Structural basis for biofilm formation via the Vibrio cholerae matrix

Structural basis for biofilm formation via the Vibrio cholerae matrix protein RbmA 1

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Krista M. Giglioa,*, Jiunn C. Fongb,*, Fitnat H. Yildizb,# and Holger Sondermanna,# 3

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a Department of Molecular Medicine, College of Veterinary Medicine, 5

Cornell University, Ithaca, NY 14853, USA 6

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b Department of Microbiology and Environmental Toxicology, 8

University of California, Santa Cruz, CA 95064, USA 9

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* These authors contributed equally to this work. 11

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# To whom correspondence should be addressed: 14

Email: [email protected], phone: 607 253 3318, fax: 607 253 3659 15

Email: [email protected], phone: 831 459 1588, fax: 831 459 3524 16

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Key words: crystal structure, adhesin, biofilm, fibronectin fold 18

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Running title: Crystal structure of V. cholerae RbmA 20

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00374-13 JB Accepts, published online ahead of print on 17 May 2013

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Abstract 24

During the transition from a free-swimming, single-cell life-style to a sessile, 25

multicellular state called a biofilm, bacteria produce and secrete an extracellular matrix 26

comprised of nucleic acids, exopolysaccharides and adhesion proteins. The Vibrio 27

cholerae biofilm matrix contains three major protein components, RbmA, Bap1 and 28

RbmC, which are unique to Vibrio cholerae and appear to support biofilm formation at 29

particular steps in the process. Here, we focus on RbmA, a structural protein with an 30

unknown fold. RbmA participates in the early cell-cell adhesion events, and is found 31

throughout the biofilm where it localizes to cell-cell contact sites. We determined crystal 32

structures of RbmA and revealed that the protein folds into tandem fibronectin III (FnIII) 33

folds. The protein is dimeric in solution and in crystals, with the dimer interface 34

displaying a surface groove that is lined with several positively charged residues. 35

Structure-guided mutagenesis studies establish a crucial role for this surface patch for 36

RbmA function. Based on the structure, we hypothesize that RbmA serves as a tether 37

by maintaining flexible linkages between cells and the extracellular matrix. 38

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Introduction 40

Biofilms are aggregates of microbial communities composed of microorganisms 41

and exo-polymeric substances (1). It has been documented that biofilms play critical 42

roles in the environmental survival, transmission, infectivity, protection from the host 43

immune system and antimicrobial resistance of pathogenic microorganisms (2). One 44

such pathogen is Vibrio cholerae, the causative agent of the severe diarrheal disease 45

cholera (3). V. cholerae is autochthonous to coastal and estuarine environments where 46

it resides in a free-living state or in a biofilm state (4-8). The pathogen is transmitted to 47

humans by ingestion of contaminated food and water. It has been shown that removal 48

of particles >20 µm in diameter from water reduces cholera incidence by 48%, 49

suggesting that biofilms contribute to V. cholerae transmission (9, 10). Biofilms are also 50

important in the disease process. Stool samples from cholera patients contain both 51

planktonic and biofilm-like aggregates of V. cholerae cells (11), and the latter were 52

shown to be significantly more infectious than the planktonic cells (11). In addition, 53

mutants unable to produce Vibrio exopolysaccharide (VPS) and the biofilm matrix 54

protein RbmA exhibit reduced intestinal colonization (12). Furthermore, growth in 55

biofilms induces a hyper-infectious phenotype (13). Collectively these studies establish 56

that the biofilm growth mode contributes to both the intestinal- and aquatic-phases of 57

the V. cholerae life cycle. 58

Biofilm formation and structural integrity are dependent on the production of 59

biofilm matrix components (14). Major components of the V. cholerae biofilm matrix are 60

exopolysaccharide (VPS) and the biofilm matrix proteins RbmA, RbmC, and Bap1 (12, 61

15-17). These components were first identified in a rugose variant of V. cholerae that 62


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exhibits enhanced production of biofilm matrix components. One of the biofilm matrix 63

proteins is RbmA (for rugosity and biofilm structure modulator A) (15). RbmA is 64

required for both maintenance of rugose colonial morphology and development of wild-65

type biofilm architecture. Loss of RbmA also causes biofilms to be fragile and dissolve 66

with detergent treatment (15). Recent studies have shown that in mature biofilms, 67

RbmA is present throughout the biofilms and that RbmA is required for retention of 68

daughter cells following division (18). Furthermore, in cells lacking RbmA VPS is not 69

retained at the cell surface, suggesting that RbmA could facilitate microcolony/cell 70

cluster formation by stabilizing both cell-cell and cell-VPS interactions (18). 71

Although RbmA plays such a critical role in biofilm structure and stability, the 72

mechanism by which it confers such properties to biofilms is not known. RbmA has no 73

homologs in protein sequence databases, and bioinformatics analysis yields only limited 74

clues regarding its structure (15). To better understand how the protein contributes to V. 75

cholerae biofilm formation, we determined its structure and identified critical residues 76

required for RbmA’s function and biofilm stability. 77

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Materials and Methods 80

81

Bacterial strains, plasmids, and culture conditions. The bacterial strains and 82

plasmids used in this study are listed in Table S1. Chromosomal point mutation strains 83

were generated using deletion mutant strains according to the same procedures as in-84

frame deletion (15, 16), via allele exchange between the truncated open reading frame 85


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(ORF) and the full-length ORF containing the point mutation. All V. cholerae and E. coli 86

strains were grown aerobically, at 30°C and 37°C, respectively, unless otherwise noted. 87

All cultures were grown in Luria-Bertani (LB) broth (1% Tryptone, 0.5% Yeast Extract, 88

