Purification, Characterization, and Mode of Action of Plantaricin GZ1

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Food Safety and Toxicology

Purification, Characterization, and Mode of Action of Plantaricin GZ1-27, a Novel Bacteriocin against Bacillus cereus Hechao Du, Jie Yang, Zhaoxin Lu, Xiaomei Bie, Haizhen Zhao, Chong Zhang, and Fengxia Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01124 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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Journal of Agricultural and Food Chemistry

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Purification, Characterization, and Mode of Action of

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Plantaricin GZ1-27, a Novel Bacteriocin against Bacillus cereus

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Hechao Du, Jie Yang, Xiaohong Lu, Zhaoxin Lu, XiaomeiBie, Haizhen Zhao, Chong Zhang,

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Fengxia Lu*

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College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang,

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Nanjing 210095, China

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* Correspondence:

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Corresponding author

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Tel: +86-2584395963;

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Fax: +86-2584395963;

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E-mail:[email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Bacillus cereus is an opportunistic pathogen that causes foodborne diseases.

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We isolated a novel bacteriocin, designated plantaricin GZ1-27, and elucidated its mode of

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action against B. cereus. Plantaricin GZ1-27 was purified using ammonium sulfate

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precipitation, gel-filtration chromatography, and RP-HPLC. MALDI-TOF/MS revealed that

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its molecular mass was 975 Da, and Q-TOF-MS/MS analysis predicted the amino acid

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sequence as VSGPAGPPGTH. Plantaricin GZ1-27 showed thermostability and pH stability.

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The antibacterial mechanism was investigated using flow cytometry, confocal laser-scanning

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microscopy, scanning and transmission electron microscopy, and RT-PCR, which revealed

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that GZ1-27 increased cell membrane permeability, triggered K+ leakage and pore formation,

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damaged cell membrane integrity, altered cell morphology and intracellular organization, and

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reduced the expression of genes related to cytotoxin production, peptidoglycan synthesis, and

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cell division. These results suggest that plantaricin GZ1-27 effectively inhibits B. cereus at

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both the cellular and molecular levels, and is a potential natural food preservative targeting B.

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cereus.

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KEYWORDS: Bacillus cereus, plantaricin, antibacterial mechanism, natural food

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preservative

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INTRODUCTION

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Bacillus cereus is a widely recognized opportunistic pathogen responsible for a vast

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range of food-spoilage and food-poisoning cases.1 In China, B. cereus ranks second as the

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causal pathogen of foodborne disease, accounting for 14.6% of cases. Depending on the toxin

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produced by the pathogen, ingestion of B. cereus-contaminated food can lead to two major

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types of gastrointestinal disease: emetic syndromes and diarrheal disease.2 Also, it’s related to

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many serious infections like endophthalmitis and septicemia, as well as food-related

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fatalities.3 Therefore, agents that could prevent B. cereus contamination are urgently required

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in the food industry. However, since the potential safety of chemical preservatives is a major

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concern among consumers, there is considerable demand for investigating natural and safe

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food preservatives as alternatives to chemical agents.

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Bacteriocins are ribosomally synthesized low-molecular-weight peptides or proteins.

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One of the most important benefits is their low oral toxicity to the host. This makes them

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potentially fit for use as bio-preservatives in the food industry.4 On the basis of the new

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revised classification system put forward by Cotter et al.,5 bacteriocins are divided into two

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major categories: Class I, lanthionine-containing lantibiotics (M < 5 kDa), and Class II,

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non-lanthionine-containing bacteriocins (M < 10 kDa). Class II bacteriocins are further

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subdivided into four subclasses: Class IIa (pediocin-like), Class IIb (two-peptide), Class IIc

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(cyclic), and Class IId (non-pediocin single linear). Although many bacteriocins only inhibit

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Gram-positive bacteria is noteworthy, some bacteriocins exhibiting broad-spectrum

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antimicrobial activity against both Gram-negative and Gram-positive bacteria have also been

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reported, such as sakacin C2,6 ent35-MccV,7 and lactocin MXJ 32A.8 3



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Accumulating evidence indicates that bacteriocins are effective in controlling various

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foodborne pathogens and inhibiting food spoilage, including Staphylococcus spp.,

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Streptococcus spp., Listeria monocytogenes, Brochothrix thermosphacta, and Lactobacillus

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curvatus.9 In addition, bacteriocins/cultures have been well documented to protect diverse

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types of foods against contamination such as fermented dairy products, bakery products and

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ingredients, alcoholic beverages, meat, fruit and vegetables, and seafood. For example,

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Staphylococcus aureus viable counts obviously decreased in the range of 1.8-2.7 log units

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with the presence of enterocin AS-48 in pumpkin confiture stored at 10 °C.10 Among the

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known bacteriocins, plantaricins, which are produced by Lactobacillus plantarum, have

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attracted substantial research attention because of their structural diversity and high

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antimicrobial activity. More than 20 kinds of plantaricins have been identified to date, which

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are distributed in every class of bacteriocins and range in size from 1-10 kDa. Some

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plantaricins have been reported as candidate natural food antiseptic agents in the food

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industry. For instance, plantaricin ZJ008,11 a newly identified bacteriocin produced by L.

