downstream- and upstream-located 320

elements recognized by this protein in PvirB as the MdrA-binding sites I and II, 321

respectively. 322

323


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

15

Analysis of the interaction of MdrA, IHF and HutC with the downstream region of 324

PvirB 325

The above experiments showed that MdrA specifically binds to a 71-bp region of 326

the promoter which also contains the binding sites for IHF and HutC. In previous 327

works, we found that IHF and HutC compete with one another during binding to PvirB 328

because their binding sites overlap (45-46). Accordingly, we next asked if binding of 329

MdrA to PvirB affects the interaction of IHF or HutC with the promoter. To address this 330

question, we incubated probe vir-down with each of the three pairs of proteins, or with 331

all three proteins together. We then carried out EMSA on the different protein 332

combinations. Figure 4B shows that, separately, each of the regulators generated a 333

signal corresponding to a binary protein-DNA complex, whereas no ternary complexes 334

were observed with any of the combinations of two or more proteins. These results 335

indicated that MdrA competes with IHF and HutC for the binding to the probe vir-336

down, suggesting that the MdrA-binding site I overlaps those of the two other 337

regulators. The same results were obtained when EMSAs were carried out with probe 338

289/59, whose length is the same as that of probe vir-down but the location of the 339

binding sites for MdrA, HutC and IHF are close to the center of the probe rather than 340

being near one end (data not shown).. However, the electrophoretic mobility of the 341

complex between IHF and probe 289/52 was substantially slower than that of the 342

complex between IHF and probe vir-down (Fig. 4B-C). The reduced mobility of the 343

IHF-289/52 complex is because IHF bends DNA as it binds to it, and the effect of 344

bending on migration is more pronounced when the binding site is located near the 345

center of the molecule (38). On the other hand, unlike IHF, the relative mobility of the 346

MdrA- and HutC-nucleoprotein complexes remained constant regardless of the probe 347


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

16

used, indicating that these two latter transcriptional regulators do not induce bending of 348

DNA upon binding (Fig. 4B-C). 349

350

Determination of the MdrA-binding sites in PvirB 351

To identify both MdrA-binding sites I and II, we performed DNAse I Footprinting 352

experiments using probes corresponding to different regions of PvirB: (a) a 228-bp 353

fragment containing the upstream region of the promoter that was labeled at the 5’ end 354

of the top strand; and (b) a 312-bp fragment containing the downstream region that was 355

labeled at the 5’ end of the bottom strand (Fig. 5A). As shown in Fig. 5B, it was 356

observed that MdrA bound to a 30-bp region extending from position -190 to -160, 357

which corresponds to the MdrA-binding site I (Fig. 5B). DNase I Footprinting 358

experiments performed with the probe that contains sequences of the upstream region of 359

PvirB showed that MdrA protected a 24-pb region that extends from position -335 to -360

312, which corresponds to the MdrA-binding site II (Fig. 5C). When a 370-bp fragment 361

was end-labeled at the top strand and used as probe (Fig. 6A), it was observed that 362

MdrA protected two regions corresponding to the MdrA-binding sites I and II (Fig.6B-363

C), which indicated that this transcription factor is able to bind simultaneously to both 364

operator sequences. 365

The DNAseI Footprinting experiments showed that the MdrA-binding site I 366

overlaps those of both HutC and IHF (Fig. 5A), consistently with the observation that 367

MdrA competed with these two transcriptional regulators for the binding to probe vir-368

down (Fig. 4B). The analysis of the protected regions revealed the presence of two 369

partially conserved 11-bp motifs arranged as inverted repeats in the MdrA-binding site 370

II (Fig. 7B). In contrast, the analysis of sequences corresponding to the MdrA-binding 371

site I showed no obvious dyad symmetry. However, alignment of the 11-bp motifs 372


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

17

together with the downstream protected region revealed the presence of a similar 373

sequence located at the center of the MdrA-binding site I (Fig. 7A), suggesting that 374

MdrA specifically recognizes this sequence. In agreement with this hypothesis, MdrA 375

was unable to bind a probe lacking a sequence which is comprised within the 11-bp 376

motifs found in both MdrA-binding sites (Fig. 7C), which supports the notion that the 377

sequence TAAA is part of the motif recognized by MdrA in the promoter. 378

379

MdrA dissociates from DNA in the presence of sodium deoxycholate 380

Several members of the MarR family are capable of directly bind specific ligands, 381

which dissociate the regulator from DNA with a consequent modulation of gene 382

expression (17). In Salmonella typhimurium, it was recently found that sodium 383

deoxycholate (DOC), a component of bile, interacts with MarR and interferes with its 384

ability to bind the promoter of the marRAB operon (35). Accordingly, we investigated 385

whether the DNA-binding activity of MdrA is susceptible to be affected by total bile 386

salts or bile components. We observed by EMSA that formation of the MdrA-vir-down 387

complex was impaired in the presence of total bile salts (data not shown). When 388

individual components of bile were tested, we observed that binding of MdrA to PvirB 389

was impaired by 250 μM DOC, whereas the binding of IHF or HutC was not affected 390

by this bile salt at any of the assayed concentrations (Fig. 8). Therefore, these results 391

demonstrated that like other members of the MarR-family of transcriptional regulators, 392

the interaction between MdrA and its operator sequences can be modulated by specific 393

compounds, and such ability may be involved in transduction of environmental stimuli 394

to regulate MdrA-dependent gene expression. 395

396

MdrA and HutC exert redundant roles on regulation of virB expression 397


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

18

In order to determine the role of MdrA on transcriptional regulation of the virB 398

genes, we introduced transcriptional fusions between PvirB and the lacZ reporter gene 399

into the B. abortus 2308 wild type strain or into a ΔmdrA deletion mutant. 400

Determination of β-Galactosidase activities showed no differences between these strains 401

after incubation of bacteria in rich medium (TSB) or in minimal media at different pH 402

values in the presence of different carbon sources (data not shown). Based on these 403

results, we hypothesized that the regulatory function of MdrA on PvirB is probably 404

redundant with that of other transcription factor. Given that both MdrA and HutC bound 405

to overlapping binding sites, and they appear to have similar structural roles since 406

neither of them induces DNA bending, we aimed to determine whether these two 407

proteins exert redundant roles on virB expression. To this end, β-Galactosidase activites 408

of the PvirB-lacZ fusions were also assayed in both B. abortus ΔhutC and the B. abortus 409

