Bacterial strains, plasmids, media, and growth conditions. The strains and plasmids used in the present study are shown in Table 1. Pcc strains were cultured at 28℃ on 1.4% nutrient agar (NA) or with shaking on modified Luria-Bertani (LB) medium, which contained half the recommended quantity of NaCl (5 g rather than 10 g of NaCl per litre). The IFO-802 medium was supplemented with 1% polypeptin, 0.2% yeast extract, 0.1% MgSO4 (pH 7.0), and 1.5% agar. Pcc isolates were identified to grow on the modified Drigalski agar medium (3). Pcc H-rif-8-6 and TH22-10 were used as bacteriocin producers, while Pcc Ea1068 and SP33 were used as bacteriocin indicators. E. coli DH5α was used as a cloning host and was grown at 37°C. E. coli BL21(DE3) was used as a protein expression host and was grown at 28℃. E. coli strains were grown on normal LB medium with shaking. Rifampicin (50 mg/L), kanamycin (50 mg/L), ampicillin (50 mg/L), chloramphenicol (68 mg/L), and tetracycline (12.5 mg/L) were added to NA and LB agars whenever necessary. All bacterial growth densities were monitored by using a spectrophotometer at 595nm (OD595).
Preparation of Genomic DNA, Plasmid DNA and mRNA. The procedures of plasmid preparation, genomic DNA isolation, and DNA manipulation were performed as described by Sambrook et al. (21). Exponentially growing cells of E. coli DH5α (OD595 of about 6.0) were harvested for RNA preparation. Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water. The concentration of RNA was determined by OD260 absorption, and RNA was analysed by electrophoresis on 1.5% formaldehyde-morpholinepropanesulfonic-agarose gel.
Antiserum preparation for Carocin S1K and Carocin S1I. The proteins Carocin S1K and Carocin S1I were expressed using the BL21/pAYL4 strain by overnight culture in LB medium. After centrifugation, the proteins contained in the supernatants were precipitated with 50% ethanol and loaded on preparative SDS-10% polyacrylamide gels. The bands corresponding to Carocin S1K and Carocin S1I were directly cut out, and the proteins were electroeluted for isolation. The protein was injected into a rabbit for antibody production.
Western Blotting. The western blot analysis was performed after electrophoretic transfer of proteins from SDS-PAGE gel to a poly(vinylidene difluoride) (PVDF) membrane. After electrophoresis, the proteins were electroblotted onto nitrocellulose in a semi-dry apparatus at 2 mA/cm2 for 20 min using a transfer buffer containing 40 mM glycine, 50 mM Tris, 0.4% SDS, and 10% methanol. The nitrocellulose was then saturated with gelatin, incubated with antibodies, and the blots were visualised using 3,3-diaminobenzidine (Sigma). Anti-CaroS1K or Anti-CaroS1I antibodies were diluted at 1:2500.
Nest-PCR and Reverse Transcription-PCR. In this study, oligonucleotide DNA primers synthesised by MD Bio Inc. (Taipei, Taiwan), and Go-Taq DNA polymerase manufactured by Promega, USA were used for PCR amplification. The procedures described by Sambrook et al. (21) were followed for thermal asymmetric interlaced PCR (TAIL-PCR). On the other hand, the Reverse Transcription-PCR (RT-PCR) using AMV Reverse Transcriptase (Promega, USA) was carried out based on the instructions provided by the manufacturer.
One microgram (1 µg) of RNA was subjected to RT-PCR. CaroS3_re_1 was used as a reverse primer in first-strand cDNA synthesis. The RT mixtures were diluted and used as templates in a PCR reaction with the two primers, CaroS3_re_1 and CaroS3_for_1 (Table 2).
Restriction DNA Library Screening and Southern Blots. The Southern blots were performed following the procedures described in the DIG Application Manual (Roche, USA). Using X4I_forS and X4I_forT (Table 2) as primers, the 390-bp DNA fragment (CAROS probe) from H-rif-8-6 was amplified. It was then subcloned into the pGEM-T Easy vector (Promega Inc., USA). Thereafter, it was labeled utilising a Random Primed DNA Labeling Kit (Roche Diagnostics, USA).
Digestion of the genomic DNA of the wild-type strain, H-rif-8-6, was done using different restriction endonucleases. Restriction sites were located outside the putative open reading frame. The samples were then subjected to electrophoresis and analysed with Southern blotting. After detection using the CAROS probe, the DNA from positive gel slices was purified. Subsequently, the DNA was cloned into pBR322 to provide the Carocin-producing plasmid pBRS3KI, which was isolated and detected with the CAROS probe.
Bacteriocin assays. Bacteriocin production was examined employing the double-layer agar method of Fredericq (22). The agar consists of hard (1.4%) and soft (0.65%) layers of IFO-802 agar medium. Growth inhibition zones around the colonies indicate the production of bacteriocin. Moreover, trypsin treatment was used for bacteriocin identification.
Computer Analysis of Sequence Data. The DNA nucleotide sequence and the deduced amino acid sequence of Carocin S1, Colicin E1, Colicin E2, and Colicin E3 were compared by the BLAST and FASTA programs of the National Center for Biotechnology Information server. Sequence data were compared by DNASIS MAX 3.0 software (Hitachi, Tokyo, Japan).