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plantarum ZJ008, has been studied to control the growth of Escherichia coli, Vibrio

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parahaemolyticus and Staphylococcus spp..

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Despite this wide range of bacteriocins identified, their mechanisms of action against

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bacteria remain elusive, except in a few Class I and IIa bacteriocins. Nisin, a typical Class I

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lantibiotic, is considered to act through pore formation by using lipid II as the docking

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molecule and by inhibiting peptidoglycan biosynthesis.12 And classical Class IIa bacteriocins,

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Pediocin PA-1/AcH, induces ion-selective pores of target cell membrane that cause the

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dissipation of intracellular ATP and depletion of the proton motive force.13 Although previous 4


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studies have provided a basis for understanding the action of bacteriocins, the specific

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mechanisms of action for bacteriocins need to be further elucidated, both at the cellular and

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molecular levels.

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Numerous bacteriocins have been purified and characterized to date; however, only nisin

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and pediocin PA-1 are currently commercially available in the USA and Europe, 5 and few

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studies have focused on B. cereus-targeting bacteriocins. Thus, in order to best inhibit the

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growth of B. cereus in food and develop more antibacterial products, abundant information

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on B. cereus-targeting bacteriocins need to be explored. In this study, we identified L.

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plantarum GZ1-27, isolated from a traditional Dong-nationality kipper that showed

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broad-spectrum antimicrobial activity and inhibited both bacteria of primary spoilage and

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pathogenic bacteria. We further purified the bacteriocin produced by L. plantarum GZ1-27

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toward elucidating its cellular and molecular mechanisms against B. cereus.

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MATERIALS AND METHODS

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Bacterial Strains and Media. Samples were collected from traditional kipper of Dong

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nationality in Qiandongnan Miao and Dong Autonomous Prefecture, China. Lactic acid

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bacteria (LAB) strains were statically cultivated in De Man Rogosa Sharpe (MRS) broth at 30

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°C. All indicator bacteria strains were cultured in Luria-Bertani (LB) broth at 37 °C, while the

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fungal strains were grown in potato dextrose agar at 30 °C.

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Screening for the Bacteriocin Producers. The screening of bacteriocin-producing

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LABs was performed according to antibacterial activity which was measured by the agar well

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diffusion method using Escherichia coil ATCC 25922 and B. cereus AS 1.1846 as indicator 5



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strains.14 In brief, 1 mL of culture (about 108 cells) was seeded into 100 mL of LB agar (1.5%,

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w/v) at 50 °C. The culture was then poured into a sterile 10-cm-diameter plate and dried.

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Every single colony isolated from the samples was inoculated into 10 mL of MRS medium

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and cultured at 30 °C for 48 h. To produce the cell-free supernatant (CFS), the supernatants

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were collected by centrifugated at 10,000 ×g for 10 min (4 °C), and then filtered using a

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0.22-µm pore-size water filter. Thereafter, 50 µL CFSs were added into per 5-mm-diameter

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well. The plate was incubated for 12 h at 37 °C and the antimicrobial activity was measured

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on the basis of the diameter of the inhibition zone.15

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After inoculating the positive strains into 100 mL of MRS, they were further screened for

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antibacterial activity using more indicator strains (Gram-positive strains: Bacillus pumilus, B.

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thermosphacta, S. aureus, Clostridium sporogenes, and L. monocytogenes; Gram-negative

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strains: Aeromonas hydrophila, E. coli, Pseudomonas fluorescens, and Salmonella enteritidis).

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The pH values of CFSs were adjusted to 5.5 by 5 M NaOH.

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Identification of L. plantarum GZ1-27. The bacteriocin-producing strain GZ1-27 was

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identified according to morphological, biochemical and physiological characteristics, and

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genotypic identification. One pair of commonly used 16S rRNA gene primers (27F, 1492R)

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were used for PCR amplification.16 Then the purified PCR products were sequenced by

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Sangon Biotech Co., Ltd.. The BLASTN program was used to compare the DNA sequence.

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The phylogenic tree was constructed with Mega 7.0 to determine the evolutionary

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relationship of strain GZ1-27.

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Purification of Plantaricin GZ1-27. The plantaricin GZ1-27 was purified using

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ammonium sulfate precipitation, Sephadex G-50 chromatography, and reverse-phase HPLC

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(RP-HPLC). 30 mL overnight L. plantarum GZ1-27 culture was added into 1 L of MRS and

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statically cultured at 30°C for 36 h to produce the bacteriocin. After centrifugation at 10,000

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×g for 30 min (4 °C), the CFS was concentrated to one-third of the original volume using a

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rotary evaporator. Next, ammonium sulfate was gently added to the concentrated CFS until

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reaching 50% (v/v) saturation, and, after stirring overnight at 4 °C, the precipitate was

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collected and resuspended in 20 mM disodium hydrogen phosphate-citric acid buffer (pH 3.8,

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pH of the fermentation supernatant).

