Downloaded from on May 6, 2020 by guest · 10/7/2013 · DOPA. OmpW is an outer membrane protein that belongs to a family of small outer membrane beta-barrel proteins that are widespread

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Preparation of Sticky Escherichia coli through Surface Display of Adhesive 1

Catecholamine Moiety 2

Joseph P. Park1, Min-Jung Choi

2, Se Hun Kim

3, Seung Hwan Lee

2,*, Haeshin Lee

1,3* 3

1Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of 4

Science and Technology (KAIST), 291 University Rd., Daejeon 305-701, South Korea 5 2Industrial Biochemicals Research Group, Research Center for Biobased Chemistry, Division 6

of Convergence Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 7 Gajeong-ro, Daejeon 305-600, South Korea 8 3Department of Chemistry, KAIST, 291 University Rd., Daejeon 305-701, South Korea 9

10 11 Running title: Sticky Escherichia coli for Spontaneous Cell Attachment 12

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Key words: catecholamine, bacteria cell surface display, gene expression, cell adhesion 14

*Corresponding Author: Prof. Haeshin Lee and Dr. Seung Hwan Lee 15

Email: [email protected] Phone: 82-42-350-2849 Fax: 82-42-350-2810 16

Email: [email protected] Phone: 82-42-860-7646 Fax: 82-42-861-7049 17

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AEM Accepts, published online ahead of print on 11 October 2013Appl. Environ. Microbiol. doi:10.1128/AEM.02223-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 20

Mussels attach to virtually all types of inorganic and organic surfaces in aqueous 21

environments, and catecholamines composed of 3,4-dihydroxy-L-phenylalanine (DOPA), 22

lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA 23

is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we 24

displayed the adhesive moiety of DOPA-histidine on Escherichia coli surfaces using outer 25

membrane protein W as an anchoring motif for the first time. Localization of catecholamines 26

on the cell surface was confirmed by western blot and immunofluorescence microscopy. 27

Furthermore, cell-to-cell cohesion (i.e. cellular aggregation) induced by the displayed 28

catecholamine and synthesis of gold nanoparticles on the cell surface support functional 29

display of adhesive catecholamines. The engineered E. coli exhibited significant adhesion 30

onto various material surfaces including silica and glass microparticles, gold, titanium, silicon, 31

poly(ethylene terephthalate) (PET), poly(urethane) (PU), and poly(dimethylsiloxane) 32

(PDMS). Uniqueness of this approach utilizing the engineered sticky E. coli is that no 33

chemistry for cell attachment are necessary and the ability of spontaneous E. coli attachment 34

allows one to immobilize the cells on challenging material surfaces such as synthetic 35

polymers. Therefore, we envision that mussel-inspired catecholamine displayed sticky E. coli 36

can be used as a new type of engineered microbial for various emerging fields such as whole 37

living cell attachment on versatile material surfaces, cell-to-cell communication systems, and 38

many others. 39

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INTRODUCTION 41

Microbial cell attachment is a crucial process in overall efficiency of a variety of 42

systems that exploits the whole microbial cell such as biocatalysts [1-9] or biosensors [10]. 43

Existing methods of attaching microbial cells to surfaces utilizes various chemistries such as 44

adsorption [11], covalent attachment [12,13], and entrapment within matrices [13,14]. 45

However, adsorption often results in the detachment of bacteria due to the weak bond with 46

the substrates, and covalent coupling requires cross-linking agents which results in the loss of 47

cell activity and viability. The entrapment process requires harsh condition in some cases 48

which jeopardizes the cellular activity and viability. Another important issue is that types of 49

materials that the conventional methods can be applied to are largely limited due to the 50

available surface chemistry. Therefore, cell attachment to variety of material surfaces without 51

surface modification or harsh chemical treatment remains a great challenge. The facile cell 52

attachment would circumvent the complex chemical modifications and reduce the loss of 53

cellular activity and viability of the whole cell. 54

As an emerging method for cell attachment, engineered cells expressing binding 55

affinity tags on the cell surfaces through display technology have been utilized although the 56

approach is not prevalent. Cell surface display allows peptides and proteins to be displayed 57

on the surface of microbial cells by fusing them with the anchoring motif through 58

recombinant DNA techniques. A wide range of applications of the display technology 59

includes bioadsorption [15-20], whole cell biocatalyst [21-27], peptide library screening 60

[28,29], live vaccine development [30-32], antibody production [33], and biosensor [34,35]. 61

Though cell attachment through cell surface display is not a well-known application, cell 62

attachement through displaying peptides with binding affinity has been reported on chitin, 63


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cellulose, and gold substrates. The first report of displaying affinity tags for cell attachment 64

was recombinant E. coli expressing Cex exoglucanase and cellulose-binding domain (CBD) 65

on the cell surface [36]. The engineered E. coli was found to bind tightly and rapidly to 66

cellulosic materials, which was later shown that the binding is very specific and stable 67

[36,37]. Also, recombinant E. coli with chitin-binding domain (ChBD) expressed on the 68

surface exhibited highly stable immobilization on the chitin substrate [38]. Cell attachemnt 69

for whole-cell biosensor was also reported on the gold substrate with recombinant E. coli 70

expressing gold-binding peptides on the cell surface [10]. Limitation of the aforementioned 71

cell attachments is that the engineered recombinant E. coli can only bind to a specific 72

substrate depending on the displayed peptide motif on the cell surface. In order to overcome 73

this limitation related to material versatility, we hypothesized that introduction of the 74

adhesive property of mussels can not only extend the range of materials for cell attachment, 75

but the adhesive E. coli can spontaneously bind to surfaces without chemical process. 76

