Mechanism of cell killing activity of plantaricin LD1 against Escherichia coli ATCC 25922

Mechanism of cell killing activity of plantaricin LD1 against Escherichia coli ATCC 25922 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism of cell killing activity of plantaricin LD1 against Escherichia coli ATCC 25922 Manoj Kumar Yadav, Santosh Kumar Tiwari This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3823808/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Mar, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract Plantaricin LD1 was purified from a putative probiotic Lactobacillus plantarum LD1 previously isolated from food. In this study, we have tested detailed mechanism of action against Escherichia coli ATCC 25922 considering Micrococcus luteus MTCC 106 as control. The plantaricin LD1 showed minimum inhibitory concentration (MIC) 34.57 µg/mL and minimum bactericidal concentration (MBC) 138.3 µg/mL against M. luteus MTCC 106 and MIC 69.15 µg/mL and MBC 276.6 µg/mL against E. coli ATCC 25922. The efflux of K + ions, dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) of plantaricin LD1-treated cells suggested the membrane-acting nature of plantaricin LD1. Plantaricin LD1 also caused degradation of genomic DNA of target strains tested. The cell killing was confirmed by staining with propidium iodide and visualizing under light and electron microscopes which were ruptured, smaller, swollen and elongated after treatment with plantaricin LD1. Thus, the findings in this paper indicates plantaricin LD1 kills E. coli ATCC 25922 by interacting with cell membrane resulting in efflux of intracellular contents and also caused degradation of nucleic acids leading to cell death. Plantaricin LD1. Scanning and transmission electron microscopy. Membrane potential. Transmembrane pH gradient. Propidium iodide. Nucleic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Several bacteria produce antimicrobial peptides known as bacteriocins as part of their defense mechanism inhibiting the growth of other microorganisms for food, space and successful establishment in a specific ecological niche [ 1 ]. Lactic acid bacteria (LAB) are an important group of bacteria which produce diverse range of bacteriocins and exhibit probiotic efficacy. These bacteria have been considered as generally regarded as safe (GRAS) by the U.S. food and drug administration [ 2 ]. Bacteriocin production by LAB has been recently identified as a probiotic characteristic which facilitates producer strains in competing complex microbial communities and positively influence host health [ 3 , 4 ]. Bacteriocins kill target cells by pore formation, inhibition of peptidoglycan synthesis and degradation of cellular metabolites [ 5 , 6 ]. The interaction of bacteriocin with cell membrane is mainly dependent on lipid composition of target cells [ 6 ]. It also affects several essential functions such as transcription, translation, replication and cell wall biosynthesis of the living cells [ 7 , 8 ]. The pore formation causes efflux of intracellular ions and nucleic acids from treated cells. The proton motive force (PMF) is an electrochemical proton gradient composed of two components such as membrane-potential (Δψ) and pH-gradient (ΔpH) which are involved in various biological functions such as ATP synthesis, active ions transport and phosphorylation. Bacteriocins deplete either one or both components causing efflux or hydrolysis of intracellular ATP leading to cell death [ 9 ]. Generally, LAB bacteriocins are known to inhibit/kill the bacteria related to producer strains and there are only few bacteriocins reported to inhibit non-related bacteria [ 10 ]. Therefore, mode of action of LAB bacteriocins against Gram-positive members is studies in detail whereas their exact mechanism of action against Gram-negative bacteria is not completely known. We have previously demonstrated the production of plantaricin LD1 by Lactobacillus plantarum LD1 isolated from fermented food Dosa [ 11 ] which is a thermostable and cationic peptide with molecular mass of 6.5 kDa [ 12 , 13 ]. It inhibited broad range of bacteria such as Staphylococcus aureus , Vibrio spp., Pseudomonas fluorescens , Salmonella typhi , P. aeruginosa , Shigella flexneri , E. coli , Enterococcus faecalis ATCC 29212 and certain related LAB [ 12 , 14 ]. In this study, an effort has been made to elucidate the killing mechanism of plantaricin LD1 against E. coli ATCC 25922 which is unique feature for a LAB bacteriocin. Materials and Methods Bacterial strains, growth media and culture conditions For the growth of L. plantarum LD1, de Man, Rogosa and Sharpe (MRS) medium [ 11 , 12 ] was used (HiMedia, Mumbai, India). E. coli ATCC 25922 and M. luteus MTCC 106 were grown in Luria-Bertani (HiMedia, Mumbai, India) and Nutrient Broth (NB) media (HiMedia, Mumbai, India), respectively at 37°C for 18 h in a biological oxygen demand (BOD) incubator shaker (Scigenics, Tamil Nadu, India) with continuous shaking at 200 rpm [ 15 ]. Other chemicals and reagents were purchased from Sigma-Aldrich, Missouri, United States and Sisco Research Laboratory (SRL), Mumbai, India. Preparation of plantaricin LD1 and determination of antimicrobial activity Plantaricin LD1 was purified using tangential flow filtration (TFF) and multi-step chromatography as performed previously [ 13 ]. Briefly, the cell-free supernatant (1 L) was filter-sterilized using a 0.2 µm membrane filter (Axiva, New Delhi, India) and further passed through a 10 kDa nominal molecular weight cut-off (NMWCO) hollow fibre cartridge fitted with AKTA flux s (GE Healthcare, Uppsala, Sweden). The retentate (100 mL) of 10 kDa was discarded and permeate (900 mL) was further passed using a 3 kDa NMWCO hollow fibre cartridge. The 3 kDa retentate (100 mL) was washed three times with 10 mM sodium acetate buffer (pH 4.5), collected in fresh sterile container and stored at 4 ºC. The retentate was loaded onto a HiPrep SP FF 16/10 (1.6 10 cm, 20 mL) column fitted with an AKTAprime plus system (GE Healthcare, Uppsala, Sweden) and eluted with a linear gradient of 0–1 M NaCl in the same buffer. The fractions showing antimicrobial activity were pooled and loaded on gel-filtration chromatography (GFC) using a Sephadex G-50 column (1.6 × 50 cm, 100 mL) equipped with an AKTAprime plus system. The agar well diffusion assay (AWDA) was used to determine the antimicrobial activity and Bradford assay was used to determine protein concentration of each collected 1 mL fraction. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Aliquots of 100 µL plantaricin LD1 with different concentrations were placed in each well of the microtiter plate (Tarson, West Bengal, India), already containing 200 µL of each indicator strain, E. coli ATCC 25922 and M. luteus MTCC 106 (OD 600 0.02) in their suitable broth medium. Sodium acetate buffer (100 µL) was used as control in a separate well. The microtiter plates were incubated at 37°C for 18 h with continuous shaking at 200 rpm. The net growth was calculated by subtracting the final and initial optical density (600 nm) determined using a microplate reader (Molecular Devices, San Jose, USA). The lowest bacteriocin concentration showing no observable growth (OD 600 < 0.1) of indicator strains was considered as the MIC for plantaricin LD1. The MBC was defined as concentration where cell viability (CFU/mL) was completely lost [ 15 ]. Kill kinetics of indicator strains E. coli ATCC 25922 cells with initial OD 600 0.02 were inoculated in 10 mL fresh Luria-Bertani (LB) broth medium and M. luteus MTCC 106 cells in 10 mL Nutrient Broth (NB) and incubated at 37 ºC for 18 h with continuous shaking (200 rpm) in an incubator shaker. The cells were harvested from the mid-log phase (OD 600 0.5 and ~ 10 6 CFU/mL) using centrifugation (9,391 x g for 15 min at 4 ºC) [Sigma Laborzentrifugen GmbH, Niedersachsen, Germany] and re-suspended in normal saline (0.8% NaCl). These cells were treated with different concentrations (½ x MIC, MIC and 2 x MIC) of plantaricin LD1 and incubated at 37 ºC with continuous shaking (200 rpm) in a BOD incubator shaker for 10 h. The control sets were grown with sodium acetate buffer at the place of plantaricin LD1. An aliquot of 100 µL was withdrawn at regular intervals of 2 h up to 10 h and cell viability in terms of CFU/mL was calculated and compared with untreated set as described by Tenea et al. [ 16 ]. Effect of plantaricin LD1 on potassium ion efflux The cells of E . coli ATCC 25922 and M . luteus MTCC 106 were harvested as mentioned above, washed three times (to remove medium) with 10 mM tris-acetate buffer (pH 7.4) containing 100 mM NaCl and re-suspended in same buffer and kept chilled until use. The cells were treated with different concentrations (½ x MIC, MIC and 2 x MIC) of plantaricin LD1. Nisin (250 µg/mL) was used as a positive control and diluents (0.02 N HCl and 10 mM sodium acetate buffer, pH 4.5) were used as negative control. The release of potassium ions was measured every minute after treatment up to 5 min using a potassium-selective electrode calibrated with KCl solutions (20 and 100 ppm) equipped with a digital flame photometer (ESICO International, Parwanoo, India) [ 17 ]. Effect of plantaricin LD1 on dissipation of membrane potential (∆ψ) The log-phase grown cells of each E. coli ATCC 25922 and M . luteus MTCC 106 were collected washed two times with equal volumes (10 mL) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (50 mM, pH 7.0) [Sigma-Aldrich, Missouri, United States], re-suspended in 100 µL of same buffer, stored at 4°C and used within 30 min. The fluorescent probe, 3,3'-dipropylthicarbocyanine iodide [di-S-C3-(3)] [Sigma-Aldrich] was used to observe the changes in membrane potential (∆ψ) of indicator strains as suggested by Shi et al. [ 18 ]. An aliquot of 5 µL of the di-S-C3-(3) probe (2 mM) was added to 2 mL of HEPES buffer (50 mM, pH 7.0) and the spectrum was scanned at 575 nm for 1500 seconds using a modular spectrofluorometer (Ocean optics, EW Duiven, The Netherlands) with excitation and emission wavelength 532 and 575 nm, respectively. After stabilizing the fluorescent signal, 10 µL of cells of each indicator strain, E. coli ATCC 25922 and M. luteus MTCC 106, were added with quick mixing followed by addition of 20 µL glucose (20%) after-stabilization. An aliquot of 2 µL of nigericin (5 mM) [Sigma-Aldrich] was added in the mixture after stabilizing the fluorescent signal. After fluorescent signal was stabilized, each of different concentration of plantaricin LD1 (½ x MIC, MIC and 2 x MIC) and nisin (250 µg/mL) were added to the respective fluorescent cuvette. The nisin and diluents of bacteriocins [diluent of nisin (0.02 N HCl) and diluent of plantaricin LD1 (10 mM sodium acetate buffer, pH 4.5)] were used as a positive and negative control, respectively. An aliquot of 2 µL of valinomycin (2 mM) [Sigma-Aldrich] was added after stabilizing the fluorescent signal. Effect of plantaricin LD1 on dissipation of transmembrane pH gradient (∆pH) The log-phase grown cells of each indicator strain, E. coli ATCC 25922 and M. luteus MTCC 106 were harvested using centrifugation at 9,391 x g for 15 min (4 ºC) and washed twice with 5 mL HEPES buffer (5 mM, pH 6.0). The cell pellets of indicator strains were re-suspended in same buffer and incubated on ice for 1 h. An aliquot of 1 µL (2 mM) of fluorescent probe, 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) [Sigma-Aldrich] was added to the tubes already filled with 2 mL of each indicator cells and incubated for 1 h at 37 ºC in the dark [ 18 ]. The probe-loaded cells (2 mL) were added to the fluorescent cuvette and spectrum was scanned at 535 nm for 600 seconds using a modular spectrofluorometer with excitation and emission wavelength 488 and 535 nm, respectively. After stabilizing the fluorescent signal, an aliquot of 20 µL glucose (20%) and 2 µL valinomycin (5 mM) were added in the mixture. After stabilizing the fluorescent signal, each nisin (250 µg/mL) and ½ x MIC, MIC and 2 x MIC of plantaricin LD1 were added to the fluorescent cuvette with the help of a micropipette. The nisin and diluents were used as positive and negative control, respectively. An aliquot of 2 µL of nigericin (2 mM) was added in the mixture after stabilizing the fluorescent signal. Effect of plantaricin LD1 on degradation of genomic DNA The cells of each indicator strain, E . coli ATCC 25922 and M. luteus MTCC 106 were treated with MIC, 2 x MIC and 4 x MIC of plantaricin LD1 and incubated in a BOD incubator shaker for 18 h at 37 ºC with continuous shaking at 200 rpm. The untreated cells were used as control. The cells were harvested using centrifugation at 9,391 x g for 15 min at (4 ºC) and genomic DNA was isolated from untreated and plantaricin LD1-treated cells of each indicator strain using HiMedia genomic DNA isolation kit and run on agarose gel electrophoresis. Briefly, the agarose gel (0.8%) was prepared in the electrophoresis buffer (1 x TAE). The ethidium bromide (0.5 µg/mL) was added in the cooled molten gel and mixed thoroughly by gentle swirling. The appropriate comb was used for the electrophoresis casting tray and poured warm agarose solution. The electrophoresis buffer was filled in the electrophoresis tank to cover the gel to a depth of ~ 1 mm. The DNA samples were loaded with gel-loading buffer and run at 40V for 30 min until the loading dye migrated an appropriate distance through the gel [ 19 ]. The genomic DNA of treated and untreated cells was compared by visualizing under UV-trans-illuminator (Select BioProducts, Tiawan). In separate experiments, genomic DNA of each indicator strain was isolated using HiMedia genomic DNA isolation kit and treated with MIC, 2 x MIC and 4 x MIC of plantaricin LD1, stored at room temperature for 1 h and run on gel electrophoresis as mentioned above [ 15 ] to observe DNA degradation. The untreated DNA was run as control. Effect of plantaricin LD1 on cell morphology E. coli ATCC 25922 and M. luteus MTCC 106 were grown to mid-log phase (OD 600 0.5) and treated with bacteriocins (2 x MIC of plantaricin LD1 and 250 µg/mL nisin) and incubated at 37 ºC for 18 h under continuous shaking (200 rpm) in a BOD incubator shaker. The cells were washed with normal saline (0.8% NaCl) and morphology was observed under light microscope, fluorescent microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). Light microscopy The smear of treated and untreated cells was individually prepared on a clean glass slide followed by heat fixation. The cells were stained with crystal violet, kept for 1 min and rinsed with doubled distilled water (ddH 2 O). Iodine solution was added and kept for 1 min and rinsed with ddH 2 O. Ethanol was added to the slide to remove the access stain, kept for ½ min, and rinsed with ddH 2 O. For counterstaining, safranin was added, kept for 1 min and rinsed with ddH 2 O. Slides were air-dried and examined under a light microscope (Labomed, Fremont, California, USA) with 100 x magnification in the presence of immersion oil as suggested by