research 1..5 - American Chemical Society

Subscriber access provided by RMIT University Library

Letter

Clay Catalyzed Disruption of Quorum Sensing in Vibrio harveyi Bacteria Sajo Naik, Jonathon Scholin, San Ching, Fang Chi, and Marc Herpfer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03918 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

Journal of Agricultural and Food Chemistry

1

Quorum Sensing Disruption in Vibrio harveyi

2

Bacteria by Clay Materials

3 4

Sajo P. Naik, * Jonathon Scholin, San Ching, Fang Chi, and Marc

5

Herpfer

6 7

The Oil-Dri Innovation Center

8

777 Forest Edge Road

9

Vernon Hills, IL 60061-3197

10 11 12 13 14 15 16 17 18 19 20 21 22

Corresponding Author Sajo P. Naik The Oil-Dri Innovation Center 777 Forest Edge Road Vernon Hills, IL 60061-3197 [email protected] Phone: +1- 847-537-7543

23 24 25 26

ACS Paragon Plus Environment

1


Page 2 of 21

27 28 29

ABSTRACT

30

This work describes the use of clay minerals as catalysts for the degradation of quorum sensing

31

molecule, N-(3-Oxooctanoyl)-DL-homoserine lactone. Certain clay minerals due to their surface

32

properties and porosity can catalytically degrade the quorum sensing molecule into smaller

33

fragments. The disruption of quorum sensing by clay in a growing Gram-negative Vibrio harveyi

34

bacteria culture was also studied by monitoring luminescence and population density of the

35

bacteria, wherein quenching of bacterial quorum sensing activity was observed by means of

36

luminescence reduction. The results of this study show that food grade clays can be used as

37

biocatalysts in disrupting bacterial activity in various media.

38

KEYWORDS

39

Clay, Biocatalysis, Transformations, N- (3-Oxooctanoyl)-DL-homoserine, quorum sensing

40 41 42 43 44 45 46 47 48 49


2

Page 3 of 21


50 51

1. Introduction

52

Quorum sensing is a cell-to-cell communicating system employed by bacteria to regulate

53

bacterial activity in response to various stimuli. Quorum sensing was first reported in the study of

54

a marine bacteria Vibrio fischeri which exhibited bioluminescence upon bacteria reaching a high

55

population density [1]. Typically, bacteria biosynthesize distinctive chemicals including N-

56

acylhomoserine lactones (AHLs) as quorum sensing signals; specifically, AHLs, such as N-(3-

57

Oxooctanoyl)-DL-homoserine lactone are used by some Gram-negative bacteria in quorum

58

sensing signaling. The biochemical production and effectiveness of AHLs depend primarily on

59

the activity of Lux and LuxR protein families, respectively, which together with the quorum

60

sensing molecules, constitute the overall quorum sensing signaling system. After AHLs are

61

produced by Lux enzymes, they diffuse across bacterial membranes and accumulate in the

62

surroundings until critical local concentrations are reached [2]. At a given threshold

63

concentration, the AHL binds to LuxR response regulator, forming a complex that activates gene

64

expression. Some bacterial features controlled through quorum sensing include bacteria

65

population, antibiotic generation, bioluminescence, nitrogen-fixing gene regulation, Ti plasmid

66

conjugal transfer, virulent gene expression, pigment generation, bacterial swarming, and biofilm

67

formation [3].

68

Quorum quenching is a process of disrupting the bacterial quorum system. Modulation of Lux,

69

LuxR and deactivation of quorum quenching molecules are some ways of accomplishing quorum

70

quenching. There exist several reports on the use of naturally occurring or artificially synthesized

71

antagonists as possible quorum quenchers [4,7]. Ideally, quorum sensing quenchers, inhibitors,

72

or regulators should not interfere with the normal physiological functions of bacterial cells or not

73

prompt bacteria to develop resistance. Although several different methods of quorum quenching

74

are reported, to the best of our knowledge there does not exist any biocatalytic ways involving

75

solid acid catalysts such as clays to disrupt quorum sensing in bacteria.

