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Calcium (Ca2+)-regulated exopolysaccharide biosynthesis in probiotic Lactobacillus plantarum K25 as analyzed by an omics approach

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    * These authors contributed equally to this work.
    Yunyun Jiang
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, P.R. China 100048

    Mengniu Gaoke Dairy (Beijing) Co. Ltd., Beijing, P.R. China 101100
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    * These authors contributed equally to this work.
    Min Zhang
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, P.R. China 100048
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  • Yang Zhang
    Affiliations
    Department of Neurology, Affiliated Hospital of Guizhou Medical University, Guiyang, P.R. China 550001
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  • Justyna Zulewska
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    Department of Dairy Science and Quality Management, Faculty of Food Sciences, University of Warmia and Mazury, 10-719 Olsztyn, Poland
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  • Zhennai Yang
    Correspondence
    Corresponding author
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    Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, P.R. China 100048
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  • Author Footnotes
    * These authors contributed equally to this work.
Open ArchivePublished:January 14, 2021DOI:https://doi.org/10.3168/jds.2020-19237

      ABSTRACT

      Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca2+ on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl2 at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca2+ also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca2+, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca2+ upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca2+ activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca2+, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.

      Key words

      INTRODUCTION

      Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) have been reported as having antioxidant, antimicrobial, antitumor, antiulcer activities, as well as lowering cholesterol, enhancing immunity, and serving as prebiotics (
      • van den Nieuwboer M.
      • van Hemert S.
      • Claassen E.
      • de Vos W.M.
      Lactobacillus plantarum WCFS1 and its host interaction: A dozen years after the genome.
      ). The EPS produced by Lactobacillus plantarum strains 70810, YW32, YW11, DM5, MTCC 9510, RJF4, ZDY2013, and C88 had both antitumor and antioxidant activities (
      • Ismail B.
      • Nampoothiri K.M.
      Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510.
      ;
      • Zhang L.
      • Liu C.
      • Li D.
      • Zhao Y.
      • Zhang X.
      • Zeng X.
      • Yang Z.
      • Li S.
      Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88.
      ,
      • Zhang Z.
      • Liu Z.
      • Tao X.
      • Wei H.
      Characterization and sulfated modification of an exopolysaccharide from Lactobacillus plantarum ZDY2013 and its biological activities.
      ;
      • Dilna S.V.
      • Surya H.
      • Aswathy R.G.
      • Varsha K.K.
      • Sakthikumar D.N.
      • Pandey A.
      • Nampoothiri K.M.
      Characterization of an exopolysaccharide with potential health-benefit properties from a probiotic Lactobacillus plantarum RJF4.
      ;
      • Wang J.
      • Zhao X.
      • Yang Y.
      • Zhao A.
      • Yang Z.
      Characterization and bioactivities of an exopolysaccharide produced by Lactobacillus plantarum YW32.
      ,
      • Wang K.
      • Li W.
      • Rui X.
      • Li T.
      • Chen X.
      • Jiang M.
      • Dong M.
      Chemical modification, characterization and bioactivity of a released exopolysaccharide (r-EPS1) from Lactobacillus plantarum 70810.
      ;
      • Jiang Y.
      • Yang Z.
      A functional and genetic overview of exopolysaccharides produced by Lactobacillus plantarum..
      ). The EPS from L. plantarum BR2, KF5, and RJF4 possessed cholesterol-lowering activity (
      • Wang Y.
      • Li C.
      • Liu P.
      • Ahmed Z.
      • Xiao P.
      • Bai X.
      Physical characterization of exopolysaccharide produced by Lactobacillus plantarum KF5 isolated from Tibet Kefir.
      ;
      • Sasikumar K.
      • Vaikkath D.K.
      • Devendra L.
      • Nampoothiri K.M.
      An exopolysaccharide (EPS) from a Lactobacillus plantarum BR2 with potential benefits for making functional foods.
      ;
      • Jiang Y.
      • Zhang J.
      • Zhao X.
      • Zhao W.
      • Yu Z.
      • Chen C.
      • Yang Z.
      Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.
      ). The EPS from Bifidobacterium longum W11 was able to regulate the human immune system (
      • Wu W.
      • Wang Y.
      • Zou J.
      • Long F.
      • Yan H.
      • Zeng L.
      • Chen Y.
      Bifidobacterium adolescentis protects against necrotizing enterocolitis and upregulates TOLLIP and SIGIRR in premature neonatal rats.
      ). It was of significance to increase the capability of LAB to produce EPS that was closely related to the producing strain, fermentation conditions, and culture medium composition, including calcium ion concentration (
      • Desai K.M.
      • Akolkar S.K.
      • Badhe Y.P.
      • Tambe S.S.
      • Lele S.S.
      Optimization of fermentation media for exopolysaccharide production from Lactobacillus plantarum using artificial intelligence-based techniques.
      ).
      Calcium ion, as an intracellular secondary messenger, played important roles in different cellular pathways involving almost all the cell reactions required for normal cell survival (
      • Domínguez D.C.
      • Guragain M.
      • Patrauchan M.
      Calcium binding proteins and calcium signaling in prokaryotes.
      ). The Ca2+ concentration was strictly controlled at 100 to 300 nM in bacterial cells, which used the concentration gradient to transmit information. Variations in Ca2+ concentration were connected with many bacterial cellular behaviors, for instance, spore formation of Bacillus subtilis, chemotaxis of Escherichia coli, cyanobacterial variation of Cyanobacteria, fruiting body development of Myxobacterium (
      • Verkhratsky A.
      • Parpura V.
      Calcium signalling and calcium channels: Evolution and general principles.
      ;
      • Song L.
      • Cui R.
      • Yang Y.
      • Wu X.
      Role of calcium channels in cellular antituberculosis effects: Potential of voltage-gated calcium-channel blockers in tuberculosis therapy.
      ). Physiologically, Ca2+ could induce phosphorylation to activate regulatory proteins including primary and secondary transporters (e.g., Ca2+ pumps), Ca2+ binding protein, and Ca2+ channel proteins (
      • Domínguez D.C.
      • Guragain M.
      • Patrauchan M.
      Calcium binding proteins and calcium signaling in prokaryotes.
      ). Addition of Ca2+ was found to increase the yield and biological activities of the EPS from Streptococcus thermophilus ST 1275 and ST 285, L. rhamnosus ZY, and Vibrio vulnificus (
      • Purwandari U.
      • Vasiljevic T.
      Microbial growth, EPS concentration and textural properties of fermented milk supplemented with calcium and whey protein analysed using response surface methodology.
      ;
      • Ng I.S.
      • Xue C.
      Enhanced exopolysaccharide production and biological activity of Lactobacillus rhamnosus ZY with calcium and hydrogen peroxide.
      ;
      • Pu M.
      • Storms E.
      • Chodur D.M.
      • Rowe-Magnus D.A.
      Calcium-dependent site-switching regulates expression of the atypical iam pilus locus in Vibrio vulnificus.
      ). However, the regulatory mechanism of Ca2+ on EPS biosynthesis in LAB is not clear (
      • Ng I.S.
      • Xue C.
      Enhanced exopolysaccharide production and biological activity of Lactobacillus rhamnosus ZY with calcium and hydrogen peroxide.
      ).
      Previously, EPS-producing L. plantarum K25 isolated from Tibetan kefir was characterized as a probiotic strain (
      • Jiang Y.
      • Zhang J.
      • Zhao X.
      • Zhao W.
      • Yu Z.
      • Chen C.
      • Yang Z.
      Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.
      ). Cheddar cheese made with strain K25 was shown with cholesterol-lowering effect in a mouse model fed a high-fat diet (
      • Zhang L.
      • Zhang X.
      • Liu C.
      • Li C.
      • Li S.
      • Li T.
      • Li D.
      • Zhao Y.
      • Yang Z.
      Manufacture of cheddar cheese using probiotic Lactobacillus plantarum K25 and its cholesterol-lowering effects in a mice model.
      ). In the present study, the effect of Ca2+ on the EPS biosynthesis in L. plantarum K25, the microstructure, molecular mass distribution, and monosaccharide composition of the EPS were studied. Data independent acquisition (DIA) proteomics and GC-MS-based nontargeted metabolomics were used to analyze the protein changes and regulatory mechanism of the EPS biosynthesis in L. plantarum K25 in response to Ca2+.