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The active sample was loaded onto Sephadex G-50 columns (80 × 2.0 cm) and eluted at

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a flow rate of 0.5 mL/min using disodium hydrogen phosphate-citric acid buffer. After

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collection with automatic collector, the highest active fraction was scanned to determine the

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maximal absorption wavelength. The fraction was then precipitated by mixing with methanol

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(1:1, v/v) and vortexing vigorously for 1 min. The concentrated active supernatant was

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further fractionated using an UltiMate 3000 HPLC system with photodiode array detector

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(Dionex, Sunnyvale, CA) equipped with a HC-C18 (5 µm, 250 mm × 4.6 mm i.d.) column

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(Agilent Technologies, Palo Alto, CA). The bacteriocin was eluted using a linear biphasic

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gradient of acetonitrile/Milli-Q water (10–95%) over 35 min at a flow rate of 0.6 mL/min. At

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each step of the purification process, antibacterial activity was measured with the agar well

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diffusion assay using B. cereus as the indicator strain and expressed as international units

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compared with that formed by nisin, and the protein concentration was measured using the

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Bradford method. All experiments were repeated in three times.

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Determination of the Primary Structure of Plantaricin GZ1-27. MALDI-TOF-MS 7



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(Bruker Daltonic, Bremen, Germany) was performed to determine the molecular mass of

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purified bacteriocin. And α-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix.

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Peptide sequence was analysis using Xevo G2-S Q-TOF-MS/MS instrument (Waters, Milford,

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MA) in positive-ion mode with electrospray ionization (ESI) source. The N-terminal amino

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acid sequence of purified plantaricin GZ1-27 was analyzed at Applied Protein Technology

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(Shanghai, China) using automated Edman degradation.

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Plantaricin GZ1-27 Stability: Effect of Heat, pH, and Enzymes. The purified

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plantaricin GZ1-27 was used to measure the stability of the bacteriocin. The influence of

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temperature on the activity of GZ1-27 was examined by heating the bacteriocin at 60 °C for

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10/30 min, 80 °C for 10/30 min, 100 °C for 10/30 min, or 121 °C for 20 min, and the

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long-term thermostability was tested during storage at 37 °C for 2 weeks and at 4 °C for 3

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months. To determine the effect of pH on antimicrobial activity, the pH of plantaricin GZ1-27

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was adjusted to range from 2-10 using sterile 1 M HCl and 1 M NaOH. After maintaining the

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samples at 37 °C for 30 min, the residual bacteriocin activities were measured at different pH

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values without adjustment to the initial pH.15 To assess the enzyme sensitivity of plantaricin

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GZ1-27, the samples were treated with trypsin (pH 7.5, 10 mM PBS), proteinase K (pH 7.5,

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50 mM Tris-HCl), papain (pH 7.5, 10 mM PBS), and pepsin (pH 2.0, 0.1 M HCl) at their

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optimal pH values, respectively.17 The final concentrations of all enzymes were 1.0 mg/mL.

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After 30 min of incubation at 37 °C, the reaction was stopped by heating at 100 °C for 5 min

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to inactivate the enzymes. The samples were subsequently adjusted to the control pH and

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assayed for antimicrobial activity. The plantaricin GZ1-27 at the original pH (pH 3.8) without

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any heat or enzyme treatments was used as the control sample. The agar well diffusion assay 8


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was carried out to test the remaining activity.

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Determination of Bacteriocin Activity. The minimal inhibitory concentration (MIC) of

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plantaricin GZ1-27 against B. cereus was determined on the basis of Clinical and Laboratory

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Standards Institute (CLSI) guidelines.18 Bacteria at the logarithmic growth phase were

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adjusted to an OD600 of 0.4 and diluted 100-fold in Mueller Hinton Broth (MHB), and 1024

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µg/mL GZ1-27 was 2-fold serially diluted in disodium hydrogen phosphate-citric acid buffer.

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Subsequently, 50 µL of each bacterial suspension and serially diluted bacteriocin aliquot were

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mixed at a 1:1 (v/v) ratio in sterile 96-well plates. The activity of nisin (Nisin Z, >90% purity)

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(J & K Chemical Technology, Shanghai, China) was measured for comparison. For negative

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controls, the bacteria were mixed with disodium hydrogen phosphate-citric acid buffer. After

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static incubation for 24 h at 37 °C, the MICs were determined by testing the OD600 in a

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microplate reader.

