臺灣博碩士論文加值系統

本論文主要包含兩個主題，第一個為「鑑定磷酸根轉移分子HptB在綠膿桿菌PAO1中參與的多階層訊息傳遞系統」，第二個主題為「AcoK之三磷酸腺苷結合區域為此蛋白調控克雷白氏菌acetoin代謝之必要區域」。
綠膿桿菌為格蘭氏陰性致病菌，尤其對於住院治療的病人，可導致多種急慢性感染病症。此菌共有三個可表現具有磷酸根轉移區域，卻缺乏磷酸激脢功能區域的蛋白分子之基因。磷酸根轉移分子為感應蛋白(hybrid sensor)和接受子(response regulator)之間的訊息傳遞分子，被認為在細菌、真菌以及植物界都扮演關鍵性的訊息傳遞功能。在第一個主題中的研究顯示，綠膿桿菌HptB訊息傳遞系統包括了四個游離的感應激脢蛋白、HptB分子，及一個專一的接受子PA3346。我們也證實了PA3346具有磷酸化水解能力，並能將其隔壁基因PA3347的蛋白質產物去磷酸化。最後，在基因HptB、PA3346以及PA3347突變株，其swarming和生物膜形成之能力也被加以分析。
在本論文第二個主題，為探討Aco此蛋白調控克雷白氏菌參與acetoin代謝的功能區域。許多細菌種類都可利用acetoin為其碳源。在克雷白氏肺炎桿菌中，此種化合物可被acoABCD基因群轉譯的蛋白acetoin dehydrogenase 氧化。之前的研究顯示，此基因群的表現依賴位於其上游緊鄰之基因所產生的蛋白分子AcoK。AcoK在C端具有一個LuxR形式之helix-turn-helix去氧核醣核酸附著區域，在N端則有一個推測為Walker A和Walker B之核苷三磷酸結合區域。第二個研究目標就是去了解AcoK不同功能區域，尤其是在核苷三磷酸結合區域，對於它轉錄功能的重要性。在這研究中，我們建構了數個AcoK的截短及點突變蛋白並研究其生化及轉譯功能。其中一個在推測為Walker A區域之點突變，造成AcoK對於三磷酸腺苷水解能力以及其導致之轉錄活性的喪失。雖然此轉錄因子具有專一結合至acoABCD特定-66到-36啟動子核酸區域的性質，但Walker A區域點突變所造成之三磷酸腺苷水解能力喪失，並不影響其去氧核醣核酸結合能力。總結，本研究提供另一個多功能區域之訊息傳遞腺嘌呤三磷酸水解酵素 (Signal Transduction ATPases with Numerous Domains) 家族中，如何活化其標的基因表現的範例。

This thesis contains two major topics. The first one is “Characterization of the histidine-containing phosphotransfer (Hpt) protein B�{mediated multi-step phosphorelay system in Pseudomonas aeruginosa PAO1” and the second one is “The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43”.
Pseudomonas aeruginosa is a gram-negative pathogen causing many acute and chronic infections, particularly in hospitalized individuals. It contains three genes that encode proteins with an Hpt domain but lack a kinase domain. Hpt proteins are signal mediators between hybrid sensors and response regulators. The proteins play a crucial role in directing signal transduction in bacteria, yeasts and plants. The study in first topic demonstrates that the Pseudomonas aeruginosa HptB-mediated signaling system consists of four orphan sensor kinases, HptB, and a specific response regulator, PA3346. We also present evidence of phosphatase activity of PA3346 on its neighboring gene product, PA3347. Finally, the swarming and biofilm forming activites of hptB, PA3346, and PA3347 knockout mutants are described.
The second part of this thesis is to characterize a transcriptional factor AcoK in the regulation of acetoin catabolism. Many bacterial species utilize acetoin as a carbon source. The compound is oxidized by acetoin dehydrogenase encoded by acoABCD operon in Klebsiella pneumoniae. Previously, we have shown the expression of this operon is induced by acetoin through AcoK, the product of a gene located immediately upstream of acoABCD. AcoK contains a helix-turn-helix DNA binding domain of the LuxR transcription activator family at the C-terminal region and putative Walker A and B nucleotide binding motifs at N-terminal portion. The goal of the second study is to understand the contribution of different domains, in particular the nucleotide binding motif, in AcoK on its transcriptional activity. A number of truncations and site-directed mutations were constructed on AcoK and the biochemical and trans-activation activities of the resulting proteins were determined and reported herein. A mutation in the putative Walker A motif resulted in a significant reduction of ATP hydrolysis and trans-activation activity of AcoK on acoABCD expression, presumably was due to the loss of ATP-binding ability. The transcription factor bound specifically to a region comprising nucleotide -66 to -36 of the acoABCD promoter, although the DNA binding ability was not affected by the Walker A motif mutation. Together, this study provides an additional example in how a member of the Signal Transduction ATPases with Numerous Domains family activates its target gene expression.

