Phage Bp7 was isolated by our research team and identified as a coliphage. Its complete genomic sequence was deposited in GenBank(HQ829472). Two Gram-positive strains and sixteen Gram-negative stains used in this report were kept in our lab; these strains are described in more details in Table 1. M. lysodeikticus was cultured in Luria-Bertani medium(LB, Takara, Dalian, China)at 30 ℃, and other bacterial strains were cultured in the same medium at 37 ℃. All bacterial strains were stored in 30% glycerol at -80 ℃ before use. Hen egg white lysozyme(HEWL, CW Biotech, China)was purchased from the specified suppliers.
Table 1. Host range of phage Bp7 and lytic activity of Bp7e and its mutation Bp7Δe
The Bp7e amino acid sequence was compared with that described in the NCBI GenBank database(http://www.ncbi.nlm.nih.gov/BLAST), and the phylogenetic relationships of Bp7e were analyzed using Mega5.0 software(http://www.megasoftware.net/index.php). The Bp7e secondary structure was analyzed using PredictProtein(http://www.predictprotein.org).
The Bp7e gene was amplified by PCR from a phage Bp7 genomic DNA template by a formard primer Bp7eF and a reverse primer Bp7eR containing Nde I and Xho I restriction sites, respectively(underlined in Table 2). The primers used in this work are listed in Table 2. The PCR product was purified and digested by Nde I and Xho I enzymes(Takara, Dalian, China) and cloned in a pET28a expression vector(Novagen, Shanghai, China)with a C-terminal 6×His tag. The presence of the insert in the recombinant plasmid pET28a-Bp7e was confirmed by DNA sequencing(Takara, Dalian, China).
Table 2. Sequences of PCR primers used for bacteriophage gene cloning.
The construct pET28a-Bp7e plasmid was transformed into E. coli BL21(DE3) and cultured on an LB plate with 50 μg/mL of Kana+ selection, and further confirmed by enzyme digestion, PCR and sequencing. The E. coli BL21(DE3)harboring the pET28a-Bp7e plasmid was cultured in 3 mL LB medium at 37 ℃ with 50 μg/mL of Kana+ to an optimal density of 0.6(OD600). Recombinant protein expression was induced for 12 h at 16 ℃ by the addition of isopropyl β-D-1-thiogalactopyr-anoside(IPTG)to a final concentration of 0.7 mmol/L. The culture was then centrifuged(4000 rpm, 10 min) and the cells were treated ultrasonically for 30 min(3 s pulse, 1 s pause)whilst being kept on ice. Samples of the supernatant and the precipitate were collected, and the presence of the recombinant proteins was confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Insoluble cell debris was removed by centrifugation(10, 000 rpm, 3 min, 4 ℃). The recombinant protein in the supernatant was purified using a 6× His-tagged Protein Purification Kit(CW Biotech)as described by the manufacturer, and detected by western-blot analysis with an anti-His tag monoclonal antibody as the primary Ab and an HRP-conjugated rabbit anti-mouse IgG as the secondary Ab. The protein concentration was determined using a BCA Protein Assay Kit with bovine serum albumin(BSA)as st and ard(CW Biotech).
Two amino acid sites(L99A and M102E)of Bp7e were mutated using a Muta-directTM kit, following the prescribed protocol(SBS Genetech Co., Ltd.). Two sets of overlapping mutagenic primers – Bp7Δe 99F and Bp7Δe 99R for L99A, and Bp7Δe 102F and Bp7Δe 102R for M102E – are presented in Table 2(mutation basepairs are shown as shaded bases). Plasmid pET28a-Bp7Δe was then introduced into competent E. coli BL21(DE3)for heterologous production, as described above.