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Time-killing Kinetics. The kinetics of B. cereus killing was assessed as previously

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described.19 In brief, overnight-cultured bacteria were washed with PBS (10 mM, pH 7.4) ,

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adjusted to an OD600 of 0.4, and exposed to 1× or 2× MIC plantaricin GZ1-27 or 1× MIC

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nisin at 37 °C for 0–150 min. At fixed time points, 1-mL aliquots were collected, 10-fold

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serially diluted, and plated on LB plates (200 µL/plate), and after incubation at 37 °C for 18 h,

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the colonies were counted.

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Leakage of K+. Log-phase B. cereus cells were adjusted to an OD600 of 0.4, and then the

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cell suspensions were incubated with 1× or 2× MIC plantaricin GZ1-27, or 1× MIC nisin for

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0, 5, 10, 15, 30, 60, and 90 min. The cells treated with PBS alone were used as control. After

9



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centrifugation at 5000 ×g for 10 min (4 °C), the supernatants were filtered through 0.22-µm

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filters and the K+ concentrations were measured on an Optima 8000 plasma atomic emission

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spectrometry (PerkinElmer Inc., Waltham, MA).20

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Flow Cytometry. Bacterial membrane integrity was assayed by measuring the uptake of

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propidium iodide.21 Log-phase B. cereus cells were treated with plantaricin GZ1-27 at 0.5×,

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1×, or 2× MIC. Following incubation at 37 °C for 15 min, the bacterial cells were centrifuged,

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washed, and fixed with propidium iodide at a final concentration of 10 µg/mL at 37 °C for 15

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min. After removing unbound dye, the fluorescence intensity was measured using an Accuri

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C6 flow cytometer (BD Biosciences, Ann Arbor, MI). For each sample, 40,000 events were

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collected, and detected at excitation wavelength (488 nm) and emission wavelength (525 nm).

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B. cereus treated with PBS served as control.

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Confocal Laser-scanning Microscopy (CLSM). Membrane permeability and dynamic

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changes were evaluated using the LIVE/DEAD BacLight Bacterial Viability Kit vital-staining

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probe mixture (Invitrogen, Carlsbad, CA). Late-log-phase B. cereus cells with an OD600 of

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0.4 were treated with plantaricin GZ1-27 at 1× MIC (final concentration) at 37 °C for 0, 5, 15,

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and 30 min. After washing three times with PBS, the cells were mixed with SYTO9 and

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propidium iodide stains, and incubated in the dark at 37 °C for 15 min. Finally, fluorescence

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images were collected using an ULraVIEW VoX CLSM system, (PerkinElmer Inc.

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Massachusetts) with excitation/emission wavelengths of 490/635 nm and 480/500 nm for

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propidium iodide and SYTO9, respectively.

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Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy

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(TEM). SEM and TEM were used to examine ultrastructural changes in B. cereus exposed to

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plantaricin GZ1-27. Log-phase B. cereus cells with an OD600 of 0.4 were treated with

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plantaricin GZ1-27 at 1× MIC for 30 and 60 min. After centrifugation at 5000 ×g for 10 min

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(4 °C), cell pellets were harvested, washed three times with PBS, and fixed overnight with

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2.5% glutaraldehyde at 4 °C. Subsequently, the cells were dehydrated by incubating with a

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series of cold ethanol solutions (30, 50, 70, 90%; 10 min each), and then replaced ethanol

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with tertiary butyl alcohol. After drying and coating with gold–palladium, the bacterial

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specimens were examined using an EVO-LS10 SEM system (CARL ZEISS, Oberkochen,

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Germany). For TEM analysis, after dehydration, the cells were infiltrated with acetone and

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epoxy resin over 24 h, polymerized at 60 °C for 48 h. and Then samples were sectioned with

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an ultramicrotome and stained using uranyl acetate and lead citrate. The bacterial specimens

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were examined using JEM-1011 TEM instrument (JEOL Japan Electronics Co., Ltd., Tokyo,

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Japan). The cells treated with PBS were used as control.

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Real-Time Quantitative PCR. The cytotoxins Nhe (NheA, NheB, and NheC) and Hbl

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(HblL2, HblL1, and HblB) related to the diarrheal disease are encoded by the nheABC operon

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and hblCDA operon, respectively.2 The gene glmS encodes glucosamine-6-phosphate

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synthase (GlmS), which is critical for the biosynthesis of bacterial cell wall peptidoglycan.22

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ftsZ encodes FtsZ, a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria,

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which is involved in septum synthesis and cell division.

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mechanism of plantaricin GZ1-27 against B. cereus at the molecular level, we measured the

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expression levels of thes eight genes (hblC, hblD, hblA, nheA, nheB, nheC, glmS, ftsZ) after

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incubation with plantaricin GZ1-27. 11


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To examine the antibacterial


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B. cereus cells were exposed to 0.8× MIC plantaricin GZ1-27 for 5 h, and then the total

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RNA was extracted using TRIzol reagent and reverse-transcribed using All-In-One RT Master

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Mix (Applied Biological Materials Inc., Vancouver, Canada); the obtained cDNA was

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amplified using an AceQ qPCR SYBR Green Master Kit (Vazyme Biotech, Nanjing, China).