      MATERIALS AND METHODS

      Bacterial Growth Conditions and EPS Preparation

      Lactobacillus plantarum K25 was provided by Jilin Academy of Agricultural Sciences of China (Jilin, China;
      • Zhang L.
      • Zhang X.
      • Liu C.
      • Li C.
      • Li S.
      • Li T.
      • Li D.
      • Zhao Y.
      • Yang Z.
      Manufacture of cheddar cheese using probiotic Lactobacillus plantarum K25 and its cholesterol-lowering effects in a mice model.
      ). To study the effect of calcium ion on the EPS biosynthesis in L. plantarum K25, the strain was grown at 37°C for 24 h in 100 mL of semi-defined medium (SDM) added with 0, 10, 20, 40, 60, 80, and 100 mg/L CaCl2, respectively. The culture samples were taken to determine values for the optical density measured at a wavelength of 600 nm (OD600), pH, and concentration of EPS by phenol-sulfuric acid method with glucose as a standard. To obtain the EPS for the following physicochemical analyses of the polysaccharide, L. plantarum K25 was grown at 37°C for 24 h in SDM added with selected concentration of CaCl2 or without addition of CaCl2 (control), and the EPS was extracted and purified using the described method (
      • Wang J.
      • Zhao X.
      • Tian Z.
      • Yang Y.
      • Yang Z.
      Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet kefir.
      ). For studies on proteomics and metabolomics of L. plantarum K25, the strain was grown in SDM added with the selected concentration of CaCl2 or without addition of CaCl2 (control) at 37°C for 12 h. Each treatment was repeated 3 times.

      Monosaccharide Analysis of EPS

      The EPS (2 mg) was hydrolyzed in 1 mL of 2 M trifluoroacetic acid (TFA) for 1.5 h. Then TFA was removed by rotary evaporation, and the hydrolysis products were determined for monosaccharides using GC-MS on a Hp-5 chromatographic column (Agilent 19091J-413, 300 × 0.25 × 0.32 mm; Agilent Technologies, Santa Clara, CA) with hydrogen and air at the flow rates of 30 and 400 mL/min, respectively. Helium was used as the carrier gas at a flow rate of 1 mL/min. The standard sugars were rhamnose, fucose, arabinose, xylose, mannose, glucose, and galactose.

      Infrared Spectral Analysis of EPS

      The Fourier transform infrared (FTIR) spectra of the EPS samples were acquired by using the total reflectance mode of a FTIR Spectrometer (iS10, Thermo Nicolet Corp., Waltham, MA). Samples were freeze-dried and mixed with potassium bromide at a ratio of 1:100 and then compressed into tablets. Measurements were performed in the midinfrared region (4,000–400 cm−1). The spectrometer had a resolution of 4 cm−1, a signal-to-signal ratio of 50,000:1, and 32 scans. All samples were analyzed in triplicate under the same conditions.

      Microstructure of the EPS

      Microstructure of the EPS (2 mg, lyophilized powder) from L. plantarum K25 was observed by scanning electron microscopy using the instrument (S-4800, Hitachi Ltd., Tokyo, Japan) with an acceleration voltage of 5.0 kV (
      • Ahmed Z.
      • Wang Y.
      • Anjum N.
      • Ahmad H.
      • Ahmad A.
      • Raza M.
      Characterization of new exopolysaccharides produced by coculturing of L. kefiranofaciens with yoghurt strains.
      ).

      Molecular Mass Determination of EPS

      Gel permeation chromatography (GPC) was used to determine the molecular weight of the EPS. The GPC system consisted of 3 Waters Ultra-hydrogel 250, 1000, and 2000 columns (7.8 × 300 mm; Waters, Milford, MA) in series. The column was eluted with 20 mM CH3COONH4 solution at a flow rate of 0.5 mL/min, and 20 μL of EPS was injected at a maintenance temperature of 40°C.

