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Identification and quantification of acylated homoserine lactones in Shewanella baltica, the specific spoilage organism of Pseudosciaena crocea by ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry Yanbo Wang, Xiaoxiao Zhang, Chong Wang, Linglin Fu, Yanyao Yi, and yan zhang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Journal of Agricultural and Food Chemistry

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Identification and quantification of acylated homoserine lactones in Shewanella baltica,

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the specific spoilage organism of Pseudosciaena crocea by ultra-high-performance

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liquid chromatography coupled to triple quadrupole mass spectrometry

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Yanbo Wanga, Xiaoxiao Zhanga, Chong Wanga, Linglin Fua,*, Yanyao Yib, and Yan Zhangc

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a

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Biotechnology, Zhejiang Gongshang University, 18 Xue Zheng Street, Hangzhou 310018, China

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b

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University Ave, Madison, WI 53706, USA

Key Laboratory for Food Microbial Technology of Zhejiang Province, School of Food Science and

Department of Statistics, University of Wisconsin-Madison, 1220 Medical Sciences Center, 1300

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c

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* Corresponding author

Hebei Food Inspection and Research Institute, Shijiazhuang, 050091, China

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Address: 18 Xue Zheng Street, Hangzhou 310018, Zhejiang Province, China

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Tel.: +86-0571-28008963

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Email: [email protected] (L. Fu)

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1

ACS Paragon Plus Environment


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Abstract

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Bacteria communicate with one another using chemical signal molecules called autoinducers, and the

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most prevalent autoinducers used by Gram-negative bacteria are N-acylated homoserine lactones (AHLs).

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However, AHLs are hardly detected in Shewanella baltica, the specific spoilage organism of

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Pseudosciaena crocea. In this study, we applied ultra-high-performance liquid chromatography coupled

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to triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) to determine AHLs. This method

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enabled the quantification of up to 11 AHLs within 5.6 minutes with excellent sensitivity (ng/mL level)

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and linearity (r2 > 0.99), which further detected 9 AHLs produced by Shewanella baltica. Furthermore, by

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using biofilm formation assay and statistical analysis, the biofilm-inducing activity of AHL in Shewanella

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baltica was firstly revealed. Our results elucidated the physiological role of AHL in Shewanella baltica and

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provided a satisfactory method to detect AHLs and a statistical model to predict food spoilage

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properties.

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Keywords: N-acylated homoserine lactones (AHLs), quorum sensing, UHPLC-QqQ-MS/MS, Shewanella baltica, statistical analysis, biofilm

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Introduction

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Bacteria produce and release chemical signal molecules called autoinducers, the concentration of

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which is correlated with cell-population density, and certain bacterial physiological activities are altered

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when autoinducers reach the threshold concentration.1 This progress enables bacteria to monitor cell

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density and is therefore called quorum sensing (QS), which regulates a variety of physiological functions,

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including symbiosis, virulence, competence, conjugation, antibiotic production, spoilage activity, motility,

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sporulation, and biofilm formation.2, 3 Though a variety of classes have been reported, the most widely

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known autoinducers are N-acylated homoserine lactones (AHLs) and autoinducing peptides (AIPs) in

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Gram-negative and Gram-positive bacteria, respectively.2

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Shewanella baltica is a well-known fish spoilage bacterium, usually described as the specific spoilage

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organism (SSO) for fish stored in ice.4, 5 Unlike other Gram-negative bacteria, Shewanella spp. specifically

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produce autoinducers of AI-2 (autoinducer-2) and DKPs (diketopiperazines), rather than AHLs.3, 6-8 Due to

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the limitation of detection sensitivity, the participation of AHLs in Shewanella baltica requires further

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investigation by more sensitive approaches.

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AHLs contain a homoserine lactone ring, which is N-acylated with a fatty acyl group at the C-1 position.

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Typically, the acyl chain ranges from 4 to 18 carbons, and it may contain double bonds. AHLs often

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contain an oxo or hydroxyl substituent at the C-3 position. A typical AHL-based QS system contains an 3



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autoinducer synthase (e.g., LuxM or LuxI) and an autoinducer receptor (e.g., LuxN or LuxR). When

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binding to AHL, the receptor demonstrates transcriptional activity and regulates downstream gene

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expression, thereby exhibiting QS phenotypes.1, 9

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Identification and quantification of autoinducers may elucidate approaches to monitor, predict and

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control bacterial growth, metabolism and many other physiological functions. As a result, several

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methods have been developed for detecting autoinducers, including bioassays and chemical approaches.

