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.
Highlights
Antimicrobial peptides characterization has received great attention in the recent past due to their applications as food preservatives without any toxic effects on host and therapeutic agents
The neighbour joining phylogenetic tree constructed with 16S rRNA gene sequences of other members of the genus Bacillus confirmed that strain SK.DU.4 belongs to genus Bacillus as it formed a distinct cluster along with other subspecies of B. subtilis (Figure 1)
In addition to antimicrobial peptides, many strains of bacteria are reported to produce a variety of antimicrobial biosurfactants like lipopeptide antibiotics, classified as iturins or surfactins (Chen and Hoover 2003)
Summary
Antimicrobial peptides characterization has received great attention in the recent past due to their applications as food preservatives without any toxic effects on host and therapeutic agents. Since lactic acid bacteria (LAB) produce an array of antimicrobial substances they were used as natural bio-preservatives for special applications (Holzapfel et al 1995; Cotter et al 2005; Deegan et al 2006). Other bacteria such as Enterococcus, Streptococcus etc., were reported to produce various bacteriocins and they are being considered for different applications. Considering the enormous diversity of Bacillus species in the soil ecosystem it is very important to isolate and preserve the strains producing these antimicrobial peptides followed by detailed characterization. In present study we report another antimicrobial peptide and a cyclic lipopeptide produced by a halotolerant isolate of Bacillus subtilis strain SK.DU. isolated from a rhizosphere soil sample
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