Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family

Abstract

Aims: The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny.

Methods and results: We used a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio, Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25-5000 nmol l(-1) .

Conclusions: This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles.

Significance and impact of the study: The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains.

Keywords: AHLs; HPLC-MS/MS; Vibrionaceae; acyl-homoserine lactones; quorum sensing.

Figure 1

Basic chemical structure of AHLs and their fragmentation products produced during tandem MS in SRM mode. Chain lengths of AHLs screened in this study were from C4 to C12 with an increment of two carbons. Functional groups of the third carbon were 3-oxo, 3-OH or unsubstituted.

Figure 2

Detected AHLs mapped onto a 16S rDNA ML phylogeny. Bootstrap values above internal nodes were calculated using the NJ method and the maximum composite likelihood model. Clades containing strains that were tested for AHL production are named Clades I–VI. GenBank accession numbers are shown in brackets for species that were added to increase the phylogenetic diversity in the tree. Numbers in parentheses to the left of Clades I–VI denote the number of strains in each clade that were tested, but excluded from the final tree because of redundancy in the data. Asterisks denote strains that grew slowly with a final OD₆₀₀=0·1 when harvested after 50 h of growth. Complete list of results is shown in Table S3. Concentrations of AHLs are grouped into four categories: ‘high’ >5 μmol l⁻¹ (+++), ‘medium’ 25–5000 nmol l⁻¹ (++), ‘low’<25 nmol l⁻¹ (+) and values below LOQ (-).