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

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

Structural studies of a lipid-binding peptide from tunicate hemocytes with anti-biofilm activity

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.We would like to thank CNPq, CAPES (Ciências sem Fronteiras), FAPDF and FUNDECT. D.G. acknowledges Fundação para a Ciência e a Tecnologia - Ministério da Educação e Ciência (FCT-MEC, Portugal) for fellowship SFRH/BPD/73500/2010 and A.S.V. for funding within the FCT Investigator Programme (IF/00803/2012).info:eu-repo/semantics/publishedVersio

Purification, biochemical characterization and self-assembled structure of a fengycin-like antifungal peptide from Bacillus thuringiensis strain SM1

An antifungal lipopeptide fengycin, producing strain SM1 was isolated from farm land soil sample and identified as Bacillus thuringienesis strain SM1 by using 16S rDNA analysis. Fengycin detected in the culture extract was further purified using HPLC and showed a molecular mass of 1492.8 Da by MALDI-TOF-MS analysis. Purified fengycin was allowed to construct their self-assembled structure onto a hydrophobic surface showing a clear improvement of antibacterial activity. In self-assembly, fengycin adapts a spherical micelle core shell like structure. Self-assembled fengycin may be a successful antimicrobial compound modifying its action from confined antifungal function. Besides it can open up a new area of research in supramolecullar lipopeptide based compound making. This can revealed the mode of action of this unique self-assembled structure to fully evaluate its potential for use as an antimicrobial drug to control the emergence of bacterial infection

Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., from Antarctica

Twelve strains of psychrophilic bacteria were isolated from cyanobacterial mat samples collected from various water bodies in the McMurdo Dry Valley region of Antarctica. All the isolates were Gram-negative, non-motile, coccoid, psychrophilic, halotolerant bacteria and had C16:1ω7c, C17:1ω8c and C18:1ω9c as the major fatty acids, ubiquinone-8 as the respiratory quinone and DNA G+C content of 41-46 mol%. Based on these characteristics, the isolates were assigned to the genus Psychrobacter. Based on their SDS-PAGE profiles, the 12 isolates could be categorized into three groups. Six isolates of Group I were identified as representing strains of Psychrobacter okhotskensis. However, using detailed phenotypic and chemotaxonomic characteristics and phylogenetic analysis based on their 16S rRNA gene sequences, strain CMS 39T, the only strain from Group II, and strain CMS 56T, a representative strain of Group III, were different from each other and from all recognized species of Psychrobacter. Therefore, it is proposed to classify CMS 39T (=DSM 15337T=MTCC 4208T) and CMS 56T (=DSM 15339T=MTCC 4386T) as representing the type strains of novel species of Psychrobacter, for which the names Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., respectively, are proposed

Understanding role of genome dynamics in host adaptation of gut commensal, L. reuteri

Lactobacillus reuteri is a gram-positive gut commensal and exhibits noteworthy adaptation to its vertebrate hosts. Host adaptation is often driven by inter-strain genome dynamics like expansion of insertion sequences that lead to acquisition and loss of gene(s) and creation of large dynamic regions. In this regard we carried in-house genome sequencing of large number of L. reuteri strains origination from human, chicken, pig and rodents. We further next generation sequence data in understanding invasion and expansion of an IS element in shaping genome of strains belonging to human associated lineage. Finally, we share our experience in high-throughput genomic library preparation and generating high quality sequence data of a very low GC bacterium like L. reuteri

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications

Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by <em>Brevibacillus</em> sp. Strain GI-9

<div><h3>Background</h3><p>Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.</p> <h3>Methodology/Findings</h3><p>The strain GI-9 was identified as <em>Brevibacillus</em> sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.</p> <h3>Conclusions</h3><p>We have identified a bacteriocin producing strain GI-9, belonging to the genus <em>Brevibacillus</em> sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.</p> </div

Genetic organization of 4 kb region of the genome encoding laterosporulin (A) and nucleotide sequence of the ORF (B).

<p>The putative ORF encoding laterosporulin structural gene is shown by filled arrow and flanking ORFs as shown by empty arrows (A). Panel B shows the nucleotide sequence of the laterosporulin gene (encoding the indicated amino acids) with putative start codan, stop codons and ribosome binding site (RBS) shown in bold.</p