Khodaei and Sh [ 20 ]. Fluorescence microscopy An aliquot of 10 µL (1 mg/mL) of each dye 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) was added into 1 mL each of untreated and bacteriocin-treated cells to observe the live and dead cells. The cells were incubated for 10 min at room temperature and analyzed at excitation wavelength of 330–380 nm under fluorescent microscope (DS-Fi2, Nikon Eclipse, Japan) with 40 x magnification as suggested by Sheoran and Tiwari [ 15 ]. Electron microscopy The untreated and bacteriocin-treated cells were re-suspended into 500 µL primary fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4) for 4 h at 4 ºC. The fixative solution was removed by centrifugation at 9,391 x g for 10 min (4 ºC), secondary fixative (1% osmium tetroxide) was added and incubated at 4 ºC for 1 h. The bacterial sample was dehydrated with ethanol, dried using hexamethyldisilazane (HMDS) and mounted on a SEM mount or stub as suggested previously [ 13 ]. The sample was coated with a mixture of gold and palladium (6:4) and the sputter was coated with argon gas was used to observe how bacteriocins affect. The cell morphology of bacteriocins-treated cells was observed under scanning electron microscope (Zeiss, Oberkochen, Baden-Wurttemberg, Germany) using 10250 x magnification. The untreated cells were used as control. For transmission electron microscopic analysis, the untreated and bacteriocin-treated cells were fixed using fixative, dehydrated with ethanol and dried using HMDS as mentioned above. The dehydrated bacterial samples were infiltrated and embedded in resin araldite CY 212 (TAAB, Reading, UK). The thin sections of grey-silver colour interference (70–80 nm) were cut and mounted onto 300 mesh-copper grids. The sections were stained with alcoholic uranyl acetate and alkaline lead citrate, washed gently with distilled water [ 15 ]. The transmission electron microscope (Thermo Scientific, Massachusetts, USA) was used to visualize the untreated and bacteriocin-treated cells at 14000 x magnifications. The experiment was carried out at the Sophisticated Analytical Instrumentation Facility, All India Institute of Medical Science, New Delhi, India. Statistical analysis The experiments were carried out in triplicate and mean values and standard deviation (mean SD) were plotted using SigmaPlot 11.0. A student t-test was used to determine the value of p < 0.05, which was considered as statistically significant. Results Minimum inhibitory concentration and minimum bactericidal concentration The growth of E. coli ATCC 25922 was reduced to OD 600 1.64 ± 0.03, 1.64 ± 0.01, 1.26 ± 0.01, 0.98 ± 0.06 and 0.73 ± 0.01 after treatment with 2.16, 4.32, 8.64, 17.29, 34.57 µg/mL of plantaricin LD1, respectively. It was observed that growth was completely inhibited (OD 600 0.02 ± 0.01) after treatment with 69.15 µg/mL as compared to growth (OD 600 1.70 ± 0.01) of untreated set and therefore, considered as MIC. Similarly, growth of M. luteus MTCC 106 was completely inhibited (OD 600 0.01 ± 0 .01) after treatment with 34.57 µg/mL as compared to growth (OD 600 1.63 ± 0.05) of untreated set used as control and therefore, considered as MIC (Supplementary Fig. 1a, b). Untreated E. coli ATCC 25922 cells were found to be up to log 10 9.60 ± 0.07 CFU/mL, whereas the loss of viability was recorded at log 10 8.34 ± 0.08, 7.77 ± 0.09, 7.86 ± 0.21, 7.60 ± 0.10, 7.14 ± 0.15, 5.20 ± 0.10, 2.10 ± 0.10 CFU/mL after treatment with 2.16, 4.32, 8.64, 17.29, 34.57, 69.15 and 138.3 µg/mL of plantaricin LD1, respectively and it was completely reduced as compared to untreated set (9.60 ± 0.07 CFU/mL) at 276.6 µg/mL, therefore, it was considered as MBC of plantaricin LD1 against E. coli ATCC 25922. Similarly, the viability of M. luteus MTCC 106 cells reduced to log 10 8.42 ± 0.25, 8.38 ± 0.21, 8.38 ± 0.05, 7.76 ± 0.09, 6.02 ± 0.06 and 2.50 ± 0.10 CFU/mL after treatment with 2.16, 4.32, 8.64, 17.29, 34.57 and 69.15 µg/mL of plantaricin LD1, respectively and it was completely reduced as compared to untreated set (log 10 9.60 ± 0.30 CFU/mL) at 138.3 µg/mL, therefore, it was considered as MBC of plantaricin LD1 against M. luteus MTCC 106 (Supplementary Fig. 1a, b). Kill kinetics of indicator strains The viability of untreated cells of E. coli ATCC 25922 was log 10 6.23 ± 0.05 CFU/mL throughout the incubation period up to 10 h. After treatment with ½ x MIC of plantaricin LD1, there was no substantial decrease was recorded in the cell viability, whereas cells treated with MIC, showed log 10 6.00 ± 0.09, 5.90 ± 0.05, 5.40 ± 0.09, 5.10 ± 0.06 and 4.80 ± 0.08 CFU/mL after incubation at 2, 4, 6, 8 and 10 h, respectively. Further, significant loss in cell viability log 10 5.60 ± 0.07, 5.10 ± 0.09, 4.90 ± 0.09, 4.20 ± 0.08 and 4.00 ± 0.05 CFU/mL was recorded after treatment with 2 x MIC of plantaricin LD1 at 2, 4, 6, 8 and 10 h, respectively (Fig. 1 a). Similarly, viability of untreated cells of M. luteus MTCC 106 was found to be log 10 6.10 ± 0.02 CFU/mL and retained almost similar up to 10 h. The viability of the cells treated with plantaricin LD1 with ½ x MIC decreased to log 10 6.00 ± 0.09, 5.80 ± 0.09, 5.60 ± 0.06, 5.50 ± 0.08 and 5.20 ± 0.04 CFU/mL at 2, 4, 6, 8 and 10 h, respectively. The viability of cells treated with the MIC of plantaricin LD1 was reduced up to log 10 5.80 ± 0.04, 5.20 ± 0.07, 5.10 ± 0.07, 4.80 ± 0.09 and 4.50 ± 0.06 CFU/mL at 2, 4, 6, 8 and 10 h, respectively. The cell viability was significantly reduced after treatment with 2 x MIC of plantaricin LD1 and reduced up to log 10 5.50 ± 0.05, 4.60 ± 0.04, 4.50 ± 0.08, 4.20 ± 0.07, 3.90 ± 0.06 CFU/mL at 2, 4, 6, 8 and 10 h, respectively (Fig. 1 b). Thus, a progressive decline in the cell viability was observed throughout the incubation period using MIC and 2 x MIC, whereas ½ x MIC did not affected the viability significantly. The loss of cell viability of E . coli ATCC 25922 and M . luteus MTCC 106 suggests a bactericidal mode of action of plantaricin LD1. Effects of plantaricin LD1 on potassium ion efflux The cells of E. coli ATCC 25922 also showed efflux of potassium ion after treatment with plantaricin LD1. The cells treated with ½ x MIC showed 3.00 ± 0.71 ppm, whereas 12.00 ± 0.87 ppm was recorded after treatment with MIC of plantaricin LD1 at 5 min. The release of potassium ions was recorded higher, 1.00 ± 0.54, 9.00 ± 0.65, 15.00 ± 0.75, 18.00 ± 0.85 and 18.00 ± 0.35 ppm after treatment with 2 x MIC of plantaricin LD1 at 1–5 min, respectively (Fig. 1 c). Similarly, efflux of potassium ion concentration (7.00 ± 0.74 ppm) was recorded after treatment with ½ x MIC of plantaricin LD1 at 5 min. A significant increase in the potassium ion concentration was observed in the presence of MIC and 2 x MIC at 13.00 ± 0.32 and 27.00 ± 0.87 ppm, respectively, at 3 min and thereafter remained stable for up to 5 min tested (Fig. 1 d). Dissipation of membrane potential (∆ψ) It was observed that probe-loaded cells caused reduction in the fluorescence just after their addition and after that, it was stabilized. The increase in the fluorescent signal did not change after the addition of nigericin, used as negative control whereas signal was increased after nisin treatment (used as control), suggesting membrane potential dissipation (∆ψ). Similarly, plantaricin LD1-treated (½ x MIC, MIC and 2 x MIC) cells of E. coli ATCC 25922 (Fig. 2 a) and M. luteus MTCC 106 (Fig. 2 b) resulted an increase in fluorescent signal, indicating the dissipation of ∆ψ. Such an increase in fluorescence was not observed in cells treated with diluents of bacteriocins. In addition, nisin was not able to cause dissipation of ∆ψ in E. coli ATCC 25922 cells. The addition of ionophore valinomycin, used as positive control, did not cause further increase in the signal suggesting complete dissipation of ∆ψ after treatment with plantaricin LD1 (Supplementary Fig. 2a, b). Transmembrane pH gradient (∆pH) The addition of 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) to both indicator cells showed increase in the fluorescence, whereas addition of valinomycin and diluents of bacteriocins did not cause a reduction in the fluorescence and used as negative control. The addition of nigericin, used as positive control, showed a reduction in fluorescent signal suggesting the dissipation of ∆pH. Similarly, the probe-loaded cells of E. coli ATCC 25922 (Fig. 2 c) and M. luteus MTCC 106 (Fig. 2 d) showed reduction in the fluorescent signal after treatment with and plantaricin LD1 (½ x MIC, MIC and 2 x MIC) indicating transmembrane pH gradient (pH). The nisin was able to dissipate pH gradient in M. luteus MTCC 106 cells but not in E. coli ATCC 25922 (Supplementary Fig. 3a, b). Effects of plantaricin LD1 on genomic DNA of indicator strains The genomic DNA of cells of E. coli ATCC 25922 and M. luteus MTCC 106 treated with 2 x MIC and 4 x MIC of plantaricin LD1 showed fragmented DNA as observed by smear DNA band on agarose gel as compared to the DNA isolated from untreated cells (Fig. 3 a, b). Thus, genomic DNA of plantaricin LD1-treated cells showed fragmentation whereas untreated DNA was found to be intact. Similar observation was reported when genomic DNA of E. coli ATCC 25922 and M. luteus MTCC 106 was treated with plantaricin and migrated slowly (Fig. 3 c, d). Effects of plantaricin LD1 on cell morphology The untreated cells of E. coli ATCC 25922 were observed rod-shaped with the entire boundary, whereas cells treated with bacteriocin were found to be small in size, swollen and elongated with a light stain and ruptured cell boundaries (Fig. 4 a). These observations suggested that plantaricin LD1 (69.15 µg/mL) caused cell elongation, swelling, disruption of cell boundary and cell lysis whereas nisin did not affect cells of E. coli ATCC 25922. Similarly, untreated cells of M. luteus MTCC 106 appeared cocci-shaped with the entire cell boundary, whereas cells treated with bacteriocins showed lighter colour and elongated with a rough cell boundary under light microscope. It was observed that treated cells seemed to have a broken cell boundary and small in size (Fig. 5 a). The above observations indicated that bacteriocins caused swelling, disruption of cell boundary and cell lysis after the treatment. The untreated cells of E. coli ATCC 25922 showed blue colour fluorescence with rod shape and red colour (PI) and smaller after treatment with plantaricin LD1 and some cells were found blue in colour [4',6-diamidino-2-phenylindole (DAPI)] suggesting the mixture of live and dead cells of E. coli ATCC 25922 after treatment with plantaricin LD1 (138.3 µg/mL). On the other hand, nisin had no effect on the cells of E. coli ATCC 25922 as shown in Fig. 4 (b). Similarly, in the absence of bacteriocins the cells of M. luteus MTCC 106 were found blue colour indicated the live cells, whereas the cell treated with nisin showed red colour under fluorescent microscope suggesting the dead cells. The mixtures of blue and red cells were found after treatment with plantaricin LD1 (69.15 µg/mL) [Fig. 5 b]. Under scanning electron microscope, untreated cells of E. coli ATCC 25922 appeared rod-shaped with the entire boundary, whereas swollen, with rough boundaries, smaller in size and ruptured after treated with 138.3 µg/mL of plantaricin LD1 whereas nisin did not affect the cells of E. coli ATCC 25922 (Fig. 4 c). The cells of M. luteus MTCC 106 were found cocci shaped with clear cell boundary, whereas cells treated with plantaricin LD1 (69.15 µg/mL) and nisin (250 µg/mL) showed ruptured cell boundary and smaller in size (Fig. 5 c). Similarly, leakage of intracellular contents, cell lysis, separation of the cytoplasmic and outer membrane and membrane distortion were observed in bacteriocin-treated cells under transmission electron microscope (Fig. 4 d and Fig. 5 d). Discussion Bacteriocins generally kill target strains using pore-formation and interacting with the cell membrane. The negatively charged phospholipids and teichoic acids of Gram-positive bacteria and lipopolysaccharide of Gram-negative bacteria are responsible for electrostatic interaction with positively charged bacteriocins [ 5 , 6 ]. Such interaction later leads to the insertion of bacteriocins into the cytoplasmic membrane of the target cells. These events cause formation of ion-permeable channels leading to depletion of intracellular ATP, dissipation of proton motive force, leakage of intracellular metabolites and cell death [ 21 ]. The release and stimulation of autolytic enzymes linked to teichoic, lipoteichoic and teichuronic acids influence the interaction of bacteriocin with the components of the cell wall leading to cell death [ 5 , 6 , 21 ]. In this study, we have characterized a bacteriocin, plantaricin LD1 which causes cytocidal effect on a Gram-negative bacterium, E. coli ATCC 25922 leading to efflux of potential ion, dissipation of ∆ψ and ∆pH and degradation of genomic DNA. Plantaricin LD1 showed lower MIC/MBC against M. luteus MTCC 106 as compared to E. coli ATCC 25922. In another study, plantaricin Pln1 required a higher concentration (100 µg/mL) to inhibit the growth of M. luteus CMCC 63202 and it was not able to kill E. coli strains [ 22 ]. Similarly, the bacteriocin MN047A from L. crustorum MN047 required an even higher concentration (305 µg/mL) to inhibit the growth of E. coli ATCC 25922, as suggested by Yi et al. [ 23 ]. In another study, 62.5 µg/mL of peptide F1 purified from L. paracasei subsp. tolerans FX-6 was needed for the inhibition of the growth of E. coli ATCC 25922 [ 17 ]. There are many plantaricins such as plantaricin KL-1X, plantaricin KL-1Z, Plantaricin LPL-1, plantaricin U10, and bacteriocin MBSa4 have been reported not to inhibit the growth of E. coli strains [ 24 – 27 ]. Earlier, there were few reports, such as plantaricin E, F, J and K, which inhibited E. coli when the outer membrane was weakened using physical conditions and chemical agents [ 28 ]. The efficacy of plantaricin LD1 on the viable cells of E. coli ATCC 25922 and M. luteus MTCC 106 was dependent on the time and concentration. The bactericidal effect of plantaricin LD1 against E. coli ATCC 25922 was found lesser than M. luteus MTCC 106. This effect could be due to the structure of the outer membrane in Gram-negative bacteria [ 17 , 29 ]. In another study, the peptide Gt2 from L. plantarum UTNGt2 showed the effect on cell-viability of E. coli ATCC 25922 in the dose and time-dependent manner [ 30 ]. Similarly, Yi and co-workers [ 23 ] demonstrated that the bacteriocin MN047A from L. crustorum MN047 showed the time and dose-dependent way of loss in cell-viability of E. coli ATCC 25922. The pore-formation in the cell membrane of indicator strains is one of the most considered mechanisms for bacteriocin activity [ 9 ]. In this study, plantaricin LD1 interacts with target membrane by a potential-dependent alignment which caused release of intracellular potassium ions, nucleic acids, ATP hydrolysis and dissipation of membrane potential/pH gradient leading to cell death. The plantaricin LD1 caused efflux of K + ions from the