( 您好!臺灣時間:2021/05/10 08:11
字體大小: 字級放大   字級縮小   預設字形  


研究生(外文):Kuan Yeh Huang
論文名稱(外文):Molecular and functional analysis of the Streptococcus salivarius 57.Ⅰ vic operon
指導教授(外文):Y.Y.M. Chen
外文關鍵詞:streptococcussalivariusvictwo component signal transduction system
  • 被引用被引用:0
  • 點閱點閱:214
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0

Two component signal transduction system( TCS )為細菌體內主要的訊息傳遞以及調節系統,此系統參與細菌生理代謝、壓力適應、抗藥性、勝任性,以及毒性因子表現等之調控。VicRK TCS( 在某些菌種稱為YycFG )是一廣泛存於革蘭氏陽性菌,且對於細菌的毒性,生長,壓力適應均扮演重要調控角色的系統。在唾液鏈球菌也發現到此一系統的存在,透過引子延伸實驗發現在-16發現一extended啟動子序列,且在-132位置發現一VicR-P結合的保留性序列,透過RT-PCR及EMSA實驗結果發現VicR-P可以一抑制子的角色逆向調控vic 操縱子的表現。vic啟動子的活性會在細菌生長至飽和期時降至最低,並且在pH中性環境下的活性也比酸性環境下好。在比較野生株、VicK及VicX突變株的功能分析實驗中發現,VicK去活性株在氧化壓力適應,生長及生物膜生合成的能力上都比野生株與VicX去活性株來的差。由此得知VicRK TCS對於細菌生長初期扮演著重要的角色,同時VicRK TCS也參與唾液鏈球菌的氧化壓力適應反應。由VicR-P所引導的回饋逆向調控可精確地控制vic操縱子的表現量,及由其所調控下游基因的表現。

Two-component signal transduction system (TCS) is the major signal transduction and regulation system in bacteria. Cellular functions such as metabolic activity, stress responses, drug resistance, competence, and virulence all are regulated by TCS. The VicRK (also known as YycFG) TCS, encoded by vic operon, is widely identified in Gram-positive bacteria, and its impact in cell wall biogenesis, and virulence gene expression has been demonstrated in various streptococcal species, Staphylococcus aureus, and Bacillus subtilis. Recent study in our laboratory revealed that a vicRKX operon is present in Streptococcus salivarius and the expression of this operon has been confirmed by RT-PCR. To analyze the expression of vic operon in S. salivarius, a promoter-chloramphenicol resistance gene fusion (pvic-cat) was constructed and integrated into the lacZ locus in this study. The promoter activities in cells grown at different conditions were examined by measuring CAT activity. The expression of pvic was regulated by both growth stages and culture pH, with lower expression in cells grown in the stationary stages and under acidic pH. Transcription start site was located 62 bp 5’ to the ATG. An extended -10 promoter sequence was found in the corresponding region and a regulator binding sequence proposed for S. pneumoniae VicR was located at -132 bp. Furthermore, the amount of vicR-specific message, measured by RT-PCR, was up-regulated in the vicK-deficient strain comparing to the wild-type strain grown under the same condition. These results suggested that the expression of pvic was negatively autoregulated by phosphorylated VicR. Functional analysis indicated that an intact vicK, but not the vicX, was essential for optimal oxidative stress responses and biofilm formation. Thus, it is likely that Vic TCS is involved in a global regulatory circuit in S. salivarius.