ΔhutC ΔmdrA double deletion mutant strain. Using all these genetic backgrounds, we 410

neither observed β-Galactosidase activity differences between strains when bacteria 411

were grown in TSB until stationary phase (OD600 ≥ 3.5) or in the above mentioned 412

minimal culture media (data not shown). However, in bacteria grown in TSB until 413

exponential phase (OD600 ~ 0.5-1.0), the ΔhutC ΔmdrA double deletion mutant showed 414

a decrease of about 45% in promoter activity, whereas both the ΔhutC and the ΔmdrA 415

single mutants showed the same levels of β-Galactosidase activity as the wild type 416

strain (Fig. 9). Complementation of mdrA by a knock-in strategy in the double mutant 417

restored the wild type β-Galactosidase activity levels (Fig. 9), thus confirming that 418

MdrA exerts a growth-phase dependent positive regulatory role on virB expression, 419

functionally redundant with HutC. The analyses performed in the presence of DOC 420

displayed the same pattern of β-Galactosidase activity as that observed without this bile 421

salt (data not shown), even at concentrations 3-fold higher than that dissociated MdrA 422


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

19

from the promoter. Thus, the sole presence of this bile salt was not a sufficient stimulus 423

for modulating virB expression under standard culture conditions, which suggests that 424

the capability of the MdrA-mediated regulation to respond to environmental signals 425

may be restricted to specific environmental conditions. 426

Taken together, these results revealed that MdrA controls expression of the virB 427

genes in a growth phase-dependent manner, and exerts a regulatory role functionally 428

redundant to a non-related factor. 429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

20

DISCUSSION 452

Expression of the virB operon is under the control of transcriptional regulatory 453

mechanisms that involve perception of environmental conditions related to the 454

intracellular lifestyle of Brucella (4). Over the last years, several studies revealed that 455

activity of the virB promoter is modulated by the direct action of many transcriptional 456

regulators belonging to different families, including a nucleoid associated protein (IHF), 457

QS-related transcription factors, and a regulator of histidine catabolism (6, 12, 25, 45-458

46). Here, using an approach that previously allowed us to isolate IHF and HutC, we 459

identified MdrA, a protein belonging to the MarR-family of transcription factors 460

involved in regulation of virulence genes, aromatic catabolic pathways, or bacterial 461

responses to environmental stress (53). The first evidence of interaction between MdrA 462

and PvirB indicated that this regulator binds to the promoter at sequences located far 463

upstream of the transcription start site (positions -430 to -202). Further EMSA analyses 464

showed that MdrA was also able to interact with the downstream region, indicating that 465

this regulator recognizes two distinct binding sites at PvirB (Fig. 3). These observations 466

were confirmed by DNase I Footprinting analyses that showed that MdrA is able to bind 467

simultaneously to both regions, which are centered at positions -175 and -323.5 (MarR-468

binding sites I and II, respectively). It is remarkable that all positive regulators 469

characterized so far in the MarR family act by means of displacing repressors (19, 40) 470

or through prototypical activation mechanisms by interacting with RNAP (28, 50). This 471

latter mode of regulation may not account for activity of MdrA on the virB promoter, 472

since it activates transcription from regions located far upstream of position +1. We 473

hypothesize that MdrA could interfere with the activity of an unknown repressor; a 474

possible candidate may be BabR, which is a negative regulator that affects virB 475

expression by 20% and whose binding site in the promoter has not yet been identified 476


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

21

(6). Alternatively, contact between MdrA and RNAP may be mediated by additional 477

elements which could introduce conformational changes in chromatin structure. Similar 478

mechanisms were previously suggested for other positive regulators that bind far 479

upstream from the transcription start site but whose mechanisms of activation have not 480

been yet elucidated (e.g., EspR, RutR, TodT, AlgR) (20, 26, 29, 39). It is worth 481

mentioning that the possible involvement of elements that introduce modifications of 482

the nucleoprotein structure of local sequences was also hypothesized for the LuxR-type 483

regulator VjbR, since it binds at position -94 and consequently could not activate 484

transcription of the virB promoter in a prototypical manner (2). 485

Our experiments revealed that the MdrA-binding site I overlaps those previously 486

identified for IHF and HutC, which was consistent with the fact that these three 487

transcription factors compete for binding to the promoter (Fig. 4). Similar structures 488

have been reported for other promoters regulated by members of the MarR-family (i.e, 489

CbaR, MarR, RovA), wherein the regulators bind to two or more operator sites (19, 24, 490

36). Furthermore, competition with nucleoid associated proteins (i.e, H-NS, IHF) or 491

with other global regulators (CRP) due to overlapping binding sites has also been 492

reported to be part of the regulatory mechanisms involving MarR-related regulators (19, 493