Adhesive properties are unique in that mussels can attach to virtually all types of 77

inorganic and organic surfaces in wet conditions. Therefore, the production of mussel 78

adhesive proteins (MAPs) and its’ mimetic peptides have been investigated in depth to be 79

used as bio-adhesives. Expression systems of MAP and hybrid fusion MAP in E. coli has 80

been explored through recombinant DNA technology [39-43], but there are yet no reports of 81

displaying mussel adhesive protein or adhesive mimetic motifs of mussels on the surface to 82

exploit its’ adhesive property. The major component of specialized mussel adhesive proteins, 83

Mytilus edulis foot protein-5 (Mefp-5), located at a distal side of adhesive pads were found to 84

have extensive repeats of 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine [44]. Also, 85

when the amino acid sequence of the Mefp-5 is carefully observed, repeats of DOPA and 86


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histidine are present in the protein (Fig. 1). From this observation co-existence of catechol 87

and cationic amine functional group was hypothesized to be a key factor in achieving 88

adhesion to a wide spectrum of materials. Therefore, histidine and DOPA, though has not 89

been tested for surface adhesion, is expected to exhibit adhesive property. 90

In this study, we have combined the cell surface display techniques and the mussel-91

inspired adhesive catecholamine moiety, histidine and DOPA, to prepare sticky E. coli to 92

achieve spontaneous adhesion onto various material surfaces. The engineered sticky E. coli 93

exhibited unprecedented adhesion to various material surfaces, including silica and glass 94

beads, titanium and gold substrates. Furthermore, the sticky E. coli even adhered to synthetic 95

substrates that are typically incompatible with immobilization chemistry, such as 96

polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), and polyurethane (PU). 97

This study implies that catecholamine display on living bacterial cells can be a new approach 98

for facile cell attachment bypassing complicated, harsh chemical processes. 99

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MATERIALS AND METHODS 101

Bacterial Strains and Growth Conditions. E. coli strain XL10 – Gold Kanr 102

TetrD(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac 103

Hte [F´ proAB lacIqZDM15 Tn10 (Tetr) Tn5 (Kanr) Amy] (Stratagene, UK) was used as a 104

host strain for all recombinant plasmid work in this study. All recombinant strains were 105

cultivated in Luria-Bertani medium (10 g/L bacto-tryptone, 5 g/L bacto-yeast extract, and 10 106

g/L NaCl) supplemented with 50 g/mL ampicillin. Control strains with no recombinant 107

plasmids were cultured in Luria-Bertani medium supplemented with 40 g/mL kanamycin. 108

The recombinant cells were all cultivated at 30°C and induced at an optical density at 600 nm 109

of 0.4–0.6 by addition of isopropyl-ȕ-D-1-thiogalactopyranoside (IPTG) to a final 110

concentration of 0.2 mM. After induction, the cells were grown for an additional 4 h and 111

harvested for further experiments. 112

Plasmids and DNA Manipulation. Polymerase chain reaction (PCR) was performed using 113

the primers from Table I with a PCR 5000 Thermal Cycler (Bio-Rad) using the ExPrime Taq 114

premix kit (GeNet Bio, Inc.). All DNA manipulation and cloning procedures, including 115

restriction enzyme digestion, ligation, and agarose gel electrophoresis, were carried out using 116

standard procedures [45]. Primers and plasmids used in this study are listed in Table 1 and 2. 117

Truncated E. coli OmpW was amplified from the genomic DNA of the E. coli W3110 strain, 118

and P. aeruginosa perhydrolase genes were amplified from the genomic DNA of P. 119

aeruginosa using a primer design based on the reported DNA sequences. 120

Construction of Surface Display System. Outer membrane protein W (OmpW) was used as 121

the protein platform in this study, and perhydrolase from P. aeruginosa (PerPA) was used as a 122


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spacer protein to display the mussel-inspired sticky catecholamine moiety of histidine and 123

DOPA. OmpW is an outer membrane protein that belongs to a family of small outer 124

membrane beta-barrel proteins that are widespread in Gram-negative bacteria [46, 47]. The 125

X-ray structure of OmpW showed that it forms an eight stranded beta-barrel with a 126

hydrophobic channel [48], and this trans-membrane structure made it possible for the OmpW 127

to be used as a stable anchoring motif for microbial cell surface display. To develop a strategy 128

for displaying PerPA with DOPA and histidine residues using OmpW as an anchoring motif, 129

we first searched for the potential fusion site in OmpW. Based on the predicted secondary 130

structure of OmpW, two sites were chosen, at the 139th

and 191st amino acids, to display the 131

PerPA fusion protein. The truncated OmpW genes encoding the 139 and 191 amino acids 132

from the N-terminus were amplified by PCR using the primer sets shown in Table 1 and were 133

cloned into the EcoRI and XbaI sites of pKK223-18MCS to generate pOW13F and pOW19F, 134

respectively. A variety of PerPA gene-containing inserts, such as PerPA (PerPA1), PerPA+His 135

(PerPA2), PerPA+His+1-DOPA (PerPA3), PerPA+His+2-DOPA (PerPA4), were amplified 136

and cloned into the XbaI and HindIII sites of pOW13F and pOW19F. Thus, the resulting 137

construct designated pOW13F-PerPA1 is a perhydrolase-OmpW fusion protein-encoding 138

gene, and the fusion site is the 139th

amino acid in OmpW. Similar constructs with different 139

fusion proteins include pOW13F-PerPA2, pOW13F-PerPA3, and pOW13F-PerPA4. Similarly, 140

the pOW19F-PerPA1 construct is a perhydrolase-OmpW fusion protein-encoding gene, and 141

the fusion site is the 191st amino acid in OmpW. Thus, we constructed pOW19F-PerPA2, 142

pOW19F-PerPA3, and pOW19F-PerPA4 (Table 2). Recombinant XL10-Gold cells harboring 143

the recombinant DNA were cultivated at 30°C and induced with IPTG. 144

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western 145


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Blot Analysis. Proteins in the whole-cell lysate and outer membrane fractions were analyzed 146

by 12% (wt/vol) SDS-PAGE. Outer membrane proteins were prepared as follows: cells were 147

