細菌的磷酸烯醇丙酮酸之糖類磷酸轉移系統 (PEP-PTS) 需要細胞膜及細胞質之成份參與糖類攝取以及糖類磷酸化作用。經過基因比對，在耐輻射奇異球菌 (Deinococcus radiodurans)， 是由 fruA 和 fruB 負責此系統之轉譯。耐輻射奇異球菌 擁有一個具有功能性的 fruA，負責轉譯果糖磷酸化中與細胞膜結合之成份。 而 fruB 是負責轉譯與果糖磷酸化相關的細胞質成份，但它卻是一個假基因。這株菌代謝果糖非常快速。在體外實驗發現需要同時存在細胞膜及細胞質的成份才可進行果糖磷酸化作用。之後也發現，果糖磷酸化的磷酸根供應者是腺苷三磷酸 (ATP) 而不是磷酸烯醇丙酮酸 (PEP)。不像大部份的與磷酸烯醇丙酮酸有關的糖類磷酸化作用，此果糖磷酸化作用是對氟化鈉 (激酶抑制劑) 敏感，並會被激酶磷酸化位置之抗體抑制其作用。
在生長靜止期加入二價錳離子會使得耐輻射奇異球菌有另一次的生長階段。此種因為錳而使得細胞再次生長的情形稱為錳促細胞生長 ( MnCD ) 。當細胞培養在沒有大量錳離子的培養基中，我們發現會有一個因子累積在培養基中，此因子耐蛋白質酵素 K 作用，並且分子量小於 10,000 道爾頓。即使將已經用過的培養基濃縮，加入新鮮的培養基裡面仍能抑制 D. radiodurans 的細胞生長; 並且抑制效果是隨著抑制因子增多而增加。然而錳促細胞增生也是隨著細胞生長抑制效用越強，錳促細胞生長效應越強。

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructosespecific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site.
During stationary phase, the highly radiation-resistant bacterium D. radiodurans undergoes new rounds of cell division when Mn2+ is added to the medium in a phenomenon known as manganese-induced cell division (MnCD). When cells were cultured in medium without Mn2+-enrichment, a proteinase K-resistant factor(s) with a molecular weight less than 10 kDa accumulated in the spent medium. Inclusion the concentrated spent medium to fresh medium could inhibit the growth of D. radiodurans significantly; and the degree of inhibition was dose dependent. However, the relative stimulatory effect of MnCD was also dose dependent—the higher the inhibition, the stronger the MnCD response.

論文審定書………………………………………………………………... ii
致謝………………………………………………………………………… iii
摘要………………………………………………………………................ iv
Abstrate…………………………………………………………………….. v
Index………………………………………………………………………… vi
Figures index……………………………………………………………….. ix
Tables index………………………………………………………………… x
Chapter 1 Introduction………………………………………………….. 1
Chapter 2 Articles review……………………………………………….. 4
2.1 Radiation resistance of various microorganisms………………… 5
2.1.1 DNA Repair mechanisms………………………………….. 7
2.1.2 The role of macromolecules in radiation resistance……... 11
2.2 Carbohydrate metabolism in D. radiodurans……………………. 13
2.3 The importance of Mn in D. radiodurans………………………… 19
2.4 MnCD effect………………………………………………………. 22
Chapter 3 ATP-dependent fructose uptake system in
D. radiodurans.………..…………………………………….. 24
3.1 Abstract…………………………………………………………….. 25
3.2 Introduction………………………………………………………... 26
3.3 Material and Methods……………………………………………... 28
3.3.1 Strains and growth conditions…………………………….. 28
3.3.2 Bacterial growth curves and in vivo analysis of fructose consumption………………………………………………… 28
3.3.3 Preparation of cell extracts, membranes and cytosols…... 29
3.3.4 PEP-dependent and ATP-dependent PTS Assay………… 29
3.3.5 Mutagenesis and Isolation of fruA defective mutants…… 30
3.3.6 EIIA-antiserum and PKC-antiserum…………………….. 32
3.3.7 ATP-Group Translocation assay…………………………. 32
3.4 Results……………………………………………………………… 34
3.4.1 Disruption of the fruA gene……………………………….. 34
3.4.2 Analysis of the deinococcal putative fruB (DRB0075)
gene…………………………………………………………. 34
3.4.3 Enzyme characterization………………………………….. 35
3.4.4 Complementation studies………………………………….. 37
3.4.5 Blocking phosphoryl transfer by antiserums…………….. 37
3.5 Conclusion………………………………………………………….. 39
3.6 Acknowledgment…………………………………………………… 41
3.7 Tables……………………………………………………………….. 42
3.8 Figures………………………………………………………………. 44
Chapter 4 The effect of Mn2+ on the autoinducing growth
inhibition factor in D. radiodurans………………………… 52
4.1 Abstract…………………………………………………………….. 53
4.2 Introduction………………………………………………………... 54
4.3 Material and methods……………………………………………... 55
4.3.1 Strain of cells, media, chemicals, and growth conditions.. 55
4.3.2 Spent medium preparation………………………………... 55
4.3.3 Growth curve of D. radiodurans…………………………. 55
4.3.4 Testing the effect of different concentrations of spent
medium on cell numbers…………………………………... 56
4.3.5 Proteinase K treatment……………………………………. 56
4.3.6 Estimation of the molecular size of the
growth inhibition factor………………………………… ... 57
4.4 Results………………………………………………………………. 58
4.4.1 Cell growth with and without Mn-enrichment…………... 58
4.4.2 Effect of spent medium concentration on cell numbers…. 58
4.4.3 Effect of proteinase K on MnCD effect…………………… 59
4.4.4 Estimation of the molecular size of the
growth inhibition factor……………………………………. 60
4.5 Conclusion…………………………………………………………… 61
4.6 Acknowledgment……………………………………………………. 62
4.7 Figures……………………………………………………………….. 63
Chapter 5 Conclusion…………………………………………………….. 68
5.1 Carbohydrate system……………………………………………….. 69
5.2 Manganese utility…………………………………………………… 69
Chapter 6 Future prospects……………………………………………… 71
Reference……………………………………………………………………. 74
Appendix…………………………………………………………………….. 88
Appendix I FruB gene sequences…………………………..……. 89
Appendix II Modified PTS system of D. radiodurans……………. 91
Appendix III Cell numbers vs MnCD effect……………………….. 92

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