本文選題：霍亂弧菌 + 山梨醇��； 參考：《中國疾病預防控制中心》2009年博士論文

【摘要】： 霍亂弧菌(Vibrio cholerae)是引起霍亂的病原菌。從1871年至今已經(jīng)發(fā)生過至少七次世界性大流行,其中第七次由O1群El Tor型霍亂弧菌引起,并且現(xiàn)今還處在此次大流行中。我國在霍亂防控中,建立了針對O1群El Tor型霍亂弧菌分型的噬菌體-生物分型方案,將El Tor型霍亂弧菌區(qū)分為流行株和非流行株,并進一步分成不同型別。目前研究發(fā)現(xiàn)流行株均為產(chǎn)毒株,非流行株幾乎全為非產(chǎn)毒株。這一方案對在霍亂防控過程中區(qū)別對待這兩類菌株、從而采取不同的預防控制措施有重要的意義,至今仍為霍亂弧菌分型的重要方法之一。該分型方案反映了El Tor霍亂弧菌產(chǎn)毒株和非產(chǎn)毒株的遺傳學差異,通過對分型方案的機制研究,能夠反映出產(chǎn)毒株(流行株)和非產(chǎn)毒株(非流行株)的基因組學差異。 山梨醇發(fā)酵實驗是噬菌體-生物分型方案中一個重要的生化反應,根據(jù)山梨醇發(fā)酵液pH下降的快慢(表現(xiàn)為發(fā)酵液顏色變黃的先后時間)可將El Tor型霍亂弧菌分為快發(fā)酵株和慢發(fā)酵株,其中流行株均屬于慢發(fā)酵株,非流行株均屬于快發(fā)酵株。我們先前通過蛋白質組學和基因轉錄分析,發(fā)現(xiàn)快慢發(fā)酵株中存在甘露醇磷酸烯醇式丙酮酸轉移酶系統(tǒng)(PTS)操縱子轉錄水平及其產(chǎn)物的差異,而且在產(chǎn)毒株和非產(chǎn)毒株間甘露醇發(fā)酵試驗具有與山梨醇發(fā)酵試驗相似的特征。在本研究中,我們對甘露醇PTS轉錄及其調控開展了進一步研究。為了解甘露醇誘導El Tor型菌株快發(fā)酵株(非產(chǎn)毒株)和慢發(fā)酵株(產(chǎn)毒株)的表達差異,我們利用霍亂弧菌全基因組表達譜芯片分析了快慢發(fā)酵菌株在甘露醇發(fā)酵液中的轉錄差異基因,發(fā)現(xiàn)負責甘露醇的轉運的PTS系統(tǒng)成分在快發(fā)酵株93097中的轉錄水平要高于其在慢發(fā)酵株N16961的,這可能是快慢菌株代謝甘露醇產(chǎn)酸速度差異的原因之一;另外也發(fā)現(xiàn)快發(fā)酵株中對甘露醇的利用更多表現(xiàn)為產(chǎn)酸,而在慢發(fā)酵株中則表現(xiàn)為更強的能量代謝。 我們進一步對快慢菌株中轉運甘露醇的PTS系統(tǒng)進行了分析。我們先前的實驗已經(jīng)證實了mtlA、mtlR和mtlD屬于甘露醇特異操縱子的成分,mtlA和mtlD在快發(fā)酵株中轉錄水平高于慢發(fā)酵株的。在本研究中,我們利用報告基因系統(tǒng)分析了mtl啟動子區(qū),發(fā)現(xiàn)mtl操縱子上游VCA1044對mtl啟動子活性具有很重要的作用,且快發(fā)酵株中mtl操縱子的啟動子活性要高于慢發(fā)酵株,另外在快慢發(fā)酵菌株中上調mtlA的轉錄水平,甘露醇的利用能力均增強了,這些結果說明甘露醇PTS操縱子的轉錄能力與甘露醇的快慢發(fā)酵利用相關,提示是造成快慢發(fā)酵株甘露醇發(fā)酵速度差異的機制之一。 細菌中cAMP-CRP復合物在多個調節(jié)通路中發(fā)揮作用,包括對糖醇利用的調控。啟動子活性收到轉錄調控因子的調控作用,作為細菌中重要的調控因子,對碳水化合物的代謝具有非常重要的調控作用。因此我們對El Tor型霍亂弧菌中cAMP-CRP復合物對甘露醇PTS操縱子的調節(jié)作用進行了研究。利用CRP合成基因crp和cAMP合成基因cya的缺失突變株,證實該復合物對mtl操縱子具有非常重要的正向調控作用。我們發(fā)現(xiàn)在快慢發(fā)酵株中crp基因的轉錄水平?jīng)]有明顯差異,但快發(fā)酵株中cya的轉錄水平要高于慢發(fā)酵株,且cya表達上調后,也會造成快慢發(fā)酵株甘露醇發(fā)酵液顏色變黃的時間均提前,提示cAMP-CRP復合物的差異對mtl操縱子的轉錄水平調控有差別,在快發(fā)酵株中,cAMP-CRP復合物活性更高,使mtl操縱子轉錄水平更高。預測到mtl啟動子區(qū)有CRP結合的保守位點“TGTGA……TCACA”,通過凝膠遷移實驗,證實CRP-cAMP復合物能夠與該啟動子區(qū)結合。 以上實驗是通過甘露醇發(fā)酵進行分析的。我們前期發(fā)現(xiàn)El Tor型霍亂弧菌中山梨醇發(fā)酵實驗與甘露醇發(fā)酵實驗具有相似的結果,但霍亂弧菌中沒有其他細菌中所具有的山梨醇特異的PTS基因簇。山梨醇在很多細菌中屬于PTS轉運糖醇,我們構建了PTS系統(tǒng)中非特異成分ptsⅠ的缺失突變株,結果顯示ptsⅠ缺失后的菌株失去了發(fā)酵山梨醇的能力,證實山梨醇在霍亂弧菌中是通過PTS系統(tǒng)轉運的。我們發(fā)現(xiàn)甘露醇mtl操縱子的基因缺失同樣導致山梨醇利用的阻斷,而且山梨醇能夠誘導甘露醇特異mtl操縱子的高轉錄表達,因此這些結果說明霍亂弧菌中山梨醇的PTS轉運是通過甘露醇特異PTS的。 本研究明確了cAMP-CRP復合物在霍亂弧菌中對甘露醇的代謝調節(jié)作用,將甘露醇和山梨醇快慢發(fā)酵株的調控機制研究進一步延伸,同時明確了霍亂弧菌中山梨醇與甘露醇共同通過一個PTS轉運,并且山梨醇是甘露醇PTS的誘導物,顯示了霍亂弧菌與其他細菌不同的特征。這些研究更深入了對El Tor霍亂弧菌產(chǎn)毒株和非產(chǎn)毒株在山梨醇和甘露醇發(fā)酵速率與利用上的差異機制的理解。在本研究中,也顯示山梨醇和甘露醇在霍亂弧菌產(chǎn)毒株和非產(chǎn)毒株中代謝途徑的差異,這種差異的原因和作用結果,包括在兩種菌株的不同遺傳背景下各自對代謝的調節(jié)、生長的需求、以及是否導致環(huán)境生存能力的差異,另外包括細胞內cAMP水平的差異會帶來哪些生長代謝影響等,還需要進一步的研究。
[Abstract]:Vibrio cholerae (Vibrio cholerae) is a pathogenic bacteria causing cholera. Since 1871, there have been at least seven worldwide pandemics, seventh of which are caused by the O1 group El Tor Vibrio cholerae, and are still in this pandemic. In cholera prevention and control, the phage biotype of the O1 group El Tor type cholera vibrio type is established in China. The El Tor type of Vibrio cholerae was divided into epidemic and non epidemic strains. The current study found that all the epidemic strains were all strains, and that the non epidemic strains were almost all non producing strains. This scheme treats these two types of bacteria differently in the prevention and control of cholera, thus taking different preventive and controlling measures to be heavy. The significance of it is still one of the important methods for the classification of Vibrio cholerae. The scheme reflects the genetic differences between the El Tor and non producing strains of Vibrio cholerae. Through the study of the mechanism of the classification scheme, it can reflect the genomics differences of the producing strains (epidemic strains) and non producing strains (non epidemic strains).