harvested by centrifuging the cell broth (3 mL) at 6000 rpm for 5 min at 4°C. The cell pellet 148

was washed with 1 mL of 10 mM Na2HPO4 buffer (pH 7.2), centrifuged at 6000 rpm for 5 149

min at 4°C, and resuspended in 0.2 mL of 10 mM Na2HPO4 buffer (pH 7.2). The resuspended 150

cells underwent three cycles of sonication (pulse on 5 sec, pulse off 10 sec at 20% of 151

maximum output). The sonicated cells were then centrifuged at 10,000 rpm for 2 min at room 152

temperature to remove the partially disrupted cells, and the supernatant was collected. The 153

collected supernatant was centrifuged again at 10,000 rpm for 30 min at 18°C. After 154

centrifugation, the cell pellet was resuspended in 0.5 mL of 10 mM Na2HPO4 buffer (pH 7.2) 155

containing 0.5% (wt/vol) sarcosyl and incubated for 30 min in a water bath at 37°C. After 156

incubation, the membrane proteins were obtained by washing the insoluble pellet with 10 157

mM Na2HPO4 buffer (pH 7.2) followed by resuspension in 40 ȝl of TE buffer (pH 8.0). The 158

whole-cell lysate and outer membrane protein samples were prepared by heating the protein 159

with protein sample buffer (5x) (Elpis Biotech) for 10 min at 100°C. Western blot analysis 160

was carried out following standard procedures [45]. Mouse monoclonal anti-6x His tag 161

antibody was used as the primary antibody (Abcam), and goat anti-mouse IgG –alkaline 162

phosphatase conjugate (Sigma-Aldrich) was used as the secondary antibody for 163

immunodetection of the fusion protein. The enzyme-catalyzed chromogenic reaction between 164

NBT and BCIP (Roche) was used for Western blotting signal detection. 165

Immunofluorescence Microscopy. Cells were harvested by centrifugation at 6000 rpm for 5 166

min at 4°C for analysis using immunofluorescence microscopy. Cells were then washed and 167

resuspended in 3% (wt/vol) bovine serum albumin (BSA, Sigma-Aldrich) in phosphate-168


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buffered saline (PBS). Cells were incubated with mouse monoclonal anti-6xHis tag antibody 169

(Abcam) diluted (1:1000) in PBS buffer containing 3 wt% BSA for 4 h at 4°C. After five 170

washes with PBS buffer, the cell-antibody complex was incubated overnight at 4°C with goat 171

polyclonal secondary antibody to mouse IgG conjugated with fluorescein isothiocyanate 172

(FITC), diluted (1:3,000) in PBS buffer containing 3 wt % BSA. After overnight incubation, 173

the cells were washed five times with PBS buffer prior to microscopic observation to remove 174

any unbound goat anti-mouse IgG conjugated with FITC. Cells were mounted on 175

microscopic glass slides and examined by fluorescence microscopy. Photographs were taken 176

with a Nikon fluorescence microscope. 177

Tyrosinase Treatment of Recombinant E. coli and Gold Reduction. Tyrosinase from 178

mushroom (Sigma-Aldrich) was used throughout the experiment to convert tyrosine residues 179

to DOPA. Tyrosinase was dissolved in distilled water to a concentration of 500 U/mL, where 180

one unit represents ǻA280 of 0.001 per min at pH 6.5 at 25°C in a 3-mL reaction mixture 181

containing L-tyrosine. The recombinant E. coli used for the tyrosinase treatment experiment 182

was resuspended in 10 mM phosphate buffer (pH 7.0) to an optical density at 600 nm of 2. 183

An equal amount of tyrosinase was added to the solution, and the mixture was reacted at 184

25°C overnight. A solution of 3 mM HAuCl4 (Sigma-Aldrich) was incubated with tyrosinase 185

(500 U/mL) for the gold reduction experiment. Recombinant E. coli cells were resuspended 186

in 10 mM phosphate buffer (pH 7.0) to an optical density of Cell OD 2 and incubated with 187

tyrosinase and gold solution for 1 hr to confirm the successful conversion of tyrosine to 188

DOPA. After 1 hr incubation, the cells were washed and resuspended in 10 mM phosphate 189

buffer (pH 7.0). Transmission electron microscopy (TEM) was used to analyze the surface of 190

the recombinant E. coli. The resuspended cells were placed on the TEM grid, washed and 191


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dried for TEM analysis. 192

Adhesion Test of Recombinant E. coli on Various Substrates. Recombinant E. coli cells 193

were resuspended in 10 mM phosphate buffer (pH 7.0) to an optical density at 600 nm of 2 194

and were incubated with tyrosinase (500 U/mL) and Silnos 290 silica beads (200 mg/L, ABC 195

Nanotech) or glass beads (200 mg/L, Polyscience, Inc.) overnight at 25°C with shaking in 196

Thermomixer (Eppendorf, 1000rpm). After incubation, the beads were dried on a silicon 197

wafer and washed 3 times with 10 mM phosphate buffer (pH 7.0). Scanning electron 198

microscopy (SEM) was used to analyze the surface of the beads. Further cell adhesion 199

experiment was performed using silica (Si), titanium (Ti), gold (Au) substrates as the metallic 200

substrates and polyethylene terephthalate (PET), polyurethane (PU), and 201

polydimethylsiloxane (PDMS) substrates as the polymeric substrates. For the Si substrates, Si 202

wafer was used and the Ti and Au substrates were prepared by thermal evaporation (BAK641, 203

Evatec, Switzerland) onto Si wafer (thickness = 100 nm). PET and PU films were purchased 204

from Hanmi Inc. (South Korea). PDMS substrates were prepared using SYLGARD 184 205

silicone elastomer kit (Dow Corning, USA). Recombinant E. coli cells were resuspended in 206

