臺灣博碩士論文加值系統

Superoxide dismutases (SODs) 是一群金屬酶,主要用以緩解氧化壓力,其功能為
將superoxide轉變成毒性較低的 H2O2 及 O2。依據金屬輔因子的不同分類
MnSODs 、 FeSODs 及 Cu-ZnSODs,三種 SODs 對於緩解氧化壓力扮演不同
角色。sodA 基因在大部份細菌中屬於 single copy,除了 Bacillus cereus 具有兩
個 sodA 基因。Stenotrophomonas maltophilia 為一株非發酵性革蘭氏陰性菌,常
見於自然環境中。在 S. maltophilia K279a 基因體中,具有兩個可能的 sodA 基
因,分別為 sodA1 (Smlt2828) 及 sodA2 (Smlt3238)。對於革蘭氏陰性細菌而言,
具有兩個 sodA 基因是相當特別的。本篇論文主要探討 sodA1 及 sodA2 的基因
表現以及所執行的功能角色。首先利用 promoter transcriptional fusion assay 與
qRT-PCR 的方法來分析 sodA1 及 sodA2 基因的表現與調控。從結果發現(i) 在
實驗室培養生長條件下, sodA1 基因幾乎不表現,而 sodA2 基因之表現隨菌齡
有上升的現象,並在 late logarithmic phase 時呈現最大表現量。(ii) Menadione
(MD) 及 plumbagin (PL) (superoxide generators) 的壓力之下, sodA1 會大量表
現且具有 dose-dependent的現象。soxR 及 oxyR 會參與 MD 及 PL 誘發 sodA1
基因表現。(iii) H2O2 的壓力之下,會誘發 sodA1 基因表現上升且經由 soxR 參
與, oxyR 則負調控 H2O2 誘發 sodA1 基因。(iv) 抗生素 kanamycin 、
ciprofloxacin 、 ceftazidime 與 ticarcillin 不會影響 sodA1 基因表現。Kanamycin
抑制 sodA2 基因表現,ciprofloxacin 則誘發 sodA2 基因表現; ceftazidime 與
ticarcillin 對於 sodA2 基因表現則無顯著影響。利用 mutant construction 與
function assay 分析 SODA1 及 SODA2 之功能,包括細菌生長、氧化壓力耐受
性及抗生素耐受性。結果顯示: (i) 在實驗室培養生長條件下,刪除 sodA1 及sodA2 不會影響 S. maltophilia 的生長。KJ、KJsodA1 及 KJsodA2 具有相似 的生長曲線。(ii) sodA1 的缺失使得菌株對於 MD 所造成的氧化壓力耐受性變差, sodA2 的缺失則無影響。此外, sodA1 與 sodA2 的缺失皆不會影響對於 H2O2 的耐受性。(iii) sodA1 與 sodA2 的缺失不會影響對於抗生素 ceftazidime、 ticarcillin-clavulanic acid、ciprofloxacin 與 levofloxacin之耐受性。

Superoxide dismutases (SODs), a group of metalloenzymes, are considered as key enzymes defending against oxidative stress and they convert superoxide (O2-) to H2O2 and O2. According to their metal cofactors, SODs are classified into manganese SODs (MnSODs), iron SODs (FeSODs) and copper zinc SODs (Cu-ZnSODs), and each one is developed certain unique function. sodA is a single-copy gene in majority of bacteria except Bacillus cereus. Stenotrophomonas maltophilia, a non-fermentative gram-negative bacterium, is ubiquitous in environment. Based on the sequenced genome, S. maltophilia K279a harbors two putative sodA genes, Smlt2828 (sodA1) and Smlt3238 (sodA2). It is unusual for a gram-negative bacterium with two sodA genes. This study aimed to investigate the expressions and functions of sodA1 and sodA2. The expression and the underlying regulation of sodA1 and sodA2 was assessed by promoter transcriptional fusion assay and qRT-PCR. We found that (i) sodA1 was intrinsically little expressed. The expression of sodA2 increased in a growth depend manner, highest in the late logarithmic phase. (ii) Upon the challenge of menadione (MD) or plumbagin (PL) (superoxide generators), the sodA1 was upexpressed in a concentration-dependent manner. In contrast, the expression of sodA2 was little affected. Furthermore MD- or PL- mediated sodA1 upexpression were positively regulated by OxyR and SoxR. (iii) Upon the challenge of H2O2, the sodA1 was upexpressed. In addition, H2O2-mediated sodA1 upexpression was positively regulated by SoxR but negatively regulated by OxyR. (iv) Kanamycin, ciprofloxacin, ceftazidime, ticarcillin did not trigger sodA1 expression. sodA2expression was attenuated by kanamycin, enhanced by ciprofloxacin, but little affected by ceftazidime and ticarcillin. The function of sodA1 and sodA2 was assessed by mutant construction and function assay, including bacterial growth, oxidative stress tolerance, and antibiotics susceptibility. The results showed that (i) Inactivation of sodA1 or sodA2 did not alter bacterial growth. KJ, KJsodA1 and KJsodA2 had the similar growth curve. (ii) sodA1, but not sodA2, was inovled in menadione tolerance. However, neither sodA1 nor sodA2 was linked to H2O2 susceptibility. (iii) Inactivation of sodA1 or sodA2 did not alter the susceptibility toward ceftazidime, ticarcillin-clavulanic acid, ciprofloxacin and levofloxacin.