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B. cereus cells that were not treated with plantaricin GZ1-27 were used as the control group.

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Gene samples were quantified using the StepOne-Plus RT-PCR System (Applied Biosystems,

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Carlsbad, CA). The stably expressed gene, gatB-Yqey, was used as the reference gene,24 and

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the relative quantification was analyzed by the 2-∆∆CT method.

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Statistical Analysis. The values were evaluated using IBM SPSS Statistics 20.0

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software, with significant differences (P < 0.05) determined according to one-way ANONA.

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All data are expressed as the means ± standard deviation (SD) of at least three replicates for

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each sample.

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RESULTS AND DISCUSSION

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Screening of Strain GZ1-27. Strain GZ1-27 was selected for the further study because

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of its significantly inhibitory activity against several spoilage bacteria, including B.

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thermosphacta, P. fluorescens, and A. baumannii, and pathogenic bacteria, including B.

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cereus, S. aureus, S. typhimurium, L. monocytogenes, and E. coli. However, Strain GZ1-27

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showed almost no activity against fungi, including Aspergillus niger, Penicillium notatum,

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and Rhizopus stolonifer. Moreover, lactic acid and acetic acid exhibited no antibacterial

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activity at the pH of 5.5, conforming that observed antimicrobial activities were not caused

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by organic acids.

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Identification of Strain GZ1-27. According to the Gram staining, strain GZ1-27 was a

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Gram-positive, non-spore-forming, and rod-shaped bacillus. This strain neither exhibited

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catalase activity nor produced gas. These features illustrated that GZ1-27 belonged to

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homo-fermentative Lactobacillus. Moreover, sugar fermentation assay tests indicated that

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strain GZ1-27 could ferment arabinose, cellobiose, aesculin, fructose, galactose, glucose,

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lactose, maltose, mannose, mannitol, melibiose, raffinose, ribose, sorbitol, sucrose, trehalose,

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and xylose, but not rhamnose. According to the Bergey's Manual of Systematic Bacteriology,

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strain GZ1-27 should be L. plantarum. In addition, the 16S rDNA of this strain (GenBank

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accession no. MH155966) showed 100% similarity with that of L. plantarum WCFS1 (Figure

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1). Taken together, this strain was identified as L. plantarum, and the bacteriocin produced

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was designated as plantaricin GZ1-27.

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The use of L. plantarum in food has been documented for a long period, and most studies

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support its safety aspect.25 To date, more than 200 L. plantarum strains have been isolated.

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Several strains were studied as potential food preservatives with cultured strains or in the

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production of antibacterial substances. L. plantarum GZ1-27 has been shown to be capable of

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secreting bacteriocin, and inhibit the growth of many pathogens and spoilage bacteria, which

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highlights that L. plantarum GZ1-27 is useful as a bio-preservative to extend the shelf-life of

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foods.

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Purification of Plantaricin GZ1-27. Following ammonium sulfate precipitation, the

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specific activity of the plantaricin increased to 262.44 IU/mg. After flowing though Sephadex

265

G-50 column, the fractions in tubes 33 to 37 showing antibacterial activity. The absorption

266

spectrum of the fraction showing the highest activity (fraction 35) revealed an absorbance 13



267

maximum at 233 nm, which was set as the UV detection wavelength in the final RP-HPLC

268

purification. A single clear peak showing activity was detected at a retention time of 16.41

269

min (Figure 2A). Parameters related to purification efficiency were summarized in Table 1.

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After purification, the specific activity increased from 246.59 IU/mg to 2601.24 IU/mg. The

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classical three-step method employed in this study to obtain pure plantaricin GZ1-27 is

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commonly used and has successfully purified diverse bacteriocins from culture supernatants,

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such as plantaricin 163,16 plantaricin ZJ008,11 garvicin A,26 and enterocins 7A and 7B.27

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Sequence Analysis of Plantaricin GZ1-27. Using HCCA as the matrix,

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MALDI-TOF/MS revealed the charge 1 (z = 1) molecular mass ions of m/z 998.469 [M +

276

Na]+, indicating that the molecular mass of the purified GZ1-27 was 975 Da (Figure 1A).

