氰化鉀 (KCN) 會降低Klebsiella oxytoca電子傳遞鏈上的終端氧化酶活性。先前研究證實Klebsiella oxytoca培養在KCN環境下會產生cyanide-resistant respiration pathway，而本實驗使用8-hydroxyquinoline阻斷此途徑以探討其在調節此菌在KCN環境下的呼吸作用所扮演的角色，並設計四種不同培養情形：(1) TSB (不含KCN或8-HQ)；(2) TSB + 1 mM KCN；(3) TSB + 100 μM 8-HQ；(4) TSB + 1 mM KCN + 100 μM 8-HQ。結果顯示此菌培養於100 μM 8-HQ時，其對生長產生微毒性而稍微減緩；但不及將此菌培養於同時含有KCN和8-HQ環境時，對細菌的生長抑制更加明顯且其氧呼吸率較控制組 (不含8-HQ) 下降。且在生長於100μM 8-HQ環境中的K. oxytoca的細胞膜中在加入KCN後，其H2O2產量會明顯增加，因而推測此抗氰呼吸途徑可避免細胞受到H2O2的氧化性傷害。此外為探討K. oxytoca的胞色素d氧化酶與分解KCN作用的相關性；故剔除K. oxytoca的胞色素d氧化酶的基因序列以構築成cyd-突變株。結果顯示K. oxytoca之胞色素d氧化酶並不屬於抗氰氧化酶，但其對於此菌分解KCN，扮演重要角色。

Potassium cyanide (KCN) is an inhibitor that reduces the activity of terminal oxidases in electron transport system of Klebsiella oxytoca. Previous research verified that K. oxytoca could induce cyanide-resistant respiration pathway when cells were grown in KCN condition. To address the role of cyanide-resistant pathway in regulating the respiration of bacterium in KCN incubation, 8-hydroxyquinoline (8-HQ), an inhibitor of cyanide-resistant pathway, was added to the bacterial suspension pretreated with KCN. This experiment was devised into 4 groups as below: (1). TSB (without KCN or 8-HQ), (2). TSB + 1 mM KCN, (3). TSB + 100 μM 8-HQ, and (4). TSB + 1 mM KCN+ 100 μM 8-HQ. Our results show 100 μM 8-HQ exerted it slight toxicity to bacterial growth. However, the bacterial growth was severely impaired when the cells treated with KCN and 8-HQ concurrently as evidenced by the lower oxygen uptake rate of cells in comparison with the control group (without addition of 8-HQ). Furthermore, K. oxytoca grown in growth medium containing 100 μM 8-HQ produced more significant H2O2. Thus we suggested that cyanide-resistant respiration of K. oxytoca could protect the cells from H2O2 damage. Since cytochrome d has been implicated in having an important role in KCN degradation in the K. oxytoca, we constructed cyd- mutant to explore the possible role in KCN degradation. In this study the sequence of the genes encoding this terminal oxidase (cydAB) of K. oxytoca mutant were deduced. Results showed that cytochrome d oxidase of K. oxytoca is not a cyanide-insensitive oxidase, but playing an important role in KCN degradation.

目錄

謝誌…………………………………………………………….……I
中文摘要……………………………………………………………II
英文摘要…………………………………………………………..III
目錄……………………………..…………………………………IV
圖目錄…………………………………………………………..V-VI
表目錄…………………………………………………………….VII
前言……………………………………………………………...1-18
研究目的…………………………………………………………..19
材料與方法…………………………………………………….20-29
結果與討論…………………………………………………….30-37
結論………………………………………………………………..38
參考文獻……………………………………………………….39-54
圖表…………………………………………………………….55-72









圖目錄

Fig. 1. The growth curves of K. oxytoca in TSB media with various concentrations of 8-hydroxyquinoline (8-HQ)………………...55

Fig. 2. The growth curves of K. oxytoca in TSB media with 1mM KCN and various concentrations of 8-HQ………………………..….56

Fig. 3. Effect of KCN on the oxygen uptake of the whole cell of K. oxytoca grown in present or absence of 100 μM 8-HQ..………57

Fig. 4. Effect of KCN on the oxygen uptake of the whole cell of K. oxytoca grown in the presence various inhibitors…...………....57

Fig. 5. Effect of HQNO (2-Heptyl-4-hydroxyquinoline N-oxide) on the NADH oxidase activity of K. oxytoca cells grown in TSB medium containing various inhibitors……..………..…………58

Fig. 6. H2O2 production during incubation of K. oxytoca vesicles with NADH. K. oxytoca cells grown in TSB medium containing inhibitors...……………..………………………………………59

Fig. 7. The catalase activity of the wild-type K. oxytoca grown in TSB medium containing the inhibitors...……………………………60

Fig. 8. Alignment of the amino acid sequence of CydA (a) and (b) from various bacteria……………………………………..……...63-66

Fig. 9. Schematic diagram of the construction of the cydA deletion mutant of SYSU-011……………………………………...……67

Fig.10. Restriction mapping for correctness of pCDm construction…...68

Fig. 11 PCR analysis of cdm mutants…………………………...……...69

Fig. 12. Difference spectra of membranes from wild-type, cdm mutant strains of K. oxytoca……………………………………………70

Fig. 13 The growth curve of K. oxytoca cdm mutant in TSB medium with inhibitor………………………...………………………………71

Fig.14. Growth curves of K. oxytoca and cyanide degradation in the NFG medium containing 1 mM KCN as nitrogen source..………….72
































表目錄

Table 1 Bacterial stains and plasmids used in this study……………….61

Table 2 Primers used in this study……………………………………...62

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