      Analysis of Expression of EPS Genes in L. plantarum K25 by RT-qPCR

      The effect of calcium ion on the expression level of eps genes in L. plantarum K25 was analyzed by real-time quantitative PCR (RT-qPCR). Strain K25 was grown in SDM added with 0 and appropriate concentration of CaCl2 at 37°C for 12 h, respectively. Total RNA was isolated with the RNAprep pure cell/bacteria kit following the manufacturer's instruction (DP430, Tiangen, Beijing, China). The RNA samples were used to synthesize cDNA with PrimeScript RT reagent Kit with gDNA Eraser following the manufacturer's instructions (RR047B, Takara, Dalian, China). The primers for amplifying gene fragments were listed in Supplemental Table S1 (https://figshare.com/s/1349c636cb625d2e12da). The RT-qPCR was performed using an SYBR Premix Ex Taq II (Tli RNaseH Plus; Tiangen, Beijing, China) on an ABI7500 Real-Time PCR system (Applied Biosystems, Foster City, CA). The expression level of genes was calculated according to the 2−ΔΔCt method (
      • Ahmed Z.
      • Wang Y.
      • Anjum N.
      • Ahmad H.
      • Ahmad A.
      • Raza M.
      Characterization of new exopolysaccharides produced by coculturing of L. kefiranofaciens with yoghurt strains.
      ) with 16S rRNA as the internal control. All experiments were conducted in triplicate.

      Proteomics

      To prepare protein samples, bacterial cells were dissolved in buffer solution (500 mM Tris-HCl, 50 mM EDTA, 700 mM sucrose, 100 mM KCl, 2% β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0). Tris-saturated phenol was used to extract proteins. Samples were settled by cold acetone at −20°C overnight. The precipitate was removed by centrifugation at 4,200 × g and 4°C for 10 min. Samples were redissolved in 8 M urea. Then proteins were separated by SDS-PAGE. Enzymatic hydrolysis of proteins was performed by the filter-aided sample preparation method. The peptides were taken out from each sample and classified with high pH RP method, and were detected through LC-MS/MS. A C18 column (3 µm, 100Å, 75 µm × 15 cm, EasyNano LC1000, Thermo Fisher Scientific, Waltham, MA) was used to separate the peptides. Injection volume was 9 μL, the mobile phase A was 0.1% formic acid, and B was 0.1% formic acid in acetonitrile. The time of chromatography gradient was 120 min.
      Proteomics data were analyzed using software Proteome Discoverer 2.1. Sequest HT was used to retrieve the database (11 AA sequences of synthetic peptides were added into the database).

      Metabolomics

      The original growth medium was removed by centrifugation at 4,200 × g and 4°C for 10 min. Bacterial cells were analyzed by GC-MS-based nontargeted metabolomics platform. Ethanol (60% vol/vol) was used to extract the metabolites. Nor-leucine (0.05 mg/mL) was the internal standard. Methoxyamine hydrochloride in pyridine was used to reconstitute the dried samples. The mixture was incubated at 37°C for 90 min, then 30 μL of bis(trimethylsilyl) trifluoroacetamide (with 1% trimethylchlorosilane) was added into the mixture and derivatized at 70°C for 60 min before GC-MS metabolomics analysis. Quality control samples pooled from serum samples in each group were prepared and analyzed with the same procedure as that for the experiment samples.
      Metabolomics instrumental analysis was performed on an Agilent 7890A GC system coupled to an Agilent 5975C inert MSD system (Agilent Technologies Inc.). The Rxi-5Sil MS fused-silica capillary column (30 m × 0.25 mm × 0.25μm, Agilent J &W Scientific, Folsom, CA) was used to separate the derivatives. Helium (>99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min through the column. Injection volume was 1 μL, and the solvent delay time was 6 min. The initial oven temperature was held at 70°C for 2 min, increased to 160°C at a rate of 6°C/min, to 240°C at a rate of 10°C/min, then to 300°C at a rate of 20°C/min, and finally held at 300°C for 6 min. The temperatures of injector, transfer line, and electron impact ion source were set to 250°C, 260°C, and 230°C, respectively. The impact energy was 70 eV, and data were collected in a full scan mode (m/z 50–600).
      The peak picking, alignment, deconvolution, and further processing of raw GC-MS data were referred to the previous published protocols (
      • Gao X.
      • Pujos-Guillot E.
      • Sebedio J.
      Development of a quantitative metabolomic approach to study clinical human fecal water metabolome based on trimethylsilylation derivatization and GC/MS analysis.
      ). The final data were exported as a peak table file, including observations (sample name), variables (rt_mz), and peak abundances. The data were normalized against total peak abundances before performing univariate and multivariate statistics.

      Statistical Analysis

      For all the experiments, one-way ANOVA followed by Tukey's post hoc test was used to determine significant differences (P < 0.05) among means. All analyses were performed with GraphPad Prism version 6.00 (GraphPad software, San Diego, CA).

      RESULTS

      Bacterial Growth and EPS Production of L. plantarum K25

      Effect of CaCl2 (0–100 mg/L) on the growth of L. plantarum K25 and EPS production in SDM broth at 37°C was shown in Figure 1. At different concentrations of CaCl2, the growth curves of strain K25 and the pH changes during the growth were not significantly different from those of the control group (Figure 1A), which rapidly reached a stationary phase at 8 h. With increase of the concentration of CaCl2 up to 20 mg/L, production of EPS increased (Figure 1B). At 20 to 80 mg/L of CaCl2, there was no significant change in EPS production. When the concentration of CaCl2 reached 100 mg/L, the production of EPS was inhibited. Further testing of EPS production by strain K25 at 20 mg/L of CaCl2 in SDM broth showed that the EPS yield increased with the increase of incubation time, reaching a maximum of 238.6 mg/L at 24 h (Figure 1C). Therefore, the concentration of CaCl2 at 20 mg/L was selected for preparation of EPS samples for the following studies of the EPS microstructure and monosaccharide composition, and studies of proteomics and metabolomics of strain K25 in response to Ca2+.
      Figure thumbnail gr1
      Figure 1Kinetics of growth, pH change, and exopolysaccharide (EPS) production by Lactobacillus plantarum K25 in semi-defined medium (SDM) added with different concentrations of CaCl2. (A) Kinetics of growth and pH change of strain K25 in SDM with different concentrations of CaCl2. (B) The EPS produced by strain K25 in SDM added with different concentration of CaCl2. (C) The EPS-production properties of strain K25 in SDM with CaCl2 at 20 mg/L. Each value represents the average of triplicate measurements.

