Global and phylogenetic distribution of quorum sensing signals, acyl homoserine lactones, in the family of Vibrionaceae

Abstract

Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria.

Figure 1

Detection of OH-C6 in S0203 using two different methods: A–F (standard tune) and G–J (small molecule tune). (A) BPC chromatogram; (B–F) extracted ion chromatograms of [M + H]⁺ ±0.5, [M + H]⁺ ±0.01, [M + Na]⁺ ±0.01, 102.05495 ±0.01, and 1 × ¹³C [M + Na]⁺ ±0.01; (G–I) extracted ion chromatograms of [M + H]⁺ ±0.5, [M + H]⁺ ±0.01, 102.05495 ±0.01, and [M + Na]⁺; (J) MS/HRMS trace of 216.123. M: MS/HRMS Library spectrum of OH-C6 [M + H]⁺ at 10 eV, N: simultaneously acquired MS/HRMS spectrum at 10 eV; (L) full scan spectrum (small molecule tune); (K) full scan spectrum (normal tune). Samples were analysed with a time difference of two months and thus retention was altered, but constant in the sequence and identical (±0.02 min) to reference standard.

Figure 2

Cluster analysis of AHL diversity per strain and phylogenetic affiliation using CIMminer [43]. Dark grey: AHL present; light grey: AHL absent; colours distinguish different clades, clusters marked in bold.

Figure 3

Chemical analysis of strain S1162. (A) base peak chromatogram; (B) m/z 56.04948 ± 0.02; (C) m/z 102.05495 ± 0.02; and (D) m/z 120.06552 ± 0.02, showing the extracted ion chromatograms of the three major diagnostic fragment ions with labels of six known long chain AHLs. At 3.36 min a novel open chain AHL is eluting with its MS/HRSM spectrum at 10 eV showed in E with a tentatively identified compound.

Figure 4

Neighbor joining tree of concatenated partial rpoA and recA gene sequences of 32 AHL-positive Vibrionaceae strains using the Jukes-Cantor method and Shewanella oneidensis MR-1 as outgroup. Bootstrap values are based on 100 replicates. Square brackets indicate clades [45,48].