臺灣博碩士論文加值系統

乳酸桿菌(Lactobacillus)是食品發酵產業中很重要的一種細菌，他對人體及動物具有許多種的正面效益。為了瞭解Lactobacillus rhamnosus代謝乳糖及半乳糖的機制及調控，我以L. rhamnosus TCELL-1作為研究對象，首先將代謝這兩種糖類的基因選殖出並加以定序。所定序的序列中包含了9個開放閱讀框 (ORFs)，它們的順序為lacTEGF接著galKETRM。在Northern blotting的實驗中，發現這段序列可產生4種mRNA (lacTEGF-galKETRM, lacTEGF, galKETRM和galETRM； 後面3種mRNA的產量明顯比第一種多)，其中lac operon的mRNA在菌以乳糖當做碳源時產量最多；而gal operon的mRNA則在菌以半乳糖當做碳源時產量最多。而藉由Northern blotting及primer extension的實驗，找到了四個可能的promoter，分別位於下列ORFs序列的上游區： lacT (lacTp), galK (galKp1 和 galKp2) 和galE (galEp)。實驗進一步測試了GalK, GalE和GalT在不同種糖類中的活性，發現這些酵素可被葡萄糖抑制產生，並可被半乳糖誘導產生。合併基因序列的分析結果，我們推測gal operon具有雙重的調控機制：葡萄糖的抑制是經由CcpA (Catabolite control protein A) 和它的結合序列 (cre site)；半乳糖的誘導是經由GalR和它的結合序列。另外，在L. rhamnosus TCELL-1中也可測到β-galactosidase的活性，由此推測L. rhamnosus TCELL-1在分解乳糖後，所產生的半乳糖可繼續經由Leloir pathway及 tagatose 6-phosphate pathway代謝，而直接給予的半乳糖則可經由Leloir pathway代謝。
Lactobacillus casei 也是一種很重要的益生菌，他常被用來發酵乳製品，而乳製品中最主要的碳源是乳糖，但不是所有的L. casei 都能代謝乳糖。在實驗中，我發現 L. casei ATCC 27139 無法代謝乳糖，但如將他塗佈在只含乳糖當作碳源的培養皿上，將可獲得可代謝乳糖的菌落。如將L. casei ATCC 27139和他的Lac+ 轉變株R1的乳糖代謝相關DNA序列 (lacTEGF-galKETRM) 做比較，發現這段DNA序列上的差別僅有一個核苷酸，其位於lacTEGF promoter (lacTp)的區域。而這段lacTEGF-galKETRM所產生的蛋白和之前所發表的L. casei (Lac+) 的相關蛋白有著96%~100%的相似度。接著又選殖出了41隻Lac+轉變株，經由DNA序列的比對，發現他們的lacTp promoter也皆含有一個點突變。依照突變位置的不同，可將這些轉變株分成6類。而藉由Northern blotting及primer extension的實驗，我們發現Lac+ 轉變株R1的lacTp promoter的確具有活性，但L. casei ATCC 27139的lacTp promoter活性卻偵測不到。由這些結果推測，lacTp promoter上所發生的點突變的確能讓其恢復活性，而轉錄出完整的lacTEGF operon，進而恢復L. casei ATCC 27139代謝lactose的能力。

A gene cluster containing nine ORFs involved in the metabolism of lactose and galactose in L. rhamnosus TCELL-1 was sequenced and characterized. The order of the ORFs was lacTEGF and galKETRM. Northern blotting experiments revealed that the gene cluster could be transcribed as one lacTEGF-galKETRM mRNA though there were three major transcripts (lacTEGF, galKETRM and galETRM) detected for the gene cluster. The transcription of the lac or gal operon was independently induced in the presence of lactose or galactose. Northern blotting and primer extension experiments found the presence of four putative promoters upstream from the ORFs lacT (lacTp), galK (galKp1 and galKp2) and galE (galEp). The measurements of enzymatic activities of GalK, GalE and GalT suggested that expression of the gal operon was subjected to a glucose repression and galactose activation mechanism. We suspect that the gal operon could be regulated by a dual regulation mechanism, namely glucose repression possibly mediated by CcpA (Catabolite control protein A) and galactose induction through GalR and its binding sites. Besides, the β-galactosidase activity could also be detected in L. rhamnosus TCELL-1. These results indicated that the galactose moiety of lactose in L. rhamnosus TCELL-1 could be metabolized by two alternative pathways (the Leloir and the tagatose 6-phosphate pathways) while galactose metabolism could be mediated by the Leloir pathway. This work provides important information of sugar metabolism in L. rhamnosus.
Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, under the lactose selection, the spontaneous revertant colonies (Lac+) were obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac-) and its Lac+ revertant (designated as strain R1) was sequenced and characterized. We found that only one nucleotide located in the lacTEGF promoter (lacTp) of these two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac+) strains was high namely, 96 to 100% identity was found and no premature stop codon was identified on it. A single point mutation occurred on the same lacTp promoter region was also detected for 41 other independently isolated Lac+ revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Northern blotting and primer extension experiments for the activity of the lacTp promoter further found that the lacTp promoter of strain R1 was functional while that of L. casei ATCC 27139 was not. These results suggest that a single point mutation on the lacTp promoter was able to restore the transcription of fully functional lacTEGF operon and caused a phenotype switch from Lac- to Lac+ for L. casei ATCC 27139 (Lac-).

