the tonb3 system in the human pathogen vibrio vulnificus...

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The TonB3 system in the human pathogen Vibrio vulnificus is under the 2

control of the global regulators Lrp and CRP. 3

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Alejandro F. Alice* and Jorge H. Crosa 7

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Department of Molecular Microbiology and Immunology, 10

Oregon Health and Science University, Portland, Oregon. 11

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Running title: TonB3 regulation in Vibrio vulnificus by Lrp and CRP. 13

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*Corresponding author 15

Department of Molecular Microbiology and Immunology, 16

Oregon Health and Science University. 17

Portland, OR 97239 – USA 18

Tel: 503-494-7583 19

Fax: 503-494-6862 20

[email protected] 21

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.06614-11 JB Accepts, published online ahead of print on 3 February 2012

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Abstract 25

TonB systems transduce the proton motive force of the cytoplasmic membrane to 26

energize substrate transport through a specific TonB-dependent transporter across the 27

outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a 28

fatal septicemic disease in humans and eels, possesses three TonB systems. While the 29

TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the 30

bacterium grows in human serum. In this work we have determined the essential role of 31

the leucine-responsive protein (Lrp) and cAMP-receptor protein (CRP) in the 32

transcriptional activation of this system. Whereas Lrp shows at least four very distinctive 33

DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively 34

in a region centered at position -122.5 from the start point of the transcription. Our 35

results suggest that both proteins bind simultaneously to the region closer to the RNA 36

polymerase binding site. Importantly, we report that the TonB3 system is induced not 37

only by serum but also during growth in minimal medium with glycerol as sole carbon 38

source and low concentrations of casamino acids. In addition to catabolite repression by 39

glucose, L-Leucine acts by inhibiting the binding of Lrp to the promoter region hence 40

preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct 41

control of two global regulators that can integrate different environmental signals (i.e. 42

glucose starvation and the transition between “feast and famine”). These results shed 43

light on new mechanisms of regulation for a TonB system that could be widespread in 44

other organisms. 45

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Introduction 48

In Gram-negative bacteria small molecules cross the outer membrane (OM) by 49

passive diffusion through transmembrane porins; however, substrates that are either 50

poorly permeable through porins (those greater than 600 Da) or are present at very low 51

concentrations require energized transport for their translocation across the OM (54). 52

The TonB complex transduces the proton motive force (pmf) of the cytoplasmic 53

membrane to energize substrate transport through a specific TonB-dependent transporter 54

(TBDT) across the OM (50). In Escherichia coli this TonB system is a cytoplasmic 55

transmembrane complex composed of the proteins TonB, ExbB and ExbD that spans the 56

periplasm (45, 50). Our laboratory has described that in Vibrio species the complex with 57

the TonB2 protein includes a fourth protein, TtpC(59). Until recently, TonB-dependent 58

transport was exclusively shown for the physiological uptake of iron complexes and 59

vitamin B12 (cobalamin). In recent years there have been descriptions of TBDT involved 60

in the transport of maltodextrins, zinc, nickel and sucrose in different species that suggest 61

the possibility that a more broad range of nutrients could be transported through this 62

complex (2, 34, 42, 53, 58). In Neisseria gonorrhoeae an iron regulated TBDT protein, 63

TdfF, is important in the intracellular growth of this pathogen and its role remains to be 64

elucidated(24). 65

Vibrio vulnificus is an opportunistic marine pathogen that can cause a fatal 66

septicemic disease in humans and eels (23, 27). This estuarine bacterium preferentially 67

affects individuals with underlying hepatic diseases and other compromised conditions, 68

such as hemochromatosis and beta thalassemia. In humans, this pathogen frequently 69

causes fatal sepsis with a very rapid progress, resulting in a mortality rate of more than 70


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50% within a few days (27). We have described the role of the TonB1 and TonB2 71

complexes in V. vulnificus virulence in iron-overloaded mice (1). These two systems are 72

involved in the transport of both the hydroxamate-type siderophore and vulnibactin, the 73

two endogenously produced iron chelators siderophores as well as heme and hemoglobin 74

(1) (and Alice & Crosa unpublished results). As expected the operons coding for those 75

complexes were expressed preferentially under iron limiting conditions under the control 76

of the Fur protein (1). We have also identified a TonB3 complex in this pathogen that is 77

specifically induced when V.vulnificus grows in human serum with the addition of ferric 78

ammonium citrate (1). This system consists of seven genes arranged in one operon 79

including a gene that codes for a putative TBDT as well as those encoding the essential 80

proteins of the complex TtpC3, ExbB3, ExbD3 and TonB3. Moreover this cluster of 81

genes has homologues in V. parahaemolyticus as well as in V. alginolyticus. 82

Since it was clear that both the expression of this operon as well as its genetic 83

arrangement show significant differences with the conventional iron-regulated TonB 84

complexes, we attempted to unveil the mechanisms of transcription activation of the 85

TonB3 system. This knowledge can be used to determine the nature of the substrate(s) 86

that is transported by this system. In this work we described the essential role of two 87

global regulators, Lrp and CRP, in the activation of this operon in conditions that bacteria 88

find under glucose starvation and the transition between “feast and famine”. In addition 89

we also demonstrated that the TonB3 system is involved in V. vulnificus invasion in iron-90

overloaded mice. 91

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Material and Methods 93

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Bacterial strains, plasmids and growth conditions. The strains and plasmids used in 95

this work are listed in Table 1. Bacterial strains were grown routinely in Trypticase Soy 96

Broth with the addition of 1.5% NaCl (TSBS) (V. vulnificus) or LB broth (Escherichia 97

coli) supplemented with antibiotics as appropriate: ampicillin 400 μg/ml, 98

chloramphenicol 2 μg/ml (V. vulnificus) or ampicillin 100 μg/ml, chloramphenicol 30 99

μg/ml and kanamycin 50 μg/ml (E. coli). Thiosulfate-citrate-bile salts-sucrose agar 100

(TCBS) was used as selective medium for V. vulnificus in the conjugations. M9 was used 101

as minimal medium (16) and, when it was needed, different carbon sources other than 102

glucose were used at 0.5% (w/v). Casamino acids (CAA) or L-Leucine were added at the 103

concentrations indicated in the figures and text. Human serum samples from at least two 104

healthy donors were pooled, heat inactivated, centrifuged, divided in aliquots and stored 105

at –80°C. In some experiments commercial pooled normal human serum was used (Cat # 106

IPLA-SER, Innovative Research, Novi, MI) with similar results to those obtained from 107

other source. Ferric ammonium citrate (200 μg/ml) (FAC) was added to allow V. 108

vulnificus growth (3) . 109

110

DNA manipulations and sequence analysis. Plasmid was prepared using the Qiaprep 111

miniprep kit (Qiagen, Valencia, CA). Touchdown PCR reactions were performed with 112

either Taq polymerase (Invitrogen, Carlsbad, CA) or Vent polymerase (New England 113

Biolabs® Inc., Ipswich, MA) using the following conditions: 95 °C 4 min, 30 cycles of 95 114

°C 30 sec, 63 °C 1 min (the temperature of this step was lowered 0.3 °C for each cycle) 115


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and 72 °C 1 min, and a final extension step of 72 °C 10 min. Digestions and ligations 116

were performed according to the manufacturer’s instructions (New England Biolabs® 117

Inc., Ipswich, MA). DNA sequencing reactions were carried out by the Vollum 118

Sequencing Core Facility (OHSU). 119

120

Construction and complementation of the V. vulnificus mutants. In-frame deletions 121

of the entire coding sequence of genes were generated using splicing by overlap 122

extension (SOE) PCR (55). Upstream and downstream regions (approximately 700 bp-123

800 bp) flanking each gene were amplified with specific primers, both fragments were 124

mixed and ligated in a new PCR reaction with primers 1 and 2 for each gene (Table S1). 125

The amplified fragment was cloned in the pCR®-Blunt II vector (Invitrogen, Carlsbad, 126