誌謝 IV
摘要 V
目錄 VII
圖表目錄 IX
三、 口腔鏈球菌的酸性適應( ACID ADAPTATION ) - 2 -
1. Sensor kinase (SK) - 5 -
2. Response regulator (RR) - 6 -
七、 TCS的重要性 - 7 -
八、 VICRK(又名YYCFG)TCS對於鏈球菌之重要性 - 8 -
九、 實驗目標 - 9 -
材料與方法 - 11 -
一、 菌株、質體、引子與生長環境 - 11 -
二、 VICX突變株之建構 - 11 -
三、 啟動子重組株之建構 - 12 -
四、 LYSATE的製備與CAT報導基因活性分析 - 12 -
五、 RNA萃取與反轉錄酶聚合酶連鎖生合成反應(RT-PCR) - 13 -
七、 引子延伸實驗(PRIMER EXTENSION) - 14 -
八、 環境壓力對於唾液鏈球菌生長抑制實驗 - 14 -
九、 生物膜生合成分析(BIOFILM FORMATION ASSAY) - 15 -
結果 - 16 -
一、 VIC操縱子與其啟動子的結構與比對 - 16 -
二、 PVIC活性分析 - 16 -
三、 野生株與突變株RT-PCR結果分析 - 17 -
四、 VICR與PVIC間的交互作用 - 17 -
五、 VIC TCS的功能分析 - 17 -
討論 - 19 -
圖 - 23 -
表 - 33 -


圖一:引子延伸結果 - 23 -
圖二:pvic-cat重組株(KY3)之建構與確認 - 24 -
圖三:唾液鏈球菌pvic-cat重組株在不同生長時期的CAT活性。 - 25 -
圖四:唾液鏈球菌pvic-cat重組株培養在TH-KPO4 (pH=7.5) 與TH-HCl (pH=5.5)下之CAT 活性。 - 26 -
圖五:藉由RT-PCR探討vicR在野生株與SF31的mRNA表現量 - 27 -
圖六:MBP-VicR與Pvic結合 - 28 -
圖七:VicX突變株之建構與確認 - 29 -
圖八:唾液鏈球菌57.I野生株及其突變株生長曲線分析 - 30 -
圖九:唾液鏈球菌57.I野生株及其突變株的生物膜生合成能力。 - 31 -
圖十:唾液鏈球菌 pvic調控路徑之假說 - 32 -
表一、本實驗所用之菌株與質體 - 33 -
表二、實驗所用之引子 - 34 -
表三:VicRKX同源性蛋白質比對結果 - 35 -
表四:野生株與突變株在各種壓力下所能生長的最低濃度 - 36 -
表五:唾液鏈球菌VicR目標基因上游序列以及S. pneumoniae 與B. subtilis 之VicR(YycF)目標基因已發表之上游序列 - 37 -

Albright, L.M., Huala, E., and Ausubel, F.M. (1989) Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet 23: 311-336.
Appleby, J.L., Parkinson, J.S., and Bourret, R.B. (1996) Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell 86: 845-848.
Bender, G.R., Sutton, S.V., and Marquis, R.E. (1986) Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infect Immun 53: 331-338.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Burbulys, D., Trach, K.A., and Hoch, J.A. (1991) Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64: 545-552.
Chen, Y.Y., and Burne, R.A. (1996) Analysis of Streptococcus salivarius urease expression using continuous chemostat culture. FEMS Microbiol Lett 135: 223-229.
Chen, Y.Y., Clancy, K.A., and Burne, R.A. (1996) Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun 64: 585-592.
Chen, Y.Y., Hall, T.H., and Burne, R.A. (1998a) Streptococcus salivarius urease expression: involvement of the phosphoenolpyruvate:sugar phosphotransferase system. FEMS Microbiol Lett 165: 117-122.
Chen, Y.Y., Weaver, C.A., Mendelsohn, D.R., and Burne, R.A. (1998b) Transcriptional regulation of the Streptococcus salivarius 57.I urease operon. J Bacteriol 180: 5769-5775.
Chen, Y.Y., Weaver, C.A., and Burne, R.A. (2000) Dual functions of Streptococcus salivarius urease. J Bacteriol 182: 4667-4669.
Chen, Y.Y., Betzenhauser, M.J., and Burne, R.A. (2002) cis-Acting elements that regulate the low-pH-inducible urease operon of Streptococcus salivarius. Microbiology 148: 3599-3608.
Chia, J.S., Chang, L.Y., Shun, C.T., Chang, Y.Y., Tsay, Y.G., and Chen, J.Y. (2001a) A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutans. Infect Immun 69: 6987-6998.
Chia, J.S., Lee, Y.Y., Huang, P.T., and Chen, J.Y. (2001b) Identification of stress-responsive genes in Streptococcus mutans by differential display reverse transcription-PCR. Infect Immun 69: 2493-2501.
Clausen, V.A., Bae, W., Throup, J., Burnham, M.K., Rosenberg, M., and Wallis, N.G. (2003) Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae. J Mol Microbiol Biotechnol 5: 252-260.