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Bioassays are sensitive, reliable and cheap methods, but they are always time-consuming and can only

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identify a limited range of autoinducers.7, 10, 11 In contrast, chemical approaches, such as chromatography

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and mass spectrometry, identify a broader range of autoinducers, but these traditional chemical

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approaches are still limited in speed, sensitivity and resolution. Recently, a rapid multi-analytic method

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has been developed for the simultaneous determination of compounds based on simple extraction and

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ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry

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(UHPLC-QqQ-MS/MS).12, 13 This separation and detection technique presents several benefits in terms of

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faster analytical speed, higher sensitivity and clearer exhibition of target peaks. Some applications of this

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technique have been reported for determination of traditional Chinese medicine and pesticide

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residues.14-17

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The aim of this work was to provide a method for fast and sensitive detection of AHLs, as well as a

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statistical model to predict food spoilage properties, which could be applied in microbiological research, 4


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food inspections and many other fields. In addition, by using this method, the present study also tried to

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verify the participation of AHLs in Shewanella baltica and investigate the physiological role of AHLs in

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that strain, which shed light on the control of spoilage bacteria and the improvement of fish

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low-temperature preservation.

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Materials and Methods

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Chemicals

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N-acylhomoserine lactones: N-butanoyl-homoserine lactone (C4-HSL), N-hexanoyl-homoserine

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lactone (C6-HSL), N-heptanoyl-homoserine lactone (C7-HSL), N-octanoyl-homoserine lactone (C8-HSL),

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N-decanoyl-homoserine

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N-tetradecanoyl-homoserine lactone (C14-HSL), N-(3-Oxooctanoyl)-homoserine lactone (oxo-C8-HSL),

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N-(3-Oxodecanoyl)-homoserine lactone (oxo-C10-HSL), N-(3-Oxo-dodecanoyl)-homoserine lactone

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(oxo-C12-HSL) and N-(3-Oxo-tetradecanoyl)-homoserine lactone (oxo-C14-HSL) standards (purity 95% or

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higher) were obtained from Sigma (St. Louis, MO). Acetonitrile, and methanol were chromatography

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grade. Ultrapure water was prepared by using a Milli-Q water purification system. All solutions were

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stored in a refrigerator at 4 °C.

lactone

(C10-HSL),

N-dodecanoyl-homoserine

5


lactone

(C12-HSL),


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Bacterial strains and culture conditions

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Shewanella baltica strains used in this study were isolated from large yellow croaker. Pseudomonas

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aeruginosa PAO1, which has been demonstrated to produce the autoinducers N-butanoyl homoserine

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lactone (C4-HSL) and N-(3-oxo-dodecanoyl)-homoserine lactone (3-oxo-C12-HSL), was generously

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provided by Professor Yan Zhang at the Food Quality Inspection and Quarantine Bureau.18 P. aeruginosa

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PAO1 strain was used as positive control in the assays.

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Extraction of AHLs from cultures

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S. baltica strains were cultured in LB at 30°C for 12 h, 24 h, or 36 h. The P. aeruginosa PAO1 strain was

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also inoculated in LB and then incubated at 37°C for 12 h, 24 h, or 36 h. Three parallel samples were

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taken from the same culture medium every time. Cell-free supernatants of these cultures were prepared

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by centrifugation for 4 min (10000 g, 4°C). Ten-milliliter supernatants were extracted with equivalent

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volume of acidified (0.1% formic acid) ethyl acetate. The mixture was shaken vigorously for 30 s and the

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phases were allowed to separate. The shaking was repeated three times before the ethyl

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acetate-containing fraction was removed and another 10 ml fraction was added. The whole extraction

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process was repeated three times. The combined ethyl acetate fractions were evaporated under

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nitrogen flow to dryness and reconstituted in 2 ml HPLC-grade acidified ethyl acetate, sterile filtered

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(0.22 μm), and later transferred to an HPLC vial and stored at -20°C. 6


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Instruments and chromatography conditions

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Liquid chromatography detection was achieved using a TSQ Quantiva Discovery mass spectrometer

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system (Thermo Fisher Scientific) equipped with an electrospray interface. An XBridge BEH C18 column

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(2.1×100 mm, 2.5 μm) (Waters, USA) was operated at a flow rate of 0.1 mL/min. The mobile phase

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consisted of water (solvent A) and methanol (solvent B).

