波罗的海希瓦氏菌AI-2/LuxS对生物被膜和致腐的调控

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

摘要:

[目的]波罗的海希瓦氏菌（Shewanella baltica）是冷藏海产鱼类的特定腐败菌。研究群体感应信号AI-2/LuxS对鱼源S.baltica生物被膜和致腐的调控作用。[方法]扩增SB11分离株的luxS基因，用自杀性质粒构建luxS基因缺失株，通过结晶紫染色、珠涡流法、显微镜观察和HPLC，比较分析野生株与缺失株△luxS在4℃和28℃下生物被膜形成、粘附能力、泳动性和致腐产物的差异。[结果]S.baltica SB11中扩增获得luxS基因，生物信息学分析显示LuxS蛋白由169个氨基酸构成，含有保守的His-Thr-Leu-Glu-His（HTLEH）模体和关键氨基酸位点，蛋白三维空间结构与其他细菌相似。与野生株相比，△luxS缺失株上清荧光信号散失，但不影响生长，生物被膜形成期和成熟期的含量显著低于野生株，在4℃培养96 h和28℃培养24 h被膜分别减少20.1%和27.9%。缺失株在不锈钢片的粘附能力明显减弱，其中在4℃培养72 h和28℃培养24 h后的粘附量比野生株分别减少6.48%和6.57%。荧光显微镜观察发现，野生株能快速粘附于玻璃片，聚集形成大量生物被膜，而△luxS仅形成平坦稀疏的被膜，粘附细菌降低，CLSM证实野生株和△luxS的成熟被膜厚度分别为68.95μm和36.44 μm。并且，△luxS株在4℃和28℃下泳动性均显著强于野生株。然而，野生株和△luxS株三甲胺和腐胺积累无差异。[结论]鱼源S.baltica中LuxS蛋白保守，AI-2/LuxS参与被膜、粘附能力及泳动性等多种生物被膜形成相关的调控作用，然而不是该菌致腐能力的功能性群体感应信号。

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.