cells of M. luteus MTCC 106 and E. coli ATCC 25922 in concentration dependent manner. Similarly, the other membrane-targeted peptides, such as plantaricin MG, NC8α and NC8β, also induce release of potassium ions in M. luteus 1.193 which causes cell death [ 7 ]. Miao et al. [ 17 ] also showed similar time-dependent efflux of K + ions from the cells of E. coli ATCC 25922 after treatment with antimicrobial peptide F1 isolated from L. paracasei subsp. tolerans FX-6. The release of K + ion from the cells of E. coli ATCC 25922 and M. luteus MTCC 106 provides supplementary proof that plantaricin LD1 disrupted the cell membrane of both target strains as suggested by Zhao et al. [ 31 ]. These findings prompted us to monitor the membrane potential dissipation of target bacteria. The transmembrane potential is a vital bio-energetic component of bacterial cells that is involved directly in energizing critical cellular activities such as ion transport, ATP synthesis, and motility, as suggested by Buttress et al. [ 32 ]. The cells of both indicator strains showed the complete dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) after treatment with plantaricin LD1 in concentration-dependent manner. Whereas, addition of diluents did not cause dissipation of ∆ψ and ∆pH indicating that intracellular pH was equivalent to the pH of the buffer as suggested by Shi et al. [ 18 ]. Further addition of valinomycin did not change fluorescence indicated plantaricin LD1 completely dissipated ∆ψ and ∆pH. Similarly, plantaricin NC8 purified from L. plantarum ZJ316 caused dissipation of Δѱ and ΔpH in M. luteus CGMCC 1.193 through membrane permeabilization [ 7 , 33 ]. The evidence of Δψ and pH depletion suggested that plantaricin LD1 interacted with the cytoplasmic membrane of target cells leading to formation of pores which allowed the efflux of potassium ions and nucleic acids. It has been reported that bacteriocins disintegrates the proton motive force (PMF) in the cells of target strains which leads to the degradation of intracellular components such as potassium ions, inorganic phosphate, ATP and UV-absorbing materials [ 18 , 33 ]. Thus, our findings suggest that plantaricin LD1 causes pore formation resulting in potential membrane dissipation of target cells. Plantaricin LD1 was able to interact with the DNA of E. coli ATCC 25922 and M. luteus MTCC 106 and caused fragmentation evidenced by gel electrophoresis. It suggests that plantaricin LD1 not only interacts with the cell membrane of target cells but also enters inside the cells and cause nucleic degradation. Our data is in consistent with the reported studies where antimicrobial peptides were found to be capable of inhibiting the synthesis and functions of intracellular biopolymers, particularly those that exhibited specific inhibition of DNA synthesis by binding directly to bacterial DNA [ 17 ]. Du et al. [ 34 ] suggested that DNA repair pathways were blocked after the treatment with plantaricin GZ1-27 which resulted instability of the genome of Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 causing cell damage or death. The concentration dependent DNA degradation was also confirmed in vitro where genomic DNA of target cells where isolated and then treated with plantaricin LD1. Similarly, Zhao et al. [ 31 ] demonstrated in vitro interaction of plantaricin 827 with genomic DNA of S. aureus ATCC 25923. They also demonstrated the in silico interaction of plantaricin 827 with the DNA minor groove in AT-rich regions of S. aureus ATCC 25923. The ability of these bacteriocins to interact with the DNA is unique feature indicating the arrest of cellular functions leading to cell death. The dead and live cells were also estimated after staining with a fluorescent dyes, PI and DAPI, respectively as suggested by Duan et al. [ 35 ]. The untreated cells of E. coli ATCC 25922 and M. luteus MTCC 106 were stained with DAPI and found blue, demonstrating live cells. The PI is resistant to integral membranes but can transfer through broken cell membrane and is associated with cellular DNA, making it a nucleus-staining reagent with high affinity [ 7 , 36 ]. After the treatment with plantaricin LD1, the target cells showed mixture of blue and red cells suggesting live and dead cells. These observations further indicated the bactericidal mode of action of plantaricin LD1 against Gram-positive and Gram-negative bacteria. In contrast, nisin-treated cells of E. coli were not affected also suggested by Cleveland et al. [ 37 ]. The microscopic images of plantaricin LD1-treated target bacteria confirmed several damages and cell death. The cells of M. luteus MTCC 106 were ruptured after the treatment with plantaricin LD1 suggesting the membrane-acting nature of plantaricin LD1. The cells of E. coli ATCC 25922 were also found swollen and ruptured as observed under light and scanning electron microscope. The nisin did not show such effects on cells of E. coli ATCC 25922. The damage to the cell membrane of E. coli ATCC 25922 is the distinct mode of action of bacteriocin, as also suggested by Zhang et al. [ 38 ]. Jiang et al. [ 7 ] suggested that after treatment with plantaricin NC8α, the cells of M. luteus CGMCC 1.193 showed similar disruption and shrinkage under SEM. Such damages of target cells were further confirmed under transmission electron microscope. In the presence of plantaricin LD1 cell membrane of E. coli ATCC 29522 was severely damaged and protruded. The membrane penetration on the target cells was also observed after treatment with plantaricin LD1 suggesting its bactericidal effect. Similar findings have also been reported by Jiang et al. [ 7 ] where plantaricin NC8 caused disruption of the cell membrane, widespread shrinkage of the cytoplasmic contents, and vacuolization of M. luteus CGMCC 1.193 cells observed under TEM. Conclusions The minimum inhibitory and minimum bactericidal concentrations of plantaricin LD1 was found lower against M. luteus MTCC 106 as compared to E. coli ATCC 29522. The loss of cell viability, release of K + ions, dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) in target cells suggested bactericidal mode of action of plantaricin LD1. The interaction of plantaricin LD1 with genomic DNA of target strains was also noticed. The morphological deformation/damages of target cells indicated the cell killing as observed under light, fluorescence, scanning and transmission electron microscopes. The staining of target cells with propidium iodide further confirmed the bactericidal mode of action of plantaricin LD1. Thus, the present investigation has found a unique bactericidal and membrane-acting mode of action of plantaricin LD1 against E. coli ATCC 29522. Declarations Acknowledgements We are thankful to Indian Council of Medical Research (5/9/1117/2013-NUT) and Indian National Science Academy (IA/INDO-AUST/F-4/2017/1872), New Delhi for financial assistance. The University Research scholarship, Department of Genetics, M. D. University Rohtak is acknowledged for providing fellowship to MKY. Funding The Indian Council of Medical Research (Project No. 5/9/1117/2013-NUT) and Indian National Science Academy (Project No. IA/INDO-AUST/F-4/2017/1872), New Delhi, India, provided financial support for this work. The University Grants Commission-Scheme for Trans-disciplinary Research for India’s Developing Economy Component-1 (UGC-STRIDE-1), New Delhi, India [Project No. F.2-16/2019 (STRIDE 1)] was truly acknowledged. Author contribution MKY-Performed the experiments and drafted the manuscript; SKT-Designed the experiments and provided all laboratory facilities. All authors read and approved the final version of manuscript. Availability of Data Not available. Ethics Approval The research did not include any human subjects or animal experiments. Consent to Participate Not applicable. Consent to Publish Not applicable. Competing Interests The authors declare no conflicts of interest. References Yang, S. C., Lin, C. H., Sung, C. T., & Fang, J. Y. (2014). Antibacterial activities of bacteriocins: Application in foods and pharmaceuticals. Frontiers In Microbiology , 5 , 241. https://doi.org/10.3389/fmicb.2014.00241 . Darbandi, A., Asadi, A., Mahdizade Ari, M., Ohadi, E., Talebi, M., Halaj Zadeh, M., Darb Emamie, A., Ghanavati, R., & Kakanj, M. (2022). Bacteriocins: Properties and potential use as antimicrobials. Journal Of Clinical Laboratory Analysis , 36 , e24093. https://doi.org/10.1002/jcla.24093 . Yadav, M. K., Singh, B., & Tiwari, S. K. (2019). Comparative analysis of inhibition-based and indicator-independent colorimetric assay for screening of bacteriocin-producing lactic acid bacteria. Probiotics Antimicrob Proteins , 11 , 687–695. https://doi.org/10.1007/s12602-018-9445-4 . Pato, U., Riftyan, E., Jonnaidi, N. N., Wahyuni, M. S., Feruni, J. A., & Abdel-Wahhab, M. A. (2022). Isolation, characterization, and antimicrobial evaluation of bacteriocin produced by lactic acid bacteria against Erwinia carotovora . Lebensmittel-Wissenschaft + [I.E. Und] Technologie. Food Science + Technology. Science + Technologie Alimentaire , 42 , e11922. https://doi.org/10.1590/fst.11922 . Soltani, S., Hammami, R., Cotter, P. D., Rebuffat, S., Said, L. B., Gaudreau, H., Bédard, F., Biron, E., Drider, D., & Fliss, I. (2021). Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. Fems Microbiology Reviews , 45 , fuaa039. https://doi.org/10.1093/femsre/fuaa039 . Yap, P. G., Lai, Z. W., & Tan, J. S. (2022). Bacteriocins from lactic acid bacteria: purification strategies and applications in food and medical industries: A review. Beni Suef Univ J Basic Appl Sci , 11 , 1–18. https://doi.org/10.1186/s43088-022-00227-x . Jiang, H., Tang, X., Zhou, Q., Zou, J., Li, P., Breukink, E., & Gu, Q. (2018). Plantaricin NC8 from Lactobacillus plantarum causes cell membrane disruption to Micrococcus luteus without targeting lipid II. Applied Microbiology And Biotechnology , 102 , 7465–7473. https://doi.org/10.1007/s00253-018-9182-3 . Timothy, B., Iliyasu, A. H., & Anvikar, A. R. (2021). Bacteriocins of lactic acid bacteria and their industrial application. Curr Top Lact Acid Bact Probiotics , 7 , 1–13. https://doi.org/10.35732/ctlabp.2021.7.1.1 . Perez-Ramos, A., Madi-Moussa, D., Coucheney, F., & Drider, D. (2021). Current knowledge of the mode of action and immunity mechanisms of LAB-bacteriocins. Microorganisms , 9 , 2107. https://doi.org/10.3390/microorganisms9102107 . Kubasova, I., Diep, D. B., Ovchinnikov, K. V., Laukova, A., & Strompfova, V. (2020). Bacteriocin production and distribution of bacteriocin-encoding genes in enterococci from dogs. International Journal Of Antimicrobial Agents , 55 , 105859. https://doi.org/10.1016/j.ijantimicag.2019.11.016 . Gupta, A., & Tiwari, S. K. (2014a). Probiotic potential of Lactobacillus plantarum ld1 isolated from batter of Dosa , a South Indian fermented food. Probiotics Antimicrob Proteins , 6 , 73–81. https://doi.org/10.1007/s12602-014-9158-2 . Gupta, A., & Tiwari, S. K. (2014b). Plantaricin LD1: A bacteriocin produced by food isolate of Lactobacillus plantarum LD1. Applied Biochemistry And Biotechnology , 172 , 3354–3362. https://doi.org/10.1007/s12010-014-0775-8 . Yadav, M. K., & Tiwari, S. K. (2021). Polydiacetylene vesicles acting as colorimetric sensor for the detection of plantaricin LD1. Analytical Biochemistry , 631 , 114368. https://doi.org/10.1016/j.ab.2021.114368 . Yadav, M. K., Yadav, P., Dhiman, M., Tewari, S., & Tiwari, S. K. (2022). Plantaricin LD1 purified from Lactobacillus plantarum LD1 inhibits biofilm formation of Enterococcus faecalis ATCC 29212 in tooth model. Letters In Applied Microbiology , 75 , 623–631. https://doi.org/10.1111/lam.13668 . Sheoran, P., & Tiwari, S. K. (2019). Enterocin LD3 from Enterococcus hirae LD3 causing efflux of intracellular ions and UV-absorbing materials in Gram-negative bacteria. Journal Of Applied Microbiology , 126 , 1059–1069. https://doi.org/10.1111/jam.14203 . Tenea, G. N., & Delgado Pozo, T. (2019). Antimicrobial peptides from Lactobacillus plantarum UTNGt2 prevent harmful bacteria growth on fresh tomatoes. Journal Of Microbiology And Biotechnology , 29 , 1553–1560. https://doi.org/10.4014/jmb.1904.04063 . Miao, J., Liu, G., Ke, C., Fan, W., Li, C., Chen, Y., Dixon, W., Song, M., Cao, Y., & Xiao, H. (2016). Inhibitory effects of a novel antimicrobial peptide from kefir against Escherichia coli . Food Control , 65 , 63–72. https://doi.org/10.1016/j.foodcont.2016.01.023 . Shi, F., Wang, Y., Li, Y., & Wang, X. (2016). Mode of action of leucocin K7 produced by Leuconostoc mesenteroides K7 against Listeria monocytogenes and its potential in milk preservation. Biotechnology Letters , 38 , 1551–1557. https://doi.org/10.1007/s10529-016-2127-y . Sambrook, J., & Rusell, D. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor Laboratory Press. Khodaei, M., & Sh, S. N. (2018). Isolation and molecular identification of bacteriocin-producing enterococci with broad antibacterial activity from traditional dairy products in Kerman province of Iran. Korean J Food Sci Anim Resour , 38 , 172–179. https://doi.org/10.5851/kosfa.2018.38.1.172 . Cui, Y., Luo, L., Wang, X., Lu, Y., Yi, Y., Shan, Y., Liu, B., Zhou, Y., & Lü, X. (2021). Mining, heterologous expression, purification, antibactericidal mechanism, and application of bacteriocins: A review. Comprehensive Reviews In Food Science And Food Safety , 20 , 863–899. https://doi.org/10.1111/1541-4337.12658 . Meng, F., Zhao, H., Zhang, C., Lu, F., Bie, X., & Lu, Z. (2016). Expression of a novel bacteriocin-the plantaricin Pln1-in Escherichia coli and its functional analysis. Protein Expr Purif , 119 , 85–93. https://doi.org/10.1016/j.pep.2015.11.008 . Yi, L., Dang, Y., Wu, J., Zhang, L., Liu, X., Liu, B., Zhou, Y., & Lu, X. (2016). Purification and characterization of a novel bacteriocin produced by Lactobacillus crustorum MN047 isolated from koumiss from Xinjiang. China J Dairy Sci , 99 , 7002–7015. http://dx.doi.org/10.3168/jds.2016-11166 . Rumjuankiat, K., Perez, R. H., Pilasombut, K., Keawsompong, S., Zendo, T., Sonomoto, K., & Nitisinprasert, S. (2015). Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1. World Journal Of Microbiology & Biotechnology , 31 , 983–994. https://doi.org/10.1007/s11274-015-1851-0 . Sogandi, S., Mustopa, A. Z., Artika, I. M., & Budiarto, B. R. (2015). Inhibitory activity of Lactobacillus plantarum U10 isolated from tempoyak (fermented durian) made in Indonesia against Salmonella typhi . Microbiol Indones , 9 , 5–5. https://doi.org/10.5454/mi.9.2.4 . Barbosa, M. S., Todorov, S. D., Ivanova, I. V., Belguesmia, Y., Choiset, Y., Rabesona, H., Chobert, J. M., Haertlé, T., & Franco, B. D. G. (2016). Characterization of a two-peptide plantaricin produced by Lactobacillus plantarum MBSa4 isolated from Brazilian salami. Food Control , 60 , 103–112. http://dx.doi.org/10.1016/j.foodcont.2015.07.029 . D. M. Wang, Y., Qin, Y., Xie, Q., Zhang, Y., Hu, J., & Li, P. (2018). Purification and characterization of plantaricin LPL-1, a novel class IIa bacteriocin produced by Lactobacillus plantarum LPL-1 isolated from fermented fish. Frontiers In Microbiology , 9 , 2276. https://doi.org/10.3389/fmicb.2018.02276 . Pal, G., & Srivastava, S. (2014). Inhibitory effect of plantaricin peptides (Pln E/F and J/K) against Escherichia coli . World Journal Of Microbiology & Biotechnology , 30 , 2829–2837. https://doi.org/10.1007/s11274-014-1708-y . Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology , 13 , 605–619. https://doi.org/10.1038/nrmicro3525 . Todorov, S. D., de Paula, O. A., Camargo, A. C., Lopes, D. A., & Nero, L. A. (2018). Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes . Revista Argentina De Microbiologia , 50 , 48–55. https://doi.org/10.1016/j.ram.2017.04.011 . Zhao, D., Wang, Q., Lu, F., Bie, X., Zhao, H., Lu, Z., & Lu, Y. (2022). A novel plantaricin 827 effectively inhibits Staphylococcus aureus and extends shelf life of skim milk. LWT , 154 , 112849. https://doi.org/10.1016/j.lwt.2021.112849 . Halte, B. J. A., Te Winkel, M., Erhardt, J. D., Popp, M., P. F., & Strahl, H. (2022). A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes. Microbiol (Reading England) , 168 , 001227. https://doi.org/10.1099/mic.0.001227 . Gong, H. S., Meng, X. C., & Wang, H. (2010). Mode of action of plantaricin MG, a bacteriocin active against Salmonella typhimurium . Journal Of Basic Microbiology , 50 , S37–S45. http://dx.doi.org/10.1002/jobm.201000130 . Du, H., Zhou, L., Lu, Z., Bie, X., Zhao, H., Niu, Y. D., & Lu, F. (2020). Transcriptomic and proteomic profiling response of methicillin-resistant Staphylococcus aureus (MRSA) to a novel bacteriocin, plantaricin GZ1-27 and its inhibition of biofilm formation. Applied Microbiology And Biotechnology , 104 , 7957–7970. https://doi.org/10.1007/s00253-020-10589-w . Duan, F., Xin, G., Niu, H., & Huang, W. (2017). Chlorinated emodin as a natural antibacterial agent against drug-resistant bacteria through dual influence on bacterial cell membranes and DNA. Scientific Reports , 7 , 12721. https://doi.org/10.1038/s41598-017-12905-3 . Jiang, Y., Mei, C., Huang, X., Gu, Q., & Song, D. (2020). Antibacterial activity and mechanism of a bacteriocin derived from the valine-cecropin a (1–8)-plantaricin zj5 (1–18) hybrid peptide against Escherichia coli O104. Food Biophys. 15, 442–451. https://doi.org/10.1007/s11483-020-09636-w . Cleveland, J., Chikindas, M., & Montville, T. J. (2002). Multimethod assessment of commercial nisin preparations. Journal Of Industrial Microbiology And Biotechnology , 29 , 228–232. https://doi.org/10.1038/sj.jim.7000315 . Zhang, X., Wang, Y., Liu, L., Wei, Y., Shang, N., Zhang, X., & Li, P. (2016). Two-peptide bacteriocin PlnEF causes cell membrane damage to Lactobacillus plantarum . Biochim Biophys Acta Biomembr , 1858 , 274–280. http://dx.doi.org/10.1016/j.bbamem.2015.11.018 . Supplementary Files Supplementaryinformation.doc Cite Share Download PDF Status: Published Journal Publication published 25 Mar, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Reviewers agreed at journal 22 Jan, 2024 Reviewers invited by journal 09 Jan, 2024 Editor invited by journal 02 Jan, 2024 First submitted to journal 30 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3823808","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266337238,"identity":"7e816058-f101-4b7c-a731-6b41fec5fc68","order_by":0,"name":"Manoj Kumar Yadav","email":"","orcid":"","institution":"Maharshi Dayanand University Rohtak","correspondingAuthor":false,"prefix":"","firstName":"Manoj","middleName":"Kumar","lastName":"Yadav","suffix":""},{"id":266337239,"identity":"9cc4435a-0165-4b68-9095-02192cb7a41c","order_by":1,"name":"Santosh Kumar Tiwari","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1477-1845","institution":"Maharshi Dayanand University Rohtak","correspondingAuthor":true,"prefix":"","firstName":"Santosh","middleName":"Kumar","lastName":"Tiwari","suffix":""}],"badges":[],"createdAt":"2023-12-30 11:02:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3823808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3823808/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-024-04927-1","type":"published","date":"2024-03-25T15:01:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49518406,"identity":"91d055c4-9d64-45d8-8e4d-523921797a85","added_by":"auto","created_at":"2024-01-12 09:02:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43402,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of plantaricin LD1 on cell viability (\u003cstrong\u003ea, b\u003c/strong\u003e) and efflux of K\u003csup\u003e+\u003c/sup\u003e ions (\u003cstrong\u003ec, d\u003c/strong\u003e) of \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Micrococcus luteus\u003c/em\u003e MTCC 106, respectively at different time intervals.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/8279312c1bff5d0cf40799c5.png"},{"id":49518655,"identity":"0a048020-6c9c-46c9-a306-af9b7d104ecd","added_by":"auto","created_at":"2024-01-12 09:10:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292358,"visible":true,"origin":"","legend":"\u003cp\u003eDissipation of membrane potential (∆ψ) [\u003cstrong\u003ea, b\u003c/strong\u003e] and transmembrane pH gradient (∆pH) [\u003cstrong\u003ec, d\u003c/strong\u003e] in \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and\u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106, respectively after treatment with plantaricin LD1. Nisin was used as a positive control and diluents of bacteriocins as a negative control.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/55a37e893436a78f2b179468.png"},{"id":49518405,"identity":"38dd58d9-f513-468d-a978-eeea0bea0d96","added_by":"auto","created_at":"2024-01-12 09:02:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56749,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of plantaricin LD1 on genomic DNA of \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106 \u003cem\u003ein-vivo\u003c/em\u003e (\u003cstrong\u003ea, b\u003c/strong\u003e) and \u003cem\u003ein-vitro\u003c/em\u003e (\u003cstrong\u003ec, d\u003c/strong\u003e) treatments, respectively.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/cc32efc6e516d45551f34ec5.png"},{"id":49518408,"identity":"32126088-1c71-4d17-995c-f7a37e02755a","added_by":"auto","created_at":"2024-01-12 09:02:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":598312,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of plantaricin LD1 on cell morphology of \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 under light microscope at 100 x magnification (\u003cstrong\u003ea\u003c/strong\u003e), fluorescent microscope at 40 x magnification (\u003cstrong\u003eb\u003c/strong\u003e), scanning electron microscope at 10250 x magnification (\u003cstrong\u003ec\u003c/strong\u003e) and transmission electron microscope at 14000 x magnification (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/a10b5f92b7972f460e866c72.png"},{"id":49518409,"identity":"cedaeb3f-4bbb-4901-83e2-02500c98a8b6","added_by":"auto","created_at":"2024-01-12 09:02:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":592512,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of plantaricin LD1 on cell morphology of \u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106 under light microscope at 100 x magnification (\u003cstrong\u003ea\u003c/strong\u003e), fluorescent microscope at 40 x magnification (\u003cstrong\u003eb\u003c/strong\u003e), scanning electron microscope at 10250 x magnification (\u003cstrong\u003ec\u003c/strong\u003e) and transmission electron microscope at 14000 x magnification (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/e76afb7d87f98f69b79d18c9.png"},{"id":53869922,"identity":"fa695b31-cefe-40ab-a83a-7e8f55aa2467","added_by":"auto","created_at":"2024-04-01 15:12:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2162944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/9fdc7924-6137-422e-aaf9-d0b40d6e4bc9.pdf"},{"id":49518410,"identity":"a11c71f5-1ed4-4a5f-b878-9447d88bb7aa","added_by":"auto","created_at":"2024-01-12 09:02:53","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":492032,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-3823808/v1/89a78f929d6f2a8612880286.doc"}],"financialInterests":"","formattedTitle":"Mechanism of cell killing activity of plantaricin LD1 against Escherichia coli ATCC 25922","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSeveral bacteria produce antimicrobial peptides known as bacteriocins as part of their defense mechanism inhibiting the growth of other microorganisms for food, space and successful establishment in a specific ecological niche [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lactic acid bacteria (LAB) are an important group of bacteria which produce diverse range of bacteriocins and exhibit probiotic efficacy. These bacteria have been considered as generally regarded as safe (GRAS) by the U.S. food and drug administration [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Bacteriocin production by LAB has been recently identified as a probiotic characteristic which facilitates producer strains in competing complex microbial communities and positively influence host health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacteriocins kill target cells by pore formation, inhibition of peptidoglycan synthesis and degradation of cellular metabolites [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The interaction of bacteriocin with cell membrane is mainly dependent on lipid composition of target cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It also affects several essential functions such as transcription, translation, replication and cell wall biosynthesis of the living cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The pore formation causes efflux of intracellular ions and nucleic acids from treated cells. The proton motive force (PMF) is an electrochemical proton gradient composed of two components such as membrane-potential (Δψ) and pH-gradient (ΔpH) which are involved in various biological functions such as ATP synthesis, active ions transport and phosphorylation. Bacteriocins deplete either one or both components causing efflux or hydrolysis of intracellular ATP leading to cell death [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Generally, LAB bacteriocins are known to inhibit/kill the bacteria related to producer strains and there are only few bacteriocins reported to inhibit non-related bacteria [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, mode of action of LAB bacteriocins against Gram-positive members is studies in detail whereas their exact mechanism of action against Gram-negative bacteria is not completely known.\u003c/p\u003e \u003cp\u003eWe have previously demonstrated the production of plantaricin LD1 by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e LD1 isolated from fermented food \u003cem\u003eDosa\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] which is a thermostable and cationic peptide with molecular mass of 6.5 kDa [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It inhibited broad range of bacteria such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eVibrio\u003c/em\u003e spp., \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e, \u003cem\u003eSalmonella typhi\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eShigella flexneri\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212 and certain related LAB [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this study, an effort has been made to elucidate the killing mechanism of plantaricin LD1 against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 which is unique feature for a LAB bacteriocin.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains, growth media and culture conditions\u003c/h2\u003e \u003cp\u003eFor the growth of \u003cem\u003eL. plantarum\u003c/em\u003e LD1, de Man, Rogosa and Sharpe (MRS) medium [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] was used (HiMedia, Mumbai, India). \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were grown in Luria-Bertani (HiMedia, Mumbai, India) and Nutrient Broth (NB) media (HiMedia, Mumbai, India), respectively at 37\u0026deg;C for 18 h in a biological oxygen demand (BOD) incubator shaker (Scigenics, Tamil Nadu, India) with continuous shaking at 200 rpm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Other chemicals and reagents were purchased from Sigma-Aldrich, Missouri, United States and Sisco Research Laboratory (SRL), Mumbai, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of plantaricin LD1 and determination of antimicrobial activity\u003c/h2\u003e \u003cp\u003ePlantaricin LD1 was purified using tangential flow filtration (TFF) and multi-step chromatography as performed previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, the cell-free supernatant (1 L) was filter-sterilized using a 0.2 \u0026micro;m membrane filter (Axiva, New Delhi, India) and further passed through a 10 kDa nominal molecular weight cut-off (NMWCO) hollow fibre cartridge fitted with AKTA flux s (GE Healthcare, Uppsala, Sweden). The retentate (100 mL) of 10 kDa was discarded and permeate (900 mL) was further passed using a 3 kDa NMWCO hollow fibre cartridge. The 3 kDa retentate (100 mL) was washed three times with 10 mM sodium acetate buffer (pH 4.5), collected in fresh sterile container and stored at 4 \u0026ordm;C. The retentate was loaded onto a HiPrep SP FF 16/10 (1.6 10 cm, 20 mL) column fitted with an AKTAprime plus system (GE Healthcare, Uppsala, Sweden) and eluted with a linear gradient of 0\u0026ndash;1 M NaCl in the same buffer. The fractions showing antimicrobial activity were pooled and loaded on gel-filtration chromatography (GFC) using a Sephadex G-50 column (1.6 \u0026times; 50 cm, 100 mL) equipped with an AKTAprime plus system. The agar well diffusion assay (AWDA) was used to determine the antimicrobial activity and Bradford assay was used to determine protein concentration of each collected 1 mL fraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)\u003c/h2\u003e \u003cp\u003eAliquots of 100 \u0026micro;L plantaricin LD1 with different concentrations were placed in each well of the microtiter plate (Tarson, West Bengal, India), already containing 200 \u0026micro;L of each indicator strain, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 (OD\u003csub\u003e600\u003c/sub\u003e 0.02) in their suitable broth medium. Sodium acetate buffer (100 \u0026micro;L) was used as control in a separate well. The microtiter plates were incubated at 37\u0026deg;C for 18 h with continuous shaking at 200 rpm. The net growth was calculated by subtracting the final and initial optical density (600 nm) determined using a microplate reader (Molecular Devices, San Jose, USA). The lowest bacteriocin concentration showing no observable growth (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1) of indicator strains was considered as the MIC for plantaricin LD1. The MBC was defined as concentration where cell viability (CFU/mL) was completely lost [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eKill