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The gradient elution profile was as follows: isocratic for 1.5 min, a linear gradient from 30% to 95% B

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over 1 min followed by 95% B for 3 min, returned to initial condition in 0.8 min and maintained for 0.7

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min, at a flow rate of 0.3 ml/min. The column temperature was 30°C. A sample volume of 1 μL was

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injected in all experiments. Electrospray in negative mode was used and the spray voltage was set at 3.0

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kV. The capillary temperature was set to 350°C. The auxiliary and the sheath gases were normal nitrogen.

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The collision gas was high-purity argon at a pressure of 1.5×10-3 torr in the collision cell.

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Calibration and validation

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Seven-point calibration lines were prepared for each analyte by making a series of matrix-matched

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calibration solutions in the range from 5 to 200 μg/L for each standard. The calibration curves were

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obtained by plotting the peak areas of the quantitative ion transition against the analyte concentrations

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with regression analysis. The linearity was expressed as the correlation coefficient. The average

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determination coefficient (r2) was >0.99 for all compounds except C12-HSL. The limits of detection were 7



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calculated by linear regression method based on the calibration curves, and results were < 1 ng/mL for

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all compounds.19

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Biofilm culture, quantification and statistical analysis

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The biofilm detection was performed following previous publications.7, 20 Twelve S. baltica strains (as

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mentioned in Figure. 2) were used to inoculate fresh LB culture at 30°C until the optical density at 600

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nm reached 1.0, and then they were diluted 1:10 in LB medium. For each S. baltica strain, 3 × 36 × 200

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μL medium was equally transferred into 108 wells of three polystyrene flat-bottom 96-well microtiter

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plates (36 wells per plate), and incubated at 30°C for 2 h. Next, the three plates were treated in parallel:

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the 36 wells that contained each S. baltica strain were randomly equally divided into 12 groups (i.e.,

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triplicated), and 10 μM of 11 different AHL standards (as mentioned in Table 1) were randomly added to

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11 groups thereafter; the same volume of ddH2O was added to the left group as a negative control. Each

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plate was further incubated at 30°C for the indicated time (12, 24 or 36 h) without agitation. The culture

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was then blotted out, and the wells were carefully washed three times with sterile phosphate-buffered

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saline (PBS) to remove non-adherent cells.

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For fixation of the biofilms, 100 μL 99% methanol was added (15 min), after which supernatants were

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removed and the plates were air-dried. Then, 50 μL of crystal violet (CV) solution was added to each well.

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After 20 min, the excess CV was removed by washing the plates with PBS, and the plates were air-dried 8


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again. The formation of biofilms was observed with an inverted microscope. Finally, bound CV was

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released by adding 150 μL 33% acetic acid and the absorbance was measured at 600 nm. All steps were

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performed at room temperature.

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The experiment was a randomized complete block design (RCBD) and every group filled with AHLs was

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treated as a block. The corresponding linear regression model was applied to investigate how AHLs affect

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the variation in biofilm formation (OD600 value) during different time periods. Tukey's HSD (honest

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significant difference) test was applied to compare the pairwise effect of AHLs (including the negative

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control set), especially between the AHL analogues with or without a carbonyl group. Any pair of AHLs

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with a P-value < 0.05 had a significantly different effect on the variation of OD600. The 95% level

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confidence interval for difference of variation of OD600 for every pair of AHLs was calculated. Three

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models were built with data collected from 12 h, 24 h and 36 h. The model fit well with normality, and

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transformation was not needed based on the diagnosis that was performed with the Q-Q norm and

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residual plots. Calculations were performed with R-software version 3.2.3 (12/10/2015).

