Bacterial strains, plasmids, and oligonucleotide
Bacterial strains, plasmids, and oligonucleotides used in this study were listed in Table 1. L. plantarum Zhang-LL was grown at 37 °C under static condition for 24 h using MRS-lactobacilli broth. L. monocytogenes ATCC54003 was grown at 37 °C for 16 to 24 h in TSBYE medium (Hopebio, China). B. subtilis WB800N was grown at 37 °C in 2YT medium. The upper three strains and vector pHT43 were generous gifts of Dr. Yuanhong Xie in Beijing University of Agriculture. E. coli Trans1 (TransGen biotech) was grown at 37 °C in LB medium for appropriate time. Ampicillin and chloramphenicol was added at 100 μg/ml and 5μg/ml respectively when necessarily.
Construction of plasmid
Two plasmid was constructed to express papA mature peptide in B. subtilis WB800N. E. coli-B. subtilis shuttle vector pHT43 was used for construction of recombination vector and expression. Fragment of papA1 was synthesized according to sequence of codon sequence from papA mature peptide. Sequence of His tag and thrombin reorganization site was added before papA sequence. The whole fragment papA1 was optimized according to codon usage frequency and synthesized in Genewiz (China), which was then inserted into vector pHT43 between site of BamH I and Xba I. The vector pHT43-papA1 was verified by sequence with primers p43F and p43R. In another plasmid pHT43-papA2, sequence encoded papA mature peptide was amplified with primers PAF+PAR from genome of L. platntarum Zhang-LL and used as template. The PCR product was digested with BamH I and Xba I, and ligated into pHT43 with the same enzyme-digestion site.
Preparation of competent B. subtilis WB800N and transformation
Transformation of B. subtilis WB800N was according to the protocol of Spizizen . In brief, one colony of B. subtilis WB800N was picked from petri dish and inoculated in grow medium I (GM I, 1 Ⅹminimal mineral salt (g/L, K2HPO4, 14; KH2PO4, 6, (NH4)2SO4, 2; trisodium citrate·2H2O, 1; MgSO4·7H2O) 95 mL, 50% glucose 1 mL, 5% acid hydrolyzed casein 0.08 mL, 10% yeast juice 1 mL, L-Trp (2 mg/mL) 2.5 mL) at 30°C , 100 rpm for 16 h. 2 mL o/n culture was inoculated in 18 mL fresh GMⅠ (10%) and incubated at 37°C , 200 rpm for 3 h. 10 ml above mentioned culture was inoculated in 90 mL GM II (1 Ⅹminimal mineral salt 97.5 ml, 50% glucose 1 mL, 5% acid hydrolyzed casein 0.08 mL, 10% yeast juice 0.04 mL, L-Trp (2 mg/mL) 0.5 mL, 500 mmol/L MgCl2 0.5 mL, 100 mmol/L Cacl2 0.5 mL) and grown at 37°C , 100 rpm for 90 min. Bacteria was collect by centrifuge at 5000 Ⅹ g for 10 min. The sediment was suspended in 10 mL GM II, which was competent cell of WB800N. 500 ng plasmid was added to 500 μL competent cell and mixed thorough. The system was incubated at 37 °C, 80 rpm for 30 min. the culture was spreaded on LB/CM agar and incubated at 37 °C for 12-16 h until colony appeared. Positive transformants was confirmed by colony PCR.
Inducible expression and purification of papA protein
Single colony of positive recombination strain was inoculated in 2TY/CM liquid medium and cultured at 37 °C, 200 rpm overnight. The broth was inoculated in fresh 2TY/CM liquid medium and cultured for another 3 h. IPTG was added to broth to induce protein expression at 30 °C, 200 rpm. Samples were withdrawed at 0, 2, 4 h. Samples were centrifuged at 12000 rpm for 5 min and stored at 4 °C until further assay. Fused protein was purified using Ni-NTA Rsein (Thermo scientific) according to user’s guide.
