碳源對Pantoea vagans M17表面移行及生物膜形成的影響

Abstract

表面移行是一群細胞在表面上快速運動的模式，與生物膜的形成有關，而生物膜已被證明是造成食物腐敗的主要原因。表面移行和生物膜的形成都是一種細胞群體行為，然而，兩者之間的關係仍然存在著爭議。而群聚效應 (QS) 是細菌以透過一種稱為自誘導劑的小型分子與其他微生物進行交流和協調調節表面移行和生物膜生成的關鍵機制之一。先前實驗室從香瓜篩出Pantoea spp.，這一群會導致新鮮水果腐敗的細菌。研究表明，碳源是微生物主要的營養來源，也是影響細菌表面移行及生物膜形成的主要趨化劑。本研究進一步探討香瓜之中存在碳源種類，包括葡萄糖、果糖、蔗糖、半乳糖、棉子糖和L-蘋果酸，以不同濃度之單一或混合碳源對P. vagans M17的生長、生物膜生成和表面移行能力的影響，並觀察參與上述表型的基因表現量變化。結果表明， P. vagans M17在菌數介於10^6至10^7 CFU/μL之間，其生物膜形成主要受到葡萄糖濃度的影響，而表面移行則受到果糖濃度的影響。蔗糖濃度在5至100 mM的範圍內對表面移行能力之影響無明差異；而生物膜生成則在24和36小時之間無顯著差異，即對這兩個表型的影響相對適中。另外，在 0.5 至 5 mM 的濃度範圍內，P. vagans M17的表面移行能力會隨著棉子糖或L-蘋果酸濃度的增加而增強。此外，在混合碳源的實驗中，添加5 mM的 L-蘋果酸能明顯提升P. vagans M17的表面移行和生物膜形成能力，包括生物膜形成和鞭毛相關的基因表現量。而藉由生物膜調控基因 (yceP1)、胞外多醣合成基因 (amsF) 及鞭毛蛋白基因 (fliC2) 的表現量變化趨勢，觀察到相較於無添加組別，尤其在6小時時，yceP1、amsF和fliC2基因表現分別提升了1.52 ± 0.16、5.40 ± 0.17和6.49 ± 0.03 的相對量。由此推測L-蘋果酸的添加能夠增強P. vagans M17鞭毛及胞外多醣生成的能力，有助於菌株提早進入生物膜形成週期。此外，在P. vagans M17的PCR結果中確認了自誘導劑合成基因pagI和QS轉錄激活基因 pagR 的存在。未來，希望可以此研究為基礎，探討碳源對調控細菌表面移行及生物膜形成之QS系統，並藉此找出可以應用於食品加工或包裝的天然或合成化合物，以抑制QS系統而不影響食品品質，藉此延長食品的保存期限。

Swarming is a rapid surface-driven movement of a group of cells that is related to the formation of biofilms, which have been shown to be the major cause of food spoilage. Swarming and biofilm formation are both multicellular behaviors, but the relationship between the two is still controversial. Quorum sensing (QS) is one of the key mechanisms that bacteria use to regulate swarming and biofilm formation, through communication and coordination with other microorganisms using small molecules called autoinducers. Previously we have identified Pantoea spp., a group of abundant bacteria that cause spoilage to fresh fruits. Research has shown that carbon sources are the primary nutritional source and also the main chemoattractant affecting these two phenotypes of bacteria. Here, we investigated the effects of carbon sources which can be found in melon, including glucose, fructose, sucrose, galactose, raffinose, and L-malic acid, and examined the effects of different concentrations of individual or mixed carbon sources on the growth, biofilm formation, swarming ability and the expression levels of genes associated with these targeted phenotypes for P. vagans M17. Our results indicated that with a bacterial count ranging from 10^6 to 10^7 CFU/μL, the biofilm formation of P. vagans M17 is primarily influenced by the concentration of glucose, while swarming is greatly affected by the concentration of fructose. Sucrose concentration within the range of 5 to 100 mM showed no significant impact on swarming ability, while biofilm formation showed no significant difference between 24 and 36 hours, suggesting a relatively moderate impact on these two phenotypes. Additionally, within the concentration range of 0.5 to 5 mM, the swarming ability of P. vagans M17 was enhanced with increasing concentrations of raffinose or L-malic acid. Moreover, in experiments with mixed carbon sources, adding a small amount of L-malic acid significantly enhanced the swarming and biofilm formation abilities of P. vagans M17, including the expression of biofilm-regulating genes (yceP1), extracellular polysaccharide synthesis genes (amsF), and motility-related genes (fliC2). Compared with the control group without L-malic acid, especially at 6 hours, the expression levels of yceP1, amsF, and fliC2 genes increased by 1.52 ± 0.16, 5.40 ± 0.17, and 6.49 ± 0.03 relative quantity, respectively. This speculates that the addition of L-malic acid enhances the ability of P. vagans M17 for flagellar and exopolysaccharide generation, allowing the strain to enter the early biofilm formation cycle. Furthermore, the existence of the autoinducer synthesis gene pagI and the QS transcriptional activation gene pagR were confirmed in the PCR results of P. vagans M17. In the future, based on this study, it is hoped that the role of carbon sources in regulating bacterial swarming and biofilm formation through the QS system can be explored, and natural or synthetic compounds that can be applied to food processing or packaging to inhibit the QS system without affecting food quality can be identified to extend the shelf life of food.