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Results

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Identification of AHLs by UHPLC-QqQ-MS/MS

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To identify and quantify AHLs, we applied a detection method based on ultra-high-performance liquid

9



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chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). The

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molecular structures of the 11 studied AHLs differ in the length of the acyl side-chain and the existence

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of an oxo substituent on the third carbon. This leads to variations in the molecular polarity and allows

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the separation of AHLs by reversed-phase HPLC. We used a 2.1 × 100 mm I.D. C18 column with a mobile

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phase of methanol-water, which presents a low background signal in MS detection, and elution was

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performed at a flow rate of 0.1 mL/min. We chose methanol-water as the mobile phase because when

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compared with acetonitrile-water, the earliest peaks eluted by methanol-water were wider but more

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resolved with higher signal intensities (data not shown).

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Using the experimental conditions described above, all AHL standards were completely eluted

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between 1.3 and 5.66 min, providing a rapid separation with a total run time of 7 min, including

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re-equilibration time. The analogues of a given type of structure (N-acyl- or 3-oxo-acyl-HSLs) were well

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separated. Though some N-acyl-HSLs and 3-oxo-acyl-HSLs were co-eluted (for example, C10-HSL and

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3-oxo-C12-HSL, C12-HSL and 3-oxo-C14-HSL), the specificity of the ESI-MS-MS signals allowed us to

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distinguish the co-eluted compounds by plotting specific ion chromatograms of [M+H]+ and [M+H-101]+

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(Table 1).

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Although the relative abundances of the product ions are different between N-acyl- and

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3-oxo-acyl-HSLs, the major ions were m/z = 102 and [M+H-101]+, while the relative abundance of the

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other ions were under 5%. Figure. 1 illustrates these observations. 10


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For quantification based on peak area measurements, the ion at m/z = 102 was selected because of

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the specificity and sensitivity to the AHLs and a better signal-to-noise ratio, and calibration curves and

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regression analysis were applied for calculation. The results showed that the limits of detection were < 1

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ng/mL for all compounds, and the linearity (presented as determination coefficient, r2) was >0.99 for all

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compounds except C12-HSL.

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Identification of AHLs produced by Shewanella baltica

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In our previous work, we have isolated several Shewanella baltica strains from large yellow croaker

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(Pseudosciaena crocea), which were identified as the SSOs secreting QS molecules of DKPs.3 To

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investigate whether Shewanella baltica produces AHLs, we studied the stationary phase culture

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supernatant in LB medium by UHPLC-QqQ-MS/MS and quantified AHLs with external calibration. As a

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positive control, the two main AHL signal molecules of P. aeruginosa PAO1, C4-HSL and 3-oxo-C12-HSL,

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were detected, as already noted by others, which confirms the reliability of the method in the detection

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of AHLs.21 As shown in Figure. 2, a total of 12 of the 18 S. baltica strains were identified as AHL

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producers. Most strains produced more than one AHL, with C10-HSL being the most prominent. C8-HSL,

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3-oxo-C10-HSL and 3-oxo-C12-HSL were also detected in most strains. However, C7-HSL and C14-HSL

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were not detected in any strain.

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Next, we investigated AHLs production during the whole growth period of Shewanella baltica. S. 11



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baltica 32 produced very high levels of C12-HSL in the lag phase, whereas this signal molecule was not

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detected in the following phases, implying that C12-HSL only played a role in lag phase. All of the other

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AHLs accumulated in the log phase and gradually decreased afterwards (Figure. 3). These data

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demonstrated that production of AHL signaling molecules in S. baltica is growth phase dependent, and

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different AHLs may take part in different phases, although the majority are present in the log phase.

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AHLs induce biofilm formation in Shewanella baltica

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To further verify the role of AHLs in the QS system of S. baltica, we introduced AHLs into a bioassay

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based on biofilm formation, where the activation of QS signaling would lead to biofilm formation and

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thus be quantified.7,

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concentration of 10 μM by referring to previous studies.7, 20, 23 In addition, the AHL-treated groups were

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compared with control group (without AHL treatment) firstly to counteract the effect of endogenous

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AHLs.