      Monosaccharides Composition of EPS

      The EPS from both the control and experimental groups were composed of the same major monosaccharides such as rhamnose, glucose, and galactose, but with different proportion of the sugars, being 1.4:4.7:3.8 with small amounts of xylose and mannose in the former, and 1.55:4.7:3.78 in the latter, respectively (Figure 2). Supplementation of calcium ions in the growth medium increased the content of rhamnose in the EPS.
      Figure thumbnail gr2
      Figure 2The GC-MS analysis of the monosaccharide composition of the exopolysaccharide (EPS) produced by Lactobacillus plantarum K25 grown in semi-defined medium without (Control) and with 20 mg/L CaCl2.

      FTIR Analysis of EPS

      Infrared spectrum provides structural information about important functional groups of EPS that give characteristic peaks of absorption. Figure 3 shows the FTIR spectra of the EPS samples obtained when L. plantarum K25 was grown at 37°C for 12 h in SDM added with CaCl2 from 0 to 100 mg/L. Increased addition of CaCl2 did not change the general shape of the infrared spectra of the EPS, suggesting that the functional groups of the polysaccharide were not altered by addition of Ca2+. The significant absorption peak at 1,056.53 cm was due to the C=O antisymmetric stretching vibration, representing the fingerprint band of EPS produced by bacteria. The absorption peak at 1,650.12 cm was possibly due to existence of a large number of C=O. The bands around 2,935.23 cm were stretching vibrations of the C–H bond. The absorption peaks near 3,397.39 cm were from H-bonding and OH stretching vibration in the polysaccharide.
      Figure thumbnail gr3
      Figure 3Infrared spectra of the exopolysaccharide produced by Lactobacillus plantarum K25 in semi-defined medium (SDM) with CaCl2 added at 0, 10, 20, 40, 60, 80, and 100 mg/L, respectively.

      Microstructure of the Cell Surface and EPS

      Observation of the bacterial cell surface structure of L. plantarum K25 by scanning electron microscopy showed that the cell surface of strain K25 was smoother with thinner wrappage of the slimy polysaccharide formed in SDM broth with CaCl2 (Figure 4B), compared with that of the control group (Figure 4A). The EPS from strain K25 grown with CaCl2 (Figure 4D) exhibited smooth and laminar microstructure with presence of many pores, whereas the EPS from the control group showed more compact and overlay microstructure with presence of many lumps (Figure 4C).
      Figure thumbnail gr4
      Figure 4Scanning electron microscopy images of the cell surface and the exopolysaccharide (EPS) from Lactobacillus plantarum K25. Images of strain K25 cells grown in semi-defined medium (SDM) without (A) and with 20 mg/L CaCl2 (B). Images of the EPS extracted from strain K25 grown in SDM without (C) and with 20 mg/L CaCl2 (D).

      Molecular Weight Distribution of EPS

      The GPC analysis of the EPS samples from L. plantarum K25 demonstrated 3 major peaks of the polysaccharide fractions corresponding to different molecular weights (Figure 5). The average molecular weights of the EPS fractions were 106.12, 104.3, and 103.13 with a ratio of 70:25:5 in the control group, respectively. After adding calcium ions, the average molecular weights of the EPS fractions did not change significantly, corresponding to 106.05, 104.16, and 103.26, but in a different ratio of 18:24:58, respectively. Therefore, addition of calcium ions resulted in decreased proportion of the high molecular weight fraction, and increased fraction of the small molecular weight, but the medium molecular weight fraction remained unchanged.
      Figure thumbnail gr5
      Figure 5Gel permeation chromatography analysis of molecular weight distribution of the exopolysaccharide (EPS) produced by Lactobacillus plantarum K25 grown in semi-defined medium without (Control) and with 20 mg/L CaCl2. The number represents the retention time.

      mRNA Expression of Related cps Gene Clusters

      In L. plantarum K25 chromosome, there were 10 related cps genes responsible for enzymes such as priming glycosyl-transferase (cps4E), glycosyltransferase (cps4F/G/I), polysaccharide chain-length regulators (cps4ABC), flippase (cps4J), and polysaccharide polymerase (cps4H; Supplemental Table S2, https://figshare.com/s/1349c636cb625d2e12da). The plasmid 3 in strain K25 had only 8 predicted open reading frames (ORF) containing cps2ABC genes, 4 rhamnose-related glycosyltransferases (rfbA, rfbC, rfbB, and rfbD), and a transposase. Under the stimulation of calcium ions, strain K25 exhibited different pattern of gene expression from that of the control group. Nine genes were upregulated (Figure 6), including cps4J (13.5-fold), cps4H (60.5-fold), cps4I (32.5-fold), cps4G (5.5-fold), cps4F (111-fold), cps4E (36-fold), cps4A (7-fold), cps4B (16-fold), cps4C (6-fold), rfbD (5.4-fold), and cps2C (2.5-fold). Among these genes, upregulation of cps4F or rfbD might play the major role in the increased proportion of rhamnose in the EPS of strain K25 when stimulated with calcium ions as described above.
      Figure thumbnail gr6
      Figure 6Effect of Ca2+ on the mRNA expression pattern of related cps genes in Lactobacillus plantarum K25: expression of cps genes in (A) chromosomes, and (B) plasmid 3. Red line represents an mRNA fold change of ∼2.

      DIA-Based Proteomic Analysis on L. plantarum K25 in Response to Ca2+

      Based on DIA proteomic quantitative techniques, there were 1,478 and 1,493 proteins extracted from L. plantarum K25 grown in normal SDM and added with 20 mg/L CaCl2, respectively. Altogether, there were 1,643 proteins identified, and 1,558 of them were screened by the condition of abundance ≥3 or ≤0.33-fold changes with P-value < 0.01 (Figure 7). There were total 193 significantly changed proteins (differentially expressed proteins, DEP), including 125 upregulated and 68 downregulated when stimulated with calcium ions. At the same time, these DEP were subjected to Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
      Figure thumbnail gr7
      Figure 7Diagram of the number of differentially expressed proteins in Lactobacillus plantarum K25 stimulated by Ca2+.
      The GO annotation and KEGG pathway analysis showed that most distinct proteins could be divided into the following 7 categories: biosynthesis (40), cell nitrogen compound metabolism (28), small molecule metabolism (26), transcription (17), carbohydrate metabolism (16), amino acid metabolism (11), and lipid metabolism (11). The most active enzymes were ion-binding proteins. Enzyme code matching analysis was performed for the above differentially expressed proteins, and the transfer enzyme was the most prominent in the results.
      To gain insights into the interaction network of 193 DEP, STRING software was used for the analysis (http://string-db.org/). The name of species was selected as L. plantarum, and the results are shown in Figure 8. The analysis indicated that calcium ions regulated the ion transport, ribosome synthesis, UMP synthesis, fatty acid synthesis, carbon source transport and metabolism, and amino acid synthesis in L. plantarum K25. The rho protein takes an interrelation in these 5 pathways, except fatty acid synthesis. All the functions of these proteins are summarized in Supplemental Table S3 (https://figshare.com/s/1349c636cb625d2e12da).
      Figure thumbnail gr8
      Figure 8The protein–protein interaction network for differentially expressed proteins in Lactobacillus plantarum K25 stimulated by Ca2+. Important pathways are encircled by a red line.

