Role of the luxS gene in bacteriocin biosynthesis by Lactobacillus plantarum KLDS1.0391: A proteomic analysis

Certain probiotic species of lactic acid bacteria, especially Lactobacillus plantarum, regulate bacteriocin synthesis through quorum sensing (QS) systems. In this study, we aimed to investigate the luxS-mediated molecular mechanisms of QS during bacteriocin synthesis by L. plantarum KLDS1.0391. In the absence of luxS, the ‘spot-on-the-lawn’ method showed that the bacteriocin production by L. plantarum KLDS1.0391 significantly decreased upon co-cultivation with L. helveticus KLDS1.9207 (P < 0.01) but did not change significantly when mono-cultivated. Furthermore, liquid chromatography-electrospray ionization tandem mass spectrometry analysis showed that, as a response to luxS deletion, L. plantarum KLDS1.0391 altered the expression level of proteins involved in carbohydrate metabolism, amino acid metabolism, fatty acid synthesis and metabolism, and the two-component regulatory system. In particular, the sensor histidine kinase AgrC (from the two-component system, LytTR family) was expressed differently between the luxS mutant and the wild-type strain during co-cultivation, whereas no significant differences in proteins related to bacteriocin biosynthesis were found upon mono-cultivation. In summary, we found that the production of bacteriocin was regulated by carbohydrate metabolism, amino acid metabolism, fatty acid synthesis and metabolism, and the two-component regulatory system. Furthermore, our results demonstrate the role of luxS-mediated molecular mechanisms in bacteriocin production.

1. Detection of the double chamber effect in co-cultivation of L. plantarum KLDS1.0391 and L.
helveticus KLDS1.9207 The membrane permeability was detected by the method described below. The results are shown in Table S1. Detection  "-" means that no strains are inoculated. "*" means that the number of cell in this chamber is not determined.
The bacterial growth was detected by the following method. L. plantarum KLDS1.0391 was inoculated (1%) into the double chamber and triangular flask. Sample was incubated in MRS broth at 37 °C for 24 h with gentle agitation (60 rpm). The culture was removed every 2 h, and the number of viable cell of L. plantarum KLDS1.0391 with time changes in two culture modes was detected. The results are shown in Fig. S3.
2. SDS-PAGE was carried out on vertical electrophoresis apparatus (SE260, GE-Healthcare) at 15 mA and per gel using a 12% (w/w) poly-acrylamide separating gel overlaid with a 5% stacking gel. Samples were boiled for 5 min in SDS-PAGE 5×(5:1, v/v) Sample Loading Buffer (10% SDS, 0.5% BPB, 50% Glycerol, 500 mM DTT, 250 mM Tris-HCl, pH 6.8), and centrifuged at 14000 g for 10 min. The supernatant was transferred to a new tube. Each lane on the gel received 20 μg of protein for electrophoresis at 5 mA for 60 min. After electrophoresis, the polypeptides were stained with Coomassie Brilliant Blue R250 and decolored with methanol/ acetic acid. Finally, the result of electrophoresis was analyzed by gel imaging system.
Filter-aided-sample-preparation procedure was described as followed. Briefly, each sample added DTT to a final concentration of 100 mM, boiled for 5min and cooled down to room temperature. Then, 200 μL UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) was added to remove the detergent, DTT and other low-molecular-weight components by repeated ultrafiltration (Microcon units, 30 kD) facilitated by centrifugation (14000 g, 15 min). Further, 100 μL 50 mM iodoacetamide (IAA, Bio-Rad) in UA buffer was added to prevent the reduced cysteine residues and stirred at 600 rmp for 1 min, and the samples were incubated for 30 min in total darkness and centrifuged at 14000 g for 10 min. The filter was washed twice with 100 μL UA buffer and 100 μL 25 mM NH4HCO3 buffer, respectively. Finally, the protein suspension was digested with 2 μg Trypsin (Promega) in 40 μL 25 mM NH4HCO3 buffer at 37 °C for 16-18 h, and the resulting peptides were collected as a filtrate. The peptides of each sample were first desalted with C18-SD Extraction Disk Cartridge and then estimated by UV light absorbance at 280 nm.
The detailed Q-Exactive MS requirements were described below. The result of MS were acquired higher-energy collisional dissociation (HCD) fragmentation by choosing the positive ion and precursor ions range from 300 m/z to 1800 m/z. The resolution for survey MS scans was set at a value of 70,000 with a 200 m/z, and the HCD spectra were acquired a resolution of 17,500 with m/z of 200. The m/z of peptides and peptide fragments were collected by full scan and selected the twenty data-dependent MS/MS scans. The Normalized Collision Energy of 30 eV was used to activate precursor irons, and the Underfill ratio, which indicates the Mix percentage of the target value likely to be attained to the Max fill time, was defined as 0.1%.
"-" means that no cells were detected, "+" means that cells were detected.