中文摘要……………………………………….i
Abstract…………………………………...……iii
目錄…………………………………………...…v
圖目錄………………………………………..…viii
表目錄…………………………………………x
第一章 緒論…………………………….1
第一節 研究背景………………………..1
1.1.1 Stenotrophomonas maltophilia....1
1.1.2氧化壓力(oxidative stress)……….2
1.1.3氧化壓力反應(oxidative stress response)………..2
1.1.4 Superoxide dismutases (SODs)之介紹…………....3
1.1.5 SODA之介紹……………………………………………………4
1.1.6 Stenotrophomonas maltophilia SODA之介紹…..4
第二節 研究目的…………………………….5
第二章 材料與方法…………………………6
第一節 研究材料…………………………....6
2.1.1菌株與質體……………………………..6
2.1.2培養基……………………………………..6
2.1.3 PCR引子………………………………….6
2.1.4實驗藥品………………………………….6
2.1.5抗生素………………………………………7
2.1.6試劑與緩衝溶液……………………….7
2.1.7儀器設備…………………………………..8
第二節 實驗方法………………………………8
2.2.1菌種的培養與保存…………………...8
2.2.2聚合酶鏈鎖反應 (Polymerase chain reaction；PCR)…9
2.2.3 DNA之製備……………………………….9
2.2.4勝任細胞 (Competent cell)之製備………………11
2.2.5轉型作用(Transformation)……………………….....11
2.2.6接合作用 (Conjugation)……………………………….12
2.2.7菌落快速檢驗聚合酶鏈鎖反應 (Colony PCR)……12
2.2.8 C23O (catechol 2, 3-dioxygenase)活性測試….....13
2.2.9 10小時生長曲線(growth curve)測試…………………13
2.2.10 H2O2感受性試驗…………………………13
2.2.11 menadione耐受性試驗…………………14
2.2.12抗生素與藥物感受性試驗…………...14
第三章 研究結果……………………………………15
(A) Stenotrophomonas maltophilia SODA1及SODA2
的生物資訊分析與基因表現調控................................…15
第一節 由生物資訊分析sodA1及sodA2基因群………….15
3.1.1 sodA1及sodA2基因群之分析……………………………..15
第二節 sodA1基因群及sodA2基因轉錄分析………………15
3.2.1利用promoter預測軟體找出sodA1基因群及sodA2基因中
promoter可能存在的位置……15
第三節 sodA1及sodA2基因表現及調控分析……………………………16
3.3.1 探討sodA1及sodA2基因在實驗室培養生長條件下
之表現及調控…16
3.3.2 探討sodA1及sodA2基因在有壓力條件下之表現與調控....17
(B) Stenotrophomonas maltophilia SODA1及SODA2的功能分析….20
第一節 SODA1及SODA2與細菌生長之關聯性…………………….21
第二節 SODA1及SODA2與氧化壓力耐受性之關聯性…………..……..21
第三節 SODA1及SODA2與抗生素耐受性之關聯性………………..……23
第四章 討論……………………………24
第五章 結論……………………………28
參考文獻…………………………………53
附錄…………………………………………59