CA), digested with appropriate restriction enzymes and sub-cloned in the suicide vector 127

pDM4 previously digested with the same restriction enzymes. E. coli S17-1λpir 128

transformed with the pDM4 derivatives were conjugated with V. vulnificus and 129

exconjugants were selected on TCBS agar with chloramphenicol. Clones were 130

subsequently screened for chloramphenicol sensitivity and sucrose resistance and 131

deletions within the genes of interest were confirmed by PCR. 132

For the complementation experiments, each gene was amplified by PCR with 133

primers containing restriction sites as indicated in Table S1. The fragments were cloned 134

in pCR®-Blunt II vector (Invitrogen, Carlsbad, CA), sequenced and then subcloned in 135

pMMB208 under the control of the Ptac promoter. The constructs were transferred to V. 136

vulnificus strains by triparental conjugation using the plasmid helper pRK2013. In order 137


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to induce transcription of the cloned genes, 0.1 mM or 1 mM isopropyl β-D-1-138

thiogalactopyranoside (IPTG) was added to the media. 139

140

RNA isolation and cDNA synthesis. Strains were grown to the determined OD600nm in 141

minimal medium or various times in human serum with the addition of ferric ammonium 142

citrate (HS+FAC). Total RNA was extracted from each strain using RNaesy kit (Qiagen, 143

Valencia, CA) according to the manufacturer’s instructions, treated with RNAse-free 144

DNAse, quantified and stored at -80C. Usually 1 μg of RNA thus treated was used for 145

cDNA synethsis with Quantitect Reverse transcription kit (Qiagen, Valencia, CA). 146

147

Quantitative Real Time-PCR (qRT-PCR). cDNA synthesized as described above was 148

used in qRT-PCR reactions with Power SYBR green PCR master mix (Applied 149

Biosystems, Carlsbad, CA) and 500 nM (each) forward and reverse primers (Table S1). 150

Relative expression was determined by calculating 2-ΔΔCt using the 23S rRNA gene as an 151

internal control. Reactions were conducted in triplicate in a StepOne™ Real-Time PCR 152

System (Applied Biosystems, Carlsbad, CA). Statistical analysis was performed by one-153

way analysis of variance (ANOVA) with Bonferroni’s post test. 154

155

Overexpression and purification of the V. vulnificus recombinant proteins. The 156

DNA fragments coding for Lrp and CRP were amplified using primers described in Table 157

S1 and individually cloned into the expression vector pET200 (Invitrogen, Carlsbad, 158

CA). A recombinant plasmid with the correct sequence was used for expression of each 159

protein in E. coli BL21(DE3) Star. In general 200 ml of LB supplemented with 160


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kanamycin were inoculated with an overnight culture with the strain harboring the 161

plasmid of interest and when the OD600 reached 0.6, 1 mM IPTG was added to induce the 162

expression of the protein. The culture was incubated another 4 h at 37°C, centrifuged, 163

washed and resuspended in lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM 164

imidazole, pH 8.0). Cells were sonicated, centrifuged and the supernatant passed through 165

a Ni-nitrilotriacetic acid resin (Qiagen, Valencia, CA) according to the manufacturer’s 166

instructions. After the elution from the column each protein was dialyzed overnight at 167

4°C against buffer D (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 2 mM 168

DTT, 10% glycerol) and the concentration determined by a bicinchoninic acid assay kit 169

(Pierce, Rockford, IL). The proteins were aliquoted and stored at –80°C. 170

171

Electrophoretic mobility shift assay (EMSA). The gel mobility shift assay was 172

performed using the digoxigenin (DIG) gel shift kit 2nd generation (Roche, Indianapolis, 173

IN). DNA fragments were amplified by PCR and 3’ end labeled with DIG-11-ddUTP 174

using terminal transferase as described by the manufacturer. After the labeling efficiency 175

was determined each of the labeled probes (4 nM) were incubated with increasing 176

amounts of the protein of interest in the binding buffer A (20 mM Hepes, pH = 7.6, 1 mM 177

EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% (w/v) Tween 20, 30 mM KCl and 1 μg/μl 178

poly [d(I-C)]) and incubated 20 min at 37 C temperature. When the protein CRP was 179

analyzed 0.2 mM cAMP was added to the binding buffer A. For competition 180

experiments 4 nM of the labeled probe was incubated with in the presence of increasing 181

amounts (usually 400 nM) of the unlabeled specific probe. Samples were separated by 6 182

% polyacrylamide gels (Invitrogen, Carlsbad, CA) and run in 0.5X TBE for 90 min at 183


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100 V. The DNA-protein complexes were transferred to positively charged nylon 184

membrane Hybond N+ (GE Healthcare, Piscataway, NJ) and the chemiluminescent signal 185

detected according to the manufacturer’s instructions (Roche, Indianapolis, IN) by using 186

an ImageQuant LAS4000 camera (GE Healthcare, Piscataway, NJ). 187

188

DNase I footprinting experiments. DNase I footprinting experiments were performed 189

on 32P end-labeled fragments using either γ-32P[ATP] (7,000 Ci/mmol, MP Biomedicals, 190

Solon, OH) or α-32P [dATP] (3,000 Ci/mmol, Perkin Elmer, Waltham, MA). For 5’ 191

labeling of the probe C a PCR fragment amplified with primers M13F and M13R using 192

as template plasmid p300-42B was labeled with T4 polynucleotide kinase, digested with 193

SacI and purified from 5% native polyacrylamide gels. For 3’ labeling of probe D, a 194

PCR fragment amplified using plasmid p632-300 with primers 632Pst-M13F was 195

digested with SpeI – EcoRV, end-labeled with DNA polymerase I large (Klenow) 196

fragment (Invitrogen, Carlsbad, CA) as indicated by the manufacturer and gel purified as 197

described above. Binding reactions (20μL) were carried out in the same conditions as 198

described for EMSA assays and DNase I (Promega) treatment was performed for 1 min at 199

room temperature. All samples were analyzed in either 5% or 6% polyacrylamide/8M 200

urea gels, run at 60W, dried and scanned in a PhosphorImager Storm 825 (GE 201

Healthcare, Piscataway, NJ). Gels were calibrated with Maxam-Gilbert G+A reactions 202

prepared as described (10). 203

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β-galactosidase assays. The upstream region of the VV10842 (TonB3 cluster) gene was 205

amplified with primers indicated in Table S1, cloned in the pCR®-Blunt II vector 206


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(Invitrogen, Carlsbad, CA), sequenced and subcloned in the pTL61T vector previously 207

digested with the corresponding restriction enzymes generating the plasmids described in 208

Table 1. The empty vector and plasmids thus constructed were transferred to V. 209

vulnificus ΔlacZ wild type or the various mutant strains by triparental conjugation. 210

Bacterial strains harboring the plasmids with the lacZ fusions or the empty vector were 211

grown in the conditions described in each figure. Two hundred microliters of each 212

culture were centrifuged, resuspended in buffer Z and used in the assay. β-galactosidase 213

activities from cultures growing in minimal medium were determined as described (38). 214

Due to the turbidity of the human serum used in this work, when samples obtained from 215

this source were analyzed the cfu (ml)-1 counts at each time point were used to normalize 216

the β-galactosidase activities instead of the OD600nm (1). Values of β-galactosidase 217

activities obtained from cells growing in human serum are comparable; however, they 218

were not comparable with those obtained from cells growing in minimal medium due to 219

the different units used to normalize them (cfu and OD600nm respectively). The levels of 220

β-galactosidase from lacZ fusions were compared using one-way ANOVA with Tukey’s 221

post test. All experiments were repeated at least three times (with some of them 222

performed at least ten times) with two replicates each and the results were expressed as 223

means ± standard deviations (SD) for each group examined. All tests were performed 224

with GraphPad Prism 4.0, and statistical significance was defined as p<0.05. 225

226

Fishing ligand experiment. The protocol used for the identification of the proteins 227

bound to probe A was adapted from a method previously described by Nordhoff et al., 228

1999 (46). The crude extracts used in these experiments were prepared as follows: 10 ml 229


