Hexanoyl-L-homoserine lactone

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish.

N-acyl-L-homoserine lactone (AHL) mediated quorum sensing is a widespread communication system in gram-negative bacteria which regulates a wide range of target genes in a cell density-dependent manner. Although Escherichia coli is not capable of synthesizing AHL molecules because it lacks an AHL synthase encoding gene, it does produce a predicted AHL receptor of the LuxR family, named SdiA. In this work, we used a promoter trap library to screen for E. coli MG1655 promoters whose expression was affected by synthetic N-hexanoyl-L-homoserine lactone (C6-HSL), and we identified six upregulated and nine downregulated promoters, which also responded to synthetic 3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). The AHL responsiveness of these promoters was eliminated by knock-out of sdiA, and was temperature dependent, since the identified promoters showed a response at 30 degrees C but not, or only very weakly at 37 degrees C. In addition, in line with the observed induction of gadA encoding a glutamate decarboxylase, we could demonstrate an increased acid tolerance of E. coli upon exposure to C6-HSL. In conclusion, our work shows that E. coli has the capacity to alter its pattern of gene expression and its phenotypical properties in response to AHLs by means of the AHL responsive transcriptional regulator SdiA.