Antimicriobial activity assay
Bacteriocin activity of fused proteins was assessed by agar diffuse test and standard microtiter plate assay. L. monocytogenes ATCC 54003 was grown in TSB-YE medium overnight at 37 °C and diluted 1:25 in fresh TSB-YE prior to the assay. Mueller-Hinton (MH) soft agar was added with appropriate volume L. monocytogenes ATCC 54003 solutions to cell concentration of 105 CFU/mL. The medium was plated on petri dish with Oxdord cup set beforehand. 100 μL of supernatant of expressed protein was added in the hole for agar diffuse. L. plantarum Zhang-LL was incubated at 37 °C statically for 24 h. The culture was centrifuge and supernatant were used for agar diffuse test. The petri dishes were incubated at 37 °C for 16-20 h and antibacterial activity was observed as a halo of inhibition in the bacterial lawn formed around the sample. For microtiter plate assay, two-fold dilution series of sample (100μl) were mixed with 100μl indicator strain in sterilized 96-well plates. The plates were incubated at 37℃ for 5-6 h or 21 h. Growth was monitored by measuring OD600 using Epoch plate reader (Bi-Tek). Bacteriocin activity in supernatants was determined in a semi-quantitative manner according to Holo . According to this method, one Unit of bacteriocin activity (BU) were defined as the highest dilution showing at least 50% inhibition of the indicator strain.
Cultivation in bioreactors
Fed-batch cultivation was carried out with B. subtilis DBN-SKL-PA2 in 30 L bioreactor (Baoxin, Shanghai) stirred tank bioreactor with an initial working volume of 15 L. Single colony was grown in 2YT medium supplemented with 5 μg/mL chloramphenicol in Erlenmeyer flasks with aeration at 37℃, 200rpm overnight to obtain primary seed culture, which was then inoculated in 300 mL 2YT/CM liuquid medium and grown for 8 h. The preculture was inoculated the 15 L 2YT/CM batch culture in 30 L bioreactor, stirred at 200 rpm, 37℃. IPTG was added at3 h to a final concentration 0.1 mM, and the temperature was lowered and kept at 30℃. The initial pH was 5.8, and 20% phosphoric acid was addd automatically to adjusted pH to blow 7. Dissolved oxygen was controlled by adaption of stirrer speed and aeration rate of 200~300 rpm. The culture was fed with 100 g/L glucose and double concentration 2YT from 10 h, and IPTG was added at the beginning of fed batch to a final concentration 0.1 mM. Total 1.6 L medium was fed. Dissolved oxygen and pH were recorded from the control panel of bioreactor. OD and glucose concentration were assayed respectively. Sample was centrifuged and the supernatant was stored at 4 °C for strain inhibition. Fermentation was conducted for 24 h. The broth was collected by centrifuge, and the supernatant was stored at 4 °C. To assay the dry material content in final broth, 10 g supernatant of 24 h fermentation broth was subjected to moisture measure on Moisture Analyzer (METTLER TOLEDO, HE53). The remaining material was redisolved in sterilized PBS (Thermo Fisher).
Scan electronic microscope
L. monocytogenes ATCC 54003 was grown in TSB-YE medium at 37 °C, 180 rpm overnight. Culture was inoculated into fresh TSB-YE and grew to OD600 of 0.6. Supernatant of papA fused protein was sterilized by filter and added to broth. The mixture was incubated at 37 °C for 3 h. Culture of L. monocytogenes ATCC 54003 without papA protein was grown simultaneously as control. Cells were harvested and washed with PBD, and fixed with 2.5% (V/V) glutaraldehyde and incubated at 4 °C o/n. After dehydrated by gradient alcohol solutions, samples were freeze-dried and coated with gold. The specimens were examined using scanning electronic microscope (SV8010, HITACHI).
SDS-PAGE and coomassie brilliant blue staining
SDS-PAGE electrophoresis was performed with Tris-Tricine 10-20% (Thermo Scientific) according to the manufacturer’s protocol. Samples were mixed with sample buffer and heated to 70 °C for 10 min before they were loading onto the gels. After electrophoresis, gels were soaked in fast staining solution (Tiangen, Beijing), boiled for 1 min, and incubated at room temperature for 30 min. The gels were then washed with ddH2O, boiled for 1 min, and incubated at room temperature for 30 min. The washing step was repeated until clear bands were shown.
Strain access number
Strain B. subtilis DBN-SKL-PA1 and B. subtilis DBN-SKL-PA2 were deposited in China General Microbiological Culture Collection Center (CGMCC) as CGMCC No. 23101 and CGMCC No. 23102.