      Classification of the Identified DEP of L. plantarum K25 Induced by Ca2+

      Carbohydrate transport and metabolism-related DEP (total 22 proteins) were mostly (19 proteins) upregulated in L. plantarum K25 when stimulated with Ca2+ (Supplemental Table S3). Among them, the expression level of the low-abundance β-glucosidase /β-galactosidase (B5G53_3021), phosphate transferase system IIA (3018), and α-mannosidase (B5G53_2794) in the control group, increased sharply, indicating that addition of Ca2+ could change the glycometabolism in strain K25. Lipid transport and metabolism were similar to those of carbohydrate metabolism, and the expression levels of related DEP (total 20 proteins) were also mostly (16 proteins) upregulated. Fatty acid synthase (FAS) contains a series of upregulated enzymes that control the synthesis of long-chain fatty acids (B5G53_1392∼B5G53_1403). It was suggested that adding Ca2+ could change the synthesis of long-chain fatty acids in L. plantarum K25. Amino acid synthetase such as homocysteine and asparagine synthetase; glutamine, hydroxyproline, tryptophan, and histidine hydrolase enzymes were upregulated, suggesting that the content of asparagine might increase. Membrane transporters such as ion transport and metabolism-related DEP (total 11 proteins) were mostly (8 proteins) upregulated in L. plantarum K25 in the SDM with Ca2+ (Supplemental Table S3). Among them, the expression level of a metal ion-ATPase (B5G53_2916) with low abundance in the control group, increased significantly. The expression level of 3 high-abundance proteins including Fe-, Co-, Zn-, Cd-, and Mn-ATPase, decreased sharply.
      Furthermore, increased levels of β-subunit proteins in class III ribonucleotide reductases might provide the necessary precursors for DNA repair. Adenosine deaminase catalyzes hydrolysis of adenine from amino group to generate hypoxanthine and ammonia. AzgA is a xanthine/uracil/vitamin C permease. PurN and PurL are involved in the synthesis of ADP and ATP, and PurH is an IMP hypoxanthine nucleotide hydrolytic enzyme (
      • Jelsbak L.
      • Mortensen M.I.B.
      • Kilstrup M.
      • Olsen J.E.
      The in vitro redundant enzymes PurN and PurT are both essential for systemic infection of mice in Salmonella enterica serovar typhimurium.
      ). All the DEP related to energy metabolism were upregulated. The protein FrsA (B5G53_2913) is a type of fermentation-respiration switch protein (
      • Jelsbak L.
      • Mortensen M.I.B.
      • Kilstrup M.
      • Olsen J.E.
      The in vitro redundant enzymes PurN and PurT are both essential for systemic infection of mice in Salmonella enterica serovar typhimurium.
      ), PTS is an EIIA (Glc)-binding protein, and pNP-butyrate has esterase activity. Pyruvate formate lyase (B5G53_2865) and pyruvate dehydrogenase system (B5G53_0349, B5G53_1871∼B5G53_1874) catalyze the decarboxylation of pyruvate to produce acetyl-CoA and enter the TCA cycle. The expression levels of 15 ribosomal proteins, which might be involved in synthesis of related proteins, were downregulated. Ribosomes are thought to be the sites of protein production, consisting of ribosomes and rRNA. Ribosome assembly is not only involved in ribosome assembly, but also closely related to mRNA translation efficiency (
      • McAteer S.P.
      • Sy B.M.
      • Wong J.L.
      • Tollervey D.
      • Gally D.L.
      • Tree J.J.
      Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli..
      ). The protein LysM degrades cell walls by degrading glycoside bonds between N-acetylicylic acid and N-acetylglucosamine, or recognizes glycoside bonds by extracellular LysM protein recognition receptors on membranes (
      • Maxwell K.L.
      • Fatehi Hassanabad M.
      • Chang T.
      • Paul V.D.
      • Pirani N.
      • Bona D.
      • Edwards A.M.
      • Davidson A.R.
      Structural and functional studies of gpX of Escherichia coli phage P2 reveal a widespread role for LysM domains in the baseplates of contractile-tailed phages.
      ).

      Metabolic Profile Analyses for EPS Synthesis

      Orthogonal partial least squares discrimination analysis (OPLS-DA), a reliable method to characterize metabolic differences between samples, was used to analyze the water-soluble metabolites data obtained with L. plantarum K25 grown in SDM or SDM+Ca2+ medium. SIMCA (soft independent modeling of class analogy) version 13.0 software (Umetrics, Umea, Sweden; https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca?page=5) was used to conduct OPLS-DA on the samples, and PCA model with one principal component and 2 orthogonal components was established. Scores plot is shown in Figure 9. The main mass parameters of the model were R2X = 0.743, R2Y = 0.999, and Q2 = 0.946. The PCA score diagram showed a significant separation between the control and the Ca2+ treatment groups. Therefore, OPLS-DA score diagram revealed significant metabolic differences between the samples of the 2 groups.
      Figure thumbnail gr9
      Figure 9Orthogonal partial least squares discrimination analysis (OPLS-DA) of water-soluble metabolites determined in Lactobacillus plantarum K25 grown in semi-defined medium without (control) and with 20 mg/L CaCl2. In left panel, t[1] = principal component 1; to[1] = orthogonal component 1. In right panel, R2 = measurement of fit; Q2 = measurement of prediction.
      The VIP value (threshold >1) of the first principal component of the OPLS-DA model was used to search for differentially expressed metabolites in combination with Student's t-test (threshold P < 0.05). Differential metabolites are characterized by searching the self-built standard substance databases, Fiehn GC/MS Metabolomics RTL Library, Golm Metabolome Database, and NIST commercial database. A total of 38 different substances (Supplemental Table S4, https://figshare.com/s/1349c636cb625d2e12da) were screened and identified in this project, among which 19 substances decreased, and 19 substances increased. There were 25 significant changes, 6 of which were downregulated, and 19 of which were upregulated (Figure 10). These would significantly change the TCA cycle, glucose metabolism, and propionate metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased.
      Figure thumbnail gr10
      Figure 10Effect of Ca2+ on metabolic pathways in Lactobacillus plantarum K25. Red = upregulation; blue = downregulation; gray = no change. Upregulation was defined as >1-fold change or downregulation as <0.5-fold change. Significance level: P < 0.05.