22

To compare the AHLs in parallel, all the AHLs were applied with the same

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According to statistical analyses, our results demonstrated that all AHLs more or less have an influence

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on static biofilm formation with the only exception of C7-HSL, which was never detected in any S. baltica

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strains; C10-HSL was the most prominent (Figure. 4A). Growth curves demonstrated that AHLs did not

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regulate S. baltica growth, indicating that biofilm formation was directly regulated by AHLs, not as a side

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effect of cell density differences (Figure. 4B). We can also see that the enhancement of biofilm by most 12


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AHLs increased mainly at the log and decline phases, staying constant at the stationary phase, suggesting

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that AHLs primarily regulate biofilm formation in those two culture phases.

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The effect of C10-HSL on biofilm formation was further studied by morphology. S. baltica were

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cultured in the presence or absence of C10-HSL for the indicated time periods followed by CV staining

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and microscopic observation. As demonstrated in Figure. 4C, at the early stages up to 12 h, the effect of

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C10-HSL was minimal: both groups displayed a sparse single layer that gradually grew denser. However,

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after 24 h, the C10-HSL treated group formed a slight thicker biofilm than the control group. The

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difference became most apparent at 36 h, when the C10-HSL treated group grew denser everywhere

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and formed a homogenous layer with thicker biofilms, while the control group demonstrated a thinner

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layer with loosely bound cells that covered the surface unevenly. The biofilm of the C10-HSL treated

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group reached an apex at that time and then exhibited a porous appearance and went through the

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detachment phase at 48 h, while the control group was still developing biofilms.

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To investigate the effect of carbonyl groups (3-oxo-) on biofilm formation, we compared the results

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between two AHL analogues with acyl chains of the same length but differences in the existence of the

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carbonyl functional group. No statistically significant difference was detected in those analogue pairs

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during all time periods based on Tukey's HSD test, except for the pair of C8-HSL and 3-oxo-C8-HSL at 24 h.

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However, this pair did not have a practical difference because the 95% confidence interval for the

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difference of OD600 was (-0.07, -0.01), which was negligible when compared with 0.34, the smallest 13



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variation of OD600 affected by AHLs at 24 h. In conclusion, the carbonyl group was not related to the

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enhancement of biofilm formation by AHLs (Table 2).

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Discussion

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AHLs have been identified in many Gram-negative pathogenic and symbiotic bacteria and recognized

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as a primary way to regulate gene expression in response to cell density, which is known as the QS

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system. Due to the physiological importance of QS system in bacteria, it is valuable to identify and

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quantify AHLs in a fast and sensitive way.

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The detection range and sensitivity of the AHL monitoring system determined which AHLs could be

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detected, and it certainly should be noted that an AHL signal cannot be detected unless the

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concentration is higher than the detection threshold. The most widely used monitoring systems are

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bioassays and chemical approaches. Bioassays are reliable and sensitive, but they cannot provide

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information about a wide or complete spectrum of AHL compounds. Traditional chemical approaches

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such as liquid chromatography (LC), mass spectrometry (MS) and gas chromatography (GC) can detect a

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wider spectrum of compounds, but they are somewhat limited in speed, sensitivity and resolution.24-27

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In recent years, several advanced approaches have been developed to achieve better identification of

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AHLs. By adapting an aromatics degrader Pseudomonas aeruginosa into a biosensor, low concentration

14


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of N-butyryl homoserine lactone can be detected, with an extremely low limit of detection (LOD) of 1.3

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nM (0.2 ng/mL) and a limit of quantification (LOQ) of 0.11 μM (19 ng/mL).11 While by using a green

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fluorescent protein (GFP) reporter, 9 AHLs could be detected with the LOD of about 20 nM (4.8 ng/mL

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for 3-oxo-C8-HSL), but this method cannot distinguish these AHLs from each other.28 On the other hand,

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Li et al. had developed a liquid-liquid extraction and HPLC-MS based method to quantify AHLs in upflow

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microaerobic sludge reactors samples, which identified 9 AHLS with the LOQ of 0.5 ng/mL within 6.5

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minutes.29 In addition, by using an optimized UPLC-MS/MS method, the LOQ of 1.33 ng/g was obtained

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and 11 AHLs was detected from wastewater and biofilm.30 When derivatized with Girards reagent T, the