76

Clay minerals are hydrous aluminum phyllosilicates, which may contain variable

77

amounts of iron, magnesium, alkali metals, alkaline earths and other cations. Clay minerals exist

78

in nature but must be further processed or purified such that their chemical or physical properties


3


Page 4 of 21

79

required in various applications may be brought to the fore. Clays have a very interesting history

80

as solid acid catalysts. Acid-treated clays were used as catalysts in petroleum refining in the early

81

1930s [8-9]. However, their applicability was limited due to their lack of thermal stability under

82

high temperature fluidized catalytic cracking (FCC) conditions. Consequently, clays were

83

replaced by more stable synthetic catalysts, e.g. silica-alumina, zeolites, etc. in the petrochemical

84

industry. Besides catalysis clay minerals have several other applications as adsorbents, feed

85

additives, fillers, carriers, etc. [8-13]. In several animal health products, clay minerals are used as

86

feed additives as they are non-toxic, have high porosity and presence of active sites that can bind

87

a range of fungal and microbial toxins to improve the health of the animal [10-13]. In the animal

88

feed and health industry versatility of clays to perform more than one physiological functions is

89

desired.

90

Herein, through in vitro studies we demonstrate that clays can adsorb and catalyze

91

bacterial quorum sensing molecules to break them into smaller fragments in an aqueous system.

92

We used N-(3 Oxooctanoyl)-DL-homoserine lactone, an AHL type quorum sensing molecule as

93

a model quorum sensing molecule for this in vitro study. We followed up the in vitro study by

94

directly monitoring the quorum quenching in Vibrio harveyi bacteria in the presence of clays

95

under simulated bacterial growth conditions. Bioluminescence and bacteria population were the

96

parameters employed to gauge the quorum sensing activity of the system. Clay minerals

97

functioned as adsorption/catalytic antagonists in that the AHLs responsible for quorum sensing

98

activity in some bacteria were either separated out by adsorption or were catalytically broken

99

down by the active site in clay surface.

Reducing the concentration of quorum sensing

100

molecules in the bacterial system either by physical separation or chemical transformation into

101

non-active molecules using clays demonstrate possible way of disrupting quorum sensing in

102

bacteria which could have a possible impact on controlling bacterial ability to produce toxins or

103

virulence as such functions in bacteria are control through quorum sensing.

104 105

2. Materials

106

2.1 Clays and chemicals

107

The clay samples were obtained from Amlan international/Oil-Dri corporation of America [13].

108

Calibrin-A and Z are type of processed montmorillonite clays and their details have been


4

Page 5 of 21


109

described earlier [11, 12]. All the other chemicals were obtained from Sigma-Aldrich, USA and

110

used as-received.

111 112

3. Methods

113

3.1 Clay Characterization

114

Clays were characterized by X-ray diffraction (XRD) on a Bruker D2 Phaser instrument using

115

Cukα as the X-ray source. A wet slide method of sample preparation was used for the analysis

116

(Data not shown). A Quantachrome NOVA 4200 instrument was used to measure the total pore

117

volume of Calibrin A and Calibrin Z samples by N2 adsorption/desorption method. The sample

118

was degassed at 110 oC for 12 h under vacuum followed by nitrogen adsorption/desorption study

119

at 77 K. Pore volume was estimated by Density Functional Theory (DFT) method of analysis

120

using NovaWin 11.03. Specific surface areas of the materials were evaluated using Brunner

121

Emmett Taylor (BET) method from N2 data.

122 123

The acidity of the clay samples was measured on a Chemisorb 2720, Micromeritics instrument

124

using ammonia as a probe molecule. Typically, 25–100 mg of the sample (100 mesh) was held

125

between small lumps of inert quartz wool taken in a quartz tube. The sample tube was connected

126

to the device and heated to 120oC, 12 h, to remove adsorbed water and other volatile impurities.

