Regulation of AI-2/LuxS gene on biofilm formation and spoilage of Shewanella baltica

Abstract:

[Objective] Shewanella baltica is a specific spoilage bacterium in marine fish during refrigerated storage. In the present study regulation of quorum sensing AI-2/LuxS on the biofilm formation and spoilage of S. baltica was elucidated. [Methods] LuxS gene in SB11 strain was amplified and knocked out by suicide plasmid. Biofilm formation, adhesion, swimming and amine metabolites between wild strain and △luxS mutant at 4℃ and 28℃ were comparatively measured by using crystal violet staining, bead vortexing, microscopy and HPLC, respectively. [Results] S. baltica SB11 luxS gene was amplified, and bioinformatics analysis revealed that deduced LuxS protein with 169 aa contained conserve His-Thr-Leu-Glu-His (HTLEH) motif and key amino acid functional sites. Three-dimensional structure of LuxS protein was similar to that from various bacteria. Compared with wild strain, supernatant of △luxS mutant lost bioluminance activity, although their growth was the same. △luxS exhibited lower biofilm development and maturation than the wild strain, and decreased by 20.1% and 27.9% at 4℃ for 96 h and at 28℃ for 24 h, respectively. Compared with the wild strain, △luxS reduced adherent cell by 6.48% at 4℃ for 72 h, and 6.57% at 28℃ for 24 h, indicating that the adherence of mutant on steel slide was significantly weak. The observation by fluorescence microscopy showed that the wild strain rapidly adhered to the coverslip and formed a large number of biofilm, whereas, △luxS mutant only seemed to form a flat sparse biofilm and failed to aggregate into clusters. Confocal Laser Scanning Microscope (CLSM) revealed that the thickness of maturing biofilm in the wild and mutant strains was 68.95 μm and 36.44 μm, respectively. Furthermore, the absence of luxS gene significantly promoted the bacterial swimming at 4 and 28℃. However, the production of trimethylamine and putrescine between the wild and mutant strain was similar. [Conclusion] LuxS protein in S. baltica SB11 was conserve, and AI-2/LuxS was involved in biofilm formation, adhesion, swimming, but not a functional signal in the regulation of spoilage potential.