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detection sensitivity of 3-oxo AHLs could be enhanced up to 60000 times by MALDI-MS, and the LOQ

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was lowered to 2.5 fmol (0.75 ng/mL for 3-oxo-C12-HSL with 1 μL sample volume).31 Moreover, novel

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technologies such as electrochemical sensor was also applied to AHLs detection.32

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In the present study, we developed an approach based on simple extraction and

247

ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry

248

(UHPLC-QqQ-MS/MS). The powerful and selective method enables the detection and quantification of

249

AHLs in complex matrices without fastidious sample purification, and it shortens the retention time (to

250

1.3–5.66 min) compared with previous studies.33, 34 Though may not be extraordinary in all the aspects,

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this method is satisfied as it allows the quantification of the most complex and challenging samples with

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relative high throughput (up to 11 AHLs), excellent sensitivity (ng/mL level) and linearity (r2 > 0.99). 15



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However, novel methods with higher efficiency, better sensitivity and broader spectrum (such as the

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ability to determine N-3-hydroxyacyl-HSL, DKPs, AIPs and other autoinducers at the same time) are still

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in urgent need.

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Shewanella baltica is known as a specific spoilage organism (SSO) for fish stored in ice. Albeit as a

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Gram-negative bacteria, AI-2 and DKPs rather than AHLs were identified as autoinducers of the strain.

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Several studies have reported that AHLs could not be detected in Shewanella spp., whereas several

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others pointed to a contrary conclusion.7, 8, 10, 35, 36 The seemingly irreconcilable results may due to the

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relative low concentration of AHLs produced by this strain. As shown in Figure. 2 and Figure. 3, high

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concentration of AHLs were only produced by few Shewanella baltica strains in specified growth phases,

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therefore, AHLs can hardly be detected under unfavorable conditions, especially by a less sensitive

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method.

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Notably, when applied our method to Shewanella baltica strains, nine AHLs produced by this marine

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bacterium was found, which is much more than that have ever been reported.10, 35, 36 This discovery

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further demonstrated the high sensitivity of UHPLC-QqQ-MS/MS method used in this study, and

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enriched our knowledge on Shewanella baltica as well as the quorum sensing system, which shed light

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on the control of that spoilage bacteria and further improvement of fish low-temperature preservation.

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Due to the difficulty in detection, the physiological role of AHLs in Shewanella baltica was poorly

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understood. It has been reported that in Shewanella sp., AHLs are produced in the late exponential 16


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phase, but rapidly degraded in the stationary phase, indicating a critical role of AHLs in late exponential

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phase.36 Our results also confirmed the conclusion as high concentration of most AHLs can only be

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detected in exponential phase (Figure. 3). Another study showed that AHLs produced by Pseudomonas

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fluorescens inhibit the biofilm formation and trimethylamine (TMA) production of Shewanella baltica,

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and AHLs may competitively inhibit the activity of DKPs, another kind of QS autoinducers in Shewanella

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balticaI.23 However, our work showed a seemingly contrary result that AHLs assist biofilm formation in

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Shewanella baltica (Figure. 4). Since our results showed that biofilm start to detach after too long time

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culture with AHLs stimulation (Figure. 4C, 48h), quantifying biofilm at wrong culture period may lead to

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the false appearance that AHLs exert negative effects on the biofilm formation. On the other hand, AHLs

280

production in exponential phase and degradation in stationary phase suggest different or even opposite

281

functions of AHLs under different conditions in Shewanella spp., which may also lead to variable

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conclusions in different studies. Additionally, the present of endogenous AHLs may also complicate the

283

results. More meticulous studies, such as the application of QS-related gene knock-out strains, are

284

required to confirm the conclusion and reveal the underlying mechanism.

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Since C10-HSL was found in most strains with a relatively higher concentration compared to other

286

AHLs, we concluded that it is the most prominent AHL in Shewanella baltica. C10-HSL significantly

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enhanced the capability of biofilm formation, indicating the importance in Shewanella baltica. Thus,

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C10-HSL could be a potential inhibitory target and examination index for the QS system in Shewanella 17



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baltica, which may provide new ideas to improve fish product preservation at low temperature, where

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Shewanella baltica may act as the SSO. Further studies are required to clarify the detailed mechanism of

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C10-HSL-regulated QS system in Shewanella baltica for more promising applications.

