title: Phospholipid/Polydiacetylene Vesicle-Based Colorimetric Assay for High-Throughput Screening of Bacteriocins and Halocins.
abstract: The colorimetric assay is phospholipid/polydiacetylene vesicle-based assay used for the detection of membrane-acting peptides. Bacteriocins and halocins are antimicrobial peptides known to kill target cells by membrane disruption. Therefore, the assay was applied for high-throughput (HTP) screening of bacteriocins and halocins produced by lactic acid bacteria and haloarchaea, respectively. The assay consisted of vesicles which were synthesized using four different phospholipids: dipalmitoylphosphatydilcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphoethanolamine (DMPE) and dimyristoylphosphatidylglycerol (DMPG) in combination with diacetylene monomer 10,12-tricosadiy noic acid (TRCDA). These vesicles demonstrated blue colour at 640 nm and turned pink/red after interaction with nisin. DMPE/TRCDA vesicles showed pink colour with the highest colorimetric response (CR %) after treatment with nisin and, therefore, selected for the screening of bacteriocins and halocins. The colour of the vesicles was changed within 5 min in the presence of 5 μM nisin suggesting the sensitivity of assay. The assay was applied on 54 strains of lactic acid bacteria (LAB) and 53 haloarchaea for screening of bacteriocins and halocins, respectively. Out of these strains, three strains of LAB and five strains of haloarchaea were found to be bacteriocin and halocin non-producer, respectively. The other strains demonstrated the presence of bacteriocins and halocins. The colorimetric assay was found to be rapid, specific and reliable for HTP screening of antimicrobial peptides such as bacteriocins and halocins from producer strains isolated from various natural resources.
authors.affiliation: Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Yadav
authors.affiliation: Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.
authors.forename: Bijender
authors.initials: B
authors.lastname: Singh
authors.affiliation: Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana, 124001, India. santoshgenetics@mdurohtak.ac.in.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Tiwari
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides; D001430:Bacteriocins / Q000032:analysis* / Q000096:biosynthesis; D003124:Colorimetry / Q000379:methods*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry; D006216:Halobacteriaceae / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D007777:Lactobacillaceae / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry
chemical_list: C434762:10,12-tricosadiynoic acid; D023181:Antimicrobial Cationic Peptides; D001430:Bacteriocins; D005231:Fatty Acids, Unsaturated; D010455:Peptides; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; C413338:halocin S8; D015060:1,2-Dipalmitoylphosphatidylcholine; C002773:dimyristoylphosphatidylglycerol; D004134:Dimyristoylphosphatidylcholine; C018525:1,2-dimyristoylphosphatidylethanolamine
keywords: Bacteriocins; Colorimetric response; Halocins; High-throughput screening; PDA vesicles