Cotter, P.D., and Hill, C. (2003) Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67: 429-453, table of contents.
Coykendall, A.L. (1989) Classification and identification of the viridans streptococci. Clin Microbiol Rev 2: 315-328.
Crosson, S., and Moffat, K. (2001) Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction. Proc Natl Acad Sci U S A 98: 2995-3000.
Deng, D.M., Liu, M.J., ten Cate, J.M., and Crielaard, W. (2007) The VicRK system of Streptococcus mutans responds to oxidative stress. J Dent Res 86: 606-610.
Dunny, G.M., Lee, L.N., and LeBlanc, D.J. (1991) Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol 57: 1194-1201.
Fabret, C., and Hoch, J.A. (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180: 6375-6383.
Facklam, R.R. (1977) Physiological differentiation of viridans streptococci. J Clin Microbiol 5: 184-201.
Fedorov, R., Schlichting, I., Hartmann, E., Domratcheva, T., Fuhrmann, M., and Hegemann, P. (2003) Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys J 84: 2474-2482.
Gong, W., Hao, B., Mansy, S.S., Gonzalez, G., Gilles-Gonzalez, M.A., and Chan, M.K. (1998) Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. Proc Natl Acad Sci U S A 95: 15177-15182.
Gusa, A.A., and Scott, J.R. (2005) The CovR response regulator of group A streptococcus (GAS) acts directly to repress its own promoter. Mol Microbiol 56: 1195-1207.
Havarstein, L.S., Gaustad, P., Nes, I.F., and Morrison, D.A. (1996) Identification of the streptococcal competence-pheromone receptor. Mol Microbiol 21: 863-869.
Kawamura, Y., Hou, X.G., Sultana, F., Miura, H., and Ezaki, T. (1995) Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45: 406-408.
Khorchid, A., and Ikura, M. (2006) Bacterial histidine kinase as signal sensor and transducer. Int J Biochem Cell Biol 38: 307-312.
Kreth, J., Hung, D.C., Merritt, J., Perry, J., Zhu, L., Goodman, S.D., Cvitkovitch, D.G., Shi, W., and Qi, F. (2007) The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis. Microbiology 153: 1799-1807.
Lange, R., Wagner, C., de Saizieu, A., Flint, N., Molnos, J., Stieger, M., Caspers, P., Kamber, M., Keck, W., and Amrein, K.E. (1999) Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae. Gene 237: 223-234.
Li, M., Lai, Y., Villaruz, A.E., Cha, D.J., Sturdevant, D.E., and Otto, M. (2007) Gram-positive three-component antimicrobial peptide-sensing system. Proc Natl Acad Sci U S A 104: 9469-9474.
Liu, M., Hanks, T.S., Zhang, J., McClure, M.J., Siemsen, D.W., Elser, J.L., Quinn, M.T., and Lei, B. (2006) Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR. Microbiology 152: 967-978.
Lukat, G.S., McCleary, W.R., Stock, A.M., and Stock, J.B. (1992) Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A 89: 718-722.
Majka, F.A., Gysin, W.M., and Zaayer, R.L. (1956) Streptococcus salivarius meningitis following diagnostic lumbar puncture. Nebr State Med J 41: 279-281.
Mizuno, T. (1997) Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res 4: 161-168.
Ng, W.L., Robertson, G.T., Kazmierczak, K.M., Zhao, J., Gilmour, R., and Winkler, M.E. (2003) Constitutive expression of PcsB suppresses the requirement for the essential VicR (YycF) response regulator in Streptococcus pneumoniae R6. Mol Microbiol 50: 1647-1663.
Ng, W.L., Kazmierczak, K.M., and Winkler, M.E. (2004) Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator. Mol Microbiol 53: 1161-1175.
Ng, W.L., Tsui, H.C., and Winkler, M.E. (2005) Regulation of the pspA virulence factor and essential pcsB murein biosynthetic genes by the phosphorylated VicR (YycF) response regulator in Streptococcus pneumoniae. J Bacteriol 187: 7444-7459.
Novak, R., Henriques, B., Charpentier, E., Normark, S., and Tuomanen, E. (1999) Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399: 590-593.