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The signaling molecules C7-HSL and C14-HSL cannot be detected in any Shewanella baltica strains;

293

therefore, it was not surprising that C7-HSL has no effect on biofilm formation in Shewanella baltica.

294

However, C14-HSL enhanced biofilm formation. As a result, we propose that C14-HSL is a signaling

295

molecule among different species, indicating that C14-HSL could be produced by other bacteria species

296

and then received by Shewanella baltica to generate a specific response, similar with that has been

297

reported by Zhao et al.23

298

Most AHLs were accumulated in log phase, correlating with the ability to enhance biofilm formation,

299

which was mainly in the log phase; C12-HSL was the only exception. C12-HSL was produced in the lag

300

phase, and the concentration immediately decreased afterwards, but it still enhanced biofilm formation

301

in log phase. We explain this asynchronous effect as an off target effect, that is, due to the structure

302

similarity of AHLs, C12-HSL can bind to the receptors of other AHLs and regulate the downstream

303

signaling that leads to biofilm formation. However, in physiological conditions, C12-HSL is only present in

304

the lag phase and regulates genes expression for some other QS phenotypes rather than biofilm

305

formation, which had not been evaluated yet. Further research is required to uncover the physiological

306

role of C12-HSL in Shewanella baltica, which may reveal a novel function of the QS system in the lag 18


Page 19 of 36

307


phase.

308

The existence of carbonyl groups did not show significant effects on biofilm formation, but we still

309

cannot conclude that carbonyl groups are dispensable. The carbonyl groups may endow AHLs with

310

different properties in physiological functions other than biofilm formation. Alternatively, AHL analogues

311

with different carbonyl groups may be produced relative to different situations and stages. C12-HSL, for

312

instance, is produced in the lag phase, while 3-oxo-C12-HSL is produced in the log phase, demonstrating

313

that they respond to different stimulations and work in different ways, and thus accommodate different

314

circumstances.

315

Overall, our study provided a simple, fast, sensitive and broad-spectrum approach to determine AHLs,

316

which produces accurate data and thus makes it possible to predict food spoilage properties, such as

317

biofilm formation, using statistical models. This technology has broad application prospects in

318

microbiology research, food inspection and fermentation engineering. Armed with this method, we

319

quantified up to 9 AHLs in fish low-temperature preservation SSO Shewanella baltica and firstly revealed

320

their physiological role as biofilm enhancer. These results shed light on the control of spoilage bacteria

321

and the improvement of fish low-temperature preservation.

19



322

323

Page 20 of 36

Abbreviations used

AHL,

N-acylated

homoserine

lactones;

HSL,

homoserine

lactones;

UHPLC-QqQ-MS/MS,

324

ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry;

325

QS, quorum sensing; SSO, specific spoilage organism; RCBD, randomized complete block design; HSD,

326

honest significant difference; RT, retention time; NL, normalized intensity level; LOD, low limit of

327

detection; LOQ, limit of quantification

328

Acknowledgments

329

This study was financially supported by the National Natural Science Foundation of China [grant

330

numbers 31571913, 31571770] and the Zhejiang Provincial Natural Science Foundation of China [grant

331

numbers LZ15C200001, LY14C200001].

332

333

Conflict of interest

The authors declare that they have no conflicts of interest.

334

20


Page 21 of 36


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335

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34. Ortori, C. A.; Dubern, J. F.; Chhabra, S. R.; Camara, M.; Hardie, K.; Williams, P.; Barrett,

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36. Tait, K.; Williamson, H.; Atkinson, S.; Williams, P.; Camara, M.; Joint, I., Turnover of

458

quorum sensing signal molecules modulates cross-kingdom signalling. Environ Microbiol 2009, 11, 1792-1802.

459 27



460

28


Page 28 of 36

Page 29 of 36

461


Figure captions

462

Figure. 1. Full-scan MS-MS spectra of AHLs. N-hexanoyl-homoserine lactone (C6-HSL) (A) and

463

3-oxo-decanoyl-homoserine lactone (3-oxo-C10-HSL) (B) were scanned by MS-MS, and the major ions

464

were shown.