1% NaCl), pH 7.5, unless otherwise stated. LB agar medium contains 1.5% (w/v) 89

granulated agar (Difco). Concentrations of antibiotics and additives used, where 90

appropriate, were as follows: ampicillin (100 µg/ml), rifampicin (100 µg/ml) and IPTG 91

(100 µM). 92

93

Recombinant DNA techniques. DNA manipulations were carried out by standard 94

molecular techniques according to manufacturer’s instructions. Restriction and DNA 95

modification enzymes were purchased from New England Biolabs. Polymerase chain 96

reactions (PCR) were carried out using primers purchased from Bioneer Corporation 97

(Alameda, CA) and the Phusion High-Fidelity PCR kit (NEB). Sequences of the primers 98

used in the present study are available upon request. Plasmid sequences were verified 99

by sequencing (UC Berkeley DNA Sequencing Facility, CA). 100

101

Colony morphology and pellicle formation analyses. Colony corrugation and pellicle 102

formation assays were carried out according to previous published protocols (12, 15, 103

16). Briefly, for colony morphology studies, cultures grown overnight at 30°C with 104

shaking (200 rpm) were serially-diluted with LB medium, and 100 μl of the diluted 105

cultures were plated onto LB agar medium. The cultures were incubated at 30°C for 3 106

days (2 days at 30°C and 1 day at room temperature). Analysis of pellicle formation was 107

carried out with diluted (1:200) overnight cultures in 24-well plates and in glass culture 108


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tubes. The plates and tubes were incubated at 30°C under non-shaking conditions for 2 109

days. Assays were repeated with at least three different biological replicates. For 110

complementation studies, strains were grown in the presence of ampicillin (100 µg/ml) 111

and IPTG (100 µM) or arabinose (0.01%). 112

113

Immunoblot analyses. Immunoblotting experiments were carried out according to 114

previously described protocols (15, 18), with some modifications. Briefly, overnight 115

cultures were diluted 1:200 and grown for 6 h at 30°C under shaking at 200 rpm. Whole 116

cell (WC) samples were prepared by centrifugation of 2-ml cultures and resuspension of 117

the cell pellets in 200 µl of 2% SDS. Culture supernatant (CS) (40 ml) was prepared by 118

separating the cells from the culture supernatants by centrifugation twice at 3,000 xg for 119

15 min and 30 min. The CSs were collected and filtered through 0.45-µm filters to 120

remove any residual whole cells. Bovine serum albumin (BSA, 200 µg) was added to 121

each CS as an additional loading control. Total protein in the CS was precipitated with 122

13% trichloroacetic acid (TCA) upon incubation at 4°C overnight, followed by 123

centrifugation at 45,000 xg for 30 min. The protein pellets from the CS were washed 124

with 2 ml ice-cold acetone and resuspended in 500 µl of 1x PBS. Protein concentrations 125

were estimated using a Pierce BCA Protein Assay Kit (Thermo Scientific). Equal 126

amounts of total protein in the WC and CS were loaded onto an SDS-PAGE for gel 127

electrophoresis followed by the immunoblot analysis. Polyclonal rabbit α-RbmA serum 128

(OpenBiosystem), generated against purified RbmA protein (18), was used at a dilution 129

of 1:1000 to detect RbmA in the WC and CS samples. As an additional loading control, 130

BSA in the CS was detected using 1:1000-diluted polyclonal rabbit α-BSA antibody 131


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(Thermo Scientific). Secondary goat α-rabbit IgG-HRP (Santa Cruz Biotechnology) was 132

used as a dilution of 1:2500. The SuperSignal West Pico Chemiluminescent Substrate 133

(Thermo Scientific) and a BioRad ChemiDoc MP Imaging System were used for 134

detection and capturing of the immunoblot signals. 135

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Protein expression and purification. A DNA fragment encoding V. cholerae RbmA 137

without the signal peptide (residues 31-271) was amplified from V. cholerae 138

chromosomal DNA by PCR and cloned into the pGEX-6P-2 (GE Healthcare Life 139

Sciences) bacterial expression vector, which adds an N-terminal GST fusion tag that is 140

cleavable with PreScission protease. Native RbmA protein was overexpressed in 141

Escherichia coli (E. coli) BL21 cells. Cultures were grown in terrific broth (TB) medium 142

supplemented with 100 µg/ml ampicillin at 37°C with shaking. When cultures reached 143

an optical density at 600 nm (OD600) of around 1.0, the temperature was reduced to 144

18°C and protein expression was induced by the addition of 0.5 mM IPTG. Expression 145

of selenomethionine-derivatized RbmA was carried out in T7 Express Crystal cells (New 146

England Biolabs). Cultures were grown at 37°C in M9 minimal medium supplemented 147

with 100 µg/ml ampicillin, vitamins (1 µg/ml thiamine and biotin), 0.4% glucose, trace 148

elements, and 40 µg/ml of each of the 20 amino acids with the exception of methionine, 149

for which selenomethionine was substituted. Protein expression was induced at an 150

OD600 of 0.5 using 0.5 mM IPTG. For both native and selenomethionine-derivatized 151

RbmA, protein was expressed for 16 h, after which cells were harvested by 152

centrifugation, resuspended in PBS buffer A (1 X PBS, pH 7.4; 500 mM NaCl), and flash 153

frozen in liquid nitrogen. 154


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For purification of heterologously expressed proteins, frozen cell suspensions 155

were thawed and cells were lysed by sonication. After centrifugation, clarified lysates 156

were incubated with Glutathione HiCap Matrix (Qiagen) that was equilibrated with PBS 157

buffer A. The resin was washed with 20 column volumes of PBS buffer A, followed by 158

elution of protein using 5 column volumes of PBS buffer B (PBS buffer A; 10 mM 159

reduced glutathione). Eluted proteins were buffer exchanged into cleavage buffer 160

containing 25 mM Tris-HCl (pH 7.5) and 250 mM NaCl by using a HiPrep 26/10 161

Desalting column (GE Healthcare Life Science), and incubated with PreScission 162

protease overnight at 4°C for removal of the GST tag. PreScission protease, uncleaved 163

protein and GST tags were removed by glutathione matrix affinity chromatography. 164

