Characteristics of N-Acylhomoserine Lactones Produced by Hafnia alvei H4 Isolated from Spoiled Instant Sea Cucumber

Abstract

This study aimed to identify <i>N</i>-acylhomoserine lactone (AHL) produced by <i>Hafnia alvei</i> H4, which was isolated from spoiled instant sea cucumber, and to investigate the effect of AHLs on biofilm formation. Two biosensor strains, <i>Chromobacterium violaceum</i> CV026 and <i>Agrobacterium tumefaciens</i> KYC55, were used to detect the quorum sensing (QS) activity of <i>H. alvei</i> H4 and to confirm the existence of AHL-mediated QS system. Thin layer chromatography (TLC) and high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS) analysis of the AHLs extracted from the culture supernatant of <i>H. alvei</i> H4 revealed the existence of at least three AHLs: <i>N</i>-hexanoyl-l-homoserine lactone (C6-HSL), <i>N</i>-(3-oxo-octanoyl)-l-homoserine lactone (3-oxo-C8-HSL), and <i>N</i>-butyryl-l-homoserine lactone (C4-HSL). This is the first report of the production of C4-HSL by <i>H. alvei</i>. In order to determine the relationship between the production of AHL by <i>H. alvei</i> H4 and bacterial growth, the β-galactosidase assay was employed to monitor AHL activity during a 48-h growth phase. AHLs production reached a maximum level of 134.6 Miller unites at late log phase (after 18 h) and then decreased to a stable level of about 100 Miller unites. AHL production and bacterial growth displayed a similar trend, suggesting that growth of <i>H. alvei</i> H4 might be regulated by QS. The effect of AHLs on biofilm formation of <i>H. alvei</i> H4 was investigated by adding exogenous AHLs (C4-HSL, C6-HSL and 3-oxo-C8-HSL) to <i>H. alvei</i> H4 culture. Biofilm formation was significantly promoted (<i>p</i> < 0.05) by 5 and 10 µM C6-HSL, inhibited (<i>p</i> < 0.05) by C4-HSL (5 and 10 µM) and 5 µM 3-oxo-C8-HSL, suggesting that QS may have a regulatory role in the biofilm formation of <i>H. alvei</i> H4.