Sub-Cloning and Transformation. About 4500-bp BamHI-HindIII digested DNA fragment, including the caroS3K and caroS3I genes, was amplified from pBRS3KI with primers of CarocinS3KI_for and CarocinS3KI_rev (Table 2) and a 2,906-bp DNA fragment was subcloned into pET32a to give the plasmid pEN3KI. The pES3KI was obtained by excision of the Tag element between the rbs (ribosome binding site) and start code (for caroS2K) in pEN3KI using the SLIM method as previously described (13,23,24). The 5IHT32a2KI_forT, 5IHTGT2KI_forS, 5IHT32a3KI_revT, and 5IHT32a4KI_revS primers were used. A 2,636-bp fragment of the caroS3K gene was knocked-out by PCR to form the plasmid pES3I. The X4I_forS, X4I_forT, 5IHT32a3KI_revT, and 5IHT32a4KI_revS primers were used. Subsequently, pES3KI and pES3I were introduced into E. coli BL21 (DE3) cells.
Plasmids were introduced into Pcc strains using electroporation (1.25 kV/cm, 200 Ω, 25 µF) (21). For heat-shock transformation, the competent cells of E. coli were prepared according to the method of Hanahan (20).
Bacteriocin Expression and Bacteriocin Activity Assay. Bacterial strains (BL21/pET32a, BL21/pES3kI, and BL21/pES3I) in BSM medium were incubated in a sterilised stainless-steel box with stainless steel cover at 28°C for 24 h under a dark condition. After centrifugation, the medium without cells was removed.
The nucleotidase activity was confirmed by adding 500 ng/1 μl genome DNA solution from strain Ea1068 into 10 μl of the protein solution and incubated at 28°C for 90 min. An equal quantity of genomic DNA was digested with EcoRI at 28℃ for 90 min. Samples were then subjected to electrophoresis on 1% agarose gel.
The bacteriocin activity was determined by adding 10 μl of the protein solution to an indicator plate containing Ea1068 strain growing on soft IFO-802 medium containing 0.65% agar at 28℃ for 1 day. Growth inhibition zones at the point of addition were considered an indication of Carocin S3 activity.
The ribonuclease activity was determined following an assay procedure provided by the manufacturer (Promega, USA). Firstly, total DNA was treated with calf intestinal alkaline phosphatase at 55℃ for 30 min. The reaction was then arrested by adding 5 mM nitrilotriacetic acid. Equal volumes of phenol and chloroform were used to extract the RNA. An aliquot of phosphatase-treated RNA was 5'-32P-labeled at 37℃ for 30 min. The labeling was done by incubating the RNA with a mixture of [γ-32P] ATP, T4 polynucleotide kinase (Promega Inc, USA), and reaction buffer in nuclease-free water (24). The 3'-labeling of RNA then followed using [5'-32P] Cytidine 3',5'-bisphosphate (pCp) and T4 RNA ligase (Promega, USA) (25). MicroSpin G-25 columns (GE Healthcare, USA) were used to purify the mixture. Subsequently, aliquots of the purified labelled-RNA were incubated with and without Carocin S3 at 28℃ for 60 minutes.
To measure its activity, Carocin S3I was pre-mixed with an equal amount of Carocin S3K. The mixtures were subjected to electrophoresis on a 9% polyacrylamide gel (19:1) containing 7M urea, 50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.3. All samples were electrophoresed at 15℃ by PROTEIN II xi (BioRad, USA).
Protein Purification. For rough protein isolation, bacterial strains (BL21/pET32a, BL21/pES3kI and BL21/pES3I) in BSM were incubated in the dark using a sterilised stainless-steel box with steel cover at 28°C for 24 hours. After centrifugation, the medium without cells was removed. Ammonium sulfate was added to 80% saturation to precipitate the protein, and the precipitate was collected on a 0.45-μm cellulose filter. One milligram of precipitated protein was dissolved in 100 μl of bacteriocin buffer (0.1 M Tris [pH 7.5], 0.01 M dithiothreitol, and 0.5 M MgCl2).
For bacteriocin isolation, the transformant cells of BL21, harbouring pES3KI or pES3I, were grown in 500 ml to an OD595 of 0.5~0.6. The cells were induced with isopropyl-β-D-thiogalactopyranoside (IPTG; final concentration, 0.1 mM; at 25℃ for 14 h). Subsequently, the cells were pelletised in a loading buffer (20 mM Tris-HCl, 28 mM NaCl, PH 8.0), and the pellets were sonicated (10 cycles of 9 s with 9-s intervals). The cell extract was collected and applied to a Q-Sepharose column (Merck, USA). The fraction was eluted by a 5% ~ 15% elution buffer (20 mM Tris-HCl, 1.4M NaCl, PH 8.0).
BL21/pES3KI was precipitated with 40%–50% ammonium sulfate, and was resuspended in buffer A, which contains 30 mM NaCl and 20 mM Tris-Cl, pH 8.0. On the other hand, BL21/pES3I was precipitated with 70% ammonium sulfate and likewise resuspended in buffer A. All protein concentrations were determined by the Bradford assay (Amresco, USA).
Antibiotic Activity of Carocin S3. Ea1068 were grown overnight in LB medium at 28℃. The overnight cultures were diluted to a density of approximately 105 CFU/ml. The activity of increasing concentrations of Carocin S3 was assessed by incubating it with the cells in suspension at 28℃ for 60 minutes. Equal molar ratio of Carocin S3I and Carocin S3K were pre-mixed. The reaction mixtures were then spread onto LB agar plates and incubated at 28℃ for 16 hours. Finally, the colonies were counted to determine the antibiotic activity of Carocin S3.
Electrospray Ionization Mass Spectrometry for Carocin S3 and Carocin S3I Molecular Weight Assay. ESI-MS spectra were obtained on a Fisons VG platform using 37.5% methanol as the sample solvent for Carocin S3K and Carocin S3I. Samples were dialysed against water to remove salts and mixed with the sample solvent before analysis. Approximately 1 nmol of protein was analysed per run. Data were acquired over the m/z range 800 to 2000, with ten scans averaged and processed using the supplier's MassLynx software. The standard errors quoted in the text represent the errors from three separate mass determinations for each sample.