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of a culture of V. vulnificus CMCP6 growing in HS + FAC for 6 h was centrifuged, 230

resuspended in binding buffer A (with the omission of Tween® 20), sonicated and 231

centrifuged. The protein concentration was measured as described above and the sample 232

was kept at -20 C. Four μg of a biotinylated PCR fragment amplified with primers 42-233

632 (Biotinylated) and 42-477Bam were incubated with 400 μg Dynabeads® M-280 234

Streptavidin (Invitrogen, Carlsbad, CA) for 15 min at room temperature in binding and 235

washing buffer B&W (5mM Tris-HCl, pH=7.5, 0.5 mM EDTA, 1 M NaCl). The DNA-236

beads were washed 3 times with buffer B&W and incubated 20 min at 37 C using gentle 237

rotation with 150 μg of total proteins from the V. vulnificus crude extract obtained as 238

described above in the presence of 1 μg poly [d(I-C)] and 0.2% (w/v) Tween® 20. After 239

the incubation, the mixture was washed four times with buffer W (20mM Tris-HCl 240

pH=7.5, 0.1 mM DTT, 100 μM NaCl, 1mM EDTA, 1 X Protease inhibitor). The first 241

wash also included 1 μg of poly [d(I-C)]. The magnetically separated beads were 242

resuspeded in 40 μL of 1% SDS and boiled for 10 min. The supernatant was analyzed in 243

the Proteomic Shared Resource at OHSU. A negative control sample was also prepared 244

in the same manner as described above with the omission of the biotynilated DNA that 245

was replaced for the same volume of buffer. At least two positive and two negative 246

samples were analyzed independently and proteins were identified with false discovery 247

rate of 3%. 248

249

Virulence experiments. Competitive index (CI) experiments were performed as 250

described previously (1). Briefly, bacterial strains were cultured in TSBS, and then equal 251

volumes were mixed and serial dilutions performed. A dilution (usually containing ca. 252


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5,000- 8,000 cfu) was injected subcutaneously (s.c.) in 4-6 weeks old CD-1 mice 253

(Charles River Laboratories, Wilmington, MA). In the iron-overloaded mouse model 900 254

μg of FAC in injected intraperitoneally (i.p.) 30 min prior to the bacterial challenge (1). 255

It has been previously demonstrated that mice are more susceptible to V. vulnificus 256

infections when iron is injected i.p. before the inoculation of the bacterial cells (66). 257

Animals were checked after approximately 10 h of inoculation and when they showed 258

signs of the disease (e.g. lethargy, slow movements, lack of appetite) they were 259

euthanized with CO2 according to IACUC regulations. A skin sample (1 cm x by 1 cm 260

around the inoculation site), spleen and liver were aseptically extracted and homogenized 261

by using a Seward Stomacher lab blender in presence of PBS (1 ml for skin and spleen 262

and 2 ml for liver). Serial dilutions were performed in PBS and the dilutions were plated 263

on TSAS supplemented with 5-bromo-4-chloro-indolyl-galactopyranoside (X-Gal) plates 264

to determine the cfu of the strains under analysis. CI values were obtained as described 265

in (61). The statistical analysis was performed by using one-way ANOVA with 266

Bonferroni’s post test with GraphPad Prism 4.0 software. 267

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Results 269

The TonB3 system is involved in V. vulnificus invasion in iron-overloaded mice. 270

The TonB3 system in V. vulnificus is unique in both its genetic organization and 271

the conditions when the expression is induced. As shown in Fig 1 this operon includes a 272

gene that codes for a putative TonB-dependent receptor (TBDR) (VV1_0842) as well as 273

those encoding the TonB3 protein and the accessory proteins TtpC3, ExbB3 and ExbD3 274

(VV1_0847, VV1_0844, VV1_0845 and VV1_0846 respectively). We have previously 275

shown that this operon was specifically induced when V. vulnificus was grown in human 276

serum with the addition of FAC (HS+FAC) (1). In this pathogen the TonB1 and TonB2 277

systems are important virulence factors; however, the ΔtonB3 mutant strain did not show 278

any change in the LD50 value compared to that of the wild type strain (1). To determine 279

if this system has any role during the invasion and progress of the infection of V. 280

vulnificus we performed competitive index (CI) assays that we have described as a very 281

sensitive test to examine the contribution of a gene in the virulence of this pathogen when 282

LD50 values are similar to that of the wild type strain (1). A CI test between the double 283

ΔtonB1ΔtonB2 (TonB3+) and the triple tonB (TonB3-) mutants was carried out in the 284

iron-overloaded mouse model as described in Material and Methods (s.c. inoculations). 285

We used a ΔtonB1ΔtonB2 genetic background for these experiments because we do not 286

know if these two systems could complement a tonB3 mutation in vivo. The results 287

obtained from livers and spleens from infected animals showed that the strain in which 288

the three tonB systems were mutated is affected in the invasion of V. vulnificus 289

highlighting a possible role for the TonB3 cluster in this process (Fig. 2). 290

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A region located far upstream of the promoter is essential for full activation of the 292

tonB3 operon. 293

Since the TonB3 system may have a role in V. vulnificus infection and it is 294

specifically induced when the bacteria are growing in human serum we attempted to 295

determine the function of this system under those conditions. Taking into account that 296

we could not detect a full induction of the expression of this operon neither in rich 297

medium nor CM9 minimal medium (1) we assumed that some of the component(s) of the 298

human serum were specifically important for this system induction. Considering the 299

complexity of the components of human serum we hypothesized that by determining the 300

identity of the activator(s) involved in that particular induction we could understand the 301

role of this system in V. vulnificus physiology. 302

We first determined the minimal region of the promoter needed for its expression 303

by constructing various transcriptional fusions of the promoter region of this operon to 304

the lacZ gene that were conjugated into a V. vulnificus lacZ mutant strain. The 305

expression was analyzed at various times during growth with the maximum expression 306

observed after 6 h when the cells reached the late log phase (1). Fig. 3A shows that three 307

different levels of activity were measured after 6 h of growth: very low activity as in the 308

case of the empty vector pTL61T and the p100 construct, the absence of expression in the 309

latter construct corresponds with the deletion of the -35 region and confirms our previous 310

results where we determined the start point of the transcription being at position -81bp 311

from the ATG ((1), Fig. 1). A second group of constructs from -218 (p300) to -420 312

(p498) relative to the transcription start site shows an intermediate level of activity; 313

interestingly constructs including the region from nucleotides -551 to -420 (constructs 314


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p632 and p720) showed to be essential to achieve the full activation of this promoter 315

observed in HS+FAC. Further confirmation of these results was obtained by using a 316

strain with a deletion of the region -528 to -396 in the chromosome (named here Δ498). 317

RNA extracted from this strain growing in HS+FAC was used to analyze the expression 318

of the tonB3 gene by qRT-PCR and compared to that of the wild type. As expected the 319

transcription of this operon was induced in the wild type strain growing in these 320

conditions; however, the Δ498 derivative showed a lower level of expression confirming 321

the results obtained with the lacZ fusions (Fig 3B). 322

323

The Lrp protein is needed for the activation of the tonB3 operon. 324

The results described above suggested that the region -551 to -420 was acting in 325

cis in the activation of the tonB3 promoter. We hypothesized that a transcriptional 326

regulator(s) can bind this region and activate the expression of the tonB3 operon. To 327

determine the identity of this protein(s) we performed fishing ligand experiments as 328

described in Materials and Methods by using cell extracts obtained from V. vulnificus 329

growing in HS+FAC (inducing conditions). A total of 158 proteins were identified 330

between the positive and negative samples. Peptides corresponding to the ORF 331

VV1_2951 (encoding the leucine-responsive regulator, Lrp) were highly represented in 332

the positive sample while they were absent in the negative controls. This protein belongs 333

to a group of seven transcription factors that in E. coli controls approximately 50% of the 334

regulated genes (5). To confirm the role of this protein in the activation of this operon we 335

studied the tonB3-lacZ transcriptional fusion (construct p632) under inducing conditions 336

in both the Δlrp mutant and the complemented strains. When compared with the wild 337


