Characterization of two antimicrobial peptides produced by a halotolerant Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample

Abstract

A bacterial strain producing two antimicrobial peptides was isolated from a rhizosphere soil sample and identified as Bacillus subtilis based on both phenotypic and 16S rRNA gene sequence phylogenetic analysis. It grew optimally up to 14% NaCl and produced antimicrobial peptide within 24 h of growth. The peptides were purified using a combination of chemical extraction and chromatographic techniques. The MALDI-TOF analysis of HPLC purified fractions revealed that the strain SK.DU.4 secreted a bacteriocin-like peptide with molecular mass of 5323.9 Da and a surface-active lipopeptide (m/z 1056 Da). The peptide mass fingerprinting of low-molecular-weight bacteriocin exhibited significant similarity with stretches of secreted lipoprotein of Methylomicrobium album BG8 and displayed 70% sequence coverage. MALDI MS/MS analysis elucidated the lipopeptide as a cyclic lipopeptide with a β-hydroxy fatty acid linked to Ser of a peptide with seven α-amino acids (Asp-Tyr-Asn-Gln-Pro-Asn-Ser) and assigned it to iturin-like group of antimicrobial biosurfactants. However, it differed in amino acid composition with other members of the iturin family. Both peptides were active against Gram-positive bacteria, suggesting that they had an additive effect.

Figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic relationship between strain SK.DU.4 and other members of the genus Bacillus. Bootstrap values greater than 50% are given at the nodes.

Figure 2

Growth phase-dependent bacteriocin production by Bacillus subtilis strain SK.DU.4. While filled squares represent bacterial growth as measured by absorbance at 600 nm, filled triangles indicate corresponding bacteriocin activity as determined by inhibition zone assay.

Figure 3

Separation of the antimicrobial lipopeptide from acidic methanol extract by using reversed-phase HPLC. (A) Chromatogram profile of acidic methanol extract showed four peaks (F1-F4) and among these fractions F2 (lipopeptide) and F4 (bacteriocin-like peptide) showed antimicrobial activity. Diagonal red line indicates the gradient of solvent B; inset picture shows the re-chromatogram profile of lipopeptide fraction (F2). (B) Tricine–SDS-PAGE of the purified bacteriocin-like peptide. (C) Direct overlay of the SDS-PAGE gel demonstrating a clear inhibition zone against test strain S. aureus (indicated with arrow).

Figure 4

MALDI-TOF mass spectrum of bacteriocin-like peptide (A) and lipopeptide (B) obtained by solvent extraction. Spectrum was acquired in positive ion linear mode and reproducibility of the spectrum checked several times with different spots of same sample. (C) Scanning electron micrograph of S. aureus MTCC1430 cells (I) without treatment of bacteriocin-like peptide and (II) after treatment with bacteriocin-like peptide (6.0 μM) for 4 h. (D) Antibacterial activity of the peptides using well diffusion assay. After each peptide was serially diluted 100 μl of the lowest dilution inhibiting test strain were used for bacteriocin-like peptide (B), lipopeptide (L) and 50 μl of same dilutions were used to check the combined effect (B+L) on L. monocytogenes (I) and M. luteus (II). The bacteriocin-like peptide showed 14 and 12 mm inhibition zone for L. monocytogenes and M. luteus respectively. Lipopeptide yielded 11 mm zone for both strains. However, additive effect of both peptides showed a zone of 17 and 15 mm for L. monocytogenes and M. luteus respectively. Similar results were obtained in three individual experiments.

Figure 5

(A) Peptide mass fingerprinting (pmf) analysis of bacteriocin-like antimicrobial peptide (5324 Da). The pmf followed by MASCOT (Matrix Science, London, UK) search shows the matched amino acid residues (in red bold) of the peptide fragments with the putative periplasmic or secreted lipoprotein [Methylomicrobium album BG8] following NCBI blast search. (B) Amino acid sequence of the lipopeptide obtained by solvent extraction.

Figure 6

Determination of MIC for purified bacteriocin-like antimicrobial peptide towards different Gram-positive indicator strains. The micro-titre plate assay performed in triplicates revealed that L. monocytogenes and S. aureus are highly sensitive.

Figure 7

MALDI TOF PSD (MS/ MS) spectrum of lipopeptide (fraction 2 obtained in RP-HPLC of solvent extract).