Sampling, separation, and quantification of N‐acyl homoserine lactones from marine intertidal sediments

Frederike Stock,Nico Ueberschaar,Wim Vyverman,Willem Stock,Georg Pohnert,Samarasinghe Gunasekara Liyanage Ishari Nuwanthi,Sven Mangelinckx,Emilio Cirri

doi:10.1002/lom3.10412

Abstract

AbstractN‐acyl homoserine lactones (AHLs) are molecules produced by many Gram‐negative bacteria as mediators of cell‐cell signaling in a mechanism known as quorum sensing (QS). QS is widespread in marine bacteria regulating diverse processes, such as virulence or excretion of polymers that mediate biofilm formation. Associated eukaryotes, such as microalgae, respond to these cues as well, leading to an intricate signaling network. To date, only very few studies attempted to measure AHL concentrations in phototrophic microbial communities, which are hot spots for bacteria‐bacteria as well as microalgae‐bacteria interactions. AHL quantification in environmental samples is challenging and requires a robust and reproducible sampling strategy. However, knowing about AHL concentrations opens up multiple perspectives from answering fundamental ecological questions to deriving guidelines for manipulation and control of biofilms. Here, we present a method for sampling and AHL identification and quantification from marine intertidal sediments. The use of contact cores for sediment sampling ensures reproducible sample surface area and volume at each location. Flash‐freezing of the samples with liquid nitrogen prevents enzymatic AHL degradation between sampling and extraction. After solvent extraction, samples were analyzed with an ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UHPLC‐HRMS) method that allows to baseline‐separate 16 different AHLs in less than 10 min. The sensitivity of the method is sufficient for detection and quantification of AHLs in environmental samples of less than 16 cm3.

Highlights

This technique is quite sensitive, it is not suitable for acyl homoserine lactones (AHLs) quantification. Given these limitations and technological advances in analytical chemistry, AHL detection by bacterial reporter strains is nowadays often used in combination with, or replaced by, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LCMS) methods (Huang et al 2007; Decho et al 2009; Wang et al 2017; Sun et al 2018)
UHPLC-high-resolution mass spectrometry (HRMS) Orbitrap measurements For detection and quantification of AHLs, we developed a protocol for UHPLC-HRMS by adapting a previous method from Sun et al (2018)
We focused on AHLs with even-numbered acyl-side chains in this study because the fatty acids utilized in AHL synthesis commonly have even-numbered carbon chains (Parsek et al 1999)

Summary

Introduction

AHL production was confirmed in 39 out of 67 marine Alphaproteobacteria using a screening with bacterial reporter strains followed by gas chromatography-mass spectrometry (GC-MS) analysis (Wagner-Döbler et al 2005) This finding was in line with an in silico analysis of the Global Ocean Sampling metagenome database where several luxI homologues were identified in microplankton samples suggesting that QS signaling is widespread in marine bacteria (Doberva et al 2015). The wide range of polarity, the low concentrations in environmental samples and the often complex matrix make a quantitative assessment of AHLs in field samples challenging These obstacles explain the scarcity of studies that have attempted to quantify AHLs in microbial mats (Decho et al 2009), sludge from bioreactors (Wang et al 2017; Sun et al 2018), subtidal biofilms (Huang et al 2007), and soils (Sheng et al 2017). The biomass dynamics in this upper compartment of the sediment are independent of those of the deeper sediment which makes this fraction interesting for ecological studies (Herlory et al 2004)

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