Cleaved RbmA proteins were subjected to size exclusion chromatography on a 165

Superdex 200 column (GE Healthcare Life Science) that was equilibrated in gel filtration 166

buffer (25 mM Tris-HCl [pH 7.5], 250 mM NaCl). Purified proteins were concentrated 167

using 10 KDa Amicon Ultra filters (Millipore), flash frozen in liquid nitrogen and stored at 168

-80°C. 169

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Crystallization, data collection, and structure solution. Protein crystals were 171

obtained for both native and selenomethionine-derivatized RbmA by hanging-drop 172

vapor diffusion, mixing equal volumes (1 µl) of protein (concentrations of 10-30 mg/ml) 173

and reservoir solution, followed by incubation at 20°C. For crystal form 1, the reservoir 174

solution consisted of 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M BIS-TRIS (pH 175

6.5), and 10% w/v Polyethylene glycol 10,000. The crystal form 2 reservoir solution 176

consisted of 0.1 M BIS-TRIS (pH 5.5) and 3.0 M NaCl. A third crystal form was obtained 177


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in the latter condition containing 10% of the following solution: 0.16% w/v Thiamine 178

monophosphate chlroide dihydrate, 0.16% w/v Acetylsalicylic acid, 0.16% Cholic acid, 179

0.16% w/v 1,2,3-Heptanetriol; 0.16% w/v Vanillin, 0.16% w/v N-Acetly-D-mannosamine, 180

and 0.02 M HEPES sodium pH 6.8 (Hampton Research Silver Bullets Bio screen). 181

Crystals were cryoprotected by soaking them in their respective reservoir solutions 182

supplemented with 25% xylitol, flash frozen and stored in liquid nitrogen. Data were 183

collected on beamline A1 at the Cornell High Energy Synchotron Source (CHESS; 184

Cornell University, Ithaca, NY). 185

Data reduction was carried out with the software package HKL2000 (19). 186

Experimental phases for the initial structure were obtained from single-wavelength 187

anomalous diffraction (SAD) experiments using crystals grown from selenomethionine-188

derivatized RbmA protein by using the software package Phenix (20). Refinement in 189

Phenix and COOT (21) yielded the final models. Data collection and refinement 190

statistics are summarized in Table S2. Structural illustrations were made in Pymol 191

(Schrödinger, LLC). 192

193

Small-angle X-Ray scattering (SAXS). SAXS data were collected at the CHESS, 194

beamline F1. Data were collected at 20°C, on homogeneous, monodisperse samples. 195

Protein samples were prepared by size exclusion chromatography on a Superdex 200 196

10/300 column (GE Healthcare Life Science) equilibrated in gel filtration buffer (25 mM 197

Tris-HCl [pH 7.5] and 250 mM NaCl). Fractions were collected and concentrated to final 198

concentrations from 1 to 10 mg/ml (75 to 370 µM). Concentrated protein samples were 199

centrifuged at 13,000 rpm for 10 min at 4°C prior to data collection. Scattering data were 200


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collected at 3 concentrations between 1 and 10 mg/ml, and were also collected on 201

buffer without protein for background correction. By careful inspection of the individual 202

scattering profiles of each exposure, data were selected that showed no changes in the 203

low-Q region over multiple exposures, and hence were devoid of apparent radiation 204

damage. Sample data were background corrected, averaged and scaled using the 205

program BioXtas (Soeren Nielson). Data reduction, analysis and modeling were carried 206

out using the software package ATSAS (22), as described previously (23). 207

Envelope reconstructions were calculated by using the program Gasbor (24) with 208

the distance distribution functions as the target. A total of 20 independent models were 209

generated, averaged and filtered using the program Damaver (25). In an independent 210

approach, the program Sasref (26) was used to model the bilobal structure of RbmA in 211

solution based on the SAXS data, with the crystallographic models for the separate 212

lobes as an input. During the modeling, lobe movement was constrained by accounting 213

for the flexible linkers that connect the two half-sides. 214

215

Size-exclusion chromatography-coupled multi-angle light scattering (SEC-MALS). 216

Molecular mass and polydispersity (mass distribution) of RbmA protein samples were 217

determined using SEC-MALS. Purified protein at concentrations between 1-15 mg/ml 218

(75-750 µM) was subjected to gel filtration on a Phenomenex Bio Sep-SEC-s 3000 219

column (Phenomenex) that was equilibrated in MALS buffer (25 mM Tris-HCl [pH 7.5] 220

and 200 mM NaCl). The SEC was coupled to a static 18-angle light scattering detector 221

(DAWN HELEOS-II), and a refractive index detector (Optilab T-rEX, Wyatt Technology). 222

Data were collected at 25°C each second for 30 min, at a flow rate of 1 ml/min. Data 223


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analysis was carried out using the program ASTRA V. The detectors were normalized to 224

a sample of 5 mg/ml BSA (Sigma). 225

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Results 228

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The crystal structures of Vibrio cholerae RbmA. To gain a mechanistic 230

understanding of the function of RbmA during Vibrio cholerae biofilm formation, we set 231

out to determine its crystal structure. The RbmA protein (residues 31-271; lacking the 232

signal peptide; Figure 1A) was purified to homogeneity and set up in crystallization 233

screens. While RbmA formed crystals under several conditions, we optimized two main 234

cocktails that yielded diffraction at a maximal resolution of 2.2 and 2.5 Å, respectively 235