圖目錄
Fig. 1 S. maltophilia K279a sodA1與sodA2鄰近基因組
裝示意圖……29
Fig. 2 S. maltophilia K279a sodA1和sodA2鄰近基因組裝
與E. coli K-12及P. aeruginosa PAO1 sodA鄰近基因組裝示意圖……..30
Fig. 3 sodA1及其鄰近基因上游序列
promoter預測分析示意圖…………….31
Fig. 4 sodA2上游序列promoter預測分析示意圖………………….…….32
Fig. 5 sodA1、sodA2及 sodB基因在實驗室培養生長條件下
之表現情形.…33
Fig. 6 SoxR與OxyR對sodA1及sodA2基因表現之調控情形………………34
Fig. 7 Superoxide generators (menadione及plumbagin)
對sodA1及sodA2基因表現之影響……………………………35
Fig. 8不同濃度superoxide generators (menadione及plumbagin
)對sodA1基因表現之影響……………………36
Fig. 9 SoxR與OxyR在superoxide generators(menadione及plumbagin)誘發sodA1基因表現所扮演的角色………………37
Fig. 10 Quantitative RT-PCR (qRT-PCR)分析
sodA1及sodA2基因的表現量.............38
Fig. 11 Quantitative RT-PCR (qRT-PCR)分析SoxR與OxyR
在H2O2誘發sodA1基因表現所扮演的角色………………………………………39
Fig. 12 Kanamycin及ciprofloxacin對sodA1及sodA2基因表現之影響……40
Fig. 13不同濃度kanamycin及ciprofloxacin對sodA2基因表現之影響……41
Fig. 14 Ceftazidime及ticarcillin對sodA1及sodA2基因表現之影響………42
Fig. 15 SoxR與OxyR在kanamycin抑制sodA2基因表現及ciprofloxacin
誘發sodA2基因表現所扮演的角色………………………………43
Fig. 16 KJ、KJΔsodA1和KJΔsodA2
之生長曲線(growth curve)………………44
Fig. 17 KJ、KJΔsodA1和KJΔsodA2之H2O2耐受性試驗……………………45
Fig. 18 KJ、KJΔsodA1和KJΔsodA2之menadione氧化壓力耐受性試驗…….46
Fig. 19 KJ(pRK415)、KJΔsodA1(pRK415)、KJΔsodA1(pRKsodA1)
與KJ(pRKsodA1) 之menadione氧化壓力耐受性試驗………………47
Fig. 20 Quantitative RT-PCR (qRT-PCR) 分析sodA1基因的表現量…..…48

表目錄
Table 1. 本次研究所使用之菌株、質體與引子………………………………49
Table 2. 本研究中所使用的抗生素及藥品…………………………………51
Table 3. KJ、KJΔsodA1和KJΔsodA2之抗生素耐受性試驗……………52

1. Berg G, Roskot N, Smalla K. 1999. Genotypic and phenotypic relationships between clinical and environmental isolates of Stenotrophomonas maltophilia. J Clin Microbiol 37:3594-600.
2. de Oliveira-Garcia D, Dall'Agnol M, Rosales M, Azzuz AC, Martinez MB, Girón JA. 2002. Characterization of flagella produced by clinical strains of Stenotrophomonas maltophilia. Emerg Infect Dis 8:918-23.
3. Zhang L, Li XZ, Poole K. 2000. Multiple antibiotic resistance in Stenotrophomonas maltophilia: Involvement of a multidrug efflux system. Antimicrob Agents Chemother 44:287–293.
4. Denton M, Todd NJ, Kerr KG, Hawkey PM, Littlewood JM. 1998. Molecular epidemiology of Stenotrophomonas maltophilia isolated from clinical specimens from patients with cystic fibrosis and associated environmental samples. J Clin Microbiol 36:1953-8.
5. Brooke JS. 2012. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 25:2-41.
6. Sánchez MB. 2015. Antibiotic resistance in the opportunistic pathogen Stenotrophomonas maltophilia. Front Microbiol 6:658.
7. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. 2012. Oxidative stress and antioxidant defense. World Allergy Organ J 5:9-19.
8. Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Curr Biol 24:R453-62.