10 mM phosphate buffer (pH 7.0) to an optical density at 600 nm of 2 and were then 207

incubated overnight with tyrosinase (500 U/mL) on various surfaces at 25°C with shaking 208

(220 rpm) to examine adhesive properties of the sticky E. coli. As a control experiment, 209

recombinant E. coli cells were not treated with tyrosinase. After incubation of recombinant E. 210

coli on metallic surfaces, the surfaces were washed three times with 10 mM phosphate buffer 211

(pH 7.0) and dried. SEM was then used to analyze the surfaces. After the recombinant E. coli 212

was incubated on polymeric surfaces, the surfaces were washed three times with 10 mM 213

phosphate buffer (pH 7.0) and dried. The surfaces were then dyed with acridine orange 214


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(Sigma-Aldrich) for 10 min to stain the E. coli and washed again with 10 mM phosphate 215

buffer (pH 7.0). The surfaces were then analyzed by fluorescence microscopy. 216

RESULTS 217

Construction of Surface Display System. In this study, outer membrane protein W was used 218

as the anchoring motif and perhydrolase from P. aeruginosa (PerPA) as a functional spacer 219

protein to display the mussel-inspired catecholamine moiety. Perhydrolase was chosen as a 220

spacer protein, that can provide an additional function to the sticky E. coli, based on the fact 221

that it can provide a valuable platform for developing a biocatalyst exploiting its’ practical 222

reactions such as epoxidation of carbon-carbon double bonds of alkene or reversible 223

formation of peroxyacids from carboxylic acid and peroxide. To develop a strategy for 224

displaying PerPA with 6 histidine and tyrosine residue using the OmpW as an anchoring 225

motif, we first searched for the potential fusion sites in the OmpW. Based on the predicted 226

secondary structure of OmpW, two fusion sites were chosen - at 139th

amino acid and 191th

227

amino acid – for displaying PerPA fusion protein. The truncated OmpW genes encoding the 228

139 and 191 amino acids from the N terminus were amplified by PCR using the primer sets 229

shown in Supplementary Table1 and were cloned into EcoRI and XbaI sites of pKK223-230

18MCS to make pOW13F and pOW19F. The inserts containing PerPA genes, PerPA 231

(PerPA1), PerPA+6-His (PerPA2), PerPA+6-His+2Tyr (PerPA3), PerPA+6His+1Tyr (PerPA4), 232

were amplified and cloned into the XbaI and HindIII sites of pOW13F and pOW19F each to 233

make pOW13F-PerPA1, pOW13F-PerPA2, pOW13F-PerPA3, pOW13F-PerPA4, pOW19F-234

PerPA1, pOW19F-PerPA2, pOW19F-PerPA3, and pOW19F-PerPA4, which were used to 235

display PerPA genes on the E. coli cell surface. Recombinant XL10-Gold cells harboring the 236

recombinant DNA were cultivated at 30°C and induced with IPTG. 237


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Confirmation of OmpW-PerPA fusion protein display on the E. coli Cell Surface. After 238

transforming the E. coli XL10-Gold with the recombinant plasmids, for the confirmation of 239

the fusion protein display on the cell surface, the whole cell lysates and outer membrane 240

proteins of recombinant XL10-Gold cells were analyzed by SDS-PAGE. The fusion protein 241

could hardly be detected by the Coomassie blue staining due to the low expression level, 242

therefore, western blot analysis was carried out using 6xHis tag antibody as the primary 243

antibody (abcam) and goat anti-mouse IgG –alkaline phosphatase conjugate (Sigma) was 244

used as the secondary antibody for the immunodetection of the fusion protein (Fig. 2 and Fig. 245

S1). The outer membrane protein size of truncated OmpW at the 139th

amino acid and the 246

PerPA fusion protein have estimated size around 45.3kDa calculated from the amino acid 247

sequence. From the western blot result, the protein band around that size was not detected on 248

the western blot results instead a protein band of size around 30 kDa was observed (Fig. S1). 249

The band with size of 30kDa seems to correspond with the PerPA which is 30.44kDa in size, 250

therefore, we can infer that the OmpW truncated at 139th

amino acid was eliminated or 251

excised as the recombinant DNA was translated and expressed. The fact that band around 252

30kDa was observed in the whole cell lysate and in the soluble fractions of recombinant E. 253

coli XL10-Gold harboring pOW13F-PerPA2, pOW13F-PerPA3, and pOW13F-PerPA4 and 254

not in the outer membrane fraction supports our theory. The fact that protein band size around 255

45.4 kDa was not detected shows us that the Omp W-PerPA fusion protein was not 256

successfully displayed on the surface of the E. coli. The outer membrane protein size of 257

truncated OmpW at the 191th

amino acid and the PerPA fusion protein have estimated size 258

around 51.0 kDa calculated from the amino acid sequence. The bands corresponding to the 259

51.0-kDa fusion protein were detected in whole-cell lysates and the outer membrane fraction 260


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of the recombinant XL10-Gold harboring pOW19F-PerPA2, pOW19F-PerPA3, and 261

pOW19F-PerPA4 suggesting successful display of fusion proteins on the cell surface (Fig. 2). 262

Therefore, recombinant XL10-Gold harboring pOW19F-PerPA1, pOW19F-PerPA2, 263

pOW19F-PerPA3, and pOW19F-PerPA4 were used in further studies. As another method to 264

confirm the successful display of the OmpW-PerPA fusion protein on the surface of the E. 265

coli cell surface, immunofluorescence microscopy was employed. As shown in Fig. 3, 266

recombinant E. coli harboring pOW19F-PerPA3 and pOW19F-PerPA4 became fluorescent 267

due to the binding of the anti-6xHIS antibody followed by binding of FITC-conjugated 268

secondary antibody, indicating that the OmpW-PerPA fusion protein was successfully 269

displayed on the surface of the E. coli. On the other hand, control E. coli XL10-Gold cells did 270

not exhibit any fluorescence. 271

Tyrosinase Treatment and Confirmation of Tyrosine Conversion to DOPA. After 272

confirming the successful display, the recombinant E. coli cells were treated with tyrosinase 273

to convert the tyrosine reside on the surface to DOPA since DOPA is an unnatural amino acid 274

and thus requires post-modification. XL10-Gold and recombinant E. coli XL10-Gold 275

harboring pOW19F-PerPA1, pOW19F-PerPA2, pOW19F-PerPA3, and pOW19F-PerPA4 276

were treated with tyrosinase (500U/ml). After the recombinant cells were treated with 277

tyrosinase, cell aggregation occurred only with the recombinant E. coli harboring pOW19F-278