127

After cooling to 25oC, dry ammonia gas [20 % NH3 balance He (UHP); 20 mL/min] was then

128

passed over the sample for 15 minutes. The physically adsorbed ammonia was flushed out by He

129

carrier gas at 25oC. The sample was heated from 25oC to 600oC at a heating rate of 5oC/min and

130

the desorbed ammonia was measured using a thermal conductivity (TC) detector connected to

131

the sample tube in a loop.

132 133

3.2 in vitro catalysis experiments

134

N-(3-Oxooctanoyl)-DL-homoserine lactone was used as a model compound in this study for in

135

vitro testing of clay performance. Typically, a fixed amount of aqueous lactone solution (200

136

ppm) was taken in a small vial containing a fixed amount (1mg – 500 mg) of clay or solid. The

137

formed suspension was then agitated at 100 rpm for 15 minutes, and successively centrifuged (a

138

Thermo Scientific Sorvall Legend T+ unit) at 3,500 rpm for 30 minutes. The supernatant was

139

analyzed using an Agilent 1260 Infinity HPLC equipped with diode array detection (DAD) and


5


Page 6 of 21

140

Agilent Zorbax RX-C8 column (150mm x 4.6mm x 5µm). The identity of the products was

141

confirmed on an Agilent LC/MS coupled to a Bruker AmaZon X ESI-ion trap mass

142

spectrometer.

143 144 145

3.3 Catalytic activity in biological system/disruption of quorum sensing in bacteria

146 147

Method 1

148

Five µl of Vibrio harveyi (ATCC14126, a luminous strain) bacterial culture (prepared overnight)

149

was inoculated with 5 ml of Luria-Bertani broth (LB) + (LB + 2% NaCl) broth and at different

150

clay loadings (10 to 10,000 µg/mL) in the system. A non-luminescent Vibrio parahaemolyticus

151

strain was included as an additional negative control. Bacterial culture was incubated at 30oC

152

with agitation (200 rpm) for 10 h. During the culturing period, bacterial growth was monitored

153

by both optical density measurements at 600 nm (BioPhotometer, Eppendorf) and by a viable

154

bacterial cell count on agar plates. The luminescent emission was detected and quantified by a

155

luminescence detector (MiniLumat, EG &G Berthold) every hour. Blank experiments were also

156

conducted to check if LB and clay would interfere with the detection of bacterial luminescence.

157

Luminescent bacteria were added to LB at different amounts of clay, and the mixtures were

158

immediately subjected to luminescence detection, which indicated no variation in the detected

159

luminescence upon the addition of clay to the bacterial medium, signifying no interference in

160

luminescence measurement.

161

Method 2

162

Five µl of Vibrio harveyi (ATCC14126, a luminous strain) bacterial culture prepared overnight

163

was inoculated with 5 ml of LB+ broth and incubated at 30oC for 4 h. The cultured bacteria were

164

separated (pellet) by centrifugation at 4000g for 10 min. The supernatant was collected and

165

filtered through a syringe filter with a 0.45 µm pore size (PALL). The purpose of this step was to

166

harvest quorum sensing molecules in the supernatant. Five ml of the filtrate was then mixed with


6

Page 7 of 21


167

different amounts of Calibrin Z to reach a final concentration of 0.5, 1, 10 and 50 mg/ml of

168

Calibrin Z in the system. These suspensions were then incubated at 30oC for 1 h with agitation

169

(200 rpm). A blank filtrate containing no added clay was also included as a control in each

170

experiment. After incubation, the clays were separated from the filtrate by centrifugation at 5000

171

RPM, followed by filtration of the supernatant through a 0.45 µm syringe filter as described

172

above. It is expected that different amounts of clay would impact the separation of quorum

173

sensing molecules differently, thereby, delaying the bioluminescence from a culture containing

174

the least number of quorum sensing molecules. A 4 µl aliquot of a new and growing Vibrio

175

harveyi bacterial culture (not yet luminescing) was inoculated into 4 ml of above filtrates, and

176

the mixture was incubated at 30oC. The luminescent emission and the bacterial growth were

177

quantified by a luminescence detector (MiniLumat, EG &G Berthold) and the optical density at

178

600 nm (BioPhotometer, Eppendorf), respectively, at various time of the inoculation intervals.