277

According to Q-TOF-MS/MS analysis, the fragmentation of the m/z 976.4774 ion yielded–y

278

fragment ions (y8 and y3) at m/z 733.3490 and m/z 314.1800 and a series of -b fragment ions

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(b2–b10) at m/z 821.3860 → 720.3390 → 663.3110 → 566.2270 → 469.2043 → 412.1939 →

280

341.1910 → 244.1610 → 187.1290 (Figure 2B). According to the peptide-fragmentation

281

nomenclature proposed by Roepstorff and Fohlman,28 the amino acid sequence of plantaricin

282

GZ1-27

283

(VSGPAGPPGTH) starting from the N-terminus. The sequence was calculated for

284

C43H65N11O15, with the theoretical molecular mass of 976.4740 [M + H]+. This result was

285

consistent with that obtained through N-terminal sequencing performed using Edman

286

degradation.

was

determined

to

be

Val-Ser-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Thr-His

287

Although the molecular weight of bacteriocins is generally greater than 2 kDa, there are

288

also some smaller bacteriocins reported, such as bifidocin A (1198.68 Da),29 plantaricin 14


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ZJ008(1334.77 Da),11 plantaricin JLA-9 (1044 Da),30 and plantaricin K25 (1772 Da).31 The

290

molecular mass of plantaricin GZ1-27 is different from that of other bacteriocins, and the

291

sequence is not the same as the known bacteriocins in the NCBI protein BLAST analysis.

292

Thus, it represents a novel bacteriocin. The single linear peptide GZ1-27 contains no

293

lanthionine or YGNGVXC (the characteristic sequence of Class Ⅱa bacteriocins) and should

294

be categorized as a Class Ⅱd bacteriocin.

295

Stability of Plantaricin GZ1-27. Plantaricin GZ1-27 retained >90% of its antibacterial

296

activity after heating at 60 °C or 80 °C for 10 and 30 min. Moreover, after storage at 37 °C

297

for 14 d, it still retained 96.3% of its inhibitory activity, and >90% inhibitory activity was

298

retained even after storage for 3 months at 4 °C. These results suggested that plantaricin

299

GZ1-27 was thermostable and could be stored for a long period at room temperature.

300

Meanwhile, plantaricin GZ1-27 was considerably stable in food processing, because after

301

heating for 20 min at 121 °C, antibacterial activity retained 84.1% of its original.

302

Plantaricin GZ1-27 was stable at low pH (2.0–6.0), but lost most part of its inhibitory

303

activity at pH 7.0 and the activity completely disappeared at pH > 8.0. This agrees with

304

previous findings that neutral and alkaline conditions readily inactivate most bacteriocins,

305

including nisin, plantaricin JLA-9,30 and bacteriocin R1333.19 However, plantaricin GZ1-27

306

might be suitable for preservation because of its relatively good acid tolerance. Moreover,

307

plantaricin GZ1-27 was slightly sensitive to proteinase K and papain, and insensitive to

308

trypsin and pepsin.

309

Antibacterial Effect of Plantaricin GZ1-27 to B. cereus. According to measurements

15



310

performed as CLSI guidelines, the MIC of plantaricin GZ1-27 against B. cereus was

311

determined to be 16 µg/mL, whereas that of nisin was 32 µg/mL, which suggested that

312

plantaricin GZ1-27 and nisin exhibit antibacterial activity within the same order of magnitude.

313

Similar results were obtained in the killing kinetic analysis of B. cereus caused by the

314

bacteriocin (Figure 3). After treatment with 32 µg/mL plantaricin GZ1-27, the number of B.

315

cereus cells decreased drastically within 30 min, and the log10 CFU value dropped to less than

316

1 in 60 min. Moreover, 16 µg/mL GZ1-27 and 32 µg/mL nisin killed all the B. cereus cells

317

within 2 h. These results demonstrated that plantaricin GZ1-27 exhibited rapid and sustained

318

inhibitory activity against B. cereus and that the bactericidal effect was time- and

319

dose-dependent.

320

Numerous bacteriocins have been found to show notable antibacterial activity against

321

foodborne pathogens. However, to the best of our knowledge, only two studies have

322

examined the effect of plantaricin on B. cereus. Wen et al.31 examined the extent of cell

323

membrane damage of plantaricin K25 under SEM and TEM, while Zhao et al.30 studied the

324

mode of action of plantaricin JLA-9 contributing to the outgrowth inhibition of B. cereus

325

spores. According to the MIC values and time-killing kinetics parameters determined in the

326

present study, plantaricin GZ1-27 shows similarly high activity to plantaricin K25 and

327

plantaricin JLA-9. To gain further mechanistic insights into the mode of action of plantaricin

328

GZ1-27, we next investigated the cellular and molecular changes in B. cereus treated with

329

GZ1-27.

330

Membrane Permeability Detection. Determining the extent of potassium ions released

331

by the target bacterial cells is a classical method for studying the permeability of antibacterial 16


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332

drugs to cell membranes. Compared with control cells, plantaricin GZ1-27- and nisin-treated

333

cells showed a drastic loss of intracellular K+, which was observed after 5 min (Figure 4).

334

The extracellular K+ concentration of cells treated with 1× MIC GZ1-27 was increased up to

335

0.70 mg/mL after 1 h, and similar results were obtained after treatment with 2× MIC nisin.

336

With an increase in treatment time, the K+ concentration of the supernatant increased and

337

remained stable in 60 min. These results demonstrated that both plantaricin GZ1-27 and nisin

338

increased the permeability of the B. cereus cell membrane, which led to the efflux of K+.