Table of contents
Abstract (Chinese) ……………………………………………………… i

Abstract ……………………………………………………… ii

Acknowledgement ……………………………………………………… iii

Table of contents ……………………………………………………… iv

Abbreviation ……………………………………………………… v

Chapter I
Characterization of the histidine-containing phosphotransfer protein B�{mediated multi-step phosphorelay system in Pseudomonas aeruginosa PAO1 1
Abstract ……………………………………………………… 2
Introduction ……………………………………………………… 3
Materials and methods ……………………………………………………… 6
Results ……………………………………………………… 13
Discussion ……………………………………………………… 23
Chapter II
The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43 28
Abstract ……………………………………………………… 29
Introduction ……………………………………………………… 30
Materials and methods ……………………………………………………… 33
Results ……………………………………………………… 40
Discussion ……………………………………………………… 46
Tables ……………………………………………………… 49
Figures ……………………………………………………… 59
References ……………………………………………………… 80
Appendix ……………………………………………………… 86

1. Kohler, T., Curty, L. K., Barja, F., van Delden, C. & Pechere, J. C. (2000) Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili, J Bacteriol. 182, 5990-6.
2. Lin, C. T., Huang, Y. J., Chu, P. H., Hsu, J. L., Huang, C. H. & Peng, H. L. (2006) Identification of an HptB-mediated multi-step phosphorelay in Pseudomonas aeruginosa PAO1, Res Microbiol. 157, 169-75.
3. Yamamoto, K., Hirao, K., Oshima, T., Aiba, H., Utsumi, R. & Ishihama, A. (2005) Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli, J Biol Chem. 280, 1448-56.
4. Skerker, J. M., Prasol, M. S., Perchuk, B. S., Biondi, E. G. & Laub, M. T. (2005) Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis, PLoS Biol. 3, e334.
5. Biondi, E. G., Skerker, J. M., Arif, M., Prasol, M. S., Perchuk, B. S. & Laub, M. T. (2006) A phosphorelay system controls stalk biogenesis during cell cycle progression in Caulobacter crescentus, Mol Microbiol. 59, 386-401.
6. West, A. H. & Stock, A. M. (2001) Histidine kinases and response regulator proteins in two-component signaling systems, Trends Biochem Sci. 26, 369-76.
7. Delumeau, O., Dutta, S., Brigulla, M., Kuhnke, G., Hardwick, S. W., Volker, U., Yudkin, M. D. & Lewis, R. J. (2004) Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C, J Biol Chem. 279, 40927-37.
8. Adler, E., Donella-Deana, A., Arigoni, F., Pinna, L. A. & Stragler, P. (1997) Structural relationship between a bacterial developmental protein and eukaryotic PP2C protein phosphatases, Mol Microbiol. 23, 57-62.
9. Vijay, K., Brody, M. S., Fredlund, E. & Price, C. W. (2000) A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis, Mol Microbiol. 35, 180-8.
10. Arigoni, F., Duncan, L., Alper, S., Losick, R. & Stragier, P. (1996) SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis, Proc Natl Acad Sci U S A. 93, 3238-42.
11. Lucet, I., Borriss, R. & Yudkin, M. D. (1999) Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis, J Bacteriol. 181, 3242-5.
12. Jin, H. & Pancholi, V. (2006) Identification and biochemical characterization of a eukaryotic-type serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification, J Mol Biol. 357, 1351-72.
13. Rajagopal, L., Clancy, A. & Rubens, C. E. (2003) A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence, J Biol Chem. 278, 14429-41.
14. Gee, K. R., Sun, W. C., Bhalgat, M. K., Upson, R. H., Klaubert, D. H., Latham, K. A. & Haugland, R. P. (1999) Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases, Anal Biochem. 273, 41-8.
15. Pastula, C., Johnson, I., Beechem, J. M. & Patton, W. F. (2003) Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases, Comb Chem High Throughput Screen. 6, 341-6.
16. Chang, C. H., Zhu, J. & Winans, S. C. (1996) Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein, J Bacteriol. 178, 4710-6.
17. Munoz-Dorado, J., Inouye, S. & Inouye, M. (1991) A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium, Cell. 67, 995-1006.
18. Bork, P., Brown, N. P., Hegyi, H. & Schultz, J. (1996) The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues, Protein Sci. 5, 1421-5.
19. Mukhopadhyay, S., Kapatral, V., Xu, W. & Chakrabarty, A. M. (1999) Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa, J Bacteriol. 181, 6615-22.