PerPA3 and pOW19F-PerPA4 (Fig. 4A) that have tyrosine residue on the surface which 279

indirectly confirms the conversion of the tyrosine residue to DOPA since molecule with 280

DOPA moiety tend to form aggregation due to their specific cohesive interactions. In the 281

other recombinant E. coli cells that did not have tyrosine on the surface did not show any 282

aggregation after tyrosinase treatment (Fig. 4A). As another method to confirm the successful 283


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conversion of tyrosine residue on the surface of recombinant E. coli XL10-Gold harboring 284

plasmid pOW19F-PerPA3 and pOW19F-PerPA4 to DOPA, the recombinant E. coli cells were 285

incubated with tyrosinase with 10mM HAuCl4 solution. It is well known that the DOPA 286

moiety has metal reducing ability that reduces Au3+

to Au0+

. Therefore, we hypothesize that 287

gold nanoparticles will be reduced on the surface of the E. coli where the DOPA moiety is 288

present. E. coli XL10-Gold, recombinant E. coli XL10-Gold harboring pOW19F-PerPA1, 289

pOW19F-PerPA2, pOW19F-PerPA3, and pOW19F-PerPA4 were incubated with tyrosinase 290

and HAuCl4 for 1hr at 25°C with mild agitation. After the incubation, the cells were observed 291

for the gold nanoparticle formation on the surfaces of the recombinant E. coli cells using 292

TEM. In the controls, XL10-Gold, recombinant E. coli harboring pOW19F-PerPA1 and 293

pOW19F-PerPA2, no gold nanoparticle formation was observed on the surface (Fig.4 B,C,D). 294

Also when the gold nanoparticle formation experiments were performed with the 295

recombinant E. coli harboring pOW19-PerPA3 and –PerPA4 without the tyrosinase treatment, 296

no gold nanoparticle formation was observed (Fig. S2). Gold nanoparticles with sizes ranging 297

from 50-120nm were only observed on the surfaces of the tyrosinase treated recombinant E. 298

coli XL10-Gold harboring pOW19F-PerPA3 and pOW19F-PerPA4 which had tyrosine 299

residue on the surface before the tyrosinase treatment (Fig. 4E,F). The gold nanoparticle was 300

confirmed by the energy dispersive X-ray spectrum (EDS) taken by TEM (Fig. S3). The gold 301

nanoparticle formation therefore can be used to confirm the successful conversion of tyrosine 302

residue on the cell surface to DOPA residue in the recombinant E. coli XL10-Gold harboring 303

pOW19F-PerPA3 and pOW19F-PerPA4. Tyrosinase could not be responsible for the gold 304

nanoparticle formation since all the controls were also treated with tyrosinase as well. From 305

on in, tyrosinase treated recombinant E. coli XL10-Gold harboring pOW19F-PerPA3 and 306


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pOW19F-PerPA4 will be referred to as recombinant E. coli PerPA3-DOPA and PerPA4-307

DOPA respectively. 308

Adhesion Property of Mussel-Inspired Catecholamine Displayed Engineered E. coli. 309

After confirming the successful display of the fusion protein on the surface and the successful 310

conversion of tyrosine to DOPA, the adhesive property of the engineered E. coli was explored. 311

In order to observe the adhesive property of our mussel-inspired catecholamine surface 312

displayed recombinant E. coli, cells were incubated with variety of substrates and surfaces. 313

First microparticles were chosen as a substrate for recombinant E. coli attachment. Silnos 290, 314

a silica microparticle with average size of 9ȝm, and glass bead with size between 30-50ȝm 315

was used. Recombinant E. coli cells were incubated with tyrosinase and the beads overnight. 316

After the incubation of XL10-Gold and recombinant E. coli XL10-Gold harboring pOW19F-317

PerPA1, pOW19F-PerPA2, pOW19F-PerPA3, and pOW19F-PerPA4 with tyrosinase and 318

beads, the incubated beads were observed with SEM to examine the degree of adhesion to the 319

beads. In the case of adhesion test to the Silnos 290 silica bead, tyrosinase treated 320

recombinant E .coli harboring pOW19F-PerPA3 and pOW19F-PerPA4 showed significant 321

adhesion to the Silnos 290 silica beads (Fig. 5B, 5D). As shown in Fig. 5, recombinant E. coli 322

XL-Gold harboring pOW19F-PerPA3 and pOW19F-PerPA4 without tyrosinase treatment 323

(Fig. 5A, 5C) did not exhibit immobilization to the microparticle confirming the adhesive 324

property of the catecholamine displayed recombinant E. coli PerPA3-DOPA and PerPA4-325

DOPA. In the other control experiments, where tyrosinase was treated to XL10-Gold, 326

recombinant E. coli XL10-Gold harboring pOW19F-PerPA1 and pOW19F-PerPA2, none or 327

only a few E. coli cells adhered to the surface of the Silnos 290 silica beads (Fig. S4). From 328

this observation it is reasonable to conclude that the catecholamine displayed recombinant E. 329