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type strain we could observe a significant reduction in the activity of this promoter in the 338

mutant strain that is restored to wild type values in the complemented strain (Fig. 4A). It 339

is worth noticing that when we overexpressed the Lrp protein with 1mM IPTG the 340

protein level reached was toxic for the bacterial cells; however, lower levels of IPTG 341

(0.1mM) allowed sufficient expression to observe full activation of the operon without 342

affecting the viability of the cells (data not shown). To address whether the role of Lrp in 343

the activation was a result of a direct binding of this protein to the 632-498 region, we 344

purified the Lrp(His)6 protein and used it in EMSA assays with the corresponding labeled 345

probe. A shift in the mobility of the probe was only detected when the protein was added 346

to the reaction in the presence of poly [d(I-C)]) as a nonspecific competitor. This shift 347

could be reversed when unlabeled competitive DNA was used confirming the specificity 348

of the binding observed (Fig 5A). Usually the amino acid L-leucine might act as a 349

coeffector of Lrp and potentiates, overcomes, or has no effect on the function of Lrp upon 350

its target genes (9, 44, 60). We then tested the possibility that the presence of this amino 351

acid in the binding buffer of the EMSA assays could modify the binding properties of Lrp 352

to this DNA fragment. As can be observed in Fig. 5A Lrp doesn’t bind to the target DNA 353

in the presence of L-leucine suggesting a role for this amino acid in the regulation of this 354

operon. Additionally we also tested whether this protein could bind other regions of the 355

promoter. In Fig. 5B we show that Lrp could bind to a probe that includes the 356

transcription start point as well as the putative binding site of the RNA polymerase (probe 357

C, from -218 to +81 with respect to the ATG). Similarly when the area comprising the 358

nucleotides -420 to -218 (probe B) was used we observed a specific shift in the mobility 359


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of the probe upon the binding of Lrp. Furthermore the presence of L-Leucine affected 360

the observed binding of Lrp to these two DNA regions (Fig 4B). 361

We next determined the binding sites of Lrp in the tonB3 promoter region by 362

performing DNaseI footprinting assays. When a fragment that includes the far-upstream 363

region as well as the middle region (probe D) was used together with purified Lrp protein 364

we observed large protection at positions -509 to -443 and -415 to -398 (Fig. 5C) that 365

overlaps a sequence with similarity to the Lrp consensus (Fig. 1 and (13, 17)). The 366

protection observed also lead to phased hypersensitivity (26) that is commonly observed 367

with this protein (64). However, it should be noticed that due to the intrinsic resistance to 368

DNase I of the region comprising nucleotides -509 to -477 and -415 to -410 369

approximately, it was difficult to delimit the binding site for Lrp at those locations. 370

Interestingly another large protected region was observed in the middle region that spans 371

postions -265 to -208 with hypersensitive sites that are consistent with the DNA bending 372

or wrapping described for this protein (Fig. 5D) (64). A sequence with similarity to the 373

Lrp consensus was also identified in this position (Fig. 1 and (13, 17)). The analysis of 374

the proximal region showed an additional Lrp binding site at positions –147 to 375

approximately –59 that lays right upstream of the -35 region suggesting that this protein 376

could interact with the RNA polymerase (Fig. 5E). Again it is worth noticing the 377

presence of a region with intrinsic resistance to DNaes I between the positions -81 to -59. 378

As expected the protected region overlapped a sequence with high similarity to the Lrp 379

consensus sequence (Fig. 1 and (13, 17)). The binding of the Lrp protein to all those 380

regions is consistent with our EMSA results. 381


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Considering the previous results two new lacZ transcriptional fusions were 382

constructed and analyzed in cells growing in HS+FAC. In one of them we deleted a 383

DNA fragment between the positions -551 to -461 (construct p548) while in a second one 384

we deleted nucleotides -233 to -217 (construct pdint). According to our footprinting 385

experiments Lrp binds to these regions of the tonB3 promoter and as shown in Fig. 4B 386

when they are deleted the induction of the expression of the lacZ fusions is abolished. 387

Altogether these results confirm the role of Lrp in the control of the transcription of the 388

tonB3 operon. 389

390

Activation of the tonB3 operon in minimal medium under starving conditions. 391

It was clear from our EMSA experiments that L-Leu affected the Lrp binding to 392

the DNA target regions. To determine if this amino acid has any role in the expression of 393

the tonB3 promoter in vivo we studied its expression in cells growing in HS+FAC 394

supplemented with 10mM L-Leucine. It is worth mentioning that the concentration of L-395

Leucine in serum from healthy individuals is 123 (98-148) μmol μmol (l)-1 (51). The 396

activity of the tonB3 promoter obtained in these conditions was reduced at least 10-fold 397

compared to that of the wild type growing in the serum without the amino acid 398

supplementation (Fig 6A). 399

Although we observed induction of this operon when V. vulnificus is grown in 400

HS+FAC, our attempts to obtain similar levels of expression in both rich and minimal 401

media in the past were unsuccessful (1). With the discovery of the role of Lrp and L-402

Leucine in the control of the transcription of this operon we explored its transcription in 403

conditions were Lrp is usually expressed at high levels and/or the presence of this amino 404


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acid could play any regulatory role. In E. coli the expression of Lrp is repressed in rich 405

medium as well as in minimal medium with 1% CAA (11, 29, 32, 44). We determined 406

the expression level of the lrp gene in cells growing in TSBS determining that it was 407

aproximately 3 fold lower to that of cells growing in HS+FAC (p>0.05, no significant 408

difference was observed, Fig.7); however, in agreement with our previous observations, 409

the expression of the tonB3 gene in TSBS is ~33 fold lower to that observed in HS+FAC 410

(p<0.001, Fig. 7). Since the Lrp levels appeared not to be the main reason for these lower 411

levels of expression of the tonB3 operon in rich medium we assumed that possibly the 412

presence of L-Leucine (and other amino acids) in TSBS could be the reason for the low 413

level of expression observed in that medium. 414

We tested this hypothesis by analyzing the expression of the tonB3-lacZ fusion in 415

cells growing in M9 minimal medium with various carbon sources and CAA 416

concentrations. Lowering the CAA concentration in minimal medium with glycerol as 417

carbon source was sufficient to obtain high levels of expression of the tonB3 mRNA that 418

was comparable, although still ~ 3 fold lower, to those observed in HS+FAC (p<0.05, 419

Fig. 7). Likewise high levels of β-galactosidase units were detected in the tonB3-lacZ 420

fusion analyzed in this medium with the highest levels measured when the cells were 421

growing in the presence of glycerol and 0.02% CAA (Fig 6B). In complete agreement 422

with our previous finding in HS+FAC, when L-Leucine was added to the minimal 423

medium the expression was repressed (Fig 6B). In addition, when the Δlrp mutant strain 424

was analyzed in this medium the expression levels of the tonB3 promoter were reduced at 425

least 10 fold compared to that of the wild type and adding L-Leucine only had marginal 426

effect on it (Fig. 6C). When the complemented strain was analyzed in similar conditions 427


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β-galactosidase activity was restored to even higher levels than that of the wild type (Fig. 428

6C) indicating that overexpression of this gene activates the expression of the tonB3 429

promoter at higher levels than the wild type. It is also worth noticing that the Δlrp 430

mutant strain has longer doubling times in this medium compared to that of the wild type 431

(~3.5 h vs ~2 h) suggesting that the Lrp protein has an important role in the physiology of 432

V. vulnificus under these conditions or that a cofactor needed in the mutant is missing in 433

the minimal medium used (32). As expected the complemented strain showed similar 434

doubling time as the wild type. The results detailed above showed for the first time a 435

complete agreement between the results obtained from V. vulnificus cells growing in 436