40, 43). An additional feature shared by MdrA and most of the MarR homologs is the 494

ligand-mediated modulation of its DNA-binding activity. However, it is important to 495

highlight that MdrA is the first ligand-responsive positive regulator characterized in this 496

family, since no ligand has yet been found for the other MarR-type transcriptional 497

activators described so far (14). Curiously, MdrA is the second positive regulator of the 498

virB promoter which dissociates from DNA in response to specific signals, since VjbR 499

was previously shown to respond to acyl-homoserine lactones (2, 12). Our results 500

indicated that either bile or micromolar concentrations of DOC specifically dissociated 501


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

22

MdrA from DNA without affecting neither IHF nor HutC (Fig. 8). These observations 502

have raised the hypothesis that after ligand recognition, MdrA could act as a signal 503

transducer that modulates expression of the virB genes in response to exposure of 504

Brucella to DOC within the host environment. Previous studies have described in 505

Brucella mechanisms that confer resistance to DOC (11, 23, 33). Moreover, it was 506

recently shown that Brucella is able to infect dendritic cells from murine Peyer´s 507

patches and to negatively modulate its activation through the action of the protein Btp1 508

(41). Although these lines of evidence support the notion that Brucella is able to 509

overcome the action of bile salts and to invade antigen-presenting cells in the host’s gut, 510

we failed to observe any effect of DOC on virB expression in exponential phase cultures 511

under standard growth conditions. However, it cannot be excluded that, under some 512

type of stress experienced by the bacterium within the host, this bile salt may permeate 513

the bacterial membrane more easily and act as an environmental signal to induce an 514

MdrA-mediated response. It is worth mentioning that it cannot be deduced any additional 515

potential ligand for MdrA from the few MarR-homologs reported so far in Rhizobiales, 516

since their known ligands do not share structural similarity with DOC (5, 31). Therefore, 517

further studies will be needed to determine whether this bile salt, or a structurally 518

similar compound, can interact with MdrA in vivo and modulate virB expression. 519

The results presented here showed that MdrA and HutC, two independently 520

isolated proteins belonging to different families of transcription factors, bind to PvirB at 521

overlapping target DNA-sequences and positively modulate expression of the virB 522

genes to similar extents. Moreover, both regulators have been shown to play redundant 523

roles since the regulatory activity of MdrA could not be determined unless a ΔmdrA 524

ΔhutC double deletion mutant background was used to measure virB promoter activity 525

(Fig. 9). In addition to these evidences, our EMSA experiments indicated that, unlike 526


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

23

IHF, neither MdrA nor HutC induced DNA-bending upon binding to PvirB (Fig. 4), 527

suggesting that these two transcriptional regulators exert similar structural roles in 528

agreement with their redundant activity on modulation of virB expression. 529

The data obtained from the present work showed that the function of MdrA leads to a 530

positive regulatory effect on virB expression which does not take place in an all-or-531

nothing manner. Deletion of mdrA affected virB expression by 45 %, which was a 532

magnitude of difference detectable by quantitative measurements of β-gal activity 533

(Figure 9) but not by Western blot experiments performed with an anti-VirB7 antibody 534

(data not shown). Our results indicated that, similarly to what was previously observed 535

with HutC, MdrA also acts as an activator which enhances virB promoter activity under 536

defined conditions and appears to play an accessory regulatory role, probably acting to 537

synchronize maximal virB expression with certain metabolic and/or environmental 538

signals. It was previously observed that HutC activates intracellular expression of the 539

virB genes within J774 macrophages (45). In contrast, MdrA does not appear to play 540

any role in this experimental model, since deletion of mdrA did not produce any 541

detectable intracellular effect, and the ΔmdrA ΔhutC double mutant affected 542

intracellular virB expression to the same extent as in the single ΔhutC deletion 543

background (Supplemental Fig. S1). On the other hand, MdrA was necessary for virB 544

expression in cultured bacteria at the exponential phase of the growth in rich medium, 545

whereas in stationary phase we did not observe any MdrA-dependent effect (Fig. 9). In 546

addition, it is worth to note that the growth phase wherein MdrA exerts its regulatory 547

activity is coincident with the only condition at which it could be isolated (Fig 1), 548

indicating that this regulator is probably under the control of mechanisms that modulate 549

its expression and/or its DNA-binding activity. Taken together, our observations 550

suggest that the MdrA-mediated regulation of the virB genes may be a result of an 551


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

24

adaptation to activate virB expression at stages of the infection process where HutC is 552

not acting. However, although functionality of MdrA was demonstrated, it still remains 553

to be determined which are the conditions wherein MdrA-dependent expression is 554

achieved in vivo. Besides, it will also be important to assess whether this MarR-related 555

transcriptional regulator is able to perceive environmental signals at specific 556

tissues/organs of the mammalian host during the course of the disease. 557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

25

ACKNOWLEDGEMENTS 575

This work is dedicated to the memory of R.A.U. We thank Joseph Connolly for help 576

with mass spectrometry. This work was supported by grant PICT05-38207 to Rodrigo 577

Sieira, and grant PICT06-651 to Rodolfo A. Ugalde from Agencia Nacional de 578

Promoción Científica y Tecnológica, Buenos Aires, Argentina; grant PIP2011-00336 to 579

Rodrigo Sieira from the Argentinian Council of Research (CONICET); and grant 580

UBACyT X240 to Angeles Zorreguieta from the University of Buenos Aires, 581

Argentina. 582

583


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

26

REFERENCES 584

1. Alekshun, M. N., S. B. Levy, T. R. Mealy, B. A. Seaton, and J. F. Head. 585 2001. The crystal structure of MarR, a regulator of multiple antibiotic resistance, 586 at 2.3 A resolution. Nat Struct Biol 8:710-4. 587

2. Arocena, G. M., R. Sieira, D. J. Comerci, and R. A. Ugalde. 2010. 588 Identification of the Quorum Sensing target DNA-sequence and N-acyl 589 Homoserine Lactone responsiveness in the Brucella abortus virB promoter. J 590 Bacteriol. 591