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coli have adhesive property. As a different microparticle with different size for the adhesion 330

test, glass bead of size 30~50um was used. In the case of adhesion test to the glass bead, 331

recombinant E. coli PerPA3-DOPA and PerPA4-DOPA showed significant adhesion to the 332

glass beads (Fig. 5F, 5H). As can be seen in Fig. 5, control recombinant E. coli harboring 333

pOW19F-PerPA3 and pOW19F-PerPA4 without tyrosinase treatment showed little or no cell 334

adhesion to the microparticles. In the other control experiments, where tyrosinase was treated 335

to XL10-Gold and recombinant E. coli harboring pOW19F-PerPA1 and pOW19F-PerPA2, 336

some cells were observed to be adhered onto the surface of the glass beads but degree of 337

adhesion was still significantly low compared with recombinant E. coli PerPA3-DOPA and 338

PerPA4-DOPA (Fig. S4). These results also confirm the adhesive property of mussel-inspired 339

catecholamine displayed recombinant E. coli. The number of engineered E. coli cells adhered 340

to various microparticles were counted (cells per surface area of the microparticles) as shown 341

in Fig. 5I. When the adhesion level of PerPA3-DOPA and PerPA4-DOPA were compared 342

using the quantified data, no significant differences were observed. Based on this observation, 343

we used the recombinant E. coli PerPA4-DOPA throughout this study. 344

Two different types of surface substrates, metallic and polymeric, were used for the 345

further adhesion test of the engineered recombinant E. coli. Au, Ti and Si surfaces were 346

chosen as the metallic surfaces for the cell adhesion experiment. Those metallic surfaces were 347

incubated with the recombinant E. coli XL10-Gold harboring pOW19F-PerPA4 with and 348

without tyrosinase. In the control group without tyrosinase treatment recombinant E. coli, in 349

order to counterpart the addition of tyrosinase, equal amount of 10mM phosphate buffer (pH 350

7.0) was added on. After the recombinant E. coli XL10-Gold harboring pOW19F-PerPA4 351

with and without tyrosinase was incubated on various metallic surfaces overnight, SEM was 352


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used to observe the adhesion of recombinant E. coli on the surfaces. SEM images of surfaces 353

incubated with tyrosinase treated and untreated recombinant E. coli harboring pOW19F-354

PerPA4 were compared (Fig. 6A,C,E). As can be seen from the Fig. 6, recombinant E. coli 355

PerPA4-DOPA, significantly adhered more to the metallic surfaces which demonstrates the 356

adhesive property of the engineered E .coli that has mussel-inspired catecholamine on the 357

surface. To test the adhesion of catecholamine displayed recombinant E. coli on different 358

surfaces, PET, PU and PDMS surfaces were selected as the polymeric surfaces for the cell 359

adhesion experiment. After the recombinant E. coli XL10-Gold harboring pOW19F-PerPA4 360

with and without tyrosinase were incubated on PET, PU, and PDMS surfaces, the surfaces 361

were stained with acridine orange dye and fluorescence microscopy was used to observe the 362

adhesion of E. coli on the surfaces. Fluorescence images of surface incubated with tyrosinase 363

treated and untreated recombinant E. coli harboring pOW19F-PerPA4 were compared (Fig. 364

6B,D,F). As can be seen from the Fig. 6, recombinant E. coli PerPA4-DOPA significantly 365

adhered more to the PET, PU, and PDMS surfaces which demonstrate the adhesive property 366

of the catecholamine cell-surface displayed recombinant E. coli. The adhered E. coli cells to 367

various substrates were quantified per unit area (Fig. 6G). The number of cell adhered to the 368

metallic surfaces can be directly counted from the SEM images. In the cases of the polymeric 369

surfaces, we obtained a linear relationship between the fluorescent intensity and the cell 370

number using many sampling areas, which was subsequently utilized to convert the 371

fluorescence intensity into number of cells. The quantification result showed a greater degree 372

of adhesion to metallic surfaces compared to the adhesion on polymeric ones. The number of 373

attached E. coli per mm2 area was: Ti (48812 ± 24232) > Au (30541 ± 2203) > Si (21583 ± 374

7042) > PET (17490 ± 7696) > PDMS (14793 ± 3363) > PU (7457 ± 2398) in descending 375


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order. 376

DISCUSSION 377

Cell attachment through microbial cell-surface display is a fairly new method, and a 378

few studies have been reported for attachment of a limited range of material substrates such 379

as cellulose, chitin, or gold [10,36-38]. Such limitation in surface adhesion is largely due to 380

adhesion peptide identification by screening process for a given material. In order to expand 381

the type of materials to which the engineered E. coli is able to bind without labor-intensive, 382

time-consuming screening processes, we turned our attention to interesting adhesive features 383

of the mussel adhesive proteins. It is well known that the catecholamine group in MAPs plays 384

a crucial role in the adhesion of mussels. For example, mefp-5, which is responsible for the 385

robust, material-independent adhesion, shows the extensive repeat of lysine and DOPA [44]. 386

Furthermore, synthetic versions of catecholamine polymers such as poly(dopamine) [49], 387

poly(norepinephrine) [50], poly(ethylenimine)-catechol [51], and chitosan-catechol [52,53] 388

have demonstrated material-independent adhesion in aqueous conditions. In this study, the 389

histidine-DOPA moiety was chosen for preparation of sticky E. coli the following two 390

reasons: (1) the histidine-DOPA moiety is found in Mefp-5, which is expected to exhibit 391

adhesive property similar to the catecholamines mentioned earlier; and (2) for the ease of 392

purification and detection of the surface displayed peptide since poly-histidine has been 393

widely used for affinity purification of genetically modified proteins. The catecholamine 394

moiety exhibits various unique properties; it can form stable complexes with metal ions, 395

undergo redox reactions to form covalent catechol-catechol/-amine/-thiol crosslinkings. One 396

of the important properties of catechol moieties is the oxidative chemical conversion to 397

quinone, and the quinone is highly reactive with amine and thiol compounds via Michael-398