HS+FAC with those obtained from cells growing in M9 with glycerol as sole carbon 437

source and low CAA concentrations. Furthermore analysis of the expression of the 438

tonB3-lacZ fusion in M9 glycerol low CAA with the addition of 200 μg/ml FAC 439

confirmed that high iron concentrations do not play any role in the expression of this 440

operon (Fig. 6D). Analysis of the p498, p400 and p300 lacZ fusions constructs in 441

minimal medium with glycerol and low CAA gave similar results to those obtained with 442

cells growing in HS+FAC (i.e. low levels of activation) (Fig. 6E). 443

As mentioned above we tested the expression of this operon in cells growing with 444

various carbon sources, importantly in the presence of glucose the expression was 445

reduced at least 5-7 fold (p<0.001) compared to that of the cells growing with glycerol as 446

sole carbon source (Figs 7 and 8A). In addition the expression of the tonB3-lacZ fusion 447

was reduced only two fold when L-leucine was added to the medium (Figs 7 and 8A). 448

Similar results were obtained with fructose, another 449

phosphoenolpyruvate:phosphotransferase transport system (PTS) sugar, as sole carbon 450


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source (Fig. 7). However, when galactose or maltose (non-PTS sugars) were used as 451

carbon source in minimal medium there was an increase in the activity of the tonB3-lacZ 452

fusion, that in the case of galactose it was identical to that observed when glycerol was 453

used as carbon source (Fig. 7). Furthermore, when the cells were growing in the presence 454

of glucose and glycerol the activity was 5-7 fold (p<0.001) lower than that observed in 455

glycerol alone and no effect was observed when L-Leucine was present in the medium 456

(Fig. 7). In agreement with these results, the addition of glucose to the HS+FAC reduced 457

the expresion of the tonB3 promoter almost three folds (p<0.001) at 6h when the 458

maximum expression is observed in the absence of this sugar (Fig. 6A). Altogether these 459

results suggested the involvement of a mechanism of catabolite repression in the 460

expression of the tonB3 operon and/or in the expression of the lrp gene. To address 461

whether the expression of the lrp gene is affected by the presence of glucose in the 462

medium, we carried out qRT-PCR assays from RNA obtained from cells growing in M9 463

low CAA with either glucose or glycerol as carbon source. There were not significant 464

changes in the lrp transcript level when measured in the presence of glucose and 465

compared to glycerol indicating that this gene is not under catabolite repression control 466

(Fig. 8B). 467

468

CRP is also involved in the activation of the tonB3 operon. 469

The results described in the previous section strongly suggested the possibility 470

that the tonB3 operon is under catabolite repression control. It is known that the cAMP 471

receptor protein (CRP) is involved in the activation and/or repression of various genes 472

and operons in response to the presence of glucose in the medium (6, 28, 56). To test the 473


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hypothesis that this protein also acts directly on the promoter region of this operon and/or 474

indirectly through the regulation of lrp we evaluated the expression of the tonB3 operon 475

and the lrp gene in a Δcrp mutant strain. Unfortunately, the CMCP6 V. vulnificus Δcrp 476

mutant strain cannot grow with glycerol as sole carbon source in minimal medium, so we 477

could not directly address the expression of those genes in cells growing under those 478

conditions. However, this mutant strain was able to grow in HS+FAC, although the 479

doubling time was longer as compared to that of the wild type (~2.5 h vs ~1.5 h), so we 480

were able to evaluate the expression of the tonB3 system in this condition. The 481

expression of the tonB3 promoter was significantly lower in the Δcrp mutant than that of 482

the wild type as measured by β-galactosidase activity (Fig. 8A). As expected the levels 483

of the tonB3 mRNA determined by qRT-PCR were lower in this mutant strain (Fig. 8A). 484

Furthermore the complemented strain showed similar levels of expression as that of the 485

wild type (Fig. 9A) confirming the role of this protein in the regulation of this promoter 486

at the transcription initiation level. The expression of the lrp gene was not significantly 487

different in this Δcrp mutant strain growing in HS+FAC compared to that of the wild 488

type growing in the same medium (p>0.05, Fig. 8B). This is important as, in E. coli, lrp 489

is responsive to both growth rate and to CRP (29, 41). Additionally the expression of the 490

crp gene in the wild type strain growing in minimal medium with glycerol and low CAA 491

is similar to that of the same strain growing in HS+FAC (Fig. 7C). However, we 492

determined lower levels of crp mRNA when L-Leucine was added to the minimal 493

medium (Fig. 8C). This effect that was relieved in a Δlrp mutant strain growing under 494

the same conditions suggests a possible role for Lrp in the control of the crp transcription. 495


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We further confirmed the role of CRP in the activation of the tonB3 operon by 496

analyzing its expression in minimal medium with glucose as carbon source in the 497

presence of exogenously added cAMP. In the presence of glucose the intracellular levels 498

of cAMP are low (20); hence it is expected that CRP does not bind to the binding sites 499

located in the regulatory regions of the genes under its control (28). Here, when we 500

added cAMP to the minimal medium containing glucose we could recover expression 501

from the tonB3-lacZ fusion as well as the L-Leucine regulation at the levels observed in 502

the presence of glycerol (Fig. 9B). 503

To address if the observed effect in the expression of the tonB3 gene is a 504

consequence of the direct action of the CRP protein on the promoter region of this operon 505

we performed EMSA assays with purified CRP(His)6 protein. Fig. 10A shows the shift 506

detected when the probe C (primers 42-300/42Bam) was incubated with increasing 507

concentrations of this protein in the presence of cAMP in the binding buffer. This shift 508

was reversed in the presence of unlabelled competing DNA. We further demonstrated 509

that CRP does not bind to probe A (far-upstream region) nor probe B (middle region) 510

indicating that this protein only binds in the region closer to the start point of the 511

transcription (data not shown). When we splitted the probe C in two, Ci (42-300/42-512

206Rev) and Cii (42-206-For/42Bam), we established a shift only in the probe Ci (Fig 513

10B). An in silico analysis of this region showed the presence of a putative binding site 514

with high identity to the sequence consensus for this protein centered at –122.5 bp from 515

the start point of the transcription (Fig. 1C). The identification of the binding site of this 516

protein was obtained by performing DNAseI footprinting with the probe C. A footprint 517


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was identified between the nucleotides -136 to -107 that overlaps with the identified 518

consensus sequence (28) (Fig. 11A). 519

Since in our footprinting studies with purified Lrp(His)6 we observed a protection 520

in the same position were cAMP-CRP binds to the DNA, we wished to determine 521

whether these proteins can bind to this DNA fragment simultaneously. Competitive 522

EMSA experiments were not sensitive enough to discern between Lrp-DNA complexes 523

and possible Lrp-CRP-DNA complexes (data not shown). However, by using the binding 524

signatures of each protein we evaluated the titration of Lrp against a constant 525

concentration of CRP. Similar analyses were previously performed by other authors with 526

a different system (4). In general, as the Lrp concentration increased, the signatures 527

characteristic of CRP binding dissapeared, replaced by signatures characteristic of 528

complete Lrp binding (Fig. 11B, compare lane 2 with lanes 3, 4 and 5). This 529

replacement, however, occurred gradually. At lower Lrp concentrations (Fig. 11B, lanes 530

3 and 4), signatures associated with both CRP and Lrp binding co-exist. Although Lrp-531

associated protection and hypersensitivity at positions -59 and -94 were present, the CRP-532

associated hypersensitivity site (-99) remain apparent even at intermediate Lrp 533

concentrations (Fig. 11B, lanes 3 and 4). At high Lrp concentrations all signatures and 534

protection correspond to this protein alone (Fig. 11B, compare lanes 5 and 6). We then 535

performed the same type of footprinting anaysis with increasing concentrations of CRP 536

and constant Lrp concentrations. At lower concentrations of CRP, signatures associated 537

to this protein were already present and co-existed with those of Lrp (Fig. 11B, lanes 7 538

and 8). Interestingly high concentrations of CRP were not enough to completely displace 539

Lrp, as hypersensitive signatures of the latter protein were still visible at 0.75 μM CRP 540