3. Backert, S., and T. F. Meyer. 2006. Type IV secretion systems and their 592 effectors in bacterial pathogenesis. Curr Opin Microbiol 9:207-17. 593

4. Boschiroli, M. L., S. Ouahrani-Bettache, V. Foulongne, S. Michaux-594 Charachon, G. Bourg, A. Allardet-Servent, C. Cazevieille, J. P. Liautard, 595 M. Ramuz, and D. O'Callaghan. 2002. The Brucella suis virB operon is 596 induced intracellularly in macrophages. Proc Natl Acad Sci U S A 99:1544-9. 597

5. Caswell, C. C., J. E. Baumgartner, D. W. Martin, and R. M. Roop, 2nd. 598 2012. Characterization of the organic hydroperoxide resistance system of 599 Brucella abortus 2308. J Bacteriol. 600

6. Caswell, C. C., J. M. Gaines, and R. M. Roop, 2nd. 2012. The RNA 601 chaperone Hfq independently coordinates expression of the VirB type IV 602 secretion system and the LuxR-type regulator BabR in Brucella abortus 2308. J 603 Bacteriol 194:3-14. 604

7. Celli, J., C. de Chastellier, D. M. Franchini, J. Pizarro-Cerda, E. Moreno, 605 and J. P. Gorvel. 2003. Brucella evades macrophage killing via VirB-606 dependent sustained interactions with the endoplasmic reticulum. J Exp Med 607 198:545-56. 608

8. Covacci, A., J. L. Telford, G. Del Giudice, J. Parsonnet, and R. Rappuoli. 609 1999. Helicobacter pylori virulence and genetic geography. Science 284:1328-610 33. 611

9. de Barsy, M., A. Jamet, D. Filopon, C. Nicolas, G. Laloux, J. F. Rual, A. 612 Muller, J. C. Twizere, B. Nkengfac, J. Vandenhaute, D. E. Hill, S. P. 613 Salcedo, J. P. Gorvel, J. J. Letesson, and X. De Bolle. 2011. Identification of a 614 Brucella spp. secreted effector specifically interacting with human small GTPase 615 Rab2. Cell Microbiol 13:1044-58. 616

10. de Jong, M. F., Y. H. Sun, A. B. den Hartigh, J. M. van Dijl, and R. M. 617 Tsolis. 2008. Identification of VceA and VceC, two members of the VjbR 618 regulon that are translocated into macrophages by the Brucella type IV secretion 619 system. Mol Microbiol 70:1378-96. 620

11. Delpino, M. V., M. I. Marchesini, S. M. Estein, D. J. Comerci, J. Cassataro, 621 C. A. Fossati, and P. C. Baldi. 2007. A bile salt hydrolase of Brucella abortus 622 contributes to the establishment of a successful infection through the oral route 623 in mice. Infect Immun 75:299-305. 624

12. Delrue, R. M., C. Deschamps, S. Leonard, C. Nijskens, I. Danese, J. M. 625 Schaus, S. Bonnot, J. Ferooz, A. Tibor, X. De Bolle, and J. J. Letesson. 2005. 626 A quorum-sensing regulator controls expression of both the type IV secretion 627 system and the flagellar apparatus of Brucella melitensis. Cell Microbiol 628 7:1151-61. 629

13. Dozot, M., R. A. Boigegrain, R. M. Delrue, R. Hallez, S. Ouahrani-Bettache, 630 I. Danese, J. J. Letesson, X. De Bolle, and S. Kohler. 2006. The stringent 631 response mediator Rsh is required for Brucella melitensis and Brucella suis 632


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

27

virulence, and for expression of the type IV secretion system virB. Cell 633 Microbiol 8:1791-802. 634

14. Ellison, D. W., and V. L. Miller. 2006. Regulation of virulence by members of 635 the MarR/SlyA family. Curr Opin Microbiol 9:153-9. 636

15. Gerhardt, P. 1958. The nutrition of brucellae. Bacteriol Rev 22:81-98. 637 16. Godfroid, J., H. C. Scholz, T. Barbier, C. Nicolas, P. Wattiau, D. Fretin, A. 638

M. Whatmore, A. Cloeckaert, J. M. Blasco, I. Moriyon, C. Saegerman, J. B. 639 Muma, S. Al Dahouk, H. Neubauer, and J. J. Letesson. 2011. Brucellosis at 640 the animal/ecosystem/human interface at the beginning of the 21st century. Prev 641 Vet Med 102:118-31. 642

17. Grkovic, S., M. H. Brown, and R. A. Skurray. 2002. Regulation of bacterial 643 drug export systems. Microbiol Mol Biol Rev 66:671-701, table of contents. 644

18. Haine, V., A. Sinon, F. Van Steen, S. Rousseau, M. Dozot, P. Lestrate, C. 645 Lambert, J. J. Letesson, and X. De Bolle. 2005. Systematic targeted 646 mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators 647 in the control of virulence. Infect Immun 73:5578-86. 648

19. Heroven, A. K., G. Nagel, H. J. Tran, S. Parr, and P. Dersch. 2004. RovA is 649 autoregulated and antagonizes H-NS-mediated silencing of invasin and rovA 650 expression in Yersinia pseudotuberculosis. Mol Microbiol 53:871-88. 651

20. Lacal, J., A. Busch, M. E. Guazzaroni, T. Krell, and J. L. Ramos. 2006. The 652 TodS-TodT two-component regulatory system recognizes a wide range of 653 effectors and works with DNA-bending proteins. Proc Natl Acad Sci U S A 654 103:8191-6. 655