(see Materials and Methods, and Table S2). Although the crystallization conditions were 236

different, both crystals belong to the same space group (P41212; 2 237

molecules/asymmetric unit) with similar crystal packing interactions between the 238

asymmetric units. Likewise, the resulting models were virtually identical (rsmd of 0.6 Å 239

considering all atoms) except for a surface-exposed, internal loop that adopted a 240

different conformation in the two crystal forms (see below). 241

The structures revealed tandem fibronectin type III (FnIII) domains as the main 242

building block of RbmA (Figure 1). FnIII folds occur in a large number of cell surface 243

receptors and cell adhesion proteins. As also observed in RbmA, they comprise a 7-244

strand β-sandwich module, and are distinguished from Fn type I, type II and the similar 245

Immunoglobulin (Ig) folds by the lack of disulfide bonds (27, 28). The schematic of the 246


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FnIII domains of RbmA is shown in Figure 1B depicting the characteristic β-sandwich 247

comprised of 3 + 4 β-sheets, with the first sheet being formed by strands a, b and e, and 248

the second sheet by strands c, c’, f and g. In the RbmA structure, β-strand c’ is 249

separated into two halves by a short coil. 250

RbmA contains two FnIII domains, FnIII [1] and FnIII [2], which are connected by 251

a well-resolved linker segment (Figure 1). The two FnIII folds superimpose well, with a 252

root-mean-square deviation (rmsd) of 1.2 Å. As seen in the structure, the two folds run 253

antiparallel to each other within a protomer but do not form extensive interactions. In 254

contrast, the N-terminal FnIII (FnIII [1]) domain of one protomer interacts tightly with the 255

C-terminal FnIII domain (FnIII [2]*) of the second protomer in the asymmetric unit. The 256

interface is made up of the respective 4-stranded β-sheets (inset in Figure 1B), and 257

spans a total area of 1287 Å2 (2180 Å2 in the full-length, bilobal dimer). As a result, 258

RbmA forms a dimer consisting of two lobes with domain contributions from each 259

protomer. 260

To assess whether the crystallographic dimer is a biologically relevant assembly, 261

we determined the molecular mass of the purified protein in solution. Here, we used 262

static multi-angle light scattering coupled to size exclusion chromatography (SEC-263

MALS), a method that yields the absolute molecular weight of a protein as it elutes from 264

a gel filtration column, independent of the protein’s shape (29). In this setup, RbmA 265

elutes from the column as a single species with a molecular weight of ~49.1 kDa (the 266

theoretical molecular weight of a monomer based on sequence is 26.3 kDa) and a low 267

polydispersity index further indicating a homogeneous sample (Figure 2). We did not 268

observe any significant fraction of higher or lower molecular weight species over a 269


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concentration range from 1 to 15 mg/ml (data not shown). Thus, the analysis of the 270

protein’s molecular weight in solution corroborates the crystallographic data and argues 271

for the dimer as RbmA’s preferred quaternary structure in solution under the conditions 272

tested. 273

Since sequence-based homology searches failed to reveal any close relatives of 274

RbmA, we turned to structure-based comparisons. The RbmA structure was used as 275

the search model in a DALI search against the Protein Structure Database (30) to 276

identify structurally related proteins. The results of the closest structural homologs are 277

shown in Figure 3. The search identified a domain of human coagulation factor XIII, a 278

transglutaminase involved in blood coagulation during clot formation, as the closest 279

homolog (pdb: 1ggt; Z=12.1) (31). Additional homologs include a dextranase from 280

Streptococcus mutans (pdb: 3vmn; Z=10.2) (32), V. cholerae GbpA, a N-281

Acetylglucosamine (GlcNAc) binding protein (pdb: 2xwx; Z=10.2) (33), a subunit of a 282

large Mycobacterium smegmatis porin, MspA (pdb: 2v9u; Z=9.0) (34) that is believed to 283

function in protein-protein interactions with transported proteins (34, 35), and a putative 284

β-galactosidase from Bacteriodes fragilis with a deposited structure but no associated 285

publication to date (pdb: 3fn9; Z=10.3). In addition to the structural resemblances, the 286

observation that some of these homologs function as carbohydrate binding modules 287

may hint at RbmA’s native ligand, potentially exopolysaccharides, a central component 288

of the V. cholerae biofilm matrix. At the same time, no protein was identified by the 289

search, which contained two FnIII domains in the same bilobal arrangement as 290

observed in RbmA. These results prompted us to investigate the surface properties of 291

RbmA for indications of functionally relevant sites. 292


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293

Surface features of the RbmA crystal structures. As mentioned above, we noted a 294

lack of tight domain interactions between the two FnIII folds within a protomer, and in 295

extension between the two lobes of the RbmA dimer (Figure 4A). At the same time, the 296

relatively high resolution of our crystallographic data sets allowed us to observe a large 297

number of ordered water molecules at the inter-lobe interface. This is consistent with 298

the fairly even distribution of polar or charged residues across this surface of the protein 299

(Figure 4B, top) and the rather loose contact between the lobes. On the opposite, fully 300

surface-exposed face of the lobe, we observe a similar, inconspicuous distribution of 301

hydrophilic and hydrophobic residues (Figure 4B, bottom). Yet, one noteworthy 302

observation is an apparent groove that runs along the interface formed by two adjacent 303

FnIII domains that form the lobe structure (FnIII domain 1 of one protomer and FnIII 304

domain 2 of the second protomer) (Figures 4B and 5). 305

Mapping of the electrostatic potential onto the RbmA surface revealed an area of 306

positive potential that originates from several positively charged residues lining the 307

aforementioned groove (Figure 5A). In particular, we identified three arginine residues 308

