Regulation of bacteriocin production by Ruminococcus albus 7

數量感應 (Quorum sensing, QS)是細菌細胞之間溝通的過程。細菌利用小的化學分子在複雜的環境中感受族群密度，協調不同行為表現。這類訊號分子稱為自體誘導物，一旦濃度到達閾值，細菌即會反應而表現不同生理現象。反芻動物利用不同飼料的效率和瘤胃中高度多樣性的微生物生態有關。前人的研究指出，在體外培養情況下，兩株纖維分解菌 R. albus與 R. flavefaciens共培養時，纖維素消化率下降，當兩者共培養時，R. albus會產生細菌素抑制 R. flavefaciens生長。然而，R. albus 產生細菌素與數量感應機制關係的資訊非常少，因此，本試驗之目的為探討 R. albus 7產生細菌素之調控路徑。探討R. albus 7與 R. flavefaciens C94是否具有通用性之 LuxS數量感應系統，本試驗利用 Vibrio harveyi BB170生物冷光系統，測定 AI-2自體誘導物活性。R. albus 7的AI-2活性會在細菌指數生長晚期至平穩生長期達到最大值，在另一方面，R. flavefaciens C94在相同的 24小時培養過程，其 AI-2活性皆少於 R. albus 7的 1/5。當 R. albus 7與 R. flavefaciens C94共培養或是添加其上清液，R. albus 7產生細菌素之時間點皆較 R. albus 7單獨培養時提早 2小時產生。另外從 V. harveyi BB152上清液取得 AI-2，經8倍稀釋的上清液會刺激 R. albus 7產生最大細菌素活性，並且和添加 R. flavefaciens C94上清液時有相似的反應趨勢。進一步比較 4、10以及 24小時的 R. flavefaciens C94上清液，只有添加培養 24小時 R. flavefaciens C94上清液會誘導細菌素產生，但其 AI-2活性較 R. albus 7低，此項結果指出，培養 24小時之 R. flavefaciens C94上清液可能含有某些特定的誘導訊號胜肽，刺激 R. albus 7細菌素之表現。以 20、40以及 60%硫酸銨沉澱 R. flavefaciens C94 24小時上清液的蛋白質物質，利用 FPLC通過膠體過濾管柱進行純化。結果指出，R. flavefaciens C94會產生自體誘導胜肽，且具有誘導 R. albus 7細菌素表現，其胜肽分子量大約在 213至5380 Da之間。上所述，R. albus 7細菌素的產生會受到 V. harveyi BB152所產生的 AI-2所誘導，除此之外，R. albus 7亦能反應由 R. flavefaciens C94產生的自體誘導胜肽，並產生細菌素。Quorum sensing is a process of bacterial cell-cell communication that uses small molecules to coordinate diverse behaviors in response to population density. The small signal molecules are known as autoinducers, which will cause bacterial response once they reach a threshold level. The efficiency of ruminants to utilize wide variety of feeds is dependent on highly diversified rumen microbial ecosystem. Previous study showed that in vitro cellulose digestion decreased when two major cellulolytic bacteria, Ruminococcus albus and R. flavefaciens were cocultured. R. albus produced bacteriocin to inhibit the growth of R. flavefaciens when they were cocultured. There is not available information about the relationship between quorum sensing and bacteriocin production in R. albus. The purpose of this study is to study the regulation route(s) of bacteriocin production by R. albus.o investigate whether R. albus 7 and R. flavefaciens C94 possess universal LuxS quorum sensing system, we used Vibrio harveyi BB170 bioluminescence assay to test the presence of AI-2 autoinducer. AI-2 reached a maximal activity in late-exponential to early stationary phase of R. albus 7, whereas R. flavefaciens C94 produced less than 1/5 AI-2 activity of R. albus 7 during the same 24 hr incubation period. Furthermore, when R. albus 7 was cocultured with R. flavefaciens C94 or its supernatant, bacteriocin production occurred one hour earlier than R. albus cultured alone. By applying AI-2 obtained from V. harveyi BB152 supernatant, it was found that eight times diluted supernatant stimulated the highest bacteriocin activity and had a similar response pattern as with R. flavefaciens C94 supernatant. Comparing 4, 10, and 24 hr supernatants from R. flavefaciens C94, only the 24 hr supernatant could induce bacteriocin production. But, the AI-2 level was very low in this supernatant compared to the AI-2 level produced by R. albus 7 itself. These results suggested that 24 hr supernatant probably contained certain inducing signal peptide which could stimulate R. albus 7. The proteinaceous material from R. flavefaciens C94 24 hr supernatant was precipitated in sequence with 20, 40, and 60% ammonium sulfate, and purified by size exclusion using FPLC. The result showed that R. flavefaciens C94 had autuinducing peptide with molecular weight between 213 to 5380 Da which could induce bacteriocin production by R. albus 7.n conclusion, bacteriocin production by R. albus 7 could be induced by AI-2 from V. harveyi BB152. In addition, R. albus 7 also can react to the specific autoinducer peptide from R. flavefaciens C94 and produce bacteriocin.目 錄次文摘要………………………………….………………………………i文摘要………………………………………………………………...iii次………………………………………………………………………v次……………………………………………………………………...vi一章、文獻檢討………………………………………..………………1、數量感應………………………………………………………..…………………1一、 定義……………………………………………..………………………………1二、 數量感應之分類………………………………..………………………………2、動物腸道與瘤胃環境所存在之數量感應……………………..…………………9一、動物腸道環境……………………………………………………………………9二、腸道病原菌之數量感應…………………………………………………………9三、瘤胃環境……………………………………………..…………………………11四、瘤胃細菌之數量感應………………………………..…………………………11、數量感應與細菌素之關係………………………………………………………13一、乳酸菌細菌素………………………………………..…………………………13二、細菌素與纖維分解菌競爭現象之關係……………..…………………………15二章 確認 R. albus 7與 R. flavefaciens C94之抑制現象以及兩者否產生 AI -2訊號分子……………….……………………17、材料方法…………………………………………………………………………17一、R. albus 7與 R. flavefaciens C94之抑制現象………..……………..…………17 (一) 材料…………………………………………………..………………...……17 (二) 試驗方法……………………………………………..……………...………20二、檢測 luxS以及 AI-2活性……………………………………………………..22(一) 材料……………………………………………………..…………...………22 (二) 試驗方法………………………………………………..…………...………23、結果與討論………………………………………………………………………26一、R. albus 7與 R. flavefaciens C94之抑制現象……………………..…………26二、檢測 luxS以及 AI-2活性……………………………………………..………27三章 R. flavefaciens對 R. albus產生細菌素之影響路徑…...……31、材料方法…………………………………………………………………………31、結果討論…………………………………………………………………………33四章 V. harveyi BB152產生之AI-2訊號分子對 R. albus 7產生細 菌素之影響…………..……………………………......………37、材料方法…………………………………………………………………………37 結果討論…………………………………………………………………………39五章 不同細胞密度 R. flavefaciens C94之上清液對 R. albus 7產生細菌素之影響…………………..………………..…………47、材料方法…………………………………………………………………………47 結果討論…………………………………………………………………………49六章 檢測R. flavefaciens上清液中可能存在的自體誘導多胜肽...54、材料方法…………………………………………………………………………54 結果討論…………………………………………………………………………57論……………………………………………………………..………66考文獻……………………………………………………..…………6