      DISCUSSION

      Based on the results of this study, the overall mechanism of regulation for the EPS synthesis in L. plantarum K25 as stimulated with Ca2+ could be deduced. As shown in Figure 11, multivariant factors associated with carbon source transport and metabolism, FA synthesis, amino acid synthesis, ion transport, and UMP synthesis were involved in regulating EPS synthesis in response to Ca2+. The Ca2+ activated the HipB signaling pathway to inhibit the expression of manipulator repressors such as ArsR, LytR/AlgR, IscR, and RafR, and activate the expression of GntR to regulate the EPS synthesis genes. The increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, there were also significant changes of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose and 5-phosphate ribose increased, facilitating the EPS biosynthesis. More detailed discussion about the relationship between the proteins and their metabolites involved in Ca2+-regulated EPS synthesis in strain K25 was described below.
      Figure thumbnail gr11
      Figure 11The overall mechanism of regulation for the exopolysaccharide (EPS) biosynthesis in Lactobacillus plantarum K25 as stimulated with Ca2+, involving multivariant factors associated with carbon source transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, and UMP synthesis in the strain.
      With respect to changes in carbohydrate transport and metabolism in L. plantarum K25, addition of Ca2+ resulted in upregulation of most of the related proteins. Upregulation of several proteins belonging to the phosphotransferase system helped absorption of monosaccharides such as glucose, galactose, or mannose (
      • Crigler J.
      • Bannerman-Akwei L.
      • Cole A.E.
      • Eiteman M.A.
      • Altman E.
      Glucose can be transported and utilized in Escherichia coli by an altered or overproduced N-acetylglucosamine phosphotransferase system (PTS).
      ). Phosphorylated monosaccharides enter the PTS pathway of sugar transporter, and then cross the cell membrane as substrates for EPS synthesis. The complex PTS is composed of multiple proteins, including nonspecific glucose transporter I (EI), phosphohistidine carrier protein (HPr), and EII proteins. All bacterial PTS contained the same EI and HPr proteins, whereas EII was specific to different PTS. Furthermore, Ca2+ significantly increased expression of α-mannosidase that was mainly involved in protein glycation modification and glycan hydrolysis modification, thus affecting cell adhesion, inflammatory response, hormone activity, arthritis, immune monitoring, and cancer cell metastasis. Previously, α-mannosidase found in Corynebacterium and Polybacillus was able to hydrolyze the glycoside bonds of α-1,2, 1,3, 1,4, and 1,6-mannose, and the catalytic activity of the enzyme was dependent on calcium ions. The Ca2+ increased expression of β-galactosidase that was found to utilize 2-nitrobenzene-, 3-nitrobenzene-, 4-nitrobenzene-β-d-galactosidase as substrates from Bacillus circulans. The melisaccharide transporter (MelB) uses free energy of cation to transport sugar to catalyze the transfer of melisaccharide and H+/Na+ or Li+ in E. coli (
      • Ethayathulla A.S.
      • Yousef M.S.
      • Amin A.
      • Leblanc G.
      • Kaback H.R.
      • Guan L.
      Structure-based mechanism for Na+/melibiose symport by MelB.
      ). A subunit of dihydroxyacetone kinase DhaK is DhaL (B5G53_0169). Increased DhaK expression may provide substrates for different types of carbohydrate production (
      • Ethayathulla A.S.
      • Yousef M.S.
      • Amin A.
      • Leblanc G.
      • Kaback H.R.
      • Guan L.
      Structure-based mechanism for Na+/melibiose symport by MelB.
      ). In strain K25, there are DhaQ (B5G53_0168, DhaKLM), DhaK (B5G53_0170), DhaM (dha-specific IIA component), and glycerol-absorbing protein Mip (B5G53_0172). In Pseudomonas aeruginosa, LytR/AlgR (B5G53_1134) is a response regulator that regulates the production of EPS associated with chronic pneumonia.
      The Ca2+ downregulated transketolase that was involved in the pentose phosphate acid cycle (
      • Shaw J.A.
      • Henard C.A.
      • Liu L.
      • Dieckman L.M.
      • Vazquez-Torres A.
      • Bourret T.J.
      Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway.
      ), Downregulation of fructose 1,6-diphosphatase (FBPase) would cause reduced production of endogenous glucose; FBPase is the key enzyme of Calvin cycle and gluconeogenesis pathway that catalyzes the conversion of fructose-1,6-diphosphate to fructose-6-phosphate and releases a molecule of phosphate. The iron-sulfur cluster sensor IscR (B5G53_0338) may be a negative regulator in L. plantarum K25. The IscR in Streptococcus pneumoniae regulates the expression of genes related to capsule synthesis by binding to specific gene fragments in the promoter region. Raffinose RafR (B5G53_0190) is a repressor, which is able to bind to the manipulation gene and block the transcription (
      • Van Camp B.M.
      • Crow R.R.
      • Peng Y.
      • Varela M.F.
      Amino acids that confer transport of raffinose and maltose sugars in the raffinose permease (RafB) of Escherichia coli as implicated by spontaneous mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391.
      ). In strain K25, RafR was located in the raffinose operon and its expression was downregulated, indicating that under the condition of adding Ca2+, the absorption and metabolism of raffinose and sorbose in cells were enhanced.
      Calcium ion affected the lipid transport and metabolism in L. plantarum K25, and most of the differentially expressed proteins were upregulated (Supplemental Table S3, https://figshare.com/s/1349c636cb625d2e12da), which might change the structure of cell membrane, further affecting the function of EPS substrate transporters and flipase (
      • Kim J.
      • Yoo H.W.
      • Kim M.
      • Kim E.J.
      • Sung C.
      • Lee P.G.
      • Park B.G.
      • Kim B.G.
      Rewiring FadR regulon for the selective production of omega-hydroxy palmitic acid from glucose in Escherichia coli..
      ;
      • You D.
      • Zhang B.Q.
      • Ye B.C.
      GntR family regulator DasR controls acetate assimilation by directly repressing the acsA gene in Saccharopolyspora erythraea.
      ). Fatty acid synthases (B5G53_1392∼B5G53_1403) are a series of enzymes that control the synthesis of long-chain fatty acids, and upregulation of these enzymes indicated that the addition of Ca2+ could alter the formation of long-chain fatty acids in L. plantarum K25. FabA and FabZ are isoenzymes, and FabA but not FabZ has the function of isomerase and participates in the biosynthesis of unsaturated fatty acid. Alkenyl-ACP reductase (FabI/FabK/FabL) and FabI (K, L) can catalyze the formation of butyryl-S-ACP from crotonoyl-S-ACP; FabH is also a β-ketoaliac-ACP synthetase, which plays a key role in initiating the reaction. Acetyl-CoA carboxylase and malonoyl-CoA:ACP transacylase (FabD) are also essential enzymes in the biosynthesis pathway of fatty acids in E. coli. Mycolic acid is a kind of long-chain α-alkyl-β-hydroxyl fatty acid that plays an important role in the formation of outer membrane of and the toxicity of Mycobacterium (
      • Kim J.
      • Yoo H.W.
      • Kim M.
      • Kim E.J.
      • Sung C.
      • Lee P.G.
      • Park B.G.
      • Kim B.G.
      Rewiring FadR regulon for the selective production of omega-hydroxy palmitic acid from glucose in Escherichia coli..
      ;
      • You D.
      • Zhang B.Q.
      • Ye B.C.
      GntR family regulator DasR controls acetate assimilation by directly repressing the acsA gene in Saccharopolyspora erythraea.
      ). The synthesis of mycolic acid in Tubercle Bacillus occurs mainly through the synthesis pathways of FAS-I and FAS-II that involve a large number of fatty acid synthases. FadR is widely reported as a very important regulator in E. coli that plays a negative regulatory role in the degradation of saturated fatty acids, whereas it plays a positive regulatory role in the synthesis of unsaturated fatty acids. The expression of heme/steroid-binding protein of ferrous (B5G53_2502) was decreased, indicating that the steroid-regulating GntR was decreased in strain K25 with Ca2+.