53
9. Cabiscol E, Tamarit J, Ros J. 2000. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3:3-8.
10. Lushchak VI. 2011. Adaptive response to oxidative stress: Bacteria, fungi, plants and animals. Comp Biochem Physiol C Toxicol Pharmacol 153:175-90.
11. Gu M, Imlay JA. 2011. The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide. Mol Microbiol 79:1136-50.
12. Passalacqua KD, Bergman NH, Herring-Palmer A, Hanna P. 2006. The superoxide dismutases of Bacillus anthracis do not cooperatively protect against endogenous superoxide stress. J Bacteriol 188:3837-48.
13. Hewitt J, Morris JG. 1975. Superoxide dismutase in some obligately anaerobic bacteria. FEBS Lett 50:315-8.
14. Moselio S. 2001. Escherichia coli and Salmonella 2000: the View From Here. Microbiol Mol Biol Rev 65:119–130.
15. Parker MW,Blake CC. 1998. Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett 229:377-82.
16. Gregory EM, Dapper CH. 1980. Chemical and physical differentiation of superoxide dismutases in anaerobes. J Bacteriol 144:967–974.
17. Meile L, Fischer K, Leisinger T. 1995. Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg. FEMS Microbiol Lett 128:247-53.
54
18. Fridovich I. 1995. Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97-112.
19. Fee JA. 1991. Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function. Mol Microbiol 5:2599-610.
20. Benov LT, Fridovich I. 1994. Escherichia coli expresses a copper- and zinc-containing superoxide dismutase. J Biol Chem 269:25310-4.
21. Fang FC, DeGroote MA, Foster JW, Bäumler AJ, Ochsner U, Testerman T, Bearson S, Giárd JC, Xu Y, Campbell G, Laessig T. 1999. Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases. Proc Natl Acad Sci U S A 96:7502–7507.
22. Hassett DJ, Schweizer HP, Ohman DE.1995. Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism. J Bacteriol 177:6330–6337.
23. Fink RC, Scandalios JG. 2002. Molecular evolution and structure-function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases. Arch Biochem Biophys 399:19-36.
24. Wang Y, Wang H, Yang CH, Wang Q, Mei R. 2007. Two distinct manganese-containing superoxide dismutase genes in Bacillus cereus: their physiological characterizations and roles in surviving in wheat rhizosphere. FEMS Microbiol Lett 272:206-13.
55
25. Gao T, Li Y, Ding M, Chai Y, Wang Q. 2017. The phosphotransferase system gene ptsI in Bacillus cereus regulates expression of sodA2 and contributes to colonization of wheat roots. Res Microbiol S0923-2508:30085-2.
26. Vattanaviboon P, Panmanee W, Mongkolsuk S. 2003. Induction of peroxide and superoxide protective enzymes and physiological cross-protection against peroxide killing by a superoxide generator in Vibrio harveyi. FEMS Microbiol Lett 221:89-95.
27. Manchado M, Michán C, Pueyo C. 2000. Hydrogen peroxide activates the SoxRS regulon in vivo. J Bacteriol 182:6842-4.
28. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. 2007. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130:797-810.
29. Saino Y, Kobayashi F, Inoue M, Mitsuhashi S. 1982. Purification and properties of inducible penicillin beta-lactamase isolated from Pseudomonas maltophilia. Antimicrob Agents Chemother 22:564-570.
30. Saino Y, Inoue M, Mitsuhashi S. 1984. Purification and properties of an inducible cephalosporinase from Pseudomonas maltophilia GN12873. Antimicrob Agents Chemother 25:362-365.
31. Carlioz A, Touati D. 1986. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J 5: 623–630.
32. Hu RM, Huang KJ, Wu LT, Hsiao YJ, Yang TC. 2008. Induction of L1 and L2 beta-lactamases of Stenotrophomonas maltophilia. Antimicrob Agents
56
Chemother 52:1198-1200.
33. Simon R, O'Connell M, Labes M, Puhler A. 1986. Plasmid vectors for the
genetic analysis and manipulation of rhizobia and other gram-negative bacteria.
Methods Enzymol 118:640-659.
34. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. 1998. A
broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77-86.
35. Keen NT, Tamaki S, Kobayashi D, Trollinger D. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191-7.
36. Hu RM. 2008. Induction of L1 and L2 β-Lactamases of Stenotrophomonas maltophilia. Antimicrob Agents Chemother 52:1198–1200.
37. Compan I, Touati D. 1993. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J Bacteriol 175:1687-96.
38. Bernard R. 2012. Plant growth-promoting bacteria: mechanisms and applications. Scientifica (Cairo) 2012:963401.
39. Wenk J, Brenneisen P, Wlaschek M, Poswig A, Briviba K, Oberley TD, Scharffetter-Kochanek K. 1999. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated induction of matrix-degrading metalloprotease-1. J Biol Chem 274:25869-76.
57
40. Rodríguez AM, Carrico PM, Mazurkiewicz JE, Meléndez JA. 2000. Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H2O2. Free Radic Biol Med 29:801-13.
41. Sibel K, Guliz A, Ayfer Y, Cigdem A, Taner D. 2013. Effects of resveratrol on hydrogen peroxide-induced oxidative stress in embryonic neural stem cells. Neural Regen Res 8:485–495.
42. Chen CH, Huang CC, Chung TC, Hu RM, Huang YW, and Yang TC. 2011. Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia. Antimicrob Agents Chemother 55:5826–5833.