Isolation of bacteria and identification
The bacterial strain SK.DU.4 was isolated from a farmland soil sample. The sample was serially diluted and plated on different media to screen the bacteria producing antimicrobial substances. Selected colonies were streaked on to nutrient agar (NA) medium with the following composition (g/l): peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5; sodium chloride, 5.0; agar 15.0 and the pH adjusted to 7.2. The isolates were checked for purity and preserved at −70°C for further studies. The indicator strains used in this study were obtained from Microbial Type Culture Collection and Genebank (MTCC), Chandigarh, India and grown on tryptone soya agar (TSA) medium with the following composition (g/l): pancreatic digest of casein, 15.0; papaic digest of soybean meal, 5.0; sodium chloride, 5.0; agar 15.0 and the pH adjusted to 7.2. Strain SK.DU.4 was tested for various phenotypic properties including morphology, physiology and biochemical characteristics, according to the standard procedures. To confirm the identification of strain SK.DU.4, the 16S rRNA gene was amplified by PCR using universal primers and the amplified PCR product was sequenced as described earlier (Suresh et al. 2006). The 16S rRNA gene sequences of closely related strains were retrieved from EzTaxon server and aligned using CLUSTAL_W program of MEGA version 5.0 (Tamura et al. 2011). The alignment was corrected manually using BioEdit sequence alignment editor (Hall, 1999). Upon calculating the pair-wise evolutionary distances (Kimura, 1980), a neighbour-joining phylogenetic tree was constructed using the MEGA version 5.0.The strain was deposited at Microbial Type Culture Collection and Genebank, (MTCC 11460) and the 16S rRNA gene sequence was submitted to EMBL (HF544505) database.
Determination of bacteriocin activity
The bacteriocin activity was determined by well diffusion assay. The strain was grown for 24 h in 200 ml nutrient broth (NB, Himedia) using 500 ml flask and subsequently cells were removed by centrifugation (8000 rpm for 20 min, 4°C). The supernatant obtained was filtered by using 0.22 μm filter (Millipore), diluted by two fold and used to test the activity as mentioned earlier (Singh et al. 2012). The antimicrobial peptide production was also tested by growing the strain in minimal medium. To examine the antimicrobial activity, S. aureus cells were processed for scanning electron micrograph (SEM) as described by earlier (Singh et al. 2012). Growth curve was prepared to examine the bacteriocin production along with the growth of strain SK.DU4.
Production and purification of bacteriocin
To test the effect of different carbon and nitrogen sources like glucose, lactose, yeast extract, peptone and beef extract on production of antimicrobial peptides, 0.5% of each of them added to minimal medium with following composition (g/l): Na2HPO4.2H2O, 7.9; KH2PO4, 3.0; NaCl, 0.5; NH4Cl, 1.0; pH 7.2. The antimicrobial peptides production was measured as zone of inhibition against indicator strains. For the characterization of peptides, culture was grown in 1000 ml of NB using 2 l flasks to obtain large quantity of antimicrobial peptides. One percent (v/v) of overnight grown culture of SK.DU.4 was inoculated to 1 l of NB medium and grown for 24 h on a rotary shaker at 30°C. Subsequently cells were separated by centrifugation (8000 rpm, 20 min at 4°C). The supernatant was mixed with 2% (w/v) of Diaion HP-20 (Supelco, SigmaAldrich, USA) resin and the antimicrobial peptide was eluted in methanol. Methanol was evaporated using a rota vapour (BUCHI Rota vapor R-200), the antimicrobial peptide was redissolved in Milli-Q water and further purification was achieved through reverse phase HPLC (1260 Infinity, Agilent Technologies, USA) using a semi-preparative C18 column (Pursuit 10C18 250×21.2 mm). Acetonitrile and 0.1% TFA were used as mobile phase. The purified bacteriocin was tested for antimicrobial activity and applied on Tricine-SDS-PAGE (16%)/6 M urea.
Mass spectrometry and peptide mass fingerprint analysis
Matrix-assisted laser desorption ionization (MALDI) was used to primarily characterize the antimicrobial bacteriocin. The lyophilized peptide was re-suspended in methanol and 4 μl of peptide solution was mixed with 4 μl of matrix (CHCA, 10 mg/ml), 1.0 μl of this mixture solution was spotted onto the MALDI 100 well stainless steel sample plate and allowed to air dry prior to the MALDI analysis (Mandal et al. 2009). To obtain MALDI mass spectra, a Voyager time-of-flight mass spectrometer (Applied Biosystem, USA), equipped with 337 nm N2 laser was used and operated in accelerating voltage 20 kV. The spectra were recorded in positive ion linear mode. Reproducibility of the spectrum was checked several times from separately spotted samples. In order to carry out peptide mass fingerprint (pmf) analysis of the purified bacteriocin solution, the sample was lyophilized and dissolved in 50 mM ammonium bicarbonate (pH 8.0) buffer. Protein was reduced with 0.1 M DTT at 56°C for 30 min and alkylated with 0.3 M iodoacetamide at room temperature for 15 min in the dark as described by Dey et al. (2012). Subsequently, the sample was subjected to digestion with trypsin at 37°C for overnight. One microliter of the digested sample was spotted directly onto a MALDI target plate and 1 μl of CHCA matrix solution was applied on the sample spot and allowed to air dry. Mass spectra of different peptide fragments were acquired in positive ion reflector mode with a mass range of 700–5000 Da after an average of 1000 laser shots. Data was uploaded into MASCOT (Matrix Science, London, UK) database to search the protein identity. During database search the following parameters were considered: database, NCBInr; maximum missed cleavages, 1; precursor tolerance, 0.2 Da; peptide charges, +1; mass, monoisotopic.
