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This study aimed to identify N-acylhomoserine lactone (AHL) produced by Hafnia alvei H4, which was isolated from spoiled instant sea cucumber, and to investigate the effect of AHLs on biofilm formation. Two biosensor strains, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens KYC55, were used to detect the quorum sensing (QS) activity of H. alvei 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 H. alvei H4 revealed the existence of at least three AHLs: N-hexanoyl-l-homoserine lactone (C6-HSL), N-(3-oxo-octanoyl)-l-homoserine lactone (3-oxo-C8-HSL), and N-butyryl-l-homoserine lactone (C4-HSL). This is the first report of the production of C4-HSL by H. alvei. In order to determine the relationship between the production of AHL by H. alvei 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 H. alvei H4 might be regulated by QS. The effect of AHLs on biofilm formation of H. alvei H4 was investigated by adding exogenous AHLs (C4-HSL, C6-HSL and 3-oxo-C8-HSL) to H. alvei H4 culture. Biofilm formation was significantly promoted (p < 0.05) by 5 and 10 µM C6-HSL, inhibited (p < 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 H. alvei H4.
Figure 1. Screening of N-acylhomoserine lactone (AHL)–producing bacteria by (a) C. violaceum CV026 and (b) A. tumefaciens KYC55 strain. H4: bacterium isolated from spoiled sea cucumber; CV026 and KYC55: biosensor strains used to detect AHL-producing strains.
Figure 2. Phylogenetic analysis of AHL-producing bacteria isolated from spoiled ready-to-eat sea cucumber. Phylogenetic analysis was performed with MEGA4.1 software.
Figure 3. TLC bioassay of AHLs produced by H. alvei H4 with C. violaceum CV026 Lane 1: Standard AHLs of C4-HSL; Lane 2: Standard AHLs of C6-HSL; Lane 3: AHLs extracted from H. alvei H4 culture supernatant.
Figure 4. Mass spectrometry analysis of AHLs extracted from H. alvei H4 culture supernatant; (a) Spectrum of C4-HSL (m/z 172.0); (b) Spectrum of C6-HSL (m/z 200.2) (marked by arrow); (c) Spectrum of 3-oxo-C8-HSL (m/z 242.1) (marked by arrow).
Figure 5. Production of AHLs by H. alvei H4 at different stages of growth in LB medium. AHLs in the culture supernatant of H. alvei H were quantitated by the β-galactosidase assay with A. tumefaciens KYC55 as the reporter strain.
Figure 6. Effect of AHLs on the biofilm formation of H. alvei H4. (ND: No addition of AHLs). (a) Addition of 5–40 µM C4-HSL; (b) Addition of 5–40 µM C6-HSL; (c) Addition of 5–40 µM 3-oxo-C8-HSL.
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