RESUMO
The aim of this study was to evaluate the role of N-acyl homoserine lactones (AHLs) in the regulation of swimming motility of Hafnia alvei H4 and its biofilm formation on 96-well plate, glass and stainless-steel surfaces. The luxI gene, which codes for an enzyme involved in AHL synthesis, was deleted to generate a luxI mutant (ΔluxI). The mutant produced no AHL, and the relative expression of the luxR gene was significantly (P < 0.05) decreased. In addition, qRT-PCR analysis showed that the relative expression of the luxR gene in ΔluxI was stimulated by the presence of exogenous AHLs (C4-HSL, C6-HSL, and 3-o-C8-HSL) added at concentrations ranging from of 50-250 µg/ml. Among the three AHLs, C6-HSL had the strongest effect. The ability of ΔluxI to form biofilm on 96-well plate, glass and stainless-steel surfaces was significantly reduced (P < 0.05) compared with the wild type (WT), but was increased when provided with 150 µg/ml C4-HSL, whereas C6-HSL and 3-o-C8-HSL had no effect. Scanning electron microscopy analysis of the biofilm revealed less bacteria adhering to the surface of stainless-steel and fewer filaments were found binding to the cells compared with the WT. Furthermore, ΔluxI also exhibited significant (P < 0.05) decrease in the expression of biofilm- and swimming motility-related genes, flgA, motA and cheA, consistent with the results observed for biofilm formation and swimming motility. Taken together, the results suggested that in H. alvei H4, C4-HSL may act as an important molecular signal through regulating the ability of the cells to form biofilm, as well as through regulating the swimming motility of the cell, and this could provide a new way to control these phenotypes of H. alvei in food processing.
RESUMO
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 ß-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.