21. Ludwig, A., C. Tengel, S. Bauer, A. Bubert, R. Benz, H. J. Mollenkopf, and 656 W. Goebel. 1995. SlyA, a regulatory protein from Salmonella typhimurium, 657 induces a haemolytic and pore-forming protein in Escherichia coli. Mol Gen 658 Genet 249:474-86. 659

22. Marchesini, M. I., C. K. Herrmann, S. P. Salcedo, J. P. Gorvel, and D. J. 660 Comerci. 2011. In search of Brucella abortus type IV secretion substrates: 661 screening and identification of four proteins translocated into host cells through 662 VirB system. Cell Microbiol 13:1261-74. 663

23. Martin, F. A., D. M. Posadas, M. C. Carrica, S. L. Cravero, D. 664 O'Callaghan, and A. Zorreguieta. 2009. Interplay between two RND systems 665 mediating antimicrobial resistance in Brucella suis. J Bacteriol 191:2530-40. 666

24. Martin, R. G., and J. L. Rosner. 1995. Binding of purified multiple antibiotic-667 resistance repressor protein (MarR) to mar operator sequences. Proc Natl Acad 668 Sci U S A 92:5456-60. 669

25. Martinez-Nunez, C., P. Altamirano-Silva, F. Alvarado-Guillen, E. Moreno, 670 C. Guzman-Verri, and E. Chaves-Olarte. 2010. The two-component system 671 BvrR/BvrS regulates the expression of the type IV secretion system VirB in 672 Brucella abortus. J Bacteriol 192:5603-8. 673

26. Mohr, C. D., N. S. Hibler, and V. Deretic. 1991. AlgR, a response regulator 674 controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the 675 algD promoter located unusually far upstream from the mRNA start site. J 676 Bacteriol 173:5136-43. 677

27. Nagel, G., A. Lahrz, and P. Dersch. 2001. Environmental control of invasin 678 expression in Yersinia pseudotuberculosis is mediated by regulation of RovA, a 679 transcriptional activator of the SlyA/Hor family. Mol Microbiol 41:1249-69. 680

28. Navarre, W. W., T. A. Halsey, D. Walthers, J. Frye, M. McClelland, J. L. 681 Potter, L. J. Kenney, J. S. Gunn, F. C. Fang, and S. J. Libby. 2005. Co-682


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

28

regulation of Salmonella enterica genes required for virulence and resistance to 683 antimicrobial peptides by SlyA and PhoP/PhoQ. Mol Microbiol 56:492-508. 684

29. Nguyen Ple, M., I. Bervoets, D. Maes, and D. Charlier. 2010. The protein-685 DNA contacts in RutR*carAB operator complexes. Nucleic Acids Res 38:6286-686 300. 687

30. O'Callaghan, D., C. Cazevieille, A. Allardet-Servent, M. L. Boschiroli, G. 688 Bourg, V. Foulongne, P. Frutos, Y. Kulakov, and M. Ramuz. 1999. A 689 homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl 690 type IV secretion systems is essential for intracellular survival of Brucella suis. 691 Mol Microbiol 33:1210-20. 692

31. Perera, I. C., and A. Grove. 2010. Urate is a ligand for the transcriptional 693 regulator PecS. J Mol Biol 402:539-51. 694

32. Pizarro-Cerda, J., S. Meresse, R. G. Parton, G. van der Goot, A. Sola-695 Landa, I. Lopez-Goni, E. Moreno, and J. P. Gorvel. 1998. Brucella abortus 696 transits through the autophagic pathway and replicates in the endoplasmic 697 reticulum of nonprofessional phagocytes. Infect Immun 66:5711-24. 698

33. Posadas, D. M., F. A. Martin, J. V. Sabio y Garcia, J. M. Spera, M. V. 699 Delpino, P. Baldi, E. Campos, S. L. Cravero, and A. Zorreguieta. 2007. The 700 TolC homologue of Brucella suis is involved in resistance to antimicrobial 701 compounds and virulence. Infect Immun 75:379-89. 702

34. Praillet, T., W. Nasser, J. Robert-Baudouy, and S. Reverchon. 1996. 703 Purification and functional characterization of PecS, a regulator of virulence-704 factor synthesis in Erwinia chrysanthemi. Mol Microbiol 20:391-402. 705

35. Prouty, A. M., I. E. Brodsky, J. Manos, R. Belas, S. Falkow, and J. S. Gunn. 706 2004. Transcriptional regulation of Salmonella enterica serovar Typhimurium 707 genes by bile. FEMS Immunol Med Microbiol 41:177-85. 708

36. Providenti, M. A., and R. C. Wyndham. 2001. Identification and functional 709 characterization of CbaR, a MarR-like modulator of the cbaABC-encoded 710 chlorobenzoate catabolism pathway. Appl Environ Microbiol 67:3530-41. 711

37. Rambow-Larsen, A. A., G. Rajashekara, E. Petersen, and G. Splitter. 2008. 712 Putative quorum-sensing regulator BlxR of Brucella melitensis regulates 713 virulence factors including the type IV secretion system and flagella. J Bacteriol 714 190:3274-82. 715

38. Robertson, C. A., and H. A. Nash. 1988. Bending of the bacteriophage lambda 716 attachment site by Escherichia coli integration host factor. J Biol Chem 717 263:3554-7. 718

39. Rosenberg, O. S., C. Dovey, M. Tempesta, R. A. Robbins, J. S. Finer-719 Moore, R. M. Stroud, and J. S. Cox. 2011. EspR, a key regulator of 720 Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among 721 helix-turn-helix proteins. Proc Natl Acad Sci U S A 108:13450-5. 722

40. Rouanet, C., K. Nomura, S. Tsuyumu, and W. Nasser. 1999. Regulation of 723 pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: 724 involvement of the main transcriptional factors. J Bacteriol 181:5948-57. 725