In-gel activity assay for detection of bacteriocin activity
Tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) was performed for antimicrobial peptide using 16% polyacrylamide gel. The purified and concentrated antimicrobial peptide was applied onto the gel in duplicate. After the electrophoresis, a part of the gel along with molecular weight markers was stained with Coomassie Brilliant Blue R-250 to visualize the protein bands. Whereas the unstained part of the gel was used for in situ detection of bacteriocin activity upon fixing it in a mixture of 2-propanol, acetic acid and H2O (25:10:65) for 15 min and washed with sterile H2O for 30 min repeatedly. It was placed in a sterile Petri dish, overlaid with 10 ml of soft agar (0.8%) containing test strain S. aureus (about 106 cells/ml).
Determination of minimum inhibitory concentration
The MIC of antimicrobial peptide was evaluated for different strains by using a microtiter plate dilution assay. Test strains grown to logarithmic phase under optimal conditions (between 0.3-0.4 OD) and used for assay in triplicates as described earlier (Singh et al. 2012). The lowest concentration that inhibited growth of test strain and did not show any increase in absorption after 48 h incubation was considered as MIC.
Effect of pH, temperature and hydrolytic enzymes on bacteriocin activity
The antimicrobial peptide was checked for tolerance to different temperature, pH and proteases activity. To determine the tolerance to temperature and pH, the purified peptide was incubated at different temperatures such as 60°, 80°, 100° and 121°C for 15 min and pH values between 2.0–12.0. Different proteolytic enzymes including pepsin, trypsin, chymotrypsin, proteinase K and pronase E were incubated with peptide for 6 h at 37°C and the activity was terminated by heating at 80°C before the peptide was used for assay.
Extraction of lipopeptide
Lipopeptide was isolated from bacterial culture in a combination of acid and solvent extraction procedure following Vater et al. (2002). In brief, cells were removed from the 24 h growing culture broth by centrifugation (13,000 × g) for 15 min at 4°C. The supernatant was adjusted to pH 2.0 by addition of concentrated HCl and allowed to precipitate at 4°C for 16 h. Precipitate was collected after centrifugation (13,000 × g) for 20 min at 4°C and extracted with methanol by stirring for 2 h. The lipopeptide containing methanol was collected after filtration and vacuum-dried.
Purification of lipopeptide
The extracted lipopeptide dissolved in methanol and fractionated by reverse phase- HPLC (Agilent 1100 series) with a ZORBAX 300-SB18 column (4.6 mm × 250 mm, particle size 5 μm), at a flow rate of 1 ml/min. The solvent system used was 0.1% aqueous TFA (A) and acetonitrile containing 0.1% TFA (B). The gradient of solvent B used to run the column was as follows: 0-60% for 0–45 min, 60-80% for 45–55 min and 80-100% for 55–60 min. The elution from the column was monitored at 215 nm in a diode array detector and all the peaks of HPLC chromatogram were collected using a fraction collector (GILSON, France) coupled with the system. Collected fractions were concentrated by speed vacuum and antimicrobial activity was screened. The major fraction showing antibacterial activity was re-chromatographed in the same column under similar conditions, but solvent B was used as 100% ACN with a gradient of 0-10% for 30 min. Peptide concentration was determined using the RP-HPLC conditions and calibrated with surfactin (Sigma-Aldrich).
MALDI-TOF-MS and sequencing
The molecular mass of the purified active lipopeptide fraction was analysed following the protocol described above. For MS/MS sequencing, the peptide was incubated with 10% NaOH in methanol at room temperature for 16 h to cleave the lactone ring. The cleaved peptide was lyophilized, again extracted with methanol and allowed for mass spectrometry analysis. The spectra were recorded in the post-source decay (PSD) ion mode as an average of 100 laser shots with a grid voltage of 75%. The reflector voltage was reduced in 25% steps and guide wire was reduced 0.02–0.01% with an extraction delay time of 100 ns.
Fatty acid analysis by GC-MS
Acid hydrolysis of lipopeptide was performed by incubating the peptide (5 mg) with 0.5 ml of 6 M HCl at 90°C for 18 h in sealed tubes. The fatty acids were extracted with ether and esterified with 0.95 ml methanol and 0.05 ml of 98% H2SO4 at 65°C for 6 h. Fatty acid methyl esters were obtained after extraction with n-hexane and analyzed by GC-MS with a Clarus 500 GC (PerkinElmer, USA). Helium was used as carrier gas at a flow rate of 1.0 ml/min. The column temperature was maintained at 120°C for 3 min and thereafter gradually increased (8°C/min) to 260°C.