kinetics of indicator strains\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells with initial OD\u003csub\u003e600\u003c/sub\u003e 0.02 were inoculated in 10 mL fresh Luria-Bertani (LB) broth medium and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 cells in 10 mL Nutrient Broth (NB) and incubated at 37 \u0026ordm;C for 18 h with continuous shaking (200 rpm) in an incubator shaker. The cells were harvested from the mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e 0.5 and ~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) using centrifugation (9,391 x g for 15 min at 4 \u0026ordm;C) [Sigma Laborzentrifugen GmbH, Niedersachsen, Germany] and re-suspended in normal saline (0.8% NaCl). These cells were treated with different concentrations (\u0026frac12; x MIC, MIC and 2 x MIC) of plantaricin LD1 and incubated at 37 \u0026ordm;C with continuous shaking (200 rpm) in a BOD incubator shaker for 10 h. The control sets were grown with sodium acetate buffer at the place of plantaricin LD1. An aliquot of 100 \u0026micro;L was withdrawn at regular intervals of 2 h up to 10 h and cell viability in terms of CFU/mL was calculated and compared with untreated set as described by Tenea et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plantaricin LD1 on potassium ion efflux\u003c/h2\u003e \u003cp\u003eThe cells of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eluteus\u003c/em\u003e MTCC 106 were harvested as mentioned above, washed three times (to remove medium) with 10 mM tris-acetate buffer (pH 7.4) containing 100 mM NaCl and re-suspended in same buffer and kept chilled until use. The cells were treated with different concentrations (\u0026frac12; x MIC, MIC and 2 x MIC) of plantaricin LD1. Nisin (250 \u0026micro;g/mL) was used as a positive control and diluents (0.02 N HCl and 10 mM sodium acetate buffer, pH 4.5) were used as negative control. The release of potassium ions was measured every minute after treatment up to 5 min using a potassium-selective electrode calibrated with KCl solutions (20 and 100 ppm) equipped with a digital flame photometer (ESICO International, Parwanoo, India) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plantaricin LD1 on dissipation of membrane potential (∆ψ)\u003c/h2\u003e \u003cp\u003eThe log-phase grown cells of each \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eluteus\u003c/em\u003e MTCC 106 were collected washed two times with equal volumes (10 mL) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (50 mM, pH 7.0) [Sigma-Aldrich, Missouri, United States], re-suspended in 100 \u0026micro;L of same buffer, stored at 4\u0026deg;C and used within 30 min. The fluorescent probe, 3,3'-dipropylthicarbocyanine iodide [di-S-C3-(3)] [Sigma-Aldrich] was used to observe the changes in membrane potential (∆ψ) of indicator strains as suggested by Shi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. An aliquot of 5 \u0026micro;L of the di-S-C3-(3) probe (2 mM) was added to 2 mL of HEPES buffer (50 mM, pH 7.0) and the spectrum was scanned at 575 nm for 1500 seconds using a modular spectrofluorometer (Ocean optics, EW Duiven, The Netherlands) with excitation and emission wavelength 532 and 575 nm, respectively. After stabilizing the fluorescent signal, 10 \u0026micro;L of cells of each indicator strain, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106, were added with quick mixing followed by addition of 20 \u0026micro;L glucose (20%) after-stabilization. An aliquot of 2 \u0026micro;L of nigericin (5 mM) [Sigma-Aldrich] was added in the mixture after stabilizing the fluorescent signal. After fluorescent signal was stabilized, each of different concentration of plantaricin LD1 (\u0026frac12; x MIC, MIC and 2 x MIC) and nisin (250 \u0026micro;g/mL) were added to the respective fluorescent cuvette. The nisin and diluents of bacteriocins [diluent of nisin (0.02 N HCl) and diluent of plantaricin LD1 (10 mM sodium acetate buffer, pH 4.5)] were used as a positive and negative control, respectively. An aliquot of 2 \u0026micro;L of valinomycin (2 mM) [Sigma-Aldrich] was added after stabilizing the fluorescent signal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plantaricin LD1 on dissipation of transmembrane pH gradient (∆pH)\u003c/h2\u003e \u003cp\u003eThe log-phase grown cells of each indicator strain, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were harvested using centrifugation at 9,391 x g for 15 min (4 \u0026ordm;C) and washed twice with 5 mL HEPES buffer (5 mM, pH 6.0). The cell pellets of indicator strains were re-suspended in same buffer and incubated on ice for 1 h. An aliquot of 1 \u0026micro;L (2 mM) of fluorescent probe, 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) [Sigma-Aldrich] was added to the tubes already filled with 2 mL of each indicator cells and incubated for 1 h at 37 \u0026ordm;C in the dark [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The probe-loaded cells (2 mL) were added to the fluorescent cuvette and spectrum was scanned at 535 nm for 600 seconds using a modular spectrofluorometer with excitation and emission wavelength 488 and 535 nm, respectively. After stabilizing the fluorescent signal, an aliquot of 20 \u0026micro;L glucose (20%) and 2 \u0026micro;L valinomycin (5 mM) were added in the mixture. After stabilizing the fluorescent signal, each nisin (250 \u0026micro;g/mL) and \u0026frac12; x MIC, MIC and 2 x MIC of plantaricin LD1 were added to the fluorescent cuvette with the help of a micropipette. The nisin and diluents were used as positive and negative control, respectively. An aliquot of 2 \u0026micro;L of nigericin (2 mM) was added in the mixture after stabilizing the fluorescent signal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plantaricin LD1 on degradation of genomic DNA\u003c/h2\u003e \u003cp\u003eThe cells of each indicator strain, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were treated with MIC, 2 x MIC and 4 x MIC of plantaricin LD1 and incubated in a BOD incubator shaker for 18 h at 37 \u0026ordm;C with continuous shaking at 200 rpm. The untreated cells were used as control. The cells were harvested using centrifugation at 9,391 x g for 15 min at (4 \u0026ordm;C) and genomic DNA was isolated from untreated and plantaricin LD1-treated cells of each indicator strain using HiMedia genomic DNA isolation kit and run on agarose gel electrophoresis. Briefly, the agarose gel (0.8%) was prepared in the electrophoresis buffer (1 x TAE). The ethidium bromide (0.5 \u0026micro;g/mL) was added in the cooled molten gel and mixed thoroughly by gentle swirling. The appropriate comb was used for the electrophoresis casting tray and poured warm agarose solution. The electrophoresis buffer was filled in the electrophoresis tank to cover the gel to a depth of ~\u0026thinsp;1 mm. The DNA samples were loaded with gel-loading buffer and run at 40V for 30 min until the loading dye migrated an appropriate distance through the gel [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The genomic DNA of treated and untreated cells was compared by visualizing under UV-trans-illuminator (Select BioProducts, Tiawan). In separate experiments, genomic DNA of each indicator strain was isolated using HiMedia genomic DNA isolation kit and treated with MIC, 2 x MIC and 4 x MIC of plantaricin LD1, stored at room temperature for 1 h and run on gel electrophoresis as mentioned above [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] to observe DNA degradation. The untreated DNA was run as control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of plantaricin LD1 on cell morphology\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were grown to mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e 0.5) and treated with bacteriocins (2 x MIC of plantaricin LD1 and 250 \u0026micro;g/mL nisin) and incubated at 37 \u0026ordm;C for 18 h under continuous shaking (200 rpm) in a BOD incubator shaker. The cells were washed with normal saline (0.8% NaCl) and morphology was observed under light microscope, fluorescent microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLight microscopy\u003c/h2\u003e \u003cp\u003eThe smear of treated and untreated cells was individually prepared on a clean glass slide followed by heat fixation. The cells were stained with crystal violet, kept for 1 min and rinsed with doubled distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO). Iodine solution was added and kept for 1 min and rinsed with ddH\u003csub\u003e2\u003c/sub\u003eO. Ethanol was added to the slide to remove the access stain, kept for \u0026frac12; min, and rinsed with ddH\u003csub\u003e2\u003c/sub\u003eO. For counterstaining, safranin was added, kept for 1 min and rinsed with ddH\u003csub\u003e2\u003c/sub\u003eO. Slides were air-dried and examined under a light microscope (Labomed, Fremont, California, USA) with 100 x magnification in the presence of immersion oil as suggested by Khodaei and Sh [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence microscopy\u003c/h2\u003e \u003cp\u003eAn aliquot of 10 \u0026micro;L (1 mg/mL) of each dye 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) was added into 1 mL each of untreated and bacteriocin-treated cells to observe the live and dead cells. The cells were incubated for 10 min at room temperature and analyzed at excitation wavelength of 330\u0026ndash;380 nm under fluorescent microscope (DS-Fi2, Nikon Eclipse, Japan) with 40 x magnification as suggested by Sheoran and Tiwari [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eElectron microscopy\u003c/h2\u003e \u003cp\u003eThe untreated and bacteriocin-treated cells were re-suspended into 500 \u0026micro;L primary fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4) for 4 h at 4 \u0026ordm;C. The fixative solution was removed by centrifugation at 9,391 x g for 10 min (4 \u0026ordm;C), secondary fixative (1% osmium tetroxide) was added and incubated at 4 \u0026ordm;C for 1 h. The bacterial sample was dehydrated with ethanol, dried using hexamethyldisilazane (HMDS) and mounted on a SEM mount or stub as suggested previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The sample was coated with a mixture of gold and palladium (6:4) and the sputter was coated with argon gas was used to observe how bacteriocins affect. The cell morphology of bacteriocins-treated cells was observed under scanning electron microscope (Zeiss, Oberkochen, Baden-Wurttemberg, Germany) using 10250 x magnification. The untreated cells were used as control.\u003c/p\u003e \u003cp\u003eFor transmission electron microscopic analysis, the untreated and bacteriocin-treated cells were fixed using fixative, dehydrated with ethanol and dried using HMDS as mentioned above. The dehydrated bacterial samples were infiltrated and embedded in resin araldite CY 212 (TAAB, Reading, UK). The thin sections of grey-silver colour interference (70\u0026ndash;80 nm) were cut and mounted onto 300 mesh-copper grids. The sections were stained with alcoholic uranyl acetate and alkaline lead citrate, washed gently with distilled water [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The transmission electron microscope (Thermo Scientific, Massachusetts, USA) was used to visualize the untreated and bacteriocin-treated cells at 14000 x magnifications. The experiment was carried out at the Sophisticated Analytical Instrumentation Facility, All India Institute of Medical Science, New Delhi, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experiments were carried out in triplicate and mean values and standard deviation (mean SD) were plotted using SigmaPlot 11.0. A student t-test was used to determine the value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, which was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration and minimum bactericidal concentration\u003c/h2\u003e \u003cp\u003eThe growth of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was reduced to OD\u003csub\u003e600\u003c/sub\u003e 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, 1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, 0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 and 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 after treatment with 2.16, 4.32, 8.64, 17.29, 34.57 \u0026micro;g/mL of plantaricin LD1, respectively. It was observed that growth was completely inhibited (OD\u003csub\u003e600\u003c/sub\u003e 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) after treatment with 69.15 \u0026micro;g/mL as compared to growth (OD\u003csub\u003e600\u003c/sub\u003e 1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) of untreated set and therefore, considered as MIC. Similarly, growth of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 was completely inhibited (OD\u003csub\u003e600\u003c/sub\u003e 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0 .01) after treatment with 34.57 \u0026micro;g/mL as compared to growth (OD\u003csub\u003e600\u003c/sub\u003e 1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) of untreated set used as control and therefore, considered as MIC (Supplementary Fig.\u0026nbsp;1a, b).\u003c/p\u003e \u003cp\u003eUntreated \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells were found to be up to log\u003csub\u003e10\u003c/sub\u003e 9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 CFU/mL, whereas the loss of viability was recorded at log\u003csub\u003e10\u003c/sub\u003e 8.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, 7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 7.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21, 7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, 7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, 2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 CFU/mL after treatment with 2.16, 4.32, 8.64, 17.29, 34.57, 69.15 and 138.3 \u0026micro;g/mL of plantaricin LD1, respectively and it was completely reduced as compared to untreated set (9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 CFU/mL) at 276.6 \u0026micro;g/mL, therefore, it was considered as MBC of plantaricin LD1 against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. Similarly, the viability of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 cells reduced to log\u003csub\u003e10\u003c/sub\u003e 8.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25, 8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21, 8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 7.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 and 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 CFU/mL after treatment with 2.16, 4.32, 8.64, 17.29, 34.57 and 69.15 \u0026micro;g/mL of plantaricin LD1, respectively and it was completely reduced as compared to untreated set (log\u003csub\u003e10\u003c/sub\u003e 9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 CFU/mL) at 138.3 \u0026micro;g/mL, therefore, it was considered as MBC of plantaricin LD1 against \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 (Supplementary Fig.