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type addition or Schiff base formation [49]. This mechanism is able to achieve robust 399

irreversible adhesion on organic substrates [54]. Thus, display of histidine-DOPA on the 400

surface of E. coli can convert non-adhesive into be adhesive for virtually all types of material 401

surfaces. The aforementioned covalent-bond-forming mechanism can explain the result of E. 402

coli aggregation show in Fig. 4. The DOPA displayed on the cell surface can undergo 403

catechol-catechol and catechol-amine crosslinking, and the amine groups might from intrinsic 404

proteins existed in the surface E. coli. Another way to confirm the display of sticky 405

catecholamine moiety, the redox activity of catechol was exploited. As the catechol moiety is 406

oxidized to catecholquinone, electrons are released from the catechol, and gold and silver 407

ions are spontaneously reduced by the released electrons as shown in the scheme of Fig. 4E. 408

Therefore, gold reduction occurred on the surface of the sticky E. coli can be expected. 409

Successfully, gold nanoparticle formation was observed on the surface only when the 410

catecholamine, histidine-DOPA, was displayed (PerPA3-DOPA and PerPA4-DOPA). 411

After the preparation of sticky E. coli, the spontaneous attachment of the cells was 412

studied using various types of material surfaces. As the cell aggregation was already observed 413

for the sticky E. coli displaying histidine-DOPA, the E. coli is expected to have enhanced 414

ability attaching spontaneously onto surfaces. In fact, microbials with a certain tendency of 415

aggregation have shown to exhibit ability to attach onto surfaces although the adhesive force 416

might not be as strong as the one by mussel adhesive molecules [55]. The mussel-inspired 417

sticky E. coli exhibited significant adhesion to unprecedented various substrates including 418

silica, glass microparticles, Au, Ti, Si, PET, PU, and PDMS surfaces when compared with the 419

control microbes. This observation is significant in the sense that a new type of a 420

catecholamine amino acid moiety, DOPA-His, was shown to exhibit wet-resistant adhesive 421


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properties that can potentially be applied to any living organisms when adhesive properties 422

are needed. Also, it is noteworthy that the sticky E. coli was attached to those various 423

substrates without applying any surface chemistry (e.g. chemical agents for coupling). In 424

particular, glass and PDMA are challenging materials for adhesion. Catecholamine moieties 425

are found to adhere to virtually any substrates via coordination bonds, ʌ-ʌ stacking, 426

electrostatics, and/or hydrogen bonding [49,54]. For surfaces such as Si and Ti wafers, their 427

oxide forms, SiO2 and TiO2, are presented onto which catechol forms a coordination bond 428

with the –OH groups, and electrostatic interactions can be established by the interactions 429

between the amines groups along the catecholamine and the hydroxyl ions from the 430

substrates. Catecholamine is stacked onto the gold substrates via the interactions between pi 431

electrons in catechol and the electrons in the conduction bands of the Au surfaces. PET and 432

PU are composed of chains that contain phenyl rings which enable the displayed 433

catecholamine moiety to interact with those substrates by ʌ-ʌ interactions. The adhesion of 434

recombinant E. coli onto the PDMS can be accredited to the electrostatic interactions between 435

the hydroxyl ions presented on surfaces of PDMS and the amine groups of catecholamine. 436

Also, it has been suggested that the catechol also contributes to the surface adhesion but it 437

remains unclear. Thus, our results indicate that the sticky E. coli are likely to attach to a wide 438

range of material surfaces. In contrast, previous experiments regarding cell immobilization 439

achieved by surface-engineered peptide display showed that the engineered E. coli adhered 440

only to limited types of materials that have binding affinity with the displayed peptide. 441

Regarding to the robustness of the sticky E. coli, our experiment condition was typically 442

under agitation (1000rpm for microparticles and 220rpm for other substrates) and washing 443

steps were performed, in which the cells remained attached, demonstrating the robustness of 444


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the sticky chemistry indirectly. These findings suggest that E. coli displaying mussel-inspired 445

catecholamine can be used as a new method for cell immobilization on a broader range of 446

substrates. 447

ACKNOWLEDGEMENTS 448

This study was supported by Molecular-level Interface Research Center (MIRC, 2012-449

000909, H.L.), Future Medical Technology Development Program (2012-0006085, H.L.), and 450

Technology Development Program to Solve Climate Changes on Systems Metabolic 451

Engineering for Biorefineries (NRF-2012-C1AAA001 -2012M1A2A2026556, S.H.L.) from 452

National Research Foundation of S. Korea. World Premier Material Program from the 453

Ministry of Knowledge and Economy also supported this work. 454

455


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Table 1. List of primers used in PCR experiment 597

Primer Sequence*

Gene to be amplified

Template

5’-gaattcatgaaaaagttaacagtggcg

Truncated Omp W_forward E. coli W3110 gDNA

5’-tctagagccatgatcgttaaatccatt Truncated Omp W (139th amino acid)_reverse E. coli W3110 gDNA

5’-tctagatgcaccgcccagcttataat Truncated Omp W (191th amino acid)_reverse E. coli W3110 gDNA

5’-tctagaatgggttacgtgacaacgaagg Perhydrolase_forward P. aeruginosa PAO1 gDNA

5’-aagctttcagccgcggatgaaggcc Perhydrolase_reverse P. aeruginosa PAO1 gDNA

5’-aagcttttaatggtgatgatggtgatggccgcggatgaaggc Perhydrolase + 6-His_reverse P. aeruginosa PAO1 gDNA

5’-aagcttttaataatggtgatgatggtgatg Perhydrolase + 6-His + 1Tyr_reverse Perhydrolase + 6-His

PCR product

5’-aagcttttaataataatggtgatgatggtgatg Perhydorlase + 6-His + 2Tyr_reverse Perhydrolase + 6-His

PCR product

*Restriction enzyme sites are shown in bold 598

599


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Table 2. List of Plasmids used in this study. 600