339

Similar results were reported for antimicrobial peptide F1,32 antimicrobial peptide

340

AMP-jsa9,20 lactosporin, 33 and two-peptide (LtnA1 and LtnA2). 34

341

Effect of Plantaricin GZ1-27 on the Integrity and Viability of B. cereus Cells.

342

Propidium iodide is a nucleus-staining reagent that cannot pass through the intact cell

343

membrane, but can enter through damaged cell membranes and bind to cellular DNA. Thus,

344

the amount of propidium iodide uptake can reflect the extent of cell membrane damage. After

345

incubation of B. cereus with 0.5×, 1×, and 2× MIC plantaricin GZ1-27 for 15 min, the ratio of

346

stained cells reached 20.1%, 39.8%, and 90.7%, respectively (Figure 5). Given that the

347

propidium iodide uptake amount increased markedly with an increase in the MIC, these

348

results demonstrate that the B. cereus cell membrane integrity was impaired and that the

349

bacterial membrane destruction occurred in a dose-dependent manner.

350

Impairment of cell membrane integrity can lead to cell death. Thus, we next examined

351

the viability of B. cereus cells incubated with 1× MIC plantaricin GZ1-27 for 30 min using

352

the LIVE/DEAD BacLight bacterial stain. Viable cells with intact membranes are stained

353

green by SYTO9, a membrane-permeable stain, whereas cells with compromised membranes 17



354

appear red because of propidium iodide entry following membrane integrity impairment.

355

Transition from green to red fluorescence was observed with an increase of treatment time

356

(Figure 6): non-treated cells were stained green, which indicated an intact cell membrane and

357

uptake of only SYTO9, whereas red signals were enhanced at 5 and 10 min and covered

358

almost the entire field of view at 30 min, which indicated membrane integrity loss in most of

359

the treated cells. The incessantly accumulation of red ﬂuorescence in B.cereus cells strongly

360

suggests that the antibacterial activity of plantaricin GZ1-27 is mediated through disruption

361

of bacterial membrane integrity, and that the membrane destruction worsens progressively

362

which eventually causes cell death. Similar results have been observed with other

363

bacteriocins such as plantaricin K25,31 bifidocin A,29 and enterocin CRL35.35

364

Morphological and Ultrastructural Changes of B. cereus Cells after Exposure to

365

Plantaricin GZ1-27. Under the view of SEM (5,000× magnification), integrated B. cereus

366

cells were plump rods, and featured smooth and brilliant membrane surfaces, and some of the

367

cells even showed vigorous binary fission (Figure 7A). After GZ1-27 treatment at 1× MIC for

368

30 min, the cell morphology changed markedly, with creping, wrinkles, and pores evident on

369

the cell surface (Figure 7B), which suggested that GZ1-27 acted directly on the cell wall.

370

When the treatment time was extended to 60 min, most of the cells were noticeably distorted;

371

the boundary of the cell membrane became blurred and adhesion between cells occurred due

372

to the loss of cell wall integrity (Figure 7C). The loss of dissepiment also reflected a gradual

373

stoppage of bifurcation. These observations indicated that plantaricin GZ1-27 induced

374

alterations in the morphology of B. cereus cells in a time-dependent manner.

18


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375

TEM (30,000× magnification) was performed to further observe the ultrastructural and

376

intracellular changes. In the absence of GZ1-27, the bacterial cells retained an intact cell wall

377

that tightly adhered to the membrane and presented a dense and even internal organization

378

(Figure 7D). Upon exposure to GZ1-27 for 30 min, pores formed in the cell wall, the

379

intracellular components leaked out, the cytoplasm became unconsolidated, and the cells

380

clearly displayed plasmolysis (Figure 7E). At 60 min, the cellular structure and intracellular

381

organization deteriorated markedly; the cell membrane became dissolved, and the leaked

382

cytoplasmic material was found to be scattered in the area surrounding the cells, and thus the

383

cells lost their basic structure (Figure 7F). These results indicated that plantaricin GZ1-27

384

induced notable disruption of the structure and intracellular organization of B. cereus cells.

385

The SEM and TEM results implied that the mode of action of plantaricin GZ1-27

386

against B. cereus involves pore formation on the cytoplasmic membrane, which is a critical

387

process in the action of several bacteriocins.36 This, combined with the results above,

388

suggests that plantaricin GZ1-27 adsorbs on the target cell membrane, increases the

389

permeability of the membrane, and induces the leakage of K+ and other ions from sensitive

390

cells; consequently, pores form, and the cell structure deforms resulting in loss of intracellular

391

organization. According to these results, the mode of action of plantaricin GZ1-27 most

392

resembles that of Class II bacteriocins.37 Similar results were obtained in the case of E. coli

393

treated with lactocin XN8-A38 and L. monocytogenes treated with enterocin CRL35.35

394

Effect of Plantaricin GZ1-27 on B. cereus Gene Expression. RT-PCR analyses

395

revealed that, compared with the control group, the expression of all eight genes was

396

downregulated when growing in the presence of plantaricin GZ1-27 (Figure 8). The 19



397

expression levels of glmS and ftsZ showed a substantial reduction (87.9% and 76.1%,

398

respectively), and the expression of genes in the hblDCA and nheABC complexes was

399

downregulated from 31.5% to 68.0%. The downregulated expression of these genessuggested

400

that the metabolism of B. cereus was markedly inhibited by plantaricin GZ1-27.