179 180

4. Results and discussion

181

The removal of N-(3-Oxooctanoyl)-DL-homoserine lactone was studied in aqueous

182

solutions at different temperatures (5-25oC) and at varying concentrations of lactone and clay

183

amount (changing the ratio of lactone and clay), Table 1 shows one such representative data

184

collected in triplicates at clay/lactone ratio of 50 (mg/mg) at 25 oC and 30 min reaction time. The

185

concentration of the lactone in the reaction system was monitored using HPLC and LC-MS

186

instrumental techniques. The percentage removal (% R) term in Table 1 indicates the extent of

187

lactone removal from the aqueous solution. It was observed that clay catalysts degraded the

188

lactone molecule to smaller fragments on the surface acid sites on the catalyst. The structure and

189

mineralogy of Calibrin clays was described previously [11-12]. Other clays used here were

190

commercial samples and their structures and mineralogy were confirmed by standard

191

characterization methods. In the case of precipitated silica and kaolinite, the small amount of

192

lactone removal happened totally through the adsorption process only as these materials did not

193

have the required active sites/surface area for the degradation of lactone, the % lactone removal

194

was 100% due to adsorption. Seemingly, the Lewis and Bronsted acid sites on the catalyst

195

surface were active in degrading the lactone ring in the N-(3-Oxooctanoyl)-DL-homoserine

196

lactone. As an example, Table 2 shows the structures and molecular weights of the lactone


7


Page 8 of 21

197

degradation products on Calibrin Z. Although the data shown in Table 1 was collected at 25oC,

198

Calibrin clays were substantially active even at low temperature (up to 5 oC). Furthermore, there

199

existed an optimum level (amount of the solid material) to function as a catalyst, and below that

200

level the material just operated as an adsorbent. The two Calibrin products started to function as

201

catalysts at a catalyst/lactone ratio of 15 (mg/mg) and above. In fact, Calibrin Z showed near

202

100% catalytic degradation of lactone at a catalyst/lactone ratio of ~500.

203

From the percentage lactone removal shown in Table 1 the strong activity of Calibrin

204

clays for degrading the quorum sensing lactone is very clear. Generally, the surface area,

205

porosity and the surface acid sites of the clay catalyst have a stronger influence on its

206

performance in a catalytic reaction. The basic catalyst characterization data shown in Table 1

207

correlated very well with their performance in the lactone removal and degradation under the

208

given experimental conditions. The catalytic cleavage of N-(3-Oxooctanoyl)-DL-homoserine

209

lactone molecule is mostly promoted by the acid sites, the number of such sites and their

210

distribution in the solid material are crucial parameters to decide the progress of the reaction.

211

Both the Calibrin clays have higher acidity, pore volume and surface area leading to their higher

212

performance as catalysts/adsorbents in lactone removal. Silica, illite and kaolinite materials fared

213

poorly in lactone removal which was probably due to their lower surface area, pore volume and

214

weaker acidity. The N-(3-Oxooctanoyl)-DL-homoserine lactone used here is an organic

215

molecule with an amide group linked to a saturated C8 fatty acid containing an oxo group at C3

216

position (entry 6, Table 2). The molecule underwent catalytic cleavages at several positions in

217

the molecule during the degradation as evidenced from the nature of the products (Table 2)

218

formed and identified using LC-MS procedure described by Nievas et al. [14]. Some

219

polymerized products of the original AHL molecules were also observed in LC-MS of the

220

reaction mixture.

221

At the next stage, we evaluated the performance of Calibrin Z clay in disrupting

222

quorum sensing in a live/growing Vibrio harveyi bacteria. It is reported that the marine

223

bacterium Vibrio harveyi ATCC BAA-1116, for example, channels the information of three AI

224

signals into one QS cascade. Three receptors perceive these AIs, the hybrid histidine kinases

225

LuxN, Lux(P)Q and CqsS, to transduce the information to the histidine phosphotransfer (HPt)

226

protein LuxU via phosphorelay, and finally to the response regulator LuxO [15]. Recently, many


8

Page 9 of 21


227

other bacteria have been shown to control cell density-dependent functions through the excretion

228

of and response to acyl-homoserine lactone autoinducers and the quorum sensing activity of such

229

bacteria can be directly evaluated by measuring bioluminescence in bacterial cultures [16, 17].