Plasmid

Relevant characteristics

Reference

pKK223-3 Amp resistant; tac promoter; 4.5 kb Brosius and Holy, 1984

[67]

pKK223-18MCS pKK223 derivative; containing additional 18 multi-cloning site; 4.6 kb Hernan et al., 1992

[68]

pKK223-pOW13F pKK223 derivative; containing 417-bp fragment of truncated OmpW at 139th AA of

E. coli; 5.0 kb

This study

pKK223-pOW19F pKK223 derivative; containing 573-bp fragment of truncated OmpW at 191th AA of

E. coli; 5.1 kb

This study

pOW13F-PerPA1 pKK223-pOW13F derivative; P. aeruginosa perhydrolase; 5.8 kb This study

pOW13F-PerPA2 pKK223-pOW13F derivative; P. aeruginosa perhydrolase + 6-His; 5.8 kb This study

pOW13F-PerPA3 pKK223-pOW13F derivative; P. aeruginosa perhydrolase + 6-His + 1Tyr; 5.8 kb This study


pOW19F-PerPA1 pKK223-pOW19F derivative; P. aeruginosa perhydrolase; 6.0 kb This study

pOW19F-PerPA2 pKK223-pOW19F derivative; P. aeruginosa perhydrolase + 6-His; 6.0 kb This study



601

602


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Figure Legend: 603

Figure 1. Schematic illustration of engineering mussel-inspired catecholamine displayed 604 E. coli. DOPA and histidine repeats found in mussel adhesive protein, mefp-5, was displayed 605 on the E. coli surface using outer membrane protein W (Omp W) as the anchoring motif. 606 Since DOPA is an unnatural amino acid, tyrosine was displayed first and converted to DOPA 607 using tyrosinase. 608

Figure 2. SDS-PAGE Analysis and Immunoblotting Analysis (A) SDS-PAGE analysis and 609 (B) Western blot analysis of recombinant E. coli XL10-Gold cells expressing OmpW-PerPA 610 fusion proteins. Lane 1, whole-cell lysate of XL10-Gold harboring pOW19F-PerPA1; lane 2, 611 outer membrane fraction of XL10-Gold harboring pOW19F-PerPA1; Lane 3, whole-cell 612 lysate of XL10-Gold harboring pOW19F-PerPA2; lane 4, outer membrane fraction of XL10-613 Gold harboring pOW19F-PerPA2; Lane 5, whole-cell lysate of XL10-Gold harboring 614 pOW19F-PerPA3; lane 6, outer membrane fraction of XL10-Gold harboring pOW19F-615 PerPA3; lane 7, whole-cell lysate of XL10-Gold harboring pOW19F-PerPA4; lane 8, outer 616 membrane fraction of XL10-Gold harboring pOW19F-PerPA4; lane M, molecular mass 617 standards. 618

Figure 3. Immuofluorescence Analysis Differential interference micrographs (upper) and 619 immune-fluorescence micrograph (lower) of (A) XL10-Gold cell and XL10-Gold cells 620 harboring (B) pOW19F-PerPA3 and (C) pOW19F-PerPA4. Cells were incubated with mouse 621 anti-His probe antibody followed by probing with goat anti-mouse IgG-FITC conjugate. The 622 scale bars are 5ȝm. 623

Figure 4. Confirmation of tyrosine to DOPA conversion (A) Tyrosinase was treated to E. 624 coli XL10-Gold and recombinant E. coli cells harboring pOW19F-PerPA1, pOW19F-PerPA2, 625 pOW19F-PerPA3, and pOW19F-PerPA4. Aggregation was observed only for tyrosinase 626 treated recombinant E. coli harboring pOW19F-PerPA3(PerPA3-1DOPA) and pOW19F-627 PerPA4 (PerPA4-2DOPA). *mark denotes tyrosinase treatment. TEM analysis of (B) XL10-628 Gold cell and XL10-Gold cell harboring (C) pOW19-PerPA1, (D) pOW19F-PerPA2, (E) 629 pOW19F-PerpA3, and (F) pOW19F-PerPA4 after incubation with tyrosinase and gold 630 solution (HAuCl4). Gold nanoparticle formation was only observed in PerPA3-DOPA and 631 PerPA4-DOPA E. coli cells. 632

Figure 5. Cell adhesion test Cell adhesion test was performed on (A,B,C,D) silica 633 microparticles and (E,F,G,H) glass microparticles. SEM analysis was employed to observe the 634 surface of the microparticles. SEM images on the left are the control XL10-Gold harboring 635 (A,E) pOW19F-PerPA3 and (C,G) pOW19F-PerPA4 without the tyrosinase treatement. SEM 636 images on the right are tyrosinase treated recombinant E. coli (B,F) PerPA3-DOPA and (D, 637 H) PerPA4-DOPA. Only catecholamine displayed E. coli exhibited adhesive properties. (I) 638 The cells adhered onto the microparticles were quantified and expressed as cells per area in 639


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the bar graph. 19-3 and 19-4 denotes recombinant E. coli harboring pOW19F-PerPA3 and 640 pOW19F-PerPA4. Tyrosinase treated cells are labeled with T. 641

Figure 6. Cell adhesion test on various substrates Cell adhesion test was performed on 642 metallic surfaces, (A) Au, (C) Ti, (E) Si, and on polymeric surfaces, (B) PET, (D) PU, and (F) 643 PDMS, using PerPA4-DOPA recombinant cells. Inlets are the control XL10-Gold harboring 644 pOW19F-PerP4. (G) The adhered cells were quantified and expressed in cells per area in the 645 bar graph. 19-4 denotes recombinant E. coli harboring pOW19F-PerPA4. Tyrosinase treated 646 cells are labeled with T. The scale bar for the inlet controls are the same as the main images. 647


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Downloaded from on May 6, 2020 by guest · 10/7/2013 · DOPA. OmpW is an outer membrane protein that belongs to a family of small outer membrane beta-barrel proteins that are widespread