The antibacterial activity of Bp7e and Bp7Δe was assayed by measuring change in turbidity. Briefly, recombinant E. coli BL21(DE3)with plasmid pET28a-Bp7e(Group Bp7e)or pET28a-Bp7Δe(Group Bp7Δe)was grown to exponential stage in LB at 37 ℃ and then 0.7 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG)was added to the medium to induce expression. E. coli BL21(DE3)with plasmid pET28a was used as a negative control. The increase in the OD600 value of both test and control cultures was recorded every 1 h for 5 h. After 5 h induction, the cultures were then adjusted to the same OD600value, and a 100 μL sample of each culture was transferred to a 96-well plate. Then, 0.5 μL chloroform, which could work on outer membrane of E. coli and help the lysis effect of endolysin appear, was added per well and the fall in the OD600value of each well was recorded every 30 min for 3 h(Legotsky et al., 2014). All assays were performed in triplicate. The intracellular lysis activities of endolysin Bp7e and its mutant Bp7Δe were evaluated according to the changes in OD600.
The in vitro antibacterial activities of Bp7e and Bp7Δe were evaluated using different strains, as listed in Table 1, which includes two Gram-positive strains(S. aureus and M. lysodeikticus) and sixteen Gram-negative strains(fourteen E. coli strains, one S. paratyphi strain and one P. aeruginosa strain). In brief, the Gram-positive bacterial strains, cultured in LB to exponential stage, were harvested by centrifugation and resuspended in 10 mmol/L Hepes-HCl(pH 7.2)to a final concentration of 106 CFU/mL. To detect the antibacterial activities, 100 μL of Bp7e or Bp7Δe protein, at 1 μmol/L or 2 μmol/L, respectively, was mixed with 100 μL of bacterial suspension and incubated at room temperature for 2 h. After incubation, a serial, 10-fold dilution series in phosohate-buffered saline(PBS)was performed for each sample, and 100 μL of each dilution was plated on LB agar plates. After overnight incubation, the number of viable bacteria was assessed by counting the colonies that had formed in the LB agar. PBS(pH 7.2)was used as a negative control instead of Bp7e and Bp7Δe protein, and HEWL(2 μmol/L)was used as a positive control. All assays were performed in triplicate.
The antibacterial activities of the Bp7e and Bp7Δe proteins against Gram-negative bacterial strains in the presence of malic acid were also assessed. Briefly, the bacterial strains cultured at the exponential phase were harvested by centrifugation and resuspended in 10mmol/L Herpes-HCl(pH 7.2)to a final concentration of 106 CFU/ mL. To test the antibacterial activity of Bp7e and Bp7Δe, samples with the following composition were prepared:(ⅰ)100 μL of bacterial suspension, 50 μL of 20 mmol/L malic acid and 50 μL of Bp7e or Bp7Δe protein(2 μmol/L; 4 μmol/L); (ⅱ)100 μL of bacterial suspension, 50 μL of PBS and 50 μL of Bp7e or Bp7Δe protein(2 μmol/L; 4 μmol/L); (ⅲ)100 μL of bacterial suspension, 50 μL of PBS and 50 μL of HEWL(2 μmol/L; 4 μmol/L); (ⅳ)100 μL of bacterial suspension, 50 μL of 20 mmol/L malic acid and 50 μL of HEWL(2 μmol/L; 4 μmol/L); (ⅴ)100 μL of bacterial suspension, 50 μL of 20 mmol/L malic acid and 50 μL of PBS(pH 7.2);(ⅵ)100 μL of bacterial suspension and 100 μL of PBS(pH 7.2). All samples were incubated at room temperature for 30 min. After incubation, a serial, 10-fold dilution series in PBS was performed for each sample, and 100 μL of each dilution was plated on LB agar plates. After overnight incubation at 37 ℃, the number of viable bacteria was assessed by counting the colonies that had formed in the LB agar. Here, PBS, rather than malic acid or protein(Bp7e, Bp7Δe and HEWL), served as a negative control; HEWL(2 μmol/L or 4 μmol/L)served as a positive control. All assays were performed in triplicate.
The antibacterial activity against the eighteen bacterial strains was quantified as the relative inactivation in logarithmic units = log10(N0/N1), where N0 is the colony number of the untreated group(in the negative control) and N1 is the colony number of the treated groups after incubation. Averages ± st and ard deviations were calculated for n = 3 repeats. All the data were analyzed by SPSS 11.0 statistical software(SPSS, Chicago, IL, USA).