230

Besides, Vibrio harveyi is also a common pathogen causing vibriosis—a major disease of

231

shrimps, fish, and shellfish that results in serious productivity and economic losses for

232

aquaculture industry [18, 19].

233

We used two different methods (1 and 2) to directly monitor [20, 21] the amount of

234

luminescence emitted by the bacteria as the measure of catalytic quorum quenching performance

235

of Calibrin Z. As shown in Figure 1, in method 1, Calibrin Z, at and above the inclusion level of

236

1 mg/mL in the bacterial broth, starts to reduce the overall luminescence produced by the

237

bacteria in comparison to the control (system without any added clay). In fact, at the clay

238

concentrations of 10 mg/ml, the bacterial luminescence was reduced by 55% (from the area

239

under the curves), respectively, which indicated a significant catalytic quorum quenching or

240

interference in the overall quorum sensing of Vibrio harveyi by Calibrin Z clay. There already

241

exist reports linking the bacterial population and luminescence reduction because of quorum

242

quenching using different approaches [20, 21]. Importantly, as seen from Figure 2, the bacterial

243

count during the measurement period was not reduced upon clay addition, confirming

244

accomplishment of catalytic quorum quenching by transformation/separation of quorum sensing

245

molecules or interference of quorum sensing process, and not by killing (antibacterial) of the

246

bacteria in the system. Of course, the quorum sensing in bacteria is regulated by several factors

247

and the presence of N-acylhomoserine lactones play a major role in this bacterial

248

communication; clays could also have impacted chemical transformation and disruption of the

249

overall quorum sensing process in some way or the other. The elucidation of the exact role of

250

clays in impacting bioluminescence produced because of quorum sensing in Vibrio harveyi needs

251

to be further investigated to confirm clay’s role in chemical transformations but direct

252

interference of clays in impacting quorum sensing activity of the bacteria is confirmed from this

253

study as luminescence is reduced without the loss of bacterial population in the Vibrio harveyi

254

system.

255

In method 2, the just formed quorum sensing molecules or their precursors in the Vibrio harveyi

256

bacterial culture were first separated out in the form of a supernatant by centrifugation. The

257

collected supernatant containing quorum sensing molecules was treated with different amounts


9


Page 10 of 21

258

of Calibrin Z clay and the luminescence was monitored with time. As seen in Figure 3, those

259

systems that originated from clay-treated filtrates showed reduction in luminescence after 1h of

260

growth, after which the intensity began to catch up. By 3 h of culture period, the luminescence

261

recovered to the original level of the medium control. The amount of the clay impacted

262

significantly the luminescence produced by the bacteria. Higher the clay amount, lower was the

263

relative luminescence after 1 and 2 hours of bacterial cultures. The results proved that Calibrin Z

264

clay caused a delay in the emission of luminescence by selectively interfering with the quorum

265

sensing system of Vibrio harveyi without causing any change in bacterial population (Figure 4).

266

As mentioned above, the exact role of the clay in disintegrating or transforming the quorum

267

sensing molecules needs to be further explored and currently is beyond the scope of this paper.

268

To the best of our knowledge, the disruption of quorum sensing molecules by catalysis of

269

quorum sensing molecule is not reported thus far. As clays are regulatory compliant and safe for

270

inclusion in animal feed, our approach of using clays as antagonists or catalysts to quench

271

quorum sensing and thus bacterial communication could be a very promising method for

272

controlling bacterial diseases caused to toxin secretion or other bacterial secretion resulting from

273

quorum sensing activity.