401

A previous study demonstrated that the bacteriocin AS-48 reduced enterotoxin

402

production in B. cereus using a diarrheic enterotoxin-detection kit; however, it was unclear

403

whether the reduction of this toxin was due to a decrease in the total number of bacterial cells

404

or the inhibition of toxin production in B. cereus.39 Our results suggested that the bacteriocin

405

could inhibit the production of the diarrheal toxins of B. cereus according to the

406

downregulated expression of the cytotoxin production-related genes hblDCA and nheABC.

407

Some previous studies have demonstrated that the synthesis of cell wall peptidoglycan is

408

inhibited by several antibiotics such as nisin12 and lacticin 3147.40 These bacteriocins target

409

lipid II, suppress peptidoglycan synthesis, and then inhibit or kill bacteria. Since we found

410

that plantaricin GZ1-27 inhibited expression of the glmS gene, GlmS could also be a potential

411

antibacterial target.41 A similar phenomenon was observed in a study of difcidin and bacilysin,

412

which downregulated the expression of genes involved in Xanthomonas virulence, protein

413

synthesis, and cell division.42

414

In conclusion, the above results indicate that plantaricin GZ1-27 inhibited the growth of

415

B. cereus both in cellular and molecular level, and plantaricin GZ1-27 could be developed as

416

a candidate bio-preservative for use in the food industry.

417

ABBREVIATIONS USED

418

MIC, seemal inhibitory concentration; CLSI, Clinical and Laboratory Standards Institute; 20


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419

CFU, colony forming unit(s); CFS, cell-free supernatant; Hbl, hemolysin BL; Nhe,

420

nonhemolytic

421

reverse-phase HPLC; CLSM, confocal laser-scanning microscopy; SEM, scanning electron

422

microscopy; TEM, transmission electron microscopy.

423

ASSOCIATED CONTENT

424

SUPPORTING INFORMATION

425

The Supporting Information is available free of charge on the ACS Publications website at

426

DOI:

427

Table S1 showing antimicrobial activity profile of Lactobacillus plantarum GZ1-27. Table S2

428

showing stability of plantaricin GZ1-27. Table S3 showing primers used in the study. Figure

429

S1 showing the antibacterial activity of fraction samples purifed by Sephadex G-50 (A), and

430

the absorption spectrum of fraction 35 (B).

431

AUTHOR INFORMATION

432

Corresponding Author

433

enterotoxin;

HCCA,

α-cyano-4-hydroxycinnamic

acid;

RP-HPLC,

*Tel: +86-2584395963; Fax: +86-2584395963; E-mail: [email protected]

434

ORCID：：0000-0003-0934-7847

435

Funding

436

This work was financially supported by grants from the National Research Program of China

437

(No. 2015BAD16B04).

438

Notes

439

The authors declare no competing financial interests.

440

21



441

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548 549

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FIGURE CAPTIONS

551

Figure 1. The phylogenetic tree of strain GZ1-27 based on Lactobacillus spp..

552

Figure 2. RP-HPLC analysis (A) and Q-TOF-MS/MS analysis (B) of plantaricin GZ1-27.

553

Figure 3. Time-killing curves of plantaricin GZ1-27 on B. cereus cells.

554

Figure 4. Effects of plantaricin GZ1-27 on K+ concentrations in the B. cereus cell culture

555

supernatant.

556

Figure 5. (A) Effects of plantaricin GZ1-27 on the membrane integrity; and (B) Propidium

557

iodide uptake of B. cereus cells.

558

Figure 6. CLSM images of B. cereus cells.

559

Figure 7. SEM and TEM images of B. cereus cells.

560

(A) and (D) control; (B) and (E) treated with 1× MIC plantaricin GZ1-27 for 30 min; (C) and

561

(F) treated with 1× MIC plantaricin GZ1-27 for 60 min. Pores (arrows #1), wrinkles (arrows

562

#2), adhesion (arrows #3), plasmolysis (arrows #4), and cytoplasmic dispersion (arrows #5)

563

are visible.

564

Figure 8. The relative expression levels of eight genes after treatment with plantaricin

565

GZ1-27.

566

The formula 2-∆∆Ct was used to determine relative fold differences in expression levels for

567

each gene. Values were normalized to the levels of the housekeeping gene gatB-Yqey. * P

Purification, Characterization, and Mode of Action of Plantaricin GZ1

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