(R234 and R219 of the second FnIII fold of one protomer; R116 of the first FnIII fold of 309

the adjacent protomer) (Figure 5B) that contribute to the surface characteristics of the 310

lobe, and more specifically the groove it displays (Figure 5B). In addition, a strong 311

negatively charged furrow was identified at the bottom of the molecule, which is formed 312

by the linker segments that connect the FnIII domains within a molecule and hence the 313

two lobes of the RbmA dimer (Figure 5A, bottom). Together, these features could be 314


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indicative of areas of interaction with ligands of the extracellular matrix and/or cell 315

surface. 316

When we analyzed the two different crystal forms, we noticed that, while they are 317

by-and-large superimposable, the only noticeable difference pertains to the 318

conformation of a segment in the outer β-sheet spanning residues 91-108 (Figure 5C). 319

In crystal form 1, which is shown in the previous illustrations, this segment adopts an 320

extended conformation with β-strand character (Figure 5C, FnIII fold colored in green). 321

As such, it is an extension to β-strand c’ and aligns with the N-terminal half of the 322

preceding β-strand c. At the same time, β-strand c’ is not continuous, and its two halves 323

are connected by a short, 2-residue linker (residues 92-93) that does not conform to a 324

β-strand structure. In this structure, residues 101-108 form a disordered β-hairpin loop 325

connecting β-strands c’ and d (Figures 1, 4A and 5C; dotted lines). In contrast, the 326

entire segment is well resolved in the second crystal form (Figure 5C, FnIII fold colored 327

in cyan). In this alternative conformation, residues 91-103 adopt a random coil structure 328

that loops out and reaches over to the second FnIII fold of the adjacent protomer, which 329

forms the other half-site of the lobe. In this case, residues 104-106 adopt a short β-330

strand conformation, forming hydrogen bonds with the preceding β-strand. In addition to 331

these two crystal forms showing preferential conformations of the segment in both 332

lobes, we also obtained crystals in an additive screen (crystal form 3), in which the 333

resulting structure revealed a mixed state where residues 91-108 adopt one 334

conformation in one lobe, and the alternative conformation in the other lobe (data not 335

shown). Since no obvious ligand binding was observed in any of these structures, we 336

argue that this segment of RbmA is inherently flexible, able to adopt at least two distinct 337


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conformations, which are populated to a different extent in the crystals. In addition, the 338

electron density maps indicate the presence of poorly populated, alternative 339

conformations of this segment within an individual crystal (data not shown), which could 340

not be modeled reliably. While the predominant conformation trapped under the 341

individual crystallization conditions appears serendipitous, different pH’s of the solutions 342

or the presence of different precipitants (sodium chloride in one case, polyethylene 343

glycol in the other) may contribute to this phenomenon. 344

Taken together, our crystallographic studies established tandem FnIII domains 345

as the major building block of RbmA. The domains come together within the context of 346

an RbmA dimer to form a unique bilobal structure with peculiar surface properties. 347

348

Structural characterization of RbmA in solution. The high degree of solvent content 349

at the inter-lobe interface and overall loose association of the lobes (Figure 4A) 350

prompted us to investigate whether the globular, more compact structure observed in 351

crystals of RbmA is maintained in solution. We already established that RbmA forms 352

dimers in solution, similar to the oligomeric state in the crystalline state (see above; 353

Figure 2). Here, we sought structural insight by employing small-angle X-ray scattering 354

(SAXS) (36, 37). For this approach, X-ray scattering data are collected on 355

monodisperse protein solutions, in this case, purified RbmA. The scattering profiles 356

contain valuable information, albeit at low resolution, about the shape of particles, in 357

particular their radius of gyration (Rg) and maximum dimensions (Dmax). Furthermore, 358

calculation of the distance distribution function provides an intuitive way to compare the 359

solution and crystalline states. Finally, more modern approaches allow for the modeling 360


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of SAXS data with the scattering profile or distance distribution function as the target to 361

reconstruct low-resolution envelopes. The ab initio models can be directly compared to 362

high-resolution crystal structures. 363

Analysis of the scattering curves and comparison with the profiles calculated 364

based on the crystal structures indicated differences between RbmA’s solution state 365

and the conformation observed in the protein crystals (Figure 6). In particular, visual 366

inspection of the curves and Rg calculations based on the Guinier plot revealed an 367

overall larger dimension of RbmA dimers in solution versus crystals (Rgcrystal: 22.4 Å; 368

RgSAXS: 33.1 ± 0.5 Å). Similarly, the apparent maximum dimension of the scattering 369

particles in solution is ~38 Å larger than that calculated based on the crystallographic 370

dimer (Dmaxcrystal: 73.1 Å; Dmax

SAXS: 110 Å). Based on the high similarity of scattering 371

data and Guinier plots at different protein concentrations, we could rule out protein 372

aggregation and/or radiation damage as a source of the overall larger dimensions 373

(Figures 6A and 6B). The theoretical and experimental distance distribution functions 374

showed considerable agreement in the short-to-medium distance regime, indicating 375

similar domain structures, but supported the presence of wider particles based on the 376

discrepancies observed at larger distances (Figure 6C). Taken together, this analysis 377

suggests that RbmA adopts an extended conformation in solution. Considering that 378

several crystal structures determined independently from distinct conditions agreed with 379

a more compact conformation, we hypothesize that RbmA may exist in a conformational 380

equilibrium with at least a fraction of the dimers residing in a prolate state. 381

In order to obtain a glimpse of the extended state, we modeled the SAXS data by 382

simulating the data with a chain-like ensemble of dummy residues. This approach yields 383


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ab initio reconstructions independent of and not requiring other structural information. 384