274

In conclusion, clays can be used as antagonists in quorum quenching of N-(3-

275

Oxooctanoyl)-DL-homoserine lactone, a quorum sensing molecule by means of catalytic

276

degradation of the lactone. Quorum sensing disruption activity of clays was also demonstrated in

277

Vibrio harveyi wherein delaying of the bioluminescence gene expression was observed in these

278

Gram-negative bacteria. In

279

breakdown

280

physicochemical properties such as structure, surface area, porosity, acidity, etc.

of

in vitro study, the performance of the clays towards catalytic

N-(3-Oxooctanoyl)-DL-homoserine

lactone

was

correlated

with

their

281 282 283 284 285


10

Page 11 of 21


286 287 288 289 290 291 292 293

REFERENCES

294 295

1. Nealson, K.; Platt T.; J. Hastings, Cellular Control of the Synthesis and Activity of The

296

Bacterial Luminescent System J. Bacteriol. 1970,104, 313–322.

297

2. Fuqua, C.; Greenberg, E., Listening In On Bacteria: Acyl-homoserine Lactone Signalling

298

Nat. Rev. Mol. Cell. Biol., 2002, 3, 685-695.

299

3. Williams, P., Quorum Sensing, Communication and Cross-Kingdom Signalling in the

300

Bacterial World Microbiology, 2007, 153, 3923–3938.

301

4. Yang, C.; Chen, X.; Toone, E. Methods of Reducing Virulence in Bacteria US Pat., 0322769,

302

2012.

303

5.Lade, H.; Paul, D.; Kweon, J. N-Acyl Homoserine Lactone-Mediated Quorum Sensing with

304

Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control,

305

BioMed Research International, 2014, Article ID 162584, 25-50.

306

6. Suga, H.; Igarash, J. Quorum Sensing Inhibitor US Pat. 0256369, 2010.

307

7. Du, Y.; Li, T.; Wan, Y.; Long, Q.; Liao, P. Signal Molecule-Dependent Quorum-Sensing and

308

Quorum-Quenching Enzymes in Bacteria Crit Rev Eukaryot Gene Expr. 2014, 24(2),117-32.

309

8. J.A. Ballantine. In: K. Smith (Ed.), Solid Supports and Catalysts in Organic Synthesis. Ellis

310

Harwood Ltd., Great Briton, 1992, p. 100.

311

9. Chitnis, S.; Sharma M., Reactive & Functional Polymers 32 (1997) 93-l 15.

312

10. Galvano, F.; Piva, A.; Ritieni A.; Galvano, G. Dietary Strategies to Counteract the Effects of

313

Mycotoxins: A Review J. Food Prot. 2001,1, 120-131.


11


Page 12 of 21

314

11. Naik, S.; Scholin J.; Goss, B. Stabilization of Phytase Enzyme on Montmorillonite Clay J.

315

Porous Mater, 2016, 23, 401–406.

316

12. Li, Y.; Lin, Y.; Yang, Y.; Wan X.; Chi, F. Effects of Naturally Mycotoxin-Contaminated

317

Corn on Nutrient and Energy Utilization of Ducks Fed Diets With or Without Calibrin-A J. Appl.

318

Poult. Res., 2012, 21, 806-815.

319

13. www.amlan.com

320

14. Nievas, F.; Bogino, P.; Sorroche F.; Giordano, W., Detection, Characterization, and

321

Biological Effect of Quorum-Sensing Signaling Molecules in Peanut-Nodulating Bradyrhizobia

322

Sensors 2012, 12, 2851-2873.

323

15. Lorenz, N.; Shin, J.; Jung K., Activity, Abundance, and Localization of Quorum Sensing

324

Receptors in Vibrio harveyi Front Microbiol. 2017; 8, 634, 1-10.

325

16. Bassler, B.; Greenberg, E.; Stevens, A.; Cross-species induction of luminescence in the

326

quorum-sensing bacterium Vibrio harveyi, J. Bacteriol., 1997, 179,12, 4043–4045.

327

17. Fuqua, W. C., S. C. Winans, and E. P. Greenberg. Quorum sensing in bacteria: the LuxR-

328

LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 1994, 176, 2, 269–

329

275.