41. Salcedo, S. P., M. I. Marchesini, H. Lelouard, E. Fugier, G. Jolly, S. Balor, 726 A. Muller, N. Lapaque, O. Demaria, L. Alexopoulou, D. J. Comerci, R. A. 727 Ugalde, P. Pierre, and J. P. Gorvel. 2008. Brucella control of dendritic cell 728 maturation is dependent on the TIR-containing protein Btp1. PLoS Pathog 729 4:e21. 730

42. Schafer, A., A. Tauch, W. Jager, J. Kalinowski, G. Thierbach, and A. 731 Puhler. 1994. Small mobilizable multi-purpose cloning vectors derived from the 732


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

29

Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the 733 chromosome of Corynebacterium glutamicum. Gene 145:69-73. 734

43. Schielke, S., C. Huebner, C. Spatz, V. Nagele, N. Ackermann, M. Frosch, O. 735 Kurzai, and A. Schubert-Unkmeir. 2009. Expression of the meningococcal 736 adhesin NadA is controlled by a transcriptional regulator of the MarR family. 737 Mol Microbiol 72:1054-67. 738

44. Segal, G., M. Purcell, and H. A. Shuman. 1998. Host cell killing and bacterial 739 conjugation require overlapping sets of genes within a 22-kb region of the 740 Legionella pneumophila genome. Proc Natl Acad Sci U S A 95:1669-74. 741

45. Sieira, R., G. M. Arocena, L. Bukata, D. J. Comerci, and R. A. Ugalde. 742 2010. Metabolic control of virulence genes in Brucella abortus: HutC 743 coordinates virB expression and the histidine utilization pathway by direct 744 binding to both promoters. J Bacteriol 192:217-24. 745

46. Sieira, R., D. J. Comerci, L. I. Pietrasanta, and R. A. Ugalde. 2004. 746 Integration host factor is involved in transcriptional regulation of the Brucella 747 abortus virB operon. Mol Microbiol 54:808-22. 748

47. Sieira, R., D. J. Comerci, D. O. Sanchez, and R. A. Ugalde. 2000. A 749 homologue of an operon required for DNA transfer in Agrobacterium is required 750 in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 751 182:4849-55. 752

48. Stachel, S. E., and E. W. Nester. 1986. The genetic and transcriptional 753 organization of the vir region of the A6 Ti plasmid of Agrobacterium 754 tumefaciens. EMBO J 5:1445-54. 755

49. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: 756 improving the sensitivity of progressive multiple sequence alignment through 757 sequence weighting, position-specific gap penalties and weight matrix choice. 758 Nucleic Acids Res 22:4673-80. 759

50. Tran, H. J., A. K. Heroven, L. Winkler, T. Spreter, B. Beatrix, and P. 760 Dersch. 2005. Analysis of RovA, a transcriptional regulator of Yersinia 761 pseudotuberculosis virulence that acts through antirepression and direct 762 transcriptional activation. J Biol Chem 280:42423-32. 763

51. Ugalde, J. E., C. Czibener, M. F. Feldman, and R. A. Ugalde. 2000. 764 Identification and characterization of the Brucella abortus phosphoglucomutase 765 gene: role of lipopolysaccharide in virulence and intracellular multiplication. 766 Infect Immun 68:5716-23. 767

52. Whatmore, A. M. 2009. Current understanding of the genetic diversity of 768 Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol 9:1168-769 84. 770

53. Wilkinson, S. P., and A. Grove. 2006. Ligand-responsive transcriptional 771 regulation by members of the MarR family of winged helix proteins. Curr Issues 772 Mol Biol 8:51-62. 773

54. Winans, S. C., and G. C. Walker. 1985. Conjugal transfer system of the IncN 774 plasmid pKM101. J Bacteriol 161:402-10. 775

776 777

778

779


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

30

LEGENDS TO FIGURES 780

Figure 1: Identification of MdrA. EMSA performed with probe vir-up and total crude 781

protein extracts from exponential (E) or stationary-phase (S) cultures of B. abortus. 782

Arrows indicate position of the free probe or a protein-DNA complex. 783

784

Figure 2: Sequence alignment of MdrA and different representative members of the 785

MarR-family of transcriptional regulators. Relative position of secondary structure 786

elements is shown as a schematic representation of α-helices, β-sheets and the wing 787

region (W1), according to the Escherichia coli MarR crystal structure described by 788

Alekshun et al. (2001) (1). White letters identify residues identical in all (highlighted in 789

black), or in six out of the seven MarR homologs considered (highlighted in dark gray). 790

Black letters identify residues identical in five or four (highlighted in gray), or in three 791

out of the seven MarR homologs (highlighted in light gray). The sequence alignment 792

was performed using ClustalW (49). 793

794

Figure 3: Analysis of the interaction between MdrA and the virB promoter. A. 795

Schematic representation of genomic sequences corresponding to the regulatory region 796

of the virB operon, radiolabeled probes used for EMSA, or unlabeled competitors. 797

Positions relative to the transcription start site are indicated. MdrA, HutC, IHF and 798

VjbR-binding sites are indicated as boxes. Crossed box indicates the non-related 799

sequences that replace the IHF-binding site in probe vir-down-ihf-. B. EMSA performed 800

with the indicated probes and 0, 10, 20, 40 or 60 nM MdrA. C. EMSA performed with 801

probe vir-up, MdrA, and the indicated unlabeled DNA fragments as competitors. 802

Protein concentration was as follows: line 1, no protein; lines 2-8, 30 nM. Concentration 803

of unlabelled DNA competitors was as follows: lanes 1 and 2, no competitor DNA; 804