      The Ca2+ affected the amino acid transport and metabolism in L. plantarum K25. The expression of some proteins decreased, and the synthesis ability of amino acids decreased. According to the changes of protein expression, the synthesis of homocysteine and asparagine amino acids might increase in L. plantarum K25 with Ca2+ added SDM medium. The expression of asparagine synthesis enzyme AsA increased, suggesting that the content of asparagine might increase (Supplemental Table S3, https://figshare.com/s/1349c636cb625d2e12da). Asparagine is a kind of powerful ornithine decarboxylase stimulator, which can promote the expression of extracellular component genes (
      • Baruch M.
      • Belotserkovsky I.
      • Hertzog B.B.
      • Ravins M.
      • Dov E.
      • McIver K.S.
      • Le Breton Y.S.
      • Zhou Y.
      • Cheng C.Y.
      • Hanski E.
      An extracellular bacterial pathogen modulates host metabolism to regulate its own sensing and proliferation.
      ). The activity of 2 thiothiether synthases Cbs (
      • Manders A.L.
      • Jaworski A.F.
      • Ahmed M.
      • Aitken S.M.
      Exploration of structure-function relationships in Escherichia coli cystathionine gamma-synthase and cystathionine beta-lyase via chimeric constructs and site-specific substitutions.
      ) decreased, likely leading to higher Hcy. The expression levels of 2 acetyl-lactate synthases, 2 glutamine catabolism aspartate transaminases and sialic acid catabolases increased, indicating that glutamine and hydroxyproline were degraded. The EPSP synthase is an enzyme for the aromatic amino acid synthesis (
      • Li L.
      • Zhou Z.
      • Jin W.
      • Wan Y.
      • Lu W.
      A transcriptomic analysis for identifying the unintended effects of introducing a heterologous glyphosate-tolerant EPSP synthase into Escherichia coli..
      ), catalytic shikimic acid-3-phosphate and phosphate enol-pyruvic acid synthesis reaction of 5-enol-pyruvic-shikimic-3-phosphate. The DAHP synthase AroG of tryptophan synthesis pathway and 9 amino transferase enzymes of histidine synthesis including histamine phosphate alcohol HPA and HPD expression decreased.
      The Ca2+ regulated the EPS biosynthesis in L. plantarum K25 by altering its membrane ion transport and metabolism. As indicated in Supplemental Table S3, expression of one low-abundance metal ion-ATPase protein (B5G53_2916, PacL3) sharply increased, whereas the expression levels of 3 high-abundance Fe/Co/Zn/Cd/Mn-ATPase proteins (ZnuA, ZitB and MtsC) sharply decreased. The compound PacL3 was found as a cation-translocating P-type ATPase in both L. plantarum WCFS1 and B. longum NCC2705; PacL3 in L. plantarum K25 might function as a Ca2+-ATPase by pumping Ca2+ and other bivalent cations out of the cytoplasm to maintain the homeostasis balance of Ca2+ in the cytoplasm, a necessary condition for normal cellular physiological activities. Furthermore, Ca2+-induced upregulation of Ca/Zn/Hg-ATPase (B5G53_2903, B5G53_0955) and MntH3 (B5G53_2555, B5G53_3270) might help expel excess ions from the cells and maintain the cell ion homeostasis (
      • Kehl-Fie T.E.
      • Zhang Y.
      • Moore J.L.
      • Farrand A.J.
      • Hood M.I.
      • Rathi S.
      • Chazin W.J.
      • Caprioli R.M.
      • Skaar E.P.
      MntABC and MntH contribute to systemic Staphylococcus aureus infection by competing with calprotectin for nutrient manganese.
      ). Upregulation of Grx (B5G53_0740), a thiol-transferase that belongs to the thioredoxin superfamily and is widely distributed in various organisms (
      • Tao K.
      oxyR-dependent induction of Escherichia coli grx gene expression by peroxide stress.
      ), would catalyze the thiol-disulfide bond exchange reaction or the reduction of protein glutathione disulfide to maintain the intracellular redox state. In addition, Mn2+/Zn2+-ATPase (B5G53_2858, B5G53_0953) and one protein (B5G53_3102) of the co-carrier Fe/Co/Zn/Cd were rarely expressed probably due to that the transported metal cation was not Ca2+. The expression of ArsR (B5G53_1814), an arsenic anti-operon transcriptional repressor protein, also decreased, indicating that the repression was reduced and the expression of arsenic anti-operon was enhanced. The protein ArsR regulates most of the acid tolerance genes of Helicobacter pylori, such as ureABEFGHI, flaABEGH, α-carbonic anhydrase, NiKR, NixA, HypB, HypA, HspA, AspA, and virulence genes, such as cagA that is the key to the growth, reproduction, pathogenesis and drug resistance of H. pylori (
      • Gupta S.S.
      • Borin B.N.
      • Cover T.L.
      • Krezel A.M.
      Structural analysis of the DNA-binding domain of the helicobacter pylori response regulator ArsR.
      ). The Ca2+ also regulated the pathway of taurine, which was widely found in animal tissues with a variety of pharmacological effects including antioxidant activity, regulation of calcium homeostasis and related signaling pathways, and anti-apoptosis effects (
      • Clifford E.L.
      • Varela M.M.
      • De Corte D.
      • Bode A.
      • Ortiz V.
      • Herndl G.J.
      • Sintes E.
      Taurine is a major carbon and energy source for marine prokaryotes in the North Atlantic Ocean off the Iberian Peninsula.
      ;
      • Heidari R.
      • Behnamrad S.
      • Khodami Z.
      • Ommati M.M.
      • Azarpira N.
      • Vazin A.
      The nephroprotective properties of taurine in colistin-treated mice is mediated through the regulation of mitochondrial function and mitigation of oxidative stress.
      ). The effects of taurine on Ca2+ mainly depend on extracellular Ca2+ concentration by interacting with ion channels until the concentration reaches the equilibrium.
      The Ca2+ affected the nucleotide transport and metabolism in L. plantarum K25, and the expression of β-subunit of ribonucleotide reductases III increased, providing the necessary precursor for DNA repair. Adenosine deaminase catalyzes the hydrolysis of adenine to generate hypoxanthine and ammonia. The protein AzgA is a xanthine/uracil/vitamin C permease. Both PurN and PurL are involved in the synthesis of ADP and ATP, and PurH is an IMP hypoxanthine nucleotide hydrolytic enzyme. The expression of all these enzymes increased, indicating the decrease of hypoxanthine synthesis (
      • Jelsbak L.
      • Mortensen M.I.B.
      • Kilstrup M.
      • Olsen J.E.
      The in vitro redundant enzymes PurN and PurT are both essential for systemic infection of mice in Salmonella enterica serovar typhimurium.
      ). The expression levels of 15 ribosomal proteins were downregulated, which may be involved in the synthesis of related proteins (
      • Ayyub S.A.
      • Lahry K.
      • Dobriyal D.
      • Mondal S.
      • Varshney U.
      Antimicrobial activity of fusidic acid in Escherichia coli is dependent on the relative levels of ribosome recycling factor and elongation factor G.
      ;
      • McAteer S.P.
      • Sy B.M.
      • Wong J.L.
      • Tollervey D.
      • Gally D.L.
      • Tree J.J.
      Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli..
      ).
      The Ca2+ affected the energy production and conversion section in L. plantarum K25. All of the differentially expressed proteins related to energy metabolism were upregulated, including fermentation-respiration switch FrsA (B5G53_2913), PTS EIIA (Glc)-binding protein and pNP-butyrate esterase activity (
      • Koo B.M.
      • Yoon M.J.
      • Lee C.R.
      • Nam T.W.
      • Choe Y.J.
      • Jaffe H.
      • Peterkofsky A.
      • Seok Y.J.
      A novel fermentation/respiration switch protein regulated by enzyme IIA(Glc) in Escherichia coli..
      ). Pyruvate formate lyase (B5G53_2865) and pyruvate dehydrogenase system (B5G53_0349, B5G53_1871∼B5G53_1874) catalyze the decarbonation of pyruvate to produce acetyl-CoA and enter the TCA cycle. Malate dehydrogenase (B5G53_0330, B5G53_0957) can catalyze the reversible conversion of malic acid to oxaloacetic acid in TCA cycle, and the γ-subunit (B5G53_0962) of citric acid lyase catalyzes the production of citric acid.