330

18. Cao, J., Meighen, E.; Purification and Structural Identification of an Autoinducer for the

331

Luminescence System of Vibrio harueyi J. Biol. Chem. 1989, 36, 21670-21676.

332

19. Mine S., Boopathy, R, Effect of Organic Acids on Shrimp Pathogen, Vibrio Harveyi. Curr.

333

Microbiol., 2011, 63, 1, 1-7.

334

20. Heitzer, H.; Webb, O.; Thonnard, J.; Sayler, G. Specific and Quantitative Assessment of

335

Naphthalene and Salicylate Bioavailability by Using a Bioluminescent Catabolic Reporter

336

Bacterium, Appl. Environ. Microbiol. 1992, 18, 2. 1839-1846

337

21. Contag, C.; Contag, P.; Mullins, J.; Spilman, S.; Stevenson, D.; Benaron, O.; Photonic

338

detection of bacterial pathogens in living hosts, Molecular Microbiology (1995) 18(4), 593-603.

339 340 341 342 343 344


12

Page 13 of 21


345 346 347 348 349 350 351 352 353 354 355

List of Tables and Figures

356

Table 1. Physicochemical characteristics of clays and % Removal (R) of N-(3-Oxooctanoyl)-

357

DL-homoserine lactone at 25oC and 30 min reaction time (catalyst/lactone = 50).

358

Table 2. LC-MS data showing structures of various N-(3-Oxooctanoyl)-DL-homoserine lactone

359

degradation products obtained on Calibrin Z at 25oC and 30 min reaction time (clay/lactone =

360

50).

361

Figure 1. Bacterial luminescence from the culture treated at different concentrations of Calibrin

362

Z in the Vibrio harveyi culture; medium control, Vibrio parahaemolyticus. This data was

363

obtained through method 1.

364

Figure 2. Bacterial count measured as concentration (CFU/ml) of viable bacterial cells at each

365

time point from Vibrio harveyi culture at different concentrations of Calibrin Z; medium control,

366

Vibrio parahaemolyticus. This data was obtained through method 1.

367

Figure 3. Relative luminescent emission (%) Vibrio harveyi cultures treated with initial Vibrio

368

harveyi culture filtrates that were treated with various amounts of Calibrin Z. This data was

369

obtained through method 2. The relative luminesce is based of luminesce measure at zero

370

amount of clay.

371

Figure 4. Growth of Vibrio harveyi after mixing the initial Vibrio harveyi culture filtrates

372

treated with different concentrations of Calibrin Z. This data was obtained through method 2.


13


Page 14 of 21

373 374 375 376 377 378 379 380 381 382 Material

BETa

Pore volume,

surface area

DFTb

2

(m /g)

TPDc-NH3 acidity

%R

(150-350oC), mmol/g

(cm3/g)

Calibrin-A

141

0.32

0.025

53

Calibrin-Z

110

0.26

0.024

60

Illite

70

0.06

0.022

36

Kaolinite

32

0.04

0

5

Precipitated

340

1.0

0

25

SiO2 383

a

Brunauer–Emmett–Teller; b Density Functional Theory; c Temperature Programmed Desorption

384 385


14

Page 15 of 21


386 387 388 389

Table 1

390 391 392 393

Entry

m/z (amu)

1

86

2

102

3

114

4

141

5

157

6*

264

Proposed Structure

394 395 396 397 398 399 400 401 402 403 404 405 406


15


407 408

Page 16 of 21

*Starting (reactant) QS molecule Table 2

409 410

411 412

Figure 1

413


16

Page 17 of 21


414 415

Figure 2


17


Page 18 of 21

416 417

Figure 3

418


18

Page 19 of 21


419 420 421 422

Figure 4

423 424 425 426 427 428 429 430


19


Page 20 of 21

431

432 433 434 435

Figure : TOC Graphic


20

Page 21 of 21


436 437 438 439


21

research 1..5 - American Chemical Society

Recommend Documents