The experiment of sorbitol fermentation is an important biochemical reaction in the phage biotyping scheme. According to the slow decline of pH in the sorbitol fermentation broth, El Tor Vibrio cholerae can be divided into fast fermenting and slow fermenting strains, all of which belong to slow fermenting strains and non epidemic strains belong to fast fermentation. By proteomics and gene transcription analysis, we found that the transcriptional level of the mannitol phosphoenolpyruvate transferase system (PTS) and the differences in the product were found in the fast and slow fermented plants, and the mannitol fermentation test was similar to that of the sorbitol fermentation test between the producing and non producing strains. In this study, we further studied the transcription and regulation of mannitol PTS. In order to understand the difference in expression of mannitol induced El Tor strain fast fermenting strain (non producing strain) and slow fermenting strain (strain producing strain), we used the whole genome expression chip of Vibrio cholerae to analyze the transcriptional difference of fast and slow fermenting strains in mannitol fermentation broth. It was found that the transcriptional level of the PTS system components responsible for the mannitol transport in the fast fermenting strain 93097 was higher than that in the slow fermenting strain N16961, which may be one of the reasons for the difference in the acid production rate of the fast and slow strains of mannitol, and also found that the use of mannitol in the fast fermented strain is more characterized by acid production and in the slow fermented plant. It shows stronger energy metabolism.
We further analyzed the PTS system of the mannitol in fast and slow strains. Our previous experiments have confirmed that mtlA, mtlR and mtlD belong to the mannitol specific operon, and mtlA and mtlD have higher transcriptional levels in the fast fermented plants than the slow fermenting strains. In this study, we used the report gene system to analyze the MTL promoter. The upstream VCA1044 of the MTL operon was found to play an important role in the activity of MTL promoter, and the promoter activity of the MTL operon in the fast fermenting strain was higher than that of the slow fermentation plant. In addition, the transcriptional level of mtlA was up-regulated in the fast and slow fermentation strains, and the utilization of mannitol increased. These results indicate the transcriptional ability of the mannitol PTS operon. It is related to mannitol fast fermentation utilization, suggesting that it is one of the mechanisms leading to the difference of mannitol fermentation speed between fast and slow fermentation strains.