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

31

lanes 3, 5, and 7, 150 ng; lanes 4, 6 and 8, 360 ng. D. EMSA performed with probe vir-805

down, MdrA, and the indicated unlabeled DNA fragments as competitors. 806

Concentration of protein and unlabelled DNA competitors was as in (C). 807

808

Figure 4: Analysis of the interaction between MdrA, HutC and/or IHF with the 809

downstream region of the virB promoter. A. Schematic representation of probes vir-810

down and 286/59. Rectangles indicate position of the binding sites of HutC, IHF, and 811

MdrA. B. Co-incubation experiment performed by EMSA with probe vir-down and 812

different combinations of 20 nM MdrA, 20 nM HutC or 40 nM IHF. C. Co-incubation 813

experiment performed by EMSA with probe 286/59 and different combinations MdrA, 814

HutC or IHF. Protein concentration was as in (B). 815

816

Figure 5: Determination of the MdrA-binding sites in the virB promoter. A. Schematic 817

representation of probes vir-up and vir-downI used for DNase I Footprinting 818

experiments. Positions relative to the transcription start site of PvirB are indicated. MdrA, 819

HutC, IHF and VjbR-binding sites are indicated as boxes. Arrows and asterisks indicate 820

the primer used for construction of each 5’ radiolabeled probe. B. DNase I Footprinting 821

experiment performed with probe vir-downI and MdrA at the indicated concentrations. 822

Lanes A and G show sequence reactions performed by the Sanger method with primer 823

vir-downI. The protected region of the MdrA-binding site I is indicated with an open 824

rectangle. C. DNase I Footprinting experiment performed with probe vir-up and MdrA 825

at the indicated concentrations. Lanes A and G show sequence reactions performed by 826

the Sanger method with primer vir-up. The protected region of the MdrA-binding site II 827

is indicated as in (B). 828

829


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

32

Figure 6: MdrA binds simultaneously to binding sites I and II. A. Schematic 830

representation of probe vir-upIII used for DNase I Footprinting experiments shown in 831

(B) and (C). Positions relative to the transcription start site of PvirB are indicated. 832

Rectangles indicate MdrA, HutC, IHF and VjbR-binding sites. Arrow and asterisk 833

indicate the primer used for construction of the 5’ radiolabeled probe vir-upIII. B and C 834

are different runs of a DNase I Footprinting experiment performed with probe vir-upIII 835

and MdrA at the indicated concentrations. Lanes A and G show sequence reactions 836

performed by the Sanger method with primer pvirup. The protected region of the MdrA-837

binding sites I and II are indicated with open rectangles. 838

839

Figure 7: Sequence analysis of the MdrA-protected regions in the virB promoter. A. 840

Schematic representation of the MdrA-protected sequence in the binding site I. The 841

rectangle indicates regions protected from DNase I cleavage in experiments performed 842

with probe vir-upIII and vir-downII. Positions relative to the transcription start site are 843

indicated. Sequences corresponding to the HutC and IHF-binding sites are underlined. 844

Arrow indicates position of the 11-bp MdrA-binding consensus motif. Nucleotides that 845

match the 11-bp MdrA-binding consensus motif are highlighted in grey. B. Schematic 846

representation of the MdrA-protected sequence in the binding site II. The open rectangle 847

indicates the region protected from DNase I cleavage. Positions relative to the 848

transcription start site are indicated. Arrows indicate position of the 11-bp MdrA-849

binding consensus motifs. Nucleotides that match the 11-bp MdrA-binding consensus 850

motifs are highlighted in grey. Arrows indicate dyad symmetry. C. Schematic 851

representation of sequences corresponding to probe vir-down-ihf-. The HutC-binding 852

site is indicated as in (A). Nucleotides that match the 11-bp MdrA-binding consensus 853

motif are highlighted in grey. Nucleotides that replaced the IHF-binding site by a non-854


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

33

related sequence are indicated in italic. Position of nucleotides of the 11-bp MdrA-855

binding consensus motif that are not present in probe vir-down-ihf- is indicated by an 856

open rectangle. D. Alignment of sequences corresponding to the 11-bp MdrA-binding 857

consensus motif found at the protected regions of the MdrA-binding sites I and II. 858

859

Figure 8: Effect of DOC on the binding activity of MdrA. EMSA performed with probe 860

vir-down and 30 nM MdrA, 50 nM IHF or 30 nM HutC, in the presence of 100, 250, 861

500, 750, or 1000 μM DOC. 862

863

Figure 9: Role of MdrA in regulation of the virB promoter activity. Strains B. abortus 864

PvirB-lacZ (wt), B. abortus ΔhutC PvirB-lacZ, B. abortus ΔmdrA PvirB-lacZ, B. abortus 865

ΔhutC ΔmdrA PvirB-lacZ, or B. abortus ΔhutC ΔmdrA-KI PvirB-lacZ were grown in rich 866

medium (TSB) until exponential phase (OD600 0.5-1). Subsequently, bacteria were 867

harvested, and β-Galactosidase activites were determined. Values are means ± standard 868

deviations of duplicate wells from a representative of three experiments. **, P < 0.01. 869