中文摘要--------------------------------------------------i
English abstract----------------------------------------iii
Table of contents-----------------------------------------v

Chapter 1: Sequence, organization, transcription and regulation of lactose and galactose operons in Lactobacillus rhamnosus TCELL-1
1-1 Introduction------------------------------------------1
1-2 Materials and methods---------------------------------4
1-2-1 Bacterial strains and growth conditions-------------4
1-2-2 DNA isolation and library construction--------------6
1-2-3 Library screening and DNA amplification-------------6
1-2-4 Nucleotide sequencing and sequence analysis---------7
1-2-5 DNA amplification procedure-------------------------8
1-2-6 Determination of the lac-gal gene cluster in other lactobacilli----------------------------------------------9
1-2-7 RNA isolation and Northern blot analysis-----------10
1-2-8 Primer extension experiment------------------------11
1-2-9 Preparation of cell extracts, protein analysis and enzyme assay---------------------------------------------12
1-2-10 Determination of β-galactosidase activity---------14
1-2-11 Nucleotide sequence accession number--------------14
1-3 Results----------------------------------------------14
1-3-1 Existence of the lac-gal gene cluster--------------14
1-3-2 Sequence analysis of the lac-gal gene cluster------15
1-3-3 Transcription and primer extension analyses of the lac-gal gene cluster-------------------------------------16
1-3-4 Growth pattern and GalK, GalE and GalT activities measured in different carbohydrates----------------------18
1-3-5 Expression of β-galactosidase----------------------19
1-4 Discussion-------------------------------------------20
1-5 Tables-----------------------------------------------26
1-6 Figures----------------------------------------------29
1-7 Appendixes-------------------------------------------34

Chapter 2: A point mutation that causing phenotype switch of lactose metabolism in Lactobacillus casei
2-1 Introduction-----------------------------------------41
2-2 Materials and methods--------------------------------43
2-2-1 Bacterial strains and growth conditions------------44
2-2-2 Accumulation of Lac+ colonies and viability assay--44
2-2-3 Selection and identification of spontaneous mutations----------------------------------------------------------45
2-2-4 Stability of the revertants------------------------46
2-2-5 Carbohydrate fermentation--------------------------46
2-2-6 Sequencing of the lac-gal gene cluster from L. casei ATCC 27139-----------------------------------------------47
2-2-7 Nucleotide sequencing and sequence analysis--------48
2-2-8 DNA amplification procedure------------------------49
2-2-9 RNA isolation and Northern blot analysis-----------49
2-2-10 Primer extension experiment-----------------------50
2-2-11 Nucleotide sequence accession numbers-------------51
2-3 Results----------------------------------------------51
2-3-1 Accumulation of Lac+ revertants--------------------51
2-3-2 Stability of the revertant and carbohydrate fermentation---------------------------------------------52
2-3-3 Sequence analysis of the lac-gal gene cluster------53
2-3-4 Spectra of spontaneous mutations-------------------54
2-3-5 Transcription and primer extension analyses of the lacTEGF operon-------------------------------------------55
2-4 Discussion-------------------------------------------56
2-5 Tables-----------------------------------------------62
2-6 Figures----------------------------------------------64
2-7 Appendixes-------------------------------------------68
References-----------------------------------------------74
附錄-----------------------------------------------------84

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