Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides   

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Pengyuan  XiuPengyuan Xiu*Rui  LiuRui Liu*Dechao  ZhangDechao Zhang Chaomin  SunChaomin Sun  (*contributed equally to this work)
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Original research article

A brief version of this protocol appeared in:
Applied and Environmental Microbiology
Jun 2017


Lipopeptides is an important class of biosurfactants having antimicrobial and anti-adhesive activity against pathogenic bacteria. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, lichenysin, and pumilacidin (Arima et al., 1968; Naruse et al., 1990; Yakimov et al., 1995; Steller and Vater, 2000; Roongsawang et al., 2002; Vater et al., 2002). To date, none of these lipopeptides have been reported to possess any anti-motility activity. We isolated, purified and characterized two novel cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. CLPs dramatically suppress the motility of pathogenic bacterium Vibrio alginolyticus 178, and promote cellular aggregation without inducing cell death. Cell aggregation assay was performed with the modification according to methods described by Dalili for anti-biofilm assay (Dalili et al., 2015). In future, this assay can be adapted to test both the cell aggregation and anti-biofilm activity of lipopeptide-like active substances derived from bacteria.

Keywords: Cyclic lipopeptides, Cell aggregation, Vibrio alginolyticus, Bacillus sp. 176, Anti-motility, Anti-biofilm


Overuse of broad-spectrum antibiotics and the accompanying proliferation of drug-resistant bacteria have stimulated efforts to develop environment-friendly biocontrol measures to reduce health hazards and environmental pollution (Nam et al., 2016; Sajitha et al., 2016). In recent years, the anti-microbial properties of biological surfactants have been increasingly recognized and harnessed for antibacterial, antifungal, and antiviral applications (Cameotra and Makkar, 2004; Singh and Cameotra, 2004; Rodrigues et al., 2006). Lipopeptides are the most widely reported class of biosurfactants having antimicrobial and 100 anti-adhesive activity against pathogenic bacteria, due to the amphipathic nature of their peptide and fatty acid components (Das et al., 2008; Dalili et al., 2015). In this study, two cyclic lipopeptides (CLPs) derived from a competing bacterium (Bacillus sp. 176) are found to inhibit the motility and promote the aggregation of V. alginolyticus 178. We purified and characterized the active anti-motility compounds and determined their structural and functional properties. In order to explore the mechanism of action of the CLPs, their impact on cell aggregation, adherence, and the expression of flagellar assembly components in V. alginolyticus were also investigated.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Xiu, P., Liu, R., Zhang, D. and Sun, C. (2018). Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides. Bio-protocol 8(1): e2686. DOI: 10.21769/BioProtoc.2686.

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