\u0026nbsp;1a, b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKill kinetics of indicator strains\u003c/h2\u003e \u003cp\u003eThe viability of untreated cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was log\u003csub\u003e10\u003c/sub\u003e 6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 CFU/mL throughout the incubation period up to 10 h. After treatment with \u0026frac12; x MIC of plantaricin LD1, there was no substantial decrease was recorded in the cell viability, whereas cells treated with MIC, showed log\u003csub\u003e10\u003c/sub\u003e 6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 and 4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 CFU/mL after incubation at 2, 4, 6, 8 and 10 h, respectively. Further, significant loss in cell viability log\u003csub\u003e10\u003c/sub\u003e 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 CFU/mL was recorded after treatment with 2 x MIC of plantaricin LD1 at 2, 4, 6, 8 and 10 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, viability of untreated cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 was found to be log\u003csub\u003e10\u003c/sub\u003e 6.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 CFU/mL and retained almost similar up to 10 h. The viability of the cells treated with plantaricin LD1 with \u0026frac12; x MIC decreased to log\u003csub\u003e10\u003c/sub\u003e 6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 CFU/mL at 2, 4, 6, 8 and 10 h, respectively. The viability of cells treated with the MIC of plantaricin LD1 was reduced up to log\u003csub\u003e10\u003c/sub\u003e 5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 and 4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 CFU/mL at 2, 4, 6, 8 and 10 h, respectively. The cell viability was significantly reduced after treatment with 2 x MIC of plantaricin LD1 and reduced up to log\u003csub\u003e10\u003c/sub\u003e 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, 4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, 4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 CFU/mL at 2, 4, 6, 8 and 10 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Thus, a progressive decline in the cell viability was observed throughout the incubation period using MIC and 2 x MIC, whereas \u0026frac12; x MIC did not affected the viability significantly. The loss of cell viability of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eluteus\u003c/em\u003e MTCC 106 suggests a bactericidal mode of action of plantaricin LD1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of plantaricin LD1 on potassium ion efflux\u003c/h2\u003e \u003cp\u003eThe cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 also showed efflux of potassium ion after treatment with plantaricin LD1. The cells treated with \u0026frac12; x MIC showed 3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 ppm, whereas 12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 ppm was recorded after treatment with MIC of plantaricin LD1 at 5 min. The release of potassium ions was recorded higher, 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54, 9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65, 15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75, 18.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 and 18.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 ppm after treatment with 2 x MIC of plantaricin LD1 at 1\u0026ndash;5 min, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Similarly, efflux of potassium ion concentration (7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 ppm) was recorded after treatment with \u0026frac12; x MIC of plantaricin LD1 at 5 min. A significant increase in the potassium ion concentration was observed in the presence of MIC and 2 x MIC at 13.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 and 27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 ppm, respectively, at 3 min and thereafter remained stable for up to 5 min tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDissipation of membrane potential (∆ψ)\u003c/h2\u003e \u003cp\u003eIt was observed that probe-loaded cells caused reduction in the fluorescence just after their addition and after that, it was stabilized. The increase in the fluorescent signal did not change after the addition of nigericin, used as negative control whereas signal was increased after nisin treatment (used as control), suggesting membrane potential dissipation (∆ψ). Similarly, plantaricin LD1-treated (\u0026frac12; x MIC, MIC and 2 x MIC) cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) resulted an increase in fluorescent signal, indicating the dissipation of ∆ψ. Such an increase in fluorescence was not observed in cells treated with diluents of bacteriocins. In addition, nisin was not able to cause dissipation of ∆ψ in \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells. The addition of ionophore valinomycin, used as positive control, did not cause further increase in the signal suggesting complete dissipation of ∆ψ after treatment with plantaricin LD1 (Supplementary Fig.\u0026nbsp;2a, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTransmembrane pH gradient (∆pH)\u003c/h2\u003e \u003cp\u003eThe addition of 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM) to both indicator cells showed increase in the fluorescence, whereas addition of valinomycin and diluents of bacteriocins did not cause a reduction in the fluorescence and used as negative control. The addition of nigericin, used as positive control, showed a reduction in fluorescent signal suggesting the dissipation of ∆pH. Similarly, the probe-loaded cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) showed reduction in the fluorescent signal after treatment with and plantaricin LD1 (\u0026frac12; x MIC, MIC and 2 x MIC) indicating transmembrane pH gradient (pH). The nisin was able to dissipate pH gradient in \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 cells but not in \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 (Supplementary Fig.\u0026nbsp;3a, b).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of plantaricin LD1 on genomic DNA of indicator strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe genomic DNA of cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 treated with 2 x MIC and 4 x MIC of plantaricin LD1 showed fragmented DNA as observed by smear DNA band on agarose gel as compared to the DNA isolated from untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Thus, genomic DNA of plantaricin LD1-treated cells showed fragmentation whereas untreated DNA was found to be intact. Similar observation was reported when genomic DNA of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 was treated with plantaricin and migrated slowly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffects of plantaricin LD1 on cell morphology\u003c/h2\u003e \u003cp\u003eThe untreated cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 were observed rod-shaped with the entire boundary, whereas cells treated with bacteriocin were found to be small in size, swollen and elongated with a light stain and ruptured cell boundaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These observations suggested that plantaricin LD1 (69.15 \u0026micro;g/mL) caused cell elongation, swelling, disruption of cell boundary and cell lysis whereas nisin did not affect cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. Similarly, untreated cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 appeared cocci-shaped with the entire cell boundary, whereas cells treated with bacteriocins showed lighter colour and elongated with a rough cell boundary under light microscope. It was observed that treated cells seemed to have a broken cell boundary and small in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The above observations indicated that bacteriocins caused swelling, disruption of cell boundary and cell lysis after the treatment.\u003c/p\u003e\u003cp\u003eThe untreated cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 showed blue colour fluorescence with rod shape and red colour (PI) and smaller after treatment with plantaricin LD1 and some cells were found blue in colour [4',6-diamidino-2-phenylindole (DAPI)] suggesting the mixture of live and dead cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 after treatment with plantaricin LD1 (138.3 \u0026micro;g/mL). On the other hand, nisin had no effect on the cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b). Similarly, in the absence of bacteriocins the cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were found blue colour indicated the live cells, whereas the cell treated with nisin showed red colour under fluorescent microscope suggesting the dead cells. The mixtures of blue and red cells were found after treatment with plantaricin LD1 (69.15 \u0026micro;g/mL) [Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb]. Under scanning electron microscope, untreated cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 appeared rod-shaped with the entire boundary, whereas swollen, with rough boundaries, smaller in size and ruptured after treated with 138.3 \u0026micro;g/mL of plantaricin LD1 whereas nisin did not affect the cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were found cocci shaped with clear cell boundary, whereas cells treated with plantaricin LD1 (69.15 \u0026micro;g/mL) and nisin (250 \u0026micro;g/mL) showed ruptured cell boundary and smaller in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Similarly, leakage of intracellular contents, cell lysis, separation of the cytoplasmic and outer membrane and membrane distortion were observed in bacteriocin-treated cells under transmission electron microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacteriocins generally kill target strains using pore-formation and interacting with the cell membrane. The negatively charged phospholipids and teichoic acids of Gram-positive bacteria and lipopolysaccharide of Gram-negative bacteria are responsible for electrostatic interaction with positively charged bacteriocins [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Such interaction later leads to the insertion of bacteriocins into the cytoplasmic membrane of the target cells. These events cause formation of ion-permeable channels leading to depletion of intracellular ATP, dissipation of proton motive force, leakage of intracellular metabolites and cell death [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The release and stimulation of autolytic enzymes linked to teichoic, lipoteichoic and teichuronic acids influence the interaction of bacteriocin with the components of the cell wall leading to cell death [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we have characterized a bacteriocin, plantaricin LD1 which causes cytocidal effect on a Gram-negative bacterium, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 leading to efflux of potential ion, dissipation of ∆ψ and ∆pH and degradation of genomic DNA. Plantaricin LD1 showed lower MIC/MBC against \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 as compared to \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. In another study, plantaricin Pln1 required a higher concentration (100 \u0026micro;g/mL) to inhibit the growth of \u003cem\u003eM. luteus\u003c/em\u003e CMCC 63202 and it was not able to kill \u003cem\u003eE. coli\u003c/em\u003e strains [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similarly, the bacteriocin MN047A from \u003cem\u003eL. crustorum\u003c/em\u003e MN047 required an even higher concentration (305 \u0026micro;g/mL) to inhibit the growth of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, as suggested by Yi et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In another study, 62.5 \u0026micro;g/mL of peptide F1 purified from \u003cem\u003eL. paracasei\u003c/em\u003e subsp. \u003cem\u003etolerans\u003c/em\u003e FX-6 was needed for the inhibition of the growth of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There are many plantaricins such as plantaricin KL-1X, plantaricin KL-1Z, Plantaricin LPL-1, plantaricin U10, and bacteriocin MBSa4 have been reported not to inhibit the growth of \u003cem\u003eE. coli\u003c/em\u003e strains [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Earlier, there were few reports, such as plantaricin E, F, J and K, which inhibited \u003cem\u003eE. coli\u003c/em\u003e when the outer membrane was weakened using physical conditions and chemical agents [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe efficacy of plantaricin LD1 on the viable cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 was dependent on the time and concentration. The bactericidal effect of plantaricin LD1 against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was found lesser than \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106. This effect could be due to the structure of the outer membrane in Gram-negative bacteria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In another study, the peptide Gt2 from \u003cem\u003eL. plantarum\u003c/em\u003e UTNGt2 showed the effect on cell-viability of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in the dose and time-dependent manner [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, Yi and co-workers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] demonstrated that the bacteriocin MN047A from \u003cem\u003eL. crustorum\u003c/em\u003e MN047 showed the time and dose-dependent way of loss in cell-viability of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922.\u003c/p\u003e \u003cp\u003eThe pore-formation in the cell membrane of indicator strains is one of the most considered mechanisms for bacteriocin activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this study, plantaricin LD1 interacts with target membrane by a potential-dependent alignment which caused release of intracellular potassium ions, nucleic acids, ATP hydrolysis and dissipation of membrane potential/pH gradient leading to cell death. The plantaricin LD1 caused efflux of K\u003csup\u003e+\u003c/sup\u003e ions from the cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in concentration dependent manner. Similarly, the other membrane-targeted peptides, such as plantaricin MG, NC8α and NC8β, also induce release of potassium ions in \u003cem\u003eM. luteus\u003c/em\u003e 1.193 which causes cell death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Miao et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] also showed similar time-dependent efflux of K\u003csup\u003e+\u003c/sup\u003e ions from the cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 after treatment with antimicrobial peptide F1 isolated from \u003cem\u003eL. paracasei\u003c/em\u003e subsp. \u003cem\u003etolerans\u003c/em\u003e FX-6. The release of K\u003csup\u003e+\u003c/sup\u003e ion from the cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 provides supplementary proof that plantaricin LD1 disrupted the cell membrane of both target strains as suggested by Zhao et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These findings prompted us to monitor the membrane potential dissipation of target bacteria.