870


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

_ E SExtract

Freeprobe


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

α1 α2 β1 α3

α4 β2 β3w1

α5

α6

Escherichia coli MarR

Salmonella typhimurium SlyA

Yersinia enterocolitica RovA

Erwinia chrysanthemi PecS

Pseudomonas aeruginosa MexR

Brucella abortus MdrA

Sinorhizobium meliloti ExpG

MarR

SlyA

RovA

PecS

MexR

MdrA

ExpG

MarR

SlyA

RovA

PecS

MexR

MdrA

ExpG

MarR

SlyA

RovA

PecS

MexR

MdrA

ExpG

---------------------------------------MKSTSDLFNEIIPLGRLIHMV 21

---------------------------------------------MKLESPLGS-DLARL 14

-----------------------------------------------MESTLGS-DLARL 12

-------------------------MARYLEVSDIVQQWRNERPDLDVEPMLVIGTLSRV 35

-------------------------------------------MNYPVNPDLMPALMAVF 17

MRNKPPHKTGRELHRGFTKCGSGREAKEMTNTQRKMDASAPFMAPQTALRSLYLEALQLV 60

----MERGMNHRILYPFADFGDTVAILPANETQRKGLDTPVDDRDGDDSLVTYFELARVM 56

NQKKDRLLNEY------LSPLDITAAQFKVLCSIRC---AACITPVELKKVLSVDLGALT 72

VRIWRALIDHR------LKPLELTQTHWVTLHNIHQ--LPPDQSQIQLAKAIGIEQPSLV 66

VRVWRALIDHR------LKPLELTQTHWVTLHNINR--LPPEQSQIQLAKAIGIEQPSLV 64

SLLIDRALDKV------FSKYKLSAREFDILATLRRRGAPYAISPSQIVNALMINNSTLT 89

QHVRTRIQSELD-----CQRLDLTPPDVHVLKLIDE---QRGLNLQDLGRQMCRDKALIT 69

ERLHRRLLDVVKDEFDRNGRSDINATQALLLFNIGN----SELTAGELRSRGYYLGSNVS 116

ERASRRFSGLLRAELTKLGVEDIGPAQAMVLLAIGE----AELSVGELLDRGHYVGSNIS 112

RMLDRLVCKGWVERLPNPNDKRGVLVKLTTSGAAICEQCHQLVGQDLHQELTKN-LTADE 131

RTLDQLEDKGLISRQTCASDRRAKRIKLTEKAEPLIAEMEEVIHKTRGEILAG--ISSEE 124

RTLDQLEEKGLITRHTCANDRRAKRIKLTEQSSPIIEQVDGVICSTRKEILGG--ISPDE 122

SRLDRLEQAGWLRRMPIEGDRRSVNIQLTDEGFALINRVVEEHVENERDILSP--FSEEE 147

RKIRELEGRNLVRRERNPSDQRSFQLFLTDEGLAIHQHAEAIMSRVHDELFAP--LTPEE 127

YNLKKLVEMGFIHHQRSRVDRRSVRVSLTDKGNEIANQVASLYERHITSIEQVGGIQVEE 176

YYLKQLADGDYIDRIASQRDKRSARIRLSEKGRQLCAGLRQAAKGYERALSHGD-QDRRN 171

VATLEHLLKKVLP---------- 144

IELLIKLIAKLEHNIMELHSHD- 146

IELLSGLIDKLERNIIQLQSK-- 143

KTQLRALLGRVEKHLVNNR---- 166

QATLVHLLDQCLAAQPLEDI--- 147

FMAMNKSLQRLDRFWNDSIAYRL 199

LETAFQTLHRLELVWGNAARYGI 194


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

B10 vir-upProbe

MdrA

vir-downvir-

down ihf

Free probe

MdrA-up

MdrA-down

C D vir-up

Competitor

MdrA

__

_ + +++++

Probe

up 77144

B

+1 8764 532

Free probe

MdrA-up

Free probe

MdrA-down

vir-down

Competitor

MdrA

__

_ + +++++

Probe

down B1A1

+1 8764 532

____

-430

-353

-286 -202

-201

up

77

144

vir-up

-94

-130

+24

down

A1

-59

B1

virB1

vir-down ihf-

vir-down Labelledprobes

Unlabelledcompetitors

+1VjbR

IHFHutC

MdrAsite I

-323

MdrAsite II

A


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

_

_

+

+_

_ _

+_ _

++

+_

_ +

+

+

+

+

+

_

_

_

Free probe

IHF-vir-down

B C

vir-downProbe

A

MdrA-vir-down

HutC-vir-down

MdrA-286/59HutC-286/59

Free probe

IHF-286/59

HutC

MdrA

IHF

286/59

_

_

+

_

+_

+_

__

_

_

_

Probe

HutC

MdrA

IHF

vir-down

286/59I


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

A G _ _40 60

MdrA (nM)

2010AG _ _10 20 1206040

MdrA (nM)B

+1III

A

*

*-335

-312

-190

-160

C

I

II

-430 -202

-286 +24

vir-upII

vir-downII


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

+1III

A

pvir-upIII

A G _ _20 50A G _ _20 50

CB

-190

-160

-335

-312

MdrA (nM)MdrA (nM)

II

I

*

-430 -60


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

agcaagttcggtaaaaatttagccagcttgga

tcgttcaagccatttttaaatcggtcgaacct

-335 -312

cgcataccacttgtatataagattttgttaaaaaagaattttctaatagagcgtatggtgaacatatattctaaaacaattttttcttaaaagattatct

IHFHutC

cgcataccacttgtatataagattttgtgccaggtgccaactgacagggagcgtatggtgaacatatattctaaaacacggtccacggttgagctgtcct

-190 -160

MdrA-binding site I

MdrA-binding site II

attttgttaaa

agttcggtaaa

agctggctaaa

MdrA-binding site I

MdrA-binding site II

B

C D

A

HutC


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

DOC (µM)

MdrA

___ ++_ __ __ _

IHF HutC

MdrA-vir-downHutC-vir-down

Free probe

IHF-vir-down

++ ++ ++++ ++ ++++ ++


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

0

20

40

60

80

100

120

2308 ∆hutC ∆mdrA ∆hutC

∆mdrA

∆hutC

∆mdrA-KI

β-G

al. A

ctivity (

M.U

.)

**


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Documents

A MarR-type regulator directly activates transcription from the