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(sites -59 and -94, lane 9). These results argue that Lrp when present at higher 541

concentrations (0.5 μM), may displace CRP from its site in this promoter; however, CRP 542

could displace Lrp from one of the binding sites (approximately -147 to -109) but could 543

not displace it from the binding site residing downstream of the CRP binding site even at 544

high concentrations (Fig. 11B, lanes 8 and 9). Importantly, they also support the 545

possibility that Lrp and CRP bind simultaneously to form a CRP-Lrp-DNA complex that 546

could drive transcription under the conditions studied in this work. 547

548


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Discussion 549

TonB systems are responsible for transducing the energy from the proton motive 550

force from the inner membrane to the outer membrane of Gram-negative bacteria for the 551

transport of iron-siderophore complexes, cobalamin or colicins by TBDT. Recently other 552

substrates for the TonB systems have also been identified in different species including 553

zinc, nickel, maltodextrins and sucrose (2, 34, 42, 53, 58). In addition to the control of the 554

expression of the TBDTs involved in iron-siderophore transport by the Fur protein, other 555

regulatory mechanisms have been described including: AraC-like transcriptional 556

regulators (FetR, MpeR), extracytoplasmic sigma factors (e.g. FecI, PvdS, MbaS), a 557

riboswitch (e.g. btuB), small RNAs (e.g. omrA and omrB) as well as the nickel-sensing 558

transcriptional regulator NikR (see for a review (45)). 559

In this work we show that two global regulators, Lrp and CRP, regulate 560

expression of the TonB3 operon, that includes the genes encoding the TBDT protein as 561

well as the structural proteins TtpC3, ExbB3, ExbD3 and TonB3, in response to L-562

Leucine and glucose starvation. We also demonstrated that theTonB3 system is involved 563

in V. vulnificus invasion in the iron-overloaded mouse model. Recently the gene 564

encoding the heme-receptor HupA in V. vulnificus was demonstrated to have a binding 565

site for CRP in the promoter region and that this protein controls the transcription of 566

hupA when the cells are growing at 40 C; however Fur (and iron) constitute the main 567

regulator under physiological conditions (47). Likewise, in the same pathogen the iron 568

regulated vulnibactin receptor is also under the control of CRP (14). Zhang et al. (69) 569

also showed the possible involvement of cAMP-CRP in the control of the transcription of 570

the fecA, fepA, cirA and fiu genes in a fur mutant strain in E. coli. It is still not clear 571


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whether either a direct or an indirect mechanism controls the changes in the expression 572

observed in the crp mutant in the latter two cases (14, 69). 573

Lrp is a protein that has an important role in the cell physiology and has been 574

designated a physiological barometer (21). Its main function is to control the expression 575

of target genes and operons according to the nutritional status of the cell and in E. coli 576

controls approximately 400 genes which at least 130 involve direct interactions (13, 60). 577

This protein ties bacterial metabolism to environmental signals, mediating transitions 578

between “feast and famine”. Therefore, Lrp upregulates specific genes during famine 579

and downregulates other genes during feast (9, 13, 60). Most of the genes regulated by 580

Lrp are involved in small-molecule transport and amino acid metabolism (13, 60). 581

According to our genetic and molecular analysis, in the absence of L-Leucine, Lrp binds 582

the promoter region of the tonB3 operon in at least four very distinct locations. An 583

essential binding site for the Lrp protein resulting in full activation of the tonB3 operon is 584

located in the far-upstream position -509 to -443 (with a second small region at positions 585

-415 to -398). The footprints showed phased hypersensitivity (26) that is consistent with 586

Lrp bending or wrapping of large regions of DNA. It has been described that this protein 587

can form multimers (dimers, octamers and hexadecamers) (12, 19). The activation at 588

long distance observed for Lrp in this work is not uncommon for this protein; however, 589

the location of this essential binding site at those positions in the tonB3 promoter makes 590

this regulatory region one of the farthest described so far for Lrp (48, 64). A third 591

binding site located in the middle region of the promoter, comprising positions -265 to -592

208, presented a large footprint also characteristic for this protein. It is worth noticing 593

that in some regions we could not determine the exact boundaries of the binding sites for 594


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this protein due to the intrinsic resistance to DNase I observed. A partial deletion of this 595

binding site demonstrated that it was also essential for full activation of the tonB3 operon. 596

Finally, a third binding site for Lrp was located upstream of the putative binding site for 597

the RNA polymerase and right downstream of the binding site for cAMP-CRP. 598

While in class I and class II promoters CRP interacts directly with the RNA 599

polymerase and other activators are not needed, complex promoters require a regulator in 600

addition to CRP and have been designed as class III promoters (7, 8). In those cases 601

CRP-induced DNA bending can reposition an activator into a productive complex with 602

RNA polymerase (e.g. MalT), or CRP can modulate the binding of an activator to the 603

DNA (e.g. AraC) (5, 52, 68). Since Lrp can induce bending of the DNA (62), according 604

to our results in the tonB3 promoter region Lrp might help to form a nucleoprotein 605

complex that facilitates interaction between cAMP-CRP and the RNA polymerase. A 606

similar situation has also been described in E. coli in the papBA operon, where Lrp 607

interacts with the DNA region located between the cAMP-CRP binding site and the RNA 608

polymerase (63). The location of Lrp between those two proteins helps the cAMP-CRP 609

protein to interact with the α-CTD region of the RNA polymerase (63). In another 610

example, CRP binds at -121.5 bp upstream of the transcription start site of the proP P2 611

promoter and coactivation is achieved by Fis and CRP independently contacting each of 612

the two α-CTDs (36). Further work is needed to establish whether in the tonB3 promoter 613

CRP interacts directly with the RNA polymerase (with or without Lrp help), or CRP 614

helps in the interaction between the RNA polymerase and the far located Lrp proteins or 615

another unrecognized activator. Although our genetic and DNaseI footprinting 616

experiments support the hypothesis of these two proteins acting in concert to drive 617


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transcription, it is also possible that in some growth conditions Lrp might displace CRP 618

from the promoter region. 619

Thus, we were able to identify not only two direct activators of the TonB3 system 620

but also growth conditions where it is expressed at high levels in addition to the 621

previously recognized HS+FAC. The interesting nutritional parallelism between human 622

serum and M9 with low CAA concentration and glycerol as sole carbon source suggests 623

that when V. vulnificus is growing in the former condition is under a deprivation of amino 624

acids (a signal sensed as low levels of L-Leucine) and a rapid utilization of the glucose 625

(the second signal). In addition to L-Leucine other amino acids such as L-Alanine, L-626

Isoleucine, L-Valine and L-Methionine repressed the expression of the TonB3 system 627

when added to the minimal medium (data not shown). This result is consistent with a 628

recent discovery that E. coli Lrp responds to several of these amino acids (25). In line 629

with those observations, when we performed a microarray analysis with total RNA 630

obtained from V. vulnificus cells growing in HS + FAC we found that the genes that 631

encode enzymes involved in the the glyoxylate shunt as well as glycerol metabolism were 632

clearly induced after 4 to 6 h of growth in serum (Alice & Crosa unpublished) indicating 633

the utilization of glyerol and/or lipids as carbon sources after the glucose depletion. 634

According to the Human Serum Metabolome Database (HMDB) the concentration of L-635

Leucine in normal human serum is 123 (98-148) μmol (l)-1 while the concentration of 636

glucose is 4440.0 +/- 370.0 μmol (l)-1 (51). These values indicate that Lrp could bind the 637

promoter region of the tonB3 operon when V. vulnificus grows in human serum and, if 638

glucose is depleted, cAMP-CRP could bind and allow full activation of this system. In 639

agreement with this hypothesis, when we added glucose or 10mM L-Leucine to the 640


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HS+FAC used throughout this work we observed a reduction in the expression of the 641

tonB3-lacZ fusion without affecting notably the growth (Fig. 6A). This TonB3 complex 642

appears to play a role during V. vulnificus invasion suggesting that the substrate(s) being 643

transported for this system inside the host is relevant for that process. It is tempting to 644

speculate that the transition between “feast and famine” (9), (43) sensed as L-Leucine 645

starvation (and may be the stringent response) constitues an important signal when this 646

pathogen grows in serum or inside the host. The study of the physiology of V. vulnificus 647

growing in human serum could lead to the discovery of new mechanisms used by this 648

pathogen during the rapid invasion observed in infected susceptible individuals. 649