The cAMP-CRP complex in bacteria plays a role in multiple regulatory pathways, including regulation of the use of sugar alcohols. Promoter activity receives the regulatory role of transcriptional regulators. As an important regulator in bacteria, it has a very important regulatory role in the metabolism of carbohydrates. Therefore, we have a cAMP-CRP complex in El Tor Vibrio cholerae. The regulatory effect of the compound on the mannitol PTS operon was studied. The deletion mutant strain of the gene CRP and cAMP synthesis gene CyA was synthesized by CRP. It was proved that the complex had a very important positive regulation on the MTL operon. We found that there was no significant difference in the transcription of the CRP gene in the fast and slow fermenting strains, but the CyA in the fast fermented strain was CyA. The transcriptional level was higher than that of the slow fermentation plant, and after the expression of CyA was up-regulated, the time of the color change of the mannitol fermentation broth in the fast and slow fermenting strain was early, suggesting that the difference in the cAMP-CRP complex was different to the regulation of the transcription level of the MTL operon. In the fast fermenting strain, the cAMP-CRP complex was more active and the MTL operon transcriptional level was higher. The CRP binding site "TGTGA.TCACA" was found in the promoter region of MTL, which confirmed that CRP-cAMP complex could be combined with the promoter region by gel mobility test.
The above experiments were analyzed by mannitol fermentation. We found that the El Tor Vibrio cholerae in Zhongshan was similar to the mannitol fermentation experiment, but there were no sorbitol specific PTS gene clusters in Vibrio cholerae, and sorbitol was PTS transporter in many bacteria. The deletion mutant of the non specific component PTS I in the PTS system was constructed. The results showed that the strain after PTS I loss lost the ability to ferment the sorbitol, and confirmed that sorbitol was transported through the PTS system in Vibrio cholerae. We found that the deletion of the gene of the mannitol MTL operon also leads to the blocking of sorbitol utilization, and sorbitol can be used. The results suggest that the high transcription of mannitol specific MTL operon is responsible for the PTS transport of Vibrio cholerae Zhongshan, which is mannitol specific PTS.
In this study, the metabolic regulation of cAMP-CRP complex in Vibrio cholerae was clarified, and the regulation mechanism of mannitol and sorbitol was further extended. At the same time, it was clear that Vibrio cholerae Zhongshan sorbitol and mannitol were transported through a PTS, and sorbitol was the inducer of mannitol PTS. The difference between Vibrio and other bacteria. These studies have further understood the differences in the mechanism of the difference in the fermentation rate and utilization of sorbitol and mannitol of Vibrio cholerae El Tor and non producing strains of Vibrio cholerae. In this study, the metabolic pathways of sorbitol and mannitol in Vibrio cholerae and non producing strains of Vibrio cholerae were also shown in this study. The causes and results of the differences include the regulation of metabolism, the demand for growth, and the differences in the survival ability of the two strains, as well as the effects of differences in intracellular cAMP levels on growth and metabolism, and further research.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R378

【引證文獻】

相關期刊論文 前1條

1 黃繼紅;張新武;于紅華;王國禮;黃濤;;草本植物效素在模擬人工胃液中的解酒試驗研究[J];安徽農(nóng)業(yè)科學;2010年29期

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