20. Mougous, J. D., Gifford, C. A., Ramsdell, T. L. & Mekalanos, J. J. (2007) Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa, Nat Cell Biol. 9, 797-803.
21. Hughes, K. T. & Mathee, K. (1998) The anti-sigma factors, Annu Rev Microbiol. 52, 231-86.
22. Goodman, A. L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R. S. & Lory, S. (2004) A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa, Dev Cell. 7, 745-54.
23. Laskowski, M. A., Osborn, E. & Kazmierczak, B. I. (2004) A novel sensor kinase-response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa, Mol Microbiol. 54, 1090-103.
24. Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L., Joachimiak, G., Ordonez, C. L., Lory, S., Walz, T., Joachimiak, A. & Mekalanos, J. J. (2006) A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus, Science. 312, 1526-30.
25. Ventre, I., Goodman, A. L., Vallet-Gely, I., Vasseur, P., Soscia, C., Molin, S., Bleves, S., Lazdunski, A., Lory, S. & Filloux, A. (2006) Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes, Proc Natl Acad Sci U S A. 103, 171-6.
26. Kominek, L. A. & Halvorson, H. O. (1965) Metabolism of poly-beta-hydroxybutyrate and acetoin in Bacillus cereus, J Bacteriol. 90, 1251-9.
27. Hullin, R. P. & Hassall, H. (1962) The synthesis of cell constituents from butane-2,3-diol by Pseudomonas sp, Biochem J. 83, 298-303.
28. Juni, E. & Heym, G. A. (1956) A cyclic pathway for the bacterial dissimilation of 2, 3-butanediol, acetylmethylcarbinol, and diacetyl. I. General aspects of the 2, 3-butanediol cycle, J Bacteriol. 71, 425-32.
29. Johansen, L., Bryn, K. & Stormer, F. C. (1975) Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes, J Bacteriol. 123, 1124-30.
30. Blomqvist, K., Nikkola, M., Lehtovaara, P., Suihko, M. L., Airaksinen, U., Straby, K. B., Knowles, J. K. & Penttila, M. E. (1993) Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes, J Bacteriol. 175, 1392-404.
31. Lopez, J. M., Thoms, B. & Rehbein, H. (1975) Acetoin degradation in Bacillus subtilis by direct oxidative cleavage, Eur J Biochem. 57, 425-30.
32. Oppermann, F. B., Steinbuchel, A. & Schlegel, H. G. (1989) Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate, FEMS Microbiol Lett. 51, 113-8.
33. Huang, M., Oppermann, F. B. & Steinbuchel, A. (1994) Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway, FEMS Microbiol Lett. 124, 141-50.
34. Ali, N. O., Bignon, J., Rapoport, G. & Debarbouille, M. (2001) Regulation of the acetoin catabolic pathway is controlled by sigma L in Bacillus subtilis, J Bacteriol. 183, 2497-504.
35. Huang, M., Oppermann-Sanio, F. B. & Steinbuchel, A. (1999) Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway, J Bacteriol. 181, 3837-41.
36. Silbersack, J., Jurgen, B., Hecker, M., Schneidinger, B., Schmuck, R. & Schweder, T. (2006) An acetoin-regulated expression system of Bacillus subtilis, Appl Microbiol Biotechnol. 73, 895-903.
37. Peng, H. L., Yang, Y. H., Deng, W. L. & Chang, H. Y. (1997) Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon, J Bacteriol. 179, 1497-504.
38. Boos, W. & Shuman, H. (1998) Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation, Microbiol Mol Biol Rev. 62, 204-29.
39. Leipe, D. D., Koonin, E. V. & Aravind, L. (2004) STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer, J Mol Biol. 343, 1-28.
40. Bao, Q., Lu, W., Rabinowitz, J. D. & Shi, Y. (2007) Calcium blocks formation of apoptosome by preventing nucleotide exchange in Apaf-1, Mol Cell. 25, 181-92.
41. Joly, N., Bohm, A., Boos, W. & Richet, E. (2004) MalK, the ATP-binding cassette component of the Escherichia coli maltodextrin transporter, inhibits the transcriptional activator malt by antagonizing inducer binding, J Biol Chem. 279, 33123-30.
42. Schlegel, A., Danot, O., Richet, E., Ferenci, T. & Boos, W. (2002) The N terminus of the Escherichia coli transcription activator MalT is the domain of interaction with MalY, J Bacteriol. 184, 3069-77.
43. Larquet, E., Schreiber, V., Boisset, N. & Richet, E. (2004) Oligomeric assemblies of the Escherichia coli MalT transcriptional activator revealed by cryo-electron microscopy and image processing, J Mol Biol. 343, 1159-69.
44. Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R. & Alnemri, E. S. (1999) Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation, J Biol Chem. 274, 17941-5.
45. Schreiber, V. & Richet, E. (1999) Self-association of the Escherichia coli transcription activator MalT in the presence of maltotriose and ATP, J Biol Chem. 274, 33220-6.
46. Labbe, D., Garnon, J. & Lau, P. C. (1997) Characterization of the genes encoding a receptor-like histidine kinase and a cognate response regulator from a biphenyl/polychlorobiphenyl-degrading bacterium, Rhodococcus sp. strain M5, J Bacteriol. 179, 2772-6.
47. Valdez, F., Gonzalez-Ceron, G., Kieser, H. M. & Servin-Gonzalez, L. (1999) The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator, Microbiology. 145 ( Pt 9), 2365-74.
48. Poon, K. K., Chu, J. C. & Wong, S. L. (2001) Roles of glucitol in the GutR-mediated transcription activation process in Bacillus subtilis: glucitol induces GutR to change its conformation and to bind ATP, J Biol Chem. 276, 29819-25.
49. De Schrijver, A. & De Mot, R. (1999) A subfamily of MalT-related ATP-dependent regulators in the LuxR family, Microbiology. 145 ( Pt 6), 1287-8.
50. Deng, W. L., Chang, H. Y. & Peng, H. L. (1994) Acetoin catabolic system of Klebsiella pneumoniae CG43: sequence, expression, and organization of the aco operon, J Bacteriol. 176, 3527-35.
51. Kondo, T., Strayer, C. A., Kulkarni, R. D., Taylor, W., Ishiura, M., Golden, S. S. & Johnson, C. H. (1993) Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria, Proc Natl Acad Sci U S A. 90, 5672-6.
52. Sambrook, J. & Russell, D. W. (2001) Molecular cloning : a laboratory manual, 3rd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
53. Peng, H. L., Shiou, S. R. & Chang, H. Y. (1999) Characterization of mdcR, a regulatory gene of the malonate catabolic system in Klebsiella pneumoniae, J Bacteriol. 181, 2302-6.
54. Barik, S. (1996) Site-directed mutagenesis in vitro by megaprimer PCR, Methods Mol Biol. 57, 203-15.
55. Shiue, S. J., Kao, K. M., Leu, W. M., Chen, L. Y., Chan, N. L. & Hu, N. T. (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL, Embo J. 25, 1426-35.
56. Lai, Y. C., Peng, H. L. & Chang, H. Y. (2003) RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level, J Bacteriol. 185, 788-800.
57. Yoshida, M. & Amano, T. (1995) A common topology of proteins catalyzing ATP-triggered reactions, FEBS Lett. 359, 1-5.
58. Marquenet, E. & Richet, E. (2007) How integration of positive and negative regulatory signals by a STAND signaling protein depends on ATP hydrolysis, Mol Cell. 28, 187-99.
59. Danot, O. (2001) A complex signaling module governs the activity of MalT, the prototype of an emerging transactivator family, Proc Natl Acad Sci U S A. 98, 435-40.
60. Lee, P. C., Umeyama, T. & Horinouchi, S. (2002) afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2), Mol Microbiol. 43, 1413-30.
61. Hu, Y., Ding, L., Spencer, D. M. & Nunez, G. (1998) WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation, J Biol Chem. 273, 33489-94.
62. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E. S. (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization, Mol Cell. 1, 949-57.
63. Dardonville, B. & Raibaud, O. (1990) Characterization of malT mutants that constitutively activate the maltose regulon of Escherichia coli, J Bacteriol. 172, 1846-52.
64. Richet, E. & Raibaud, O. (1987) Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon, J Biol Chem. 262, 12647-53.
65. Richet, E. & Raibaud, O. (1989) MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator, Embo J. 8, 981-7.
66. Furste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector, Gene. 48, 119-31.
67. Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. (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.
68. Osuna, R. & Bender, R. A. (1991) Klebsiella aerogenes catabolite gene activator protein and the gene encoding it (crp), J Bacteriol. 173, 6626-31.
69. Schwacha, A. & Bender, R. A. (1990) Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes, J Bacteriol. 172, 5477-81.
70. Chang, A. C. & Cohen, S. N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid, J Bacteriol. 134, 1141-56.