臺灣博碩士論文加值系統

在幽門桿菌（Helicobacter pylori）中已被證實有luxS同源基因的存在，luxS 基因普遍存在於許多格蘭氏陽性和陰性菌中，在不同的生長時期藉由quorum sensing system調控毒性基因的表現、生物膜（biofilm）的生成、抑菌素（bacteriocin）的產生等等。在對於幽門桿菌luxS基因的研究中，我們已知道它會降低flaA基因的表現和生物膜的生成。幽門桿菌在培養過程中或在環境壓力下會形成球狀體（coccoid form）且有研究指出它在生長過程中會進行自裂解（autolysis）作用。本研究藉由建構幽門桿菌luxS基因的突變株來進行luxS基因功能分析與觀察它在幽門桿菌中所扮演的角色。結果顯示luxS基因的突變株生長速率較野生株慢，而LuxS蛋白質的表現量與生長時期有關；在對活菌與死菌的螢光染色觀察中發現突變株在60小時的培養後（死亡時期（death phase））才出現死亡細胞，但在停滯期（lag phase）就會出現球狀體。自裂解實驗中則顯示突變株會導致細胞自裂解的速率較野生株快。另外在對於環境壓力的適應如環境中存在濃度過高的過渡元素離子可能對細胞造成毒性的情形下，突變株明顯表現出較佳的耐受性。由本研究結果顯示luxS基因與細胞生長、細胞自裂解作用的調控有關；且可能也參與一些過渡元素離子的運輸。在本研究中我們期望以flaA promoter來表現luxS基因以建構互補株，但卻沒有成功表現LuxS蛋白質。我們認為是由於luxS基因突變降低了flaA基因的表現以致於flaA promoter無法成功表現LuxS蛋白質。

Helicobacter pylori possesses a homologue of luxS gene, luxS gene exists in numerous gram-positive and gram-negative bacteria. It regulates virulence genes expression, biofilm formation, and bacteriocin production through quorum sensing system with a growth phase-dependent manner. Previous studies have revealed that the flaA expression and biofilm formation were regulated by luxS gene. During the cultivation, H. pylori changed its morphology from rod to coccoid form. Studies have shown that H. pylori cells underwent spontaneous autolysis during cultivation. In our study, we constructed a luxS mutant to characterize its function and elucidate what important role it may play in H. pylori. In this report, the luxS mutant was grown slower than wild type. The LuxS expression was growth phase dependent. Live and dead staining revealed that in the luxS mutant the dead cells appeared after 60h cultivation (death phase), but the coccoid forms appeared at lag phase. The autolysis assay demonstrated that luxS mutation would induce autolysis. The disc diffusion assay have shown that when incubated with high concentrations of transition elements which was toxic to cells, the luxS mutant presented significantly better tolerance than wild type. We suggested that luxS was associated with growth and autolysis regulation and may participate in the transportation of transition elements. We constructed a luxS complementary strain in which the luxS gene was under the control of flaA promoter, but the LuxS protein was not expressed. We considered it resulted from the luxS mutation would repress the the flaA expression.

ABSTRACT......................................................................3摘要.........................................................................4 CONTENT.......................................................................5INTRODUCTION..................................................................91. Overview of Helicobacter pylori...........................................9
2. luxS and quorum sensing..................................................10 3. luxS regulates biofilm formation in staphylococci........................11
4. Quorum sensing in streptococci...........................................12
5. Quorum sensing regulates bioluminescence in vibrios......................14
6. Quorum sensing in E. coli and Salmonella.................................15
7. Quorum sensing in Helicobacter pylori....................................16 MATERIALS...................................................................17
1. Bacterial strains........................................................17
2. Bacterial culture medium.................................................17
3. Primers..................................................................17
4. Cloning vectors..........................................................17
5. Kits.....................................................................17 METHODS.....................................................................18
1. Bacterial culture conditions.............................................18
2. Helicobacter pylori genomic DNA extraction...............................18
3. DNA manipulation.........................................................19
4. Protein extraction.......................................................19
5. Overexpression and purification of LuxS protein..........................20
6. Natural transformation...................................................20
7. SDS-PAGE and Western blotting............................................21
8. Antibody production......................................................21
9. Antibody purification....................................................22
10. Growth curve............................................................23 11. Live and dead staining..................................................23
12. Urease assay............................................................23
13. Disc diffusion assay....................................................24 RESULTS.....................................................................25
1. Construction of H. pylori 11637 luxS mutant..............................25
2. Complementation of luxS mutant, HPYL.....................................26
3. Sequence alignment of luxS gene..........................................26
4. Gene organization of H. pylori 11637 luxS gene flanking region...........27 5. Growth of luxS mutant....................................................28 6. Expression of LuxS was growth-dependent..................................28 7. Monitor the viable rate of H. pylori during cultivation..................28 8. Mutation of luxS would induce autolysis of H. pylori.....................29 9. luxS gene may regulate the uptake or transportation of microelements.....30
DISCUSSION..................................................................32 REFERENCE...................................................................35
Table 1. Bacterial strains used in this study...............................42 Table 2. The oligonucleotides used in this study............................43 Table 3. Vectors used in this study.........................................44 Table 4. Names of species used in the LuxS protein sequence alignment analysis....................................................................45 Table 5. Deduced ORFs and the relative genes in sequenced H. pylori strains.....................................................................46 Table 6. Effects of different compounds on the inhibition of H. pylori11637 and HPYL growth is..........................................................47 Fig 1. Strategy of luxS mutant Construction.................................48 Fig 2. Southern blot analysis of 11637, HPYL, and HPYLC strains.............49 Fig 3. Strategy of luxS gene complementary strain HPYLC construction........50 Fig 4. Sequences alignment of LuxS homologues of different species..........53 Fig 5. Phylogenetic analysis of LuxS portein ...............................54 Fig 6.Cloning strategy......................................................58 Fig 7. Growth curves of NCTC 11637 and HPYL.................................59 Fig 8. Western blot to monitor the expression of LuxS in 11637..............60 Fig 9. Time course of live/dead fluorescence microscope.....................64 Fig 10. Analysis of autolysis of 11637 and HPYL during cultivation..........65 Fig 11. Disc diffusion assay................................................66

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