Using the distance distribution function as the target, we obtained models that show a 385

rather flat and extended envelope (Figure 6D; grey envelope). In an independent 386

approach, we modeled the SAXS data by changing the angle between the two RbmA 387

lobes and their relative position, yielding a pseudo-high-resolution structure. The 388

resulting model with the best fit and comparable dimensions to the scattering data 389

resembled the ab initio reconstructions, collectively suggesting that the two lobes open 390

up completely in the most extreme case (Figure 6D). Since the modeling is likely biased 391

by the experimentally determined Dmax value, which is encoded in the distance 392

distribution function, other intermediate conformations or the crystalline, compact state 393

may be present in the same sample, which likely contributes to the good but not perfect 394

fits between crystal structure-based models and the SAXS data. At the same time, 395

given that the interdomain linkers are unstructured and the inter-lobal space is well 396

hydrated, opening up of the structure and separation of the two lobes would come at 397

little energetic cost. 398

Thus far, we elucidated the structure of RbmA and, based on the models, 399

identified regions in the protein that might be important for its biological function as a 400

scaffold in the V. cholerae biofilm matrix. 401

402

Phenotypic characterization of RbmA mutant strains. To investigate whether the 403

surface features identified from the crystal structures play a role in RbmA function 404

during biofilm formation, structure-guided mutations in surface-exposed residues at 405

these areas were designed. Specifically, we designed single amino acid substitutions in 406


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the positively charged putative ligand binding pocket (R116A, R219A, R234A) and in 407

the negatively charged furrow at the bottom of the dimer (E84A, E84R). In addition, we 408

constructed a deletion of 8 amino acids (101-108; Δloop), removing the flexible β-hairpin 409

linker that is disordered in crystal form 1 (see above). We argue that the latter mutation 410

would prevent the protein from adopting a conformation observed in crystal form 2 (see 411

Figure 5C), eliminating structural heterogeneity. Since the loop would consequentially 412

adopt an extended conformation as observed in crystal form 1, which ultimately 413

contributes to the formation of the surface groove with positive electrostatic potential, 414

we should be able to address whether the alternative conformation is relevant for 415

RbmA’s biogenesis, transport and/or function during biofilm formation. 416

Chromosomal mutations were made in rugose V. cholerae (see Table S1 for 417

strains) and the resulting strains were tested in colony corrugation and pellicle 418

formation, assays that are indicative of adhesion and biofilm formation. The phenotypes 419

of strains harboring RbmA point mutations were compared to that of a rugose variant 420

(reference strain) that forms robust biofilms, and to that of a strain lacking RbmA 421

(ΔrbmA). Production and secretion of RbmA in all mutant strains (with the exception of 422

the ΔrbmA mutant) was confirmed to be comparable to that of rugose cells (Figure 7). 423

The two mutants that either removed a negative charge (E84A) or introduced a charge-424

reversal (E84R) at the bottom furrow of the RbmA dimer exhibited colony corrugation 425

and pellicle formation similar to those of rugose cells (Figure 8). Likewise, the Δloop 426

mutation did not produce any obvious colony corrugation or pellicle formation 427

phenotypes, suggesting that the conformation observed in crystal form 1 (Figure 5C) is 428

a biologically relevant state. In contrast, individual mutations of the three arginine 429


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residues that line the positively charged groove at the side of the lobes comprising 430

RbmA resulted in strains with phenotypes that were reduced in colony corrugation 431

and/or pellicle formation (Figure 8). Specifically, a strain harboring the R234A mutation, 432

produced colonies and formed pellicles that were indistinguishable from those of a 433

ΔrbmA mutant, although RbmA-R234A protein was produced and secreted (Figures 7 434

and 8). Strains carrying either the R116A or R219A mutation produced pellicles 435

resembling those of the ΔrbmA and R234A mutant strains. Colonies formed by the 436

R116A and R219A mutants exhibited reduced colony corrugation, albeit less 437

pronounced, when compared to the rugose strain (Figure 8). The apparent differences 438

in severity between pellicle and rugose phenotypes are likely due to the different 439

physical properties of the particular growth media (formation of a biofilm at the liquid-air 440

interface versus bacterial colony formation on plates), which may render cell-cell and 441

cell-matrix interactions more or less sensitive to perturbation of the RbmA protein. 442

The defects caused by these mutations with regard to colony morphology can be 443

rescued by complementation with WT rbmA expressed from a plasmid under the control 444

of the Ptac promoter (Figure 9A). Heterologous expression of RbmA from this plasmid 445

resulted in increased colony corrugation in the ΔrbmA strains as well as strains carrying 446

the R234A, R116A, or R219A chromosomal mutations indicating that the mutations 447

created are indeed causing a loss of function in RbmA. Furthermore, the analysis also 448

indicates that these mutants do not function as dominant-negative alleles, given that 449

their phenotypes can be rescued by wild-type RbmA. To further show that the mutated 450

versions of RbmA proteins are not dominant-negative, we introduced mutated versions 451

of RbmA into the rugose strain. Colony morphologies of these strains are 452


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indistinguishable from strains harboring either the empty vector or wild-type RbmA 453

(Figure 9B). Collectively, these data show that the RbmA-R116A, RbmA-R219A and 454

RbmA-R234A alleles are not dominant-negative. 455

In summary, our studies revealed the structural make-up of RbmA, which was 456

enigmatic up to this point due to poor sequence homology to any other known proteins. 457