465 466

Figure. 2. AHLs produced by Shewanella baltica. Stationary phase culture supernatants of 12

467

Shewanella baltica strains were measured by UHPLC-QqQ-MS/MS with external calibration.

468 469

Figure. 3. Quantification of AHLs in different culture phases. The culture supernatant of Shewanella

470

baltica 32 strains in each growth phase was measured by UHPLC-QqQ-MS/MS.

471 472

Figure. 4. AHLs induced biofilm formation in Shewanella baltica. (A) Biofilm formation at each indicated

473

incubation time point (12 h, 24 h, or 36 h) was quantified by CV staining and spectrophotometry at 600

474

nm. Differences between experimental (treated with indicated AHL) and control groups (without AHL

475

treatment) were calculated and displayed as ΔOD600. (B) Growth curve of Shewanella baltica treated

476

with or without C10-HSL. Cell density was directly measured by spectrophotometry at 600 nm. (C) The

477

Shewanella baltica strain was cultured in the presence (lower panel) or absence (higher panel) of 29



478

479

Page 30 of 36

C10-HSL for indicated time periods. Biofilm was stained by CV and observed by microscope.

Tables

480

Table 1. Selected precursor and product ion m/z values, retention times and mass spectrometer

481

parameters used for AHLs analysis. compound

RT1

NL2

precursor ion (m/z)

product ion (m/z)

C4 -HSL

1.30

1.40E6

172.095

84.1, 102.1

C6-HSL

3.89

4.44E6

200.185

74.2, 99.2, 102.1

3-oxo-C8-HSL

4.05

4.78E6

242.085

102.0, 141.3

C7-HSL

4.21

4.78E6

214.295

102.2

C8-HSL

4.41

4.87E6

228.230

102.1

3-oxo-C10-HSL

4.46

5.18E6

270.085

84.2, 102.2, 169.1

C10-HSL

4.74

4.62E6

256.180

102.2

3-oxo-C12-HSL

4.78

7.38E6

298.290

102.2, 197.2

C12-HSL

5.18

9.09E4

284.235

102.2

3-oxo-C14-HSL

5.19

4.77E6

326.285

95.2, 102.2, 225.2

C14-HSL

5.66

3.43E6

312.235

102.2

30


Page 31 of 36


482

1

RT: retention time, minutes.

483

2

NL: normalized intensity level, counts per second.

484 485

Table 2. Shewanella baltica 100 was treated with AHL analogues with or without carbonyl groups

486

(3-oxo-), and biofilm formation was measured by CV staining and spectrophotometry at 600 nm. C8-HSL vs.

C10-HSL vs.

C12-HSL vs.

C14-HSL vs.

time

3-oxo-C8-HSL

3-oxo-C10-HSL

3-oxo-C12-HSL

3-oxo-C14-HSL

point

conf.Int 1

(ΔOD600)

P-value2

conf.Int (ΔOD600)

P-value

conf.Int (ΔOD600)

P-value

conf.Int (ΔOD600)

P-value

12 h

(-0.04, 0.02)

0.995

(-0.01, 0.06)

0.417

(-0.06, 0.01)

0.414

(-0.03, 0.04)

1

24 h

(-0.07, -0.01)

0.001∗∗∗

(-0.02, 0.04)

1

(-0.06, 0.00)

0.06

(-0.04, 0.02)

1

36 h

(-0.10, 0.01)

0.276

(-0.04, 0.06)

1

(-0.07, 0.04)

0.998

(-0.08, 0.03)

0.938

487

1

488

treatment pairs.

489

2

490

between two AHLs, in terms of Tukey’s HSD test.

491

***

conf.Int (ΔOD600): 95% confidence interval for the variation of OD600 between indicated AHL

P-value: calculated probability. P-value < 0.05 indicates significant different effect on biofilm formation

P-value < 0.001.

492

31



493

Figure 1

494

32


Page 32 of 36

Page 33 of 36

495 496


Figure 2

33



497

Figure 3

498

34


Page 34 of 36

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499


Figure 4

500

35



501

For Table of Contents Only

502 503

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Identification and Quantification of Acylated ... - ACS Publications

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