\u003c/p\u003e \u003cp\u003eThe transmembrane potential is a vital bio-energetic component of bacterial cells that is involved directly in energizing critical cellular activities such as ion transport, ATP synthesis, and motility, as suggested by Buttress et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The cells of both indicator strains showed the complete dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) after treatment with plantaricin LD1 in concentration-dependent manner. Whereas, addition of diluents did not cause dissipation of ∆ψ and ∆pH indicating that intracellular pH was equivalent to the pH of the buffer as suggested by Shi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Further addition of valinomycin did not change fluorescence indicated plantaricin LD1 completely dissipated ∆ψ and ∆pH. Similarly, plantaricin NC8 purified from \u003cem\u003eL. plantarum\u003c/em\u003e ZJ316 caused dissipation of Δѱ and ΔpH in \u003cem\u003eM. luteus\u003c/em\u003e CGMCC 1.193 through membrane permeabilization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe evidence of Δψ and pH depletion suggested that plantaricin LD1 interacted with the cytoplasmic membrane of target cells leading to formation of pores which allowed the efflux of potassium ions and nucleic acids. It has been reported that bacteriocins disintegrates the proton motive force (PMF) in the cells of target strains which leads to the degradation of intracellular components such as potassium ions, inorganic phosphate, ATP and UV-absorbing materials [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, our findings suggest that plantaricin LD1 causes pore formation resulting in potential membrane dissipation of target cells.\u003c/p\u003e \u003cp\u003ePlantaricin LD1 was able to interact with the DNA of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 and caused fragmentation evidenced by gel electrophoresis. It suggests that plantaricin LD1 not only interacts with the cell membrane of target cells but also enters inside the cells and cause nucleic degradation. Our data is in consistent with the reported studies where antimicrobial peptides were found to be capable of inhibiting the synthesis and functions of intracellular biopolymers, particularly those that exhibited specific inhibition of DNA synthesis by binding directly to bacterial DNA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Du et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] suggested that DNA repair pathways were blocked after the treatment with plantaricin GZ1-27 which resulted instability of the genome of Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) ATCC 43300 causing cell damage or death. The concentration dependent DNA degradation was also confirmed \u003cem\u003ein vitro\u003c/em\u003e where genomic DNA of target cells where isolated and then treated with plantaricin LD1. Similarly, Zhao et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] demonstrated \u003cem\u003ein vitro\u003c/em\u003e interaction of plantaricin 827 with genomic DNA of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923. They also demonstrated the \u003cem\u003ein silico\u003c/em\u003e interaction of plantaricin 827 with the DNA minor groove in AT-rich regions of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923. The ability of these bacteriocins to interact with the DNA is unique feature indicating the arrest of cellular functions leading to cell death.\u003c/p\u003e \u003cp\u003eThe dead and live cells were also estimated after staining with a fluorescent dyes, PI and DAPI, respectively as suggested by Duan et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The untreated cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were stained with DAPI and found blue, demonstrating live cells. The PI is resistant to integral membranes but can transfer through broken cell membrane and is associated with cellular DNA, making it a nucleus-staining reagent with high affinity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. After the treatment with plantaricin LD1, the target cells showed mixture of blue and red cells suggesting live and dead cells. These observations further indicated the bactericidal mode of action of plantaricin LD1 against Gram-positive and Gram-negative bacteria. In contrast, nisin-treated cells of \u003cem\u003eE. coli\u003c/em\u003e were not affected also suggested by Cleveland et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe microscopic images of plantaricin LD1-treated target bacteria confirmed several damages and cell death. The cells of \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 were ruptured after the treatment with plantaricin LD1 suggesting the membrane-acting nature of plantaricin LD1. The cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 were also found swollen and ruptured as observed under light and scanning electron microscope. The nisin did not show such effects on cells of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The damage to the cell membrane of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 is the distinct mode of action of bacteriocin, as also suggested by Zhang et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Jiang et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] suggested that after treatment with plantaricin NC8α, the cells of \u003cem\u003eM. luteus\u003c/em\u003e CGMCC 1.193 showed similar disruption and shrinkage under SEM. Such damages of target cells were further confirmed under transmission electron microscope. In the presence of plantaricin LD1 cell membrane of \u003cem\u003eE. coli\u003c/em\u003e ATCC 29522 was severely damaged and protruded. The membrane penetration on the target cells was also observed after treatment with plantaricin LD1 suggesting its bactericidal effect. Similar findings have also been reported by Jiang et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] where plantaricin NC8 caused disruption of the cell membrane, widespread shrinkage of the cytoplasmic contents, and vacuolization of \u003cem\u003eM. luteus\u003c/em\u003e CGMCC 1.193 cells observed under TEM.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe minimum inhibitory and minimum bactericidal concentrations of plantaricin LD1 was found lower against \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 as compared to \u003cem\u003eE. coli\u003c/em\u003e ATCC 29522. The loss of cell viability, release of K\u003csup\u003e+\u003c/sup\u003e ions, dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) in target cells suggested bactericidal mode of action of plantaricin LD1. The interaction of plantaricin LD1 with genomic DNA of target strains was also noticed. The morphological deformation/damages of target cells indicated the cell killing as observed under light, fluorescence, scanning and transmission electron microscopes. The staining of target cells with propidium iodide further confirmed the bactericidal mode of action of plantaricin LD1. Thus, the present investigation has found a unique bactericidal and membrane-acting mode of action of plantaricin LD1 against \u003cem\u003eE. coli\u003c/em\u003e ATCC 29522.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe are thankful to Indian Council of Medical Research (5/9/1117/2013-NUT) and Indian National Science Academy (IA/INDO-AUST/F-4/2017/1872), New Delhi for financial assistance. The University Research scholarship, Department of Genetics, M. D. University Rohtak is acknowledged for providing fellowship to MKY.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe Indian Council of Medical Research (Project No. 5/9/1117/2013-NUT) and Indian National Science Academy (Project No. IA/INDO-AUST/F-4/2017/1872), New Delhi, India, provided financial support for this work. The University Grants Commission-Scheme for Trans-disciplinary Research for India\u0026rsquo;s Developing Economy Component-1 (UGC-STRIDE-1), New Delhi, India [Project No. F.2-16/2019 (STRIDE 1)] was truly acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eMKY-Performed the experiments and drafted the manuscript; SKT-Designed the experiments and provided all laboratory facilities. All authors read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data\u0026nbsp;\u003c/strong\u003eNot available.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003eThe research did not include any human subjects or animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYang, S. C., Lin, C. H., Sung, C. T., \u0026amp; Fang, J. Y. (2014). Antibacterial activities of bacteriocins: Application in foods and pharmaceuticals. \u003cem\u003eFrontiers In Microbiology\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, 241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2014.00241\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2014.00241\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarbandi, A., Asadi, A., Mahdizade Ari, M., Ohadi, E., Talebi, M., Halaj Zadeh, M., Darb Emamie, A., Ghanavati, R., \u0026amp; Kakanj, M. (2022). Bacteriocins: Properties and potential use as antimicrobials. \u003cem\u003eJournal Of Clinical Laboratory Analysis\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e, e24093. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcla.24093\u003c/span\u003e\u003cspan address=\"10.1002/jcla.24093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav, M. K., Singh, B., \u0026amp; Tiwari, S. K. (2019). Comparative analysis of inhibition-based and indicator-independent colorimetric assay for screening of bacteriocin-producing lactic acid bacteria. \u003cem\u003eProbiotics Antimicrob Proteins\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 687\u0026ndash;695. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12602-018-9445-4\u003c/span\u003e\u003cspan address=\"10.1007/s12602-018-9445-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePato, U., Riftyan, E., Jonnaidi, N. N., Wahyuni, M. S., Feruni, J. A., \u0026amp; Abdel-Wahhab, M. A. (2022). Isolation, characterization, and antimicrobial evaluation of bacteriocin produced by lactic acid bacteria against \u003cem\u003eErwinia carotovora\u003c/em\u003e. \u003cem\u003eLebensmittel-Wissenschaft + [I.E. Und] Technologie. Food Science + Technology. Science + Technologie Alimentaire\u003c/em\u003e, \u003cem\u003e42\u003c/em\u003e, e11922. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/fst.11922\u003c/span\u003e\u003cspan address=\"10.1590/fst.11922\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoltani, S., Hammami, R., Cotter, P. D., Rebuffat, S., Said, L. B., Gaudreau, H., B\u0026eacute;dard, F., Biron, E., Drider, D., \u0026amp; Fliss, I. (2021). Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. \u003cem\u003eFems Microbiology Reviews\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e, fuaa039. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsre/fuaa039\u003c/span\u003e\u003cspan address=\"10.1093/femsre/fuaa039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYap, P. G., Lai, Z. W., \u0026amp; Tan, J. S. (2022). Bacteriocins from lactic acid bacteria: purification strategies and applications in food and medical industries: A review. \u003cem\u003eBeni Suef Univ J Basic Appl Sci\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43088-022-00227-x\u003c/span\u003e\u003cspan address=\"10.1186/s43088-022-00227-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, H., Tang, X., Zhou, Q., Zou, J., Li, P., Breukink, E., \u0026amp; Gu, Q. (2018). Plantaricin NC8 from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e causes cell membrane disruption to \u003cem\u003eMicrococcus luteus\u003c/em\u003e without targeting lipid II. \u003cem\u003eApplied Microbiology And Biotechnology\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e, 7465\u0026ndash;7473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-018-9182-3\u003c/span\u003e\u003cspan address=\"10.1007/s00253-018-9182-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimothy, B., Iliyasu, A. H., \u0026amp; Anvikar, A. R. (2021). Bacteriocins of lactic acid bacteria and their industrial application. \u003cem\u003eCurr Top Lact Acid Bact Probiotics\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, 1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.35732/ctlabp.2021.7.1.1\u003c/span\u003e\u003cspan address=\"10.35732/ctlabp.2021.7.1.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Ramos, A., Madi-Moussa, D., Coucheney, F., \u0026amp; Drider, D. (2021). Current knowledge of the mode of action and immunity mechanisms of LAB-bacteriocins. \u003cem\u003eMicroorganisms\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 2107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms9102107\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms9102107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubasova, I., Diep, D. B., Ovchinnikov, K. V., Laukova, A., \u0026amp; Strompfova, V. (2020). Bacteriocin production and distribution of bacteriocin-encoding genes in enterococci from dogs. \u003cem\u003eInternational Journal Of Antimicrobial Agents\u003c/em\u003e, \u003cem\u003e55\u003c/em\u003e, 105859. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijantimicag.2019.11.016\u003c/span\u003e\u003cspan address=\"10.1016/j.ijantimicag.2019.11.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, A., \u0026amp; Tiwari, S. K. (2014a). Probiotic potential of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e ld1 isolated from batter of \u003cem\u003eDosa\u003c/em\u003e, a South Indian fermented food. \u003cem\u003eProbiotics Antimicrob Proteins\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e, 73\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12602-014-9158-2\u003c/span\u003e\u003cspan address=\"10.1007/s12602-014-9158-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, A., \u0026amp; Tiwari, S. K. (2014b). Plantaricin LD1: A bacteriocin produced by food isolate of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e LD1. \u003cem\u003eApplied Biochemistry And Biotechnology\u003c/em\u003e, \u003cem\u003e172\u003c/em\u003e, 3354\u0026ndash;3362. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12010-014-0775-8\u003c/span\u003e\u003cspan address=\"10.1007/s12010-014-0775-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav, M. K., \u0026amp; Tiwari, S. K. (2021). Polydiacetylene vesicles acting as colorimetric sensor for the detection of plantaricin LD1. \u003cem\u003eAnalytical Biochemistry\u003c/em\u003e, \u003cem\u003e631\u003c/em\u003e, 114368. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ab.2021.114368\u003c/span\u003e\u003cspan address=\"10.1016/j.ab.2021.114368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav, M. K., Yadav, P., Dhiman, M., Tewari, S., \u0026amp; Tiwari, S. K. (2022). Plantaricin LD1 purified from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e LD1 inhibits biofilm formation of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212 in tooth model. \u003cem\u003eLetters In Applied Microbiology\u003c/em\u003e, \u003cem\u003e75\u003c/em\u003e, 623\u0026ndash;631. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/lam.13668\u003c/span\u003e\u003cspan address=\"10.1111/lam.13668\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheoran, P., \u0026amp; Tiwari, S. K. (2019). Enterocin LD3 from \u003cem\u003eEnterococcus hirae\u003c/em\u003e LD3 causing efflux of intracellular ions and UV-absorbing materials in Gram-negative bacteria. \u003cem\u003eJournal Of Applied Microbiology\u003c/em\u003e, \u003cem\u003e126\u003c/em\u003e, 1059\u0026ndash;1069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jam.14203\u003c/span\u003e\u003cspan address=\"10.1111/jam.14203\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTenea, G. N., \u0026amp; Delgado Pozo, T. (2019). Antimicrobial peptides from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e UTNGt2 prevent harmful bacteria growth on fresh tomatoes. \u003cem\u003eJournal Of Microbiology And Biotechnology\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e, 1553\u0026ndash;1560. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4014/jmb.1904.04063\u003c/span\u003e\u003cspan address=\"10.4014/jmb.1904.04063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao, J., Liu, G., Ke, C., Fan, W., Li, C., Chen, Y., Dixon, W., Song, M., Cao, Y., \u0026amp; Xiao, H. (2016). Inhibitory effects of a novel antimicrobial peptide from kefir against \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eFood Control\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e, 63\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodcont.2016.01.023\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2016.01.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, F., Wang, Y., Li, Y., \u0026amp; Wang, X. (2016). Mode of action of leucocin K7 produced by \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e K7 against \u003cem\u003eListeria monocytogenes\u003c/em\u003e and its potential in milk preservation. \u003cem\u003eBiotechnology Letters\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, 1551\u0026ndash;1557. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10529-016-2127-y\u003c/span\u003e\u003cspan address=\"10.1007/s10529-016-2127-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSambrook, J., \u0026amp; Rusell, D. (2001). \u003cem\u003eMolecular cloning: A laboratory manual\u003c/em\u003e (3rd ed.). Cold Spring Harbor Laboratory Press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhodaei, M., \u0026amp; Sh, S. N. (2018). Isolation and molecular identification of bacteriocin-producing enterococci with broad antibacterial activity from traditional dairy products in Kerman province of Iran. \u003cem\u003eKorean J Food Sci Anim Resour\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e, 172\u0026ndash;179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5851/kosfa.2018.38.1.172\u003c/span\u003e\u003cspan address=\"10.5851/kosfa.2018.38.1.172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, Y., Luo, L., Wang, X., Lu, Y., Yi, Y., Shan, Y., Liu, B., Zhou, Y., \u0026amp; L\u0026uuml;, X. (2021). Mining, heterologous expression, purification, antibactericidal mechanism, and application of bacteriocins: A review. \u003cem\u003eComprehensive Reviews In Food Science And Food Safety\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e, 863\u0026ndash;899. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1541-4337.12658\u003c/span\u003e\u003cspan address=\"10.1111/1541-4337.12658\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, F., Zhao, H., Zhang, C., Lu, F., Bie, X., \u0026amp; Lu, Z. (2016). Expression of a novel bacteriocin-the plantaricin Pln1-in \u003cem\u003eEscherichia coli\u003c/em\u003e and its functional analysis. \u003cem\u003eProtein Expr Purif\u003c/em\u003e, \u003cem\u003e119\u003c/em\u003e, 85\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pep.2015.11.008\u003c/span\u003e\u003cspan address=\"10.1016/j.pep.2015.11.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi, L., Dang, Y., Wu, J., Zhang, L., Liu, X., Liu, B., Zhou, Y., \u0026amp; Lu, X. (2016). Purification and characterization of a novel bacteriocin produced by \u003cem\u003eLactobacillus crustorum\u003c/em\u003e MN047 isolated from koumiss from Xinjiang. \u003cem\u003eChina J Dairy Sci\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e, 7002\u0026ndash;7015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.3168/jds.2016-11166\u003c/span\u003e\u003cspan address=\"10.3168/jds.2016-11166\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRumjuankiat, K., Perez, R. H., Pilasombut, K., Keawsompong, S., Zendo, T., Sonomoto, K., \u0026amp; Nitisinprasert, S. (2015). Purification and characterization of a novel plantaricin, KL-1Y, from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e KL-1. \u003cem\u003eWorld Journal Of Microbiology \u0026amp; Biotechnology\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e, 983\u0026ndash;994. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-015-1851-0\u003c/span\u003e\u003cspan address=\"10.1007/s11274-015-1851-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSogandi, S., Mustopa, A. Z., Artika, I. M., \u0026amp; Budiarto, B. R. (2015). Inhibitory activity of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e U10 isolated from tempoyak (fermented durian) made in Indonesia against \u003cem\u003eSalmonella typhi\u003c/em\u003e. \u003cem\u003eMicrobiol Indones\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 5\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5454/mi.9.2.4\u003c/span\u003e\u003cspan address=\"10.5454/mi.9.2.4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbosa, M. S., Todorov, S. D., Ivanova, I. V., Belguesmia, Y., Choiset, Y., Rabesona, H., Chobert, J. M., Haertl\u0026eacute;, T., \u0026amp; Franco, B. D. G. (2016). Characterization of a two-peptide plantaricin produced by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e MBSa4 isolated from Brazilian salami. \u003cem\u003eFood Control\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e, 103\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.foodcont.2015.07.029\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2015.07.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. D. M.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., Qin, Y., Xie, Q., Zhang, Y., Hu, J., \u0026amp; Li, P. (2018). Purification and characterization of plantaricin LPL-1, a novel class IIa bacteriocin produced by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e LPL-1 isolated from fermented fish. \u003cem\u003eFrontiers In Microbiology\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 2276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2018.02276\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2018.02276\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePal, G., \u0026amp; Srivastava, S. (2014). Inhibitory effect of plantaricin peptides (Pln E/F and J/K) against \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eWorld Journal Of Microbiology \u0026amp; Biotechnology\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e, 2829\u0026ndash;2837. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-014-1708-y\u003c/span\u003e\u003cspan address=\"10.1007/s11274-014-1708-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwechheimer, C., \u0026amp; Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 605\u0026ndash;619. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro3525\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro3525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTodorov, S. D., de Paula, O. A., Camargo, A. C., Lopes, D. A., \u0026amp; Nero, L. A. (2018). Combined effect of bacteriocin produced by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by \u003cem\u003eListeria monocytogenes\u003c/em\u003e. \u003cem\u003eRevista Argentina De Microbiologia\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e, 48\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ram.2017.04.011\u003c/span\u003e\u003cspan address=\"10.1016/j.ram.2017.04.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, D., Wang, Q., Lu, F., Bie, X., Zhao, H., Lu, Z., \u0026amp; Lu, Y. (2022). A novel plantaricin 827 effectively inhibits \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and extends shelf life of skim milk. \u003cem\u003eLWT\u003c/em\u003e, \u003cem\u003e154\u003c/em\u003e, 112849. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2021.112849\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2021.112849\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalte, B. J. A., Te Winkel, M., Erhardt, J. D., Popp, M., P. F., \u0026amp; Strahl, H. (2022). A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes. \u003cem\u003eMicrobiol (Reading England)\u003c/em\u003e, \u003cem\u003e168\u003c/em\u003e, 001227. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.001227\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.001227\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong, H. S., Meng, X. C., \u0026amp; Wang, H. (2010). Mode of action of plantaricin MG, a bacteriocin active against \u003cem\u003eSalmonella typhimurium\u003c/em\u003e. \u003cem\u003eJournal Of Basic Microbiology\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e, S37\u0026ndash;S45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1002/jobm.201000130\u003c/span\u003e\u003cspan address=\"10.1002/jobm.201000130\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, H., Zhou, L., Lu, Z., Bie, X., Zhao, H., Niu, Y. D., \u0026amp; Lu, F. (2020). Transcriptomic and proteomic profiling response of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) to a novel bacteriocin, plantaricin GZ1-27 and its inhibition of biofilm formation. \u003cem\u003eApplied Microbiology And Biotechnology\u003c/em\u003e, \u003cem\u003e104\u003c/em\u003e, 7957\u0026ndash;7970. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-020-10589-w\u003c/span\u003e\u003cspan address=\"10.1007/s00253-020-10589-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan, F., Xin, G., Niu, H., \u0026amp; Huang, W. (2017). Chlorinated emodin as a natural antibacterial agent against drug-resistant bacteria through dual influence on bacterial cell membranes and DNA. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, 12721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-017-12905-3\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-12905-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, Y., Mei, C., Huang, X., Gu, Q., \u0026amp; Song, D. (2020). Antibacterial activity and mechanism of a bacteriocin derived from the valine-cecropin a (1\u0026ndash;8)-plantaricin zj5 (1\u0026ndash;18) hybrid peptide against \u003cem\u003eEscherichia coli\u003c/em\u003e O104. Food Biophys. 15, 442\u0026ndash;451. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11483-020-09636-w\u003c/span\u003e\u003cspan address=\"10.1007/s11483-020-09636-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCleveland, J., Chikindas, M., \u0026amp; Montville, T. J. (2002). Multimethod assessment of commercial nisin preparations. \u003cem\u003eJournal Of Industrial Microbiology And Biotechnology\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e, 228\u0026ndash;232. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.jim.7000315\u003c/span\u003e\u003cspan address=\"10.1038/sj.jim.7000315\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X., Wang, Y., Liu, L., Wei, Y., Shang, N., Zhang, X., \u0026amp; Li, P. (2016). Two-peptide bacteriocin PlnEF causes cell membrane damage to \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. \u003cem\u003eBiochim Biophys Acta Biomembr\u003c/em\u003e, \u003cem\u003e1858\u003c/em\u003e, 274\u0026ndash;280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.bbamem.2015.11.018\u003c/span\u003e\u003cspan address=\"10.1016/j.bbamem.2015.11.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plantaricin LD1. Scanning and transmission electron microscopy. Membrane potential. Transmembrane pH gradient. Propidium iodide. Nucleic acid","lastPublishedDoi":"10.21203/rs.3.rs-3823808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3823808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlantaricin LD1 was purified from a putative probiotic \u003cem\u003eLactobacillus plantarum\u003c/em\u003e LD1 previously isolated from food. In this study, we have tested detailed mechanism of action against \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 considering \u003cem\u003eMicrococcus luteus\u003c/em\u003e MTCC 106 as control. The plantaricin LD1 showed minimum inhibitory concentration (MIC) 34.57 \u0026micro;g/mL and minimum bactericidal concentration (MBC) 138.3 \u0026micro;g/mL against \u003cem\u003eM. luteus\u003c/em\u003e MTCC 106 and MIC 69.15 \u0026micro;g/mL and MBC 276.6 \u0026micro;g/mL against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The efflux of K\u003csup\u003e+\u003c/sup\u003e ions, dissipation of membrane potential (∆ψ) and transmembrane pH gradient (∆pH) of plantaricin LD1-treated cells suggested the membrane-acting nature of plantaricin LD1. Plantaricin LD1 also caused degradation of genomic DNA of target strains tested. The cell killing was confirmed by staining with propidium iodide and visualizing under light and electron microscopes which were ruptured, smaller, swollen and elongated after treatment with plantaricin LD1. Thus, the findings in this paper indicates plantaricin LD1 kills \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 by interacting with cell membrane resulting in efflux of intracellular contents and also caused degradation of nucleic acids leading to cell death.\u003c/p\u003e","manuscriptTitle":"Mechanism of cell killing activity of plantaricin LD1 against Escherichia coli ATCC 25922","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 09:02:48","doi":"10.21203/rs.3.rs-3823808/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-22T05:30:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-09T15:55:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2024-01-02T12:50:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2023-12-30T05:58:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6b96eee4-72c2-494c-a6b1-46989a2efedb","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-01T15:09:05+00:00","versionOfRecord":{"articleIdentity":"rs-3823808","link":"https://doi.org/10.1007/s12010-024-04927-1","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2024-03-25 15:01:38","publishedOnDateReadable":"March 25th, 2024"},"versionCreatedAt":"2024-01-12 09:02:48","video":"","vorDoi":"10.1007/s12010-024-04927-1","vorDoiUrl":"https://doi.org/10.1007/s12010-024-04927-1","workflowStages":[]},"version":"v1","identity":"rs-3823808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3823808","identity":"rs-3823808","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}