The structure-informed functional analysis identified a surface groove with strong 458

positive electrostatic potential as being important for RbmA’s biological function during 459

biofilm formation. 460

461

462

Discussion 463

V. cholerae RbmA has been implicated in cell-cell interactions and plays a crucial 464

role in cell adhesion within the VPS (12, 15, 18, 38). Here, we developed a structural 465

framework for its function in vivo. Given the nature of the RbmA surface area that was 466

sensitive to point mutations, we speculate that the groove formed by two adjacent FnIII 467

domains of an RbmA dimer provides a functional interaction site for extracellular 468

ligands. Possible candidates that would favor such surface properties could be 469

carbohydrates that are an essential component of the Vibrio biofilm matrix. While the 470

exact binding sites and modes may differ, this notion is supported by the observation 471

that structural homologs of RbmA include several sugar-binding proteins (Figure 3). 472

One such example is the V. cholerae colonization factor GbpA (33). GbpA is essential 473

for intestinal colonization and pathogenesis in mouse models by providing stable host-474

bacterial connections (33). Its structure and binding studies revealed a modular 475


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structure, with several domains contributing to GbpA’s interaction with chitin and mucin. 476

In analogy, we speculate that RbmA utilizes similar domains to interact with its ligands, 477

yet built into a unique, bilobal domain arrangement. 478

At the same time, other proteins or even nucleic acids cannot be excluded as 479

ligands for RbmA. Protein and nucleic acids are components of the bacterial 480

exopolysaccharide matrix, and several FnIII domains are known to bind to other 481

proteins and mediate protein-protein interactions (39). Additionally, since RbmA has 482

been implicated in cell-cell interactions within the biofilm, it is also possible that RbmA 483

can interact with ligands on the surface of cells. A model where RbmA can interact both 484

with cell surface features and with components of the extracellular matrix would support 485

a functional role where RbmA tethers cells within the biofilm matrix (18). 486

FnIII folds are common modules in extracellular proteins (although they are also 487

present in intracellular proteins) and are found in 2% of all animal proteins (40). 488

Previously thought to be exclusive to eukaryotes, this fold has been shown to be 489

common to bacterial proteins (40) and studies suggest that the acquisition of this fold in 490

bacteria was due to horizontal gene transfer from animal hosts (40, 41). This is 491

supported by data showing that bacterial proteins with FnIII folds share greater 492

homology with animal FnIII domain-containing proteins than they do with those of other 493

bacteria, as is also the case with RbmA, whose closest structural homolog is a domain 494

from mammalian tissue transglutaminases (Figure 3). 495

One documented role for tandem FnIII folds is to provide resistance to 496

mechanical tension through elasticity and recoil (42). This is thought to be accomplished 497

through unfolding and refolding of the FnIII domain, and is unusual given the expected 498


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kinetics due to the complex multiple β-sheet structure of these modules (43). A supreme 499

example of the elasticity of unfolding and rapid refolding of these domains has been 500

demonstrated in the case of the muscle protein titin, which can elongate up to four times 501

its length with rapid recoil via its more than 100 tandem FnIII modules (44). Similar 502

resistance to applied tension has been observed in fibronectin with stretch and recoil 503

accomplished via partial unfolding and refolding of the FnIII modules (43, 45). In the 504

Staphylococcus extracellular matrix protein tenascin, the elasticity provided by the FnIII 505

modules, which also provide binding to the host cells, allows for bonds with ligands that 506

can persist and provide resistance to force (46, 47). Even in the absence of 507

unfolding/refolding transitions of FnIII domains, the inter-domain flexibility inherent to 508

FnIII fold-containing (mutli-domain) proteins also plays a functional role. One example 509

pertains to the transglutaminase, human coagulation factor XIII, the protein with 510

structural features bearing the strongest homology to RbmA (Figure 3) (31). It has been 511

speculated that the FnIII folds in coagulation factor XIII undergo rigid-body 512

rearrangements via flexible linkers in order to expose or occlude the active site and thus 513

control enzyme activity (48). 514

Data from our solution structures showing larger, more elongated molecular 515

species in solution than the one seen in our crystal structures would suggest that 516

flexibility also plays a role in the function of RbmA. The extensive solvation of the two 517

RbmA lobes and flexible linkage connecting the FnIII folds would support a model 518

where RbmA dimers can adopt a large range of conformations in solution, which 519

ultimately could provide elastic scaffolds. In such a model, RbmA-mediated contacts in 520


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the biofilm could stabilize cell-cell connections and cell-matrix interactions and confer 521

resistance to shearing forces. 522

523

524

Acknowledgements 525

We thank members of the Sondermann and Yildiz lab for helpful discussions. We 526

are grateful to the staff and scientists at the Cornell High Energy Synchrotron Source 527

(CHESS) for their support during data collection. The CHESS facility is supported by 528

Grant DMR-0225180 from the National Science Foundation (NSF) and Grant 529

GM103485 from the National Institutes of Health (NIH). Our work was supported by the 530

NIH under Grants R01-AI097307 (to H.S.) and R01-AI055987 (to F.H.Y.), and by a 531

PEW scholar award in Biomedical Sciences (H.S.). 532

533

534

Accession numbers 535

Coordinates and structure factors have been deposited in the PDB with accession 536

numbers 4KKP, 4KKQ, and 4KKR. 537

538

539

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Figure Legends 682

683

Figure 1. Structure of V. cholerae RbmA. (A) Domain organization of full-length 684

RbmA. Two molecules are shown as their domains are arranged in a crystallographic 685

dimer. (B) Crystal structure. Two perpendicular views of an asymmetric unit are shown. 686