      CONCLUSIONS

      Calcium ions had significant effects on the EPS biosynthesis in L. plantarum K25. The yield of the EPS increased by 40%, reaching 238.6 mg/L when added with CaCl2 at 20 mg/L in SDM. The molecular weight distribution and monosaccharide composition of the EPS from strain K25 were also changed with decreased proportion of the high molecular weight fraction and more content of rhamnose, though the functional groups of the polymer were not affected. The Ca2+ stimulated the cps4 gene cluster on chromosome, and the expression levels of glycosyltransferase Cps4F, Cps4I and chain-length protein significantly changed. The increased expression of MelB, phosphotransferase system, α-mannose glycosidase, mannose phosphotransferase system IICD, Bgl, GH1, MalFGK, DhaK and FBPase facilitated supplying more substrates for the EPS synthesis. Furthermore, Ca2+ activated ArsR, LytR/AlgR, RafR, IscR, HipB, and GntR, which perceived the environmental changes with appropriate cellular responses to regulate EPS synthesis genes. Future work will be focused on more detailed studies of compositional and structural changes of the EPS produced by probiotic L. plantarum in response to Ca2+, and manipulation of the EPS biosynthesis for exploring its application in probiotic dairy products.

      ACKNOWLEDGMENTS

      This work was supported by National Key Research and Development Program (No. 2017YFE0131800), National Natural Science Foundation of China (Project No. 31871823), and the joint training program of overseas cooperative advisor for doctoral students in 2020 (19008020144). All authors read and approved the final manuscript. The authors have not stated any conflicts of interest.

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