As mentioned above Lrp and CRP appear to act in concert in the activation of the 650

TonB3 operon sensing, at least, two different environmental signals: low concentrations 651

of L-Leucine and glucose. In E. coli, a number of genes are controlled by both Lrp and 652

Crp (15, 18, 35, 49, 65, 67, 70). A question that remains to be answered is which other(s) 653

protein(s) bind this DNA when Lrp and CRP are not bound (i.e. when the operon is not 654

actively transcribed). Recently Choi et al (30) in a search of binding sites for the quorum 655

sensing regulator SmcR in the V. vulnificus genome identified and confirmed a binding 656

site for this protein in front of the tonB3 operon. More interestingly, the binding site 657

sequence identified by DNase I footprinting for SmcR lies immediately upstream of the 658

CRP binding region identified in this work. Since the binding of these two proteins to the 659

promoter region seems to be mutually exclusive (Alice & Crosa unpublished data) we are 660

currently exploring the hypothesis that this operon responds to different environmental 661

signals in addition or together with those described here. 662


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Since the sequence of the tonB3 promoter region is longer than usual and the fact 663

that Lrp and CRP binding might occur at defined moments of growth, the most plausible 664

situation when the cells are growing rapidly (i.e. when glucose and/or high CAA 665

concentrations are present) is that other transcriptional factors and/or nucleoid-associated 666

proteins (NAP) such as Fis, H-NS, or HU could bind this region afecting the 667

conformation and/or the supercoiling of the chromosmal DNA and consequently the 668

expression of the tonB3 operon. Of course, some of those NAPs could also work in 669

concert with Lrp and/or CRP for the activation of this operon. We are currently pursuing 670

these hypotheses in what we believe is an interesting model to study the timely 671

expression of an orphan TonB system that is mainly induced under conditions of 672

nutritional deprivation together with possible changes in the organization of the 673

chromosome. 674

675

Acknowledgements 676

This work was supported by grant AI065981 from the National Institutes of 677

Health to J.H.C. and a Beginning Grant-in-Aid Program Award (10BGIA2560022) from 678

the American Hearth Association to A.F.A. 679

The authors thank David Lewinshon and Gwendolyn Swarbrick for the generous 680

provision of pooled healthy human serum and Larry David and John Klimek at Proteomic 681

Shared Resource at OHSU. 682

683


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Table 1. Strains and plasmids used in this work. 684

685

Strain or plasmid

Relevant genotype or relevant characteristics

Origin or reference

V. vulnificus

CMCP6 wild type J. Rhee

ALE-LAC CMCP6 ΔlacZ (33)

AA-100 ΔlacZ Δlrp This study

AA-101 ΔlacZ Δcrp This study

Δ498 ΔlacZ Δ528-396 This study

AA-9 ΔtonB1ΔtonB2 (1)

AA-12 ΔtonB1ΔtonB2ΔtonB3ΔlacZ (1)

E. coli

Top 10 F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139

Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG

Invitrogen

S17-1λpir λ-pir lysogen; thi pro hsdR hsdM+recA RP4-2 Tc::Mu-Km::Tn7(Tpr Smr)

(57)

BL21(DE3) Star

F-ompT hsdSB(rB- mB

-) gal dcm rne131 (DE3) Invitrogen

MM294 F– endA1 hsdR17 supE44 thi-1 spoT1 λ– harboring plasmid pRK2013

(37)

Plasmids

pCR-Blunt II- TOPO

Blunt end cloning vector, Kmr Invitrogen

pMMB208 Broad-host range expression vector, Cmr Ptac (40)

pTL61T Contains a promoterless lacZ gene used for (31)


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transcriptional gene fusions studies, Ampr

pDM4 suicide vector with oriR6K, Cmr sacB (39)

pET200 Expression vector, Kmr Invitrogen

pRK2013 Helper plasmid Kmr (22)

p732 (p42TL) pTL61T containing the VV10842 promoter region from 42Pst-42Bam fused to lacZ

(1)

p632 pTL61T containing the fragment 42-632-42Bam fused to lacZ

this work


this work


this work


this work


this work

pdint pTL61T containing the fragment 42-632-42Bam with the deletion between position -

233 to -217 fused to lacZ

this work

pMMLrp pMMB208 derivative containing the lrp gene this work

pMMCrp pMMB208 derivative containing the crp gene this work

pETLrp pET200 derivative containing the lrp gene this work

pETCrp pET200 derivative containing the crp gene this work

p632-477 PCR fragment amplified with primers 42-632 and 42-477Bam cloned in pCR-Blunt II-

TOPO

this work

p632-300 PCR fragment amplified with primers 42-632 and 42-300Rev cloned in pCR-Blunt II-

TOPO

this work

p300-42B PCR fragment amplified with primers 42-300 and 42-Bam cloned in pCR-Blunt II- TOPO

this work

686

687


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Legends to Figures. (these legends can be also found accompanying each Figure) 688

Figure 1. Organization of the tonB3 operon and the regulatory region. Panel A: genetic 689

arrangement of the tonB3 operon; the gene names are based on homology to the 690

counterpart of the TonB2 system of V. vulnificus and other Vibrios. Panel B: close up of 691

the regulatory region analyzed in this work. The different lines indicate the approximate 692

locations of the probes used throughout this work. The length of the promoter region as 693

well as the size of the probes in this panel are not drawn to scale. Panel C: sequence of 694

the promoter region from – 551 [relative to the transcription start site (+1)] to the position 695

+81 of the partial coding sequence of the VV1_0842 gene. The likely -10 and -35 696

elements are underlined and the identified start point of the transcription as well as the 697

predicted start codon are shown in bold case. The extent of the protections identified by 698

DNase I footprintings for each protein are indicated with black (Lrp) and gray (CRP) 699

boxes, and the locations of the predicted binding sites as determined by similarity to 700

consensus motifs (13, 17, 28) are indicated in bold case. Binding sites positions that 701

could not be delimited by DNase I footprinting are indicated with dotted lines. The 702

positions of the primers used for amplification of the probes used in EMSA and DNase I 703

footprintings as well as those used in the construct of the lacZ fusions are indicated with 704

double underlines. Relevant nucleotide positions relative to the transcription start site are 705

also indicated. 706

707

Figure 2. Competitive index for tonB mutant strains. Mixtures of the strains analyzed 708

(0.1ml) were injected s.c. in iron-overloaded animals as described in Material and 709

Methods. When animals showed signs of the disease, organs were removed, 710

homogenized and bacterial cells plated on TSBS + 1.5% agar (TSAS) X-Gal plates. Bars 711


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represent the geometric mean CI value for each organ. Experiments were performed 712

twice. ***, p< 0.001 based on one-way ANOVA with Bonferroni’s post test. 713

714

Figure 3. Expression of various tonB3 promoter deletions in human serum. Panel A: the 715

deletions of the promoter region of the TonB3 operon were cloned in front of the 716

promoterless lacZ gene in the pTL61T vector and plasmids conjugated into the ΔlacZ 717

strain. Cells were grown in human serum + FAC 200 μg/ml and samples taken at 718

different times to determine β-galactosidase activities (white bars T2, 2h; grey bars T4, 719

4h and black bars T6, 6h after inoculation). Due to the turbidity of the human serum, 720

OD600nm was not recorded and activity was normalized to 106 cfu(ml)-1. The results 721

shown are the means of three to seven independent experiments with their standard 722

deviations. Those columns showing no significant difference are labeled with the same 723

letters, and those showing significant difference (p<0.05) are labeled with different letters 724

based on one-way ANOVA with Tukey’s post test. 725

Panel B: expression of the tonB3 gene in the wild type and Δ498 strains growing in 726