The crystal structure revealed an RbmA dimer comprised of two tandem FnIII folds (top 687

panel). Color coding of the individual FnIII folds is consistent with the color scheme 688

introduced in (A). The schematic (bottom left) depicts a half-site consisting of a FnIII fold 689

donated by each protomer, which align in an antiparallel fashion. In the structure 690

cartoon and the schematic, the β-strands of individual FnIII folds are labeled following a 691

commonly used nomenclature (β-strands a-g). Unless otherwise stated, all illustrations 692

were made with the model generated from crystal form 1 (Table S2). 693

694

Figure 2. Oligomeric state of RbmA in solution. SEC-MALS was used to determine 695

the absolute molecular mass of RbmA in solution. Light scattering signal (red line) and 696

refractive index detector signal (hashed black line) are shown on the x-axis. Molecular 697

mass determinations across the protein elution peak are shown (black dots; right y-698

axis). The theoretical molecular weights for a dimer and monomer were calculated 699

based on their amino acid sequence and indicated as vertical, dashed lines. The 700

average experimental molecular mass is 49.1 ± 1.0 kDa. 701

702

Figure 3. Structural neighbors of RbmA. A DALI search against the protein data bank 703

revealed structurally conserved features between RbmA (center) and (from top right, 704


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clockwise) human transglutaminase (Z score 12.1, rmsd 6.1 Å); a putative b-705

galactosidase (Z score 10.3, rmsd 7.0 Å); a subunit of the Mycobacterium smegmatis 706

porin MspA (Z score 9.0, rmsd 2.5 Å); a Streptococcus mutans dextranase (Z score 707

10.2, rmsd 2.8 Å); and V. cholerae GbpA (Z score 10.2, rmsd 9.1 Å). Monomeric 708

structures were arranged adjacent to the most homologous FnIII fold of RbmA identified 709

in this search. The aligning domains in the individual proteins are shown in color. 710

711

Figure 4. Characteristics of the RbmA lobe interface. (A) Crystallographic water 712

molecules. A large number of water molecules were resolved in the crystal structure 713

and many of them fill the inter-lobe interface within an RbmA dimer. Water molecules 714

are shown as blue spheres. (B) Mapping of the distribution of polar and hydrophobic 715

residues at the inter-lobe interface (top) and at the surface-exposed face of an individual 716

lobe (bottom). Orientation with the molecule in (A) is shown in the circled inset. 717

718

Figure 5. Surface properties of RbmA. (A) Electrostatic potential. The electrostatic 719

potential of the RbmA dimer was mapped onto its molecular surface (right panel), with 720

red representing negative potential and blue representing positive potential (-4 to +4 721

kBT). Electrostatic potentials were calculated by using the program Adaptive Poisson-722

Boltzmann Solver (APBS). The left panel is color-coded as shown in Figure 1. Two 723

perpendicular views are shown. (B) Surface groove. A putative binding pocket at a site 724

located at the FnIII domain dimer interface at which two protomers come together is 725

shown as a close-up view. Residues contributing to the positive potential are shown as 726


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sticks. (C) Crystal forms 1 vs 2. The alternative loop conformation of residues 91-108 as 727

observed in crystal form 2 (and 3) is shown in cyan. 728

729

Figure 6. Low-resolution solution structure of RbmA. (A) Primary solution scattering 730

profiles. Averaged and buffer-subtracted SAXS intensity curves of RbmA solutions at 2 731

(green), 5 (blue), and 10 (red) mg/ml are shown. Radius of gyration (Rg) and the 732

maximum diameter of the dimeric protein (Dmax) was calculated based on the scattering 733

data or from the crystal structure (see main text). (B) Guinier plot. The Guinier plot 734

based on the data collected at the three different protein concentrations is shown for the 735

low-angle region. (C) Distance distribution functions. The distance distribution functions 736

were computed based on the scattering profile (red line; 10 mg/ml data set) or the 737

crystal structure of dimeric RbmA (dashed line). (D) Envelope reconstructions. SAXS 738

data was modeled ab initio using dummy residues (applying P2 symmetry during the 739

modeling). 20 individual models were superimposed, averaged and filtered. The filtered 740

envelope is shown as grey surface representation in two perpendicular views. 741

Independently, the SAXS data was modeled using two individual lobes taken from the 742

high-resolution RbmA structure as the input. Relative lobe orientations were restricted 743

during the positional modeling only by distance constraints accounting for the coil-like 744

linker segment connecting the two FnIII of an RbmA protomer. The best-fitting model 745

was docked manually into the low-resolution envelope. 746

747

Figure 7. Production of RbmA in V. Cholerae. Western blot analysis of RbmA 748

production in whole-cell (WC) lysates (top panel), and secretion in culture supernatant 749


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(CS) fractions (middle panel) in rugose, ΔrbmA, and chromosomal rbmA mutants 750

(E84A, E84R, R116A, R219A, R234A, Δloop). Equal amounts of total protein were 751

loaded in all the blots and BSA was used as an additional loading control for the CS 752

fractions (lower panel). 753

754

Figure 8. Phenotypes of chromosomal rbmA mutants. (A) Colony morhphology and 755

(B) pellicle formation (top view, upper panel; side view, lower panel) of rugose, ΔrbmA, 756

and chromosomal rbmA mutants (E84A, E84R, R116A, R219A, R234A, Δloop). 757

Scalebar, 0.5 mm. 758

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Figure 9. Complementation of rbmA chromosomal mutant phenotypes and 760

overexpression of rbmA point mutants. (A) Colony morphology of rugose, ΔrbmA, 761

and chromosomal rbmA mutants (E84A, E84R, R116A, R219A, R234A, Δloop) 762

harboring either the empty vector pMMB67EH (upper panel), or complementation 763

plasmid prbmA (lower panel). Scalebar, 0.5 mm. (B) Colony morphologis of rugose 764

strain carrying the vector or the pBAD overexpression plasmids with wild-type rbmA, 765

rbmA-R116A, rbmA-R219A or rbmA-R234A. Scalebar, 0.5 mm. 766

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