Pantoea vagans M17 之群體感應調節對運動性和生物膜形成的影響

Abstract

群體感應 (quorum sensing, QS) 允許細菌感知自誘導劑的變化，在細菌間溝通並因應環境變動。自誘導劑的濃度反映族群密度，細菌據以調節基因表現並展現特定集體行為，包含生物膜生成與表面移行。這兩個表型可能促進細菌導致食品腐敗。研究表示 Pantoea spp. 的 QS 系統以 N-酰基-L-高絲氨酸內酯 (N-acyl-L-homoserine lactones, AHLs) 作為自誘導劑。實驗室先前從腐敗香瓜中篩選出 Pantoea vagans M17，具明顯生物膜生成與表面移行能力。為探討 P. vagans M17 的 QS 系統，本研究先利用多重序列比對確認 P. vagans M17 的合成酶 PagI 及受體 PagR 序列，與 P. vagans C9-1 的序列高度相似，且含有保守胺基酸。同時，以 HPLC-MS/MS 鑑定 P. vagans M17 產生的 QS 自誘導劑為 N-butyryl-L-homoserine lactone (BHL)，並添加 BHL 與潛在的 QS 抑制劑 2(5H)-呋喃酮。在 P. vagans M17 的培養液中，BHL 濃度隨著培養時間增加而上升。添加 40 - 140 ppm 的 BHL 雖不影響 P. vagans M17 生長，但可以提升 24 小時的生物膜生成。添加 8 - 40 mM 的範圍之 2(5H)-呋喃酮減緩 P. vagans M17 生長，而當濃度達 32 mM 以上則會抑制生物膜生成。添加 BHL 與 2(5H)-呋喃酮於養分較缺乏之 M9 培養基，可以分別促進與降低 P. vagans M17 表面移行的能力。此外，在第 6 與 24 小時，BHL 與 2(5H)-呋喃酮抑制 pagI 的表現。pagR 與鞭毛蛋白基因 fliC2 的表現情況不受兩者改變。2(5H)-呋喃酮會抑制並延後生物膜調節基因 bssS1 的表達。這些結果顯示，P. vagans M17 的 QS 對生物膜生成及表面移行的調控具有重要性，但其效果會受到環境中營養成分等因素影響。後續研究可以探討 QS 與環境的交互作用對細菌的適應性之貢獻，以及 QS 對生物膜及表面移行的其他重要因子之影響。對 QS 系統更深入的了解，有助於未來尋找合適的方法進行抑制，以減緩細菌導致的食品腐敗並促進食品安全。

Quorum sensing (QS) is used by bacteria to detect changes in the concentration of autoinducers, communicate with each other, and respond to environmental variations. The concentration of autoinducers reflects population density, enabling bacteria to regulate gene expression and exhibit specific collective behaviors including biofilm formation and swarming motility. These two phenotypes may contribute to food spoilage. Studies have shown that the QS system of Pantoea spp. utilizes N-acyl-L-homoserine lactones (AHLs) as autoinducers. Previously, Pantoea vagans M17, isolated from spoiled cantaloupe, was found to have strong biofilm formation and swarming motility. To investigate the QS system of P. vagans M17, we used multiple sequence alignment to confirm that the sequences of AHL synthase PagI and receptor PagR in P. vagans M17 were highly similar to those in P. vagans C9-1 and contained conserved amino acids. Using HPLC-MS/MS, the autoinducer produced by P. vagans M17 was identified as N-butyryl-L-homoserine lactone (BHL). BHL and the potential QS inhibitor 2(5H)-furanone were further added to observe their impact. Concentration of BHL in the culture medium of P. vagans M17 increased with cultivation time. Exogenous addition of 40 to 140 ppm of BHL did not affect the growth of P. vagans M17 but enhanced 24-hour biofilm formation. 2(5H)-furanone suppressed growth within the range of 8 to 40 mM, and inhibited biofilm formation above 32 mM. Addition of BHL and 2(5H)-furanone to the nutrient-deficient M9 medium significantly promoted and diminished the swarming motility of P. vagans M17, respectively. Furthermore, BHL and 2(5H)-furanone reduced the expression of pagI at 6 and 24 hours. The expression of pagR and the flagellin gene fliC2 were not affected by either compound. 2(5H)-furanone decreased and postponed the expression of the biofilm regulatory gene bssS1. These findings suggest that QS is pivotal in governing biofilm formation and swarming motility in P. vagans M17, while other factors such as environmental nutrients may influence the regulation. Future research can investigate the contribution of interactions between QS and the environment to bacterial adaptability. Besides, the effects of QS on other key factors in biofilm formation and swarming motility are worth further study. A comprehensive understanding of QS is expected to aid in devising strategies to mitigate food spoilage caused by bacteria and enhance food safety.