HS+FAC as determined by qRT-PCR. RNA extracted from the wild type and Δ498 727

strains growing in HS+FAC was reverse transcribed and cDNAs used in qRT-PCR as 728

described in Materials and Methods. Relative expression of the tonB3 gene was 729

determined by calculating 2-ΔΔCt using the 23S rRNA gene as an internal control. *** 730

p<0.001 based on one-way ANOVA with Bonferroni’s post test for values of wild type 731

compared to the mutant strain. 732

733


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Figure 4. The lrp gene is essential for tonB3 induction in HS+FAC. Panel A: wild type, 734

Δlrp and Δlrp pMMlrp strains harboring the plasmid p632 containing the tonB3-lacZ 735

fusion were grown in HS+FAC. Samples were removed at various time points and 736

analyzed as described in Figure 3A. Panel B: Constructs p548 and pdint were conjugated 737

into the V. vulnificus ΔlacZ strain, samples were removed at various times and β-738

galactosidase activity measured as described above. 739

The bars represent means ± standard deviations; n =3. Those columns showing no 740

significant difference are labeled with the same letters, and those showing significant 741

difference (p<0.01 panel A and p<0.001 panel B) are labeled with different letters based 742

on one-way ANOVA with Tukey’s post test. 743

744

Figure 5. Analysis of the binding of the Lrp(His)6 protein to the tonB3 promoter region. 745

Panels A and B: EMSA with different promoter regions of this operon that were 746

amplified, labeled as described in Materials and Methods (probes concentration 4 nM), 747

and incubated with increasing concentrations of Lrp(His)6 as described in each panel. A: 748

Probe A amplified with primers 42-632 and 42-477Bam; B: probe B amplified with 749

primers 42-498 and 42-300Rev and probe C amplified with primers 42-300 and 42Bam. 750

Probes are illustrated in Fig 1B. In each panel it is indicated when 10mM L-Leucine was 751

added to the binding buffer as well as when unlabeled probe DNA (400 nM) was used for 752

competition. Free-labeled DNA and Lrp(His)6-DNA complexes are indicated. 753

Panels C, D and E: DNase I footprinting analysis of the tonB3 promoter with purified 754

Lrp(His)6. End labeled probes D (panels C and D) and C (panel E) were incubated with 755

various concentrations of Lrp(His)6 as indicated in each panel and treated with DNase I. 756


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Gels were calibrated using G+A sequencing reactions (G+A) and relevant positions are 757

indicated. The locations of DNA binding sites for Lrp(His)6 are shown by black lines, 758

putative binding sites but with intrinsic resistance to DNAse I are shown with dotted 759

lines. Hypersensitive sites due to Lrp(His)6 binding are labeled with asterisks. 760

761

Figure 6. Effects of L-Leucine and glucose in the tonB3 promoter expression. Panel A: 762

V. vulnificus ΔlacZ strain harboring the p632 construct was grown in HS+FAC or the 763

same medium with the addition of 10mM L-Leucine or 0.5% glucose. Samples were 764

removed at various times and β-galactosidase activity measured as described in Figure 765

3A. Values obtained for time 6h after the inoculation are shown. Panel B: V. vulnificus 766

ΔlacZ strain harboring the p632 construct was grown in M9 0.5% glycerol and various 767

concentrations of CAA in the presence or absence of 10mM L-Leucine up to an OD600nm 768

~0.7, samples were removed and β-galactosidase activity measured as described in 769

Materials and Methods. Panel C: wild type, Δlrp and Δlrp pMMlrp strains harboring the 770

plasmid p632 containing the tonB3-lacZ fusion were grown in M9 0.5% glycerol 0.02% 771

CAA in the presence or absence of 10mM L-Leucine and β-galactosidase activity 772

measured as described above. Panel D: V. vulnificus ΔlacZ strain harboring the p632 773

construct was grown in M9 0.5% glycerol 0.02% CAA or the same media with the 774

addition of 10mM L-Leucine. When indicated 200 μg/ml FAC was addded. Samples 775

were removed at an OD600nm ~0.7 and β-galactosidase activity measured as described. 776

Panel E: V. vulnificus ΔlacZ strains harboring the p300, p400, p498 or p632 constructs 777

were grown in M9 0.5% glycerol 0.02% CAA or the same media with the addition of 778


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10mM L-Leucine. Samples were removed at an OD600nm ~0.7 and β-galactosidase 779

activity measured as described. 780

The bars represent means ± standard deviations; n =3. Those columns showing no 781

significant difference are labeled with the same letters, and those showing significant 782

difference (p<0.001 panels A, C, D and E, p<0.05 panel B) are labeled with different 783

letters based on one-way ANOVA with Tukey’s post test. 784

785

Figure 7. Expression of the tonB3 promoter with various carbon sources. V. vulnificus 786

ΔlacZ strain harboring the p632 construct was grown in M9 with various carbon sources 787

(0.5% each) and 0.02% CAA in the presence or absence of 10mM L-Leucine and β-788

galactosidase activity measured as described above. The bars represent means ± standard 789

deviations; n =3. Those columns showing no significant difference are labeled with the 790

same letters, and those showing significant difference p<0.01 are labeled with different 791


793

Figure 8. Expression of the tonB3, lrp and crp genes under various conditions. Total 794

RNA was extracted from the different strains growing under the indicated conditions. 795

The mRNA levels (represented by 2-ΔΔCt values) of various genes in the different strains 796

then were measured by qRT-PCR and expressed as the fold change from that measured in 797

the wild-type strain growing in M9 Glycerol 0.02% CAA without the addition of L-798

Leucine. Panels A: tonB3; B: lrp and C: crp. The bar represents means ± standard 799

deviations; n = 3. The significance of difference was analyzed by one-way ANOVA with 800

Bonferroni’s post test. *, P < 0.05; **, P < 0.01; ***P < 0.001. 801


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802

Figure 9. Role of cAMP-CRP in the tonB3 promoter expression. Panel A: wild type, 803

Δcrp and Δcrp pMMcrp strains harboring the plasmid p632 containing the tonB3-lacZ 804

fusion were grown in HS+FAC. Samples were removed at various times as indicated and 805

analyzed as described in Figure 3A. Panel B: V. vulnificus ΔlacZ strain harboring the 806

p632 construct was grown in M9 with 0.5% glucose and 0.02% CAA in the presence or 807

absence of 10mM L-Leucine. When indicated 1mM or 2mM cAMP was also added to the 808

medium, samples were removed at the indicated OD600nm and β-galactosidase activity 809

measured as described in Materials and Methods. The bars represent means ± standard 810

deviations; n =3. Those columns showing no significant difference are labeled with the 811

same letters, and those showing significant difference (p<0.05) are labeled with different 812


814

Figure 10. EMSA analysis of the promoter region of the TonB3 operon with CRP(His)6. 815

The different promoter regions of this operon were amplified and labeled as described in 816

Materials and Methods, and incubated with increasing concentrations of CRP(His)6 in the 817

presence of 0.2mM cAMP as described in each panel. Panels A: Probe C (42-818

300/42Bam); B: Probes Ci (42-300/42-206Rev) and Cii (42-206-For/42Bam). Probes are 819

illustrated in Fig 1B. Indicated are free-labeled DNA and CRP(His)6-DNA complexes. 820

Probes were used at 4nM and in each panel it is indicated when unlabeled probe DNA 821

(400 nM) was used for competition. 822

823


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Figure 11. DNase I footprint analysis of the tonB3 promoter region with purified 824

CRP(His)6 and Lrp(His)6 proteins. End labeled probe C was incubated with various 825

concentrations of CRP(His)6 (Panel A) or CRP(His)6 and Lrp(His)6 (Panel B) as indicated 826

in each panel and treated with DNase I. Gels were calibrated using G+A sequencing 827

reactions (G+A) and relevant positions are indicated. The locations of DNA binding sites 828

for CRP(His)6 are shown by full black lines while those that correspond to Lrp(His)6 are 829

shown by dotted lines. Hypersensitive sites produced by CRP(His)6 binding are labeled 830

with black stars and those due to Lrp(His)6 binding are labeled with white stars. 831

832

833


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References. 834

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the tonb3 system in the human pathogen vibrio vulnificus...

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