O'Toole, G.A., and Kolter, R. (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30: 295-304.
Parkinson, J.S. (1993) Signal transduction schemes of bacteria. Cell 73: 857-871.
Perraud, A.L., Weiss, V., and Gross, R. (1999) Signalling pathways in two-component phosphorelay systems. Trends Microbiol 7: 115-120.
Razeto, A., Ramakrishnan, V., Litterst, C.M., Giller, K., Griesinger, C., Carlomagno, T., Lakomek, N., Heimburg, T., Lodrini, M., Pfitzner, E., and Becker, S. (2004) Structure of the NCoA-1/SRC-1 PAS-B domain bound to the LXXLL motif of the STAT6 transactivation domain. J Mol Biol 336: 319-329.
Reinscheid, D.J., Gottschalk, B., Schubert, A., Eikmanns, B.J., and Chhatwal, G.S. (2001) Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B streptococcus. J Bacteriol 183: 1175-1183.
Safford, C.E., Sherman, J.M., and Hodge, H.M. (1937) Streptococcus salivarius. J Bacteriol 33: 263-274.
Senadheera, M.D., Guggenheim, B., Spatafora, G.A., Huang, Y.C., Choi, J., Hung, D.C., Treglown, J.S., Goodman, S.D., Ellen, R.P., and Cvitkovitch, D.G. (2005) A VicRK signal transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development. J Bacteriol 187: 4064-4076.
Senadheera, M.D., Lee, A.W., Hung, D.C., Spatafora, G.A., Goodman, S.D., and Cvitkovitch, D.G. (2007) The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance. J Bacteriol 189: 1451-1458.
Shaulsky, G., Escalante, R., and Loomis, W.F. (1996) Developmental signal transduction pathways uncovered by genetic suppressors. Proc Natl Acad Sci U S A 93: 15260-15265.
Shaw, W.V., Packman, L.C., Burleigh, B.D., Dell, A., Morris, H.R., and Hartley, B.S. (1979) Primary structure of a chloramphenicol acetyltransferase specified by R plasmids. Nature 282: 870-872.
Simms, S.A., Keane, M.G., and Stock, J. (1985) Multiple forms of the CheB methylesterase in bacterial chemosensing. J Biol Chem 260: 10161-10168.
Sissons, C.H., Hancock, E.M., Perinpanayagam, H.E., and Cutress, T.W. (1988) The bacteria responsible for ureolysis in artificial dental plaque. Arch Oral Biol 33: 727-733.
Smiley, K.L., Niven, C.F., and Sherman, J.M. (1943) The Nutrition of Streptococcus salivarius. J Bacteriol 45: 445-454.
Stock, A.M., Robinson, V.L., and Goudreau, P.N. (2000) Two-component signal transduction. Annu Rev Biochem 69: 183-215.
Stock, J.B., Ninfa, A.J., and Stock, A.M. (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53: 450-490.
Szurmant, H., Mohan, M.A., Imus, P.M., and Hoch, J.A. (2007) YycH and YycI interact to regulate the essential YycFG two-component system in Bacillus subtilis. J Bacteriol 189: 3280-3289.
Taylor, B.L., and Zhulin, I.B. (1999) PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63: 479-506.
Throup, J.P., Koretke, K.K., Bryant, A.P., Ingraham, K.A., Chalker, A.F., Ge, Y., Marra, A., Wallis, N.G., Brown, J.R., Holmes, D.J., Rosenberg, M., and Burnham, M.K. (2000) A genomic analysis of two-component signal transduction in Streptococcus pneumoniae. Mol Microbiol 35: 566-576.
Voskuil, M.I., and Chambliss, G.H. (1998) The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Res 26: 3584-3590.
Wagner, C., Saizieu Ad, A., Schonfeld, H.J., Kamber, M., Lange, R., Thompson, C.J., and Page, M.G. (2002) Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon. Infect Immun 70: 6121-6128.
West, A.H., and Stock, A.M. (2001) Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci 26: 369-376.
Yildiz, O., Doi, M., Yujnovsky, I., Cardone, L., Berndt, A., Hennig, S., Schulze, S., Urbanke, C., Sassone-Corsi, P., and Wolf, E. (2005) Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD. Mol Cell 17: 69-82.
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