臺灣博碩士論文加值系統

綠膿桿菌(Pseudomonas aeruginosa)，為一臨床常見的伺機性病原，對環境有很強的適應性。在已解碼的基因體序列中，發現綠膿桿菌具有123個雙分子訊息傳遞系統，藉以偵測環境的變化與刺激、調控適當的基因表現，使細菌可以適應環境、躲避免疫系統及抗生素的攻擊。目前已建立的雙分子訊息傳遞系統模式分為三類：典型系統(classic system)、非典型系統(unorthodox system)、混成系統(hybrid system)。混成系統的訊息由感應蛋白質激脢透過游離的接受模組分子(Hpt domain)傳遞給調控蛋白質。為了確認這些Hpts在綠膿桿菌中傳遞磷酸根的功能，我利用聚合脢鏈鎖反應選殖出這三個Hpt基因序列，以pET30表現系統在大腸桿菌中表現並進而純化蛋白質，再以放射性元素標定磷酸根來偵測磷酸根在雜合感應子PA1611、Hpt分子及感應蛋白質間的轉移。結果證實專一的磷酸根傳遞路徑由感應子PA1611到HptB再到調控蛋白質PA3346。然而，以酵母菌雜合系統分析卻無法證實這些蛋白質間的交互作用。經由蛋白質序列的比對，在多種單胞桿菌中，均可發現HptB基因與鞭毛生合成的基因組座落在一起。為了探討HptB的功能，以同源互換方式構築HptB基因缺損株。此HptB缺損株在營養缺乏的狀態下生長速率降低；在添加碳源及氮源的情況下，其生物膜的生成量是野生株的兩倍；缺損株的游移能力在半液態環境下較差，而在固態界面中則較佳；趨化性的分析結果顯示缺損株朝趨化物移動的能力降低。由這些特性分析及基因體序列比較結果，我們推斷HptB分子可能與鞭毛的生合成及調控有關。

Pseudomonas aeruginosa PAO1 is an opportunistic pathogen, and owns great capability to adapt versatile environment. There are 123 genes encoding two-component system components in the bacteria, which serve as a stimulus-response coupling machinery allowing the organism sense and respond to the changes in environment. The system has been classified into three types: the classical system, the unorthodox system, and the hybrid system. We focus on the hybrid system which consists of a histidine kinase (HK) without output domain, a separated Histidine-containing phosphotransfer (Hpt) molecule, and a response regulator (RR). In order to demonstrate the role of Hpt modules in signaling phosphorelay, the gene fragments corresponding to each of the Hpt domain and one hybrid sensor (PA1611) and two RRs (PA3346 and PA0034) were amplified by PCR and subcloned respectively into pET30 expression vector. The recombinant proteins were expressed in E. coli and the proteins purified by His-Bind nickel column for the phosphorylation assays. The assay demonstrated a specific phosphoryl transfer from sensor kinase PA1611 to HptB and then to PA3346. However, using yeast two-hybrid screening failed to identify the protein interactions. Sequence comparisons of several Pseudomonas species revealed that the genome DNA contains the HptB homolog flanking by a flagella biosynthesis gene cluster. To identify the functional role of HptB, hptB mutant was constructed by homologous recombination. The hptB mutant showed a decrease of growth rate decreased in a nutrient limiting condition. The hptB mutation also affected the biofilm formation with a 2-fold higher activity than that of the wild type strain in a minimal medium supplemented with carbon and nitrogen source. A reducing swimming capability and an increase of twitching motility were also found in hptB mutant. Taken together, the HptB is likely mediated the signaling involved in flagella biosynthesis and regulation.

Contents
Abstract--------------2
Introduction----------4
Materials and Methods---10
Results---------------17
Discussion------------22
References------------27
Table-----------------33
Figure----------------45
Appendix--------------66

Reference


黃致翔，國立交通大學碩士論文，中華民國九十二年，綠膿桿菌PAO1中雜合感應子的特性分析。

Bodey, G., R. Bolivar, V. Fainstein, and L. Jadeja. 1983. Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5: 297-313

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.

Chang, M., and I. P. Crawford. 1990. The role of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res. 18:979-988.

Craven, R., and T. C. Montie. 1985. Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source. J. Bacteriol. 164:544-549.

Dazins, A. 1993. The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY. J. Bacteriol. 175:5934-5944.

De-Lorenzo, V., and K. N. Timmis. 1994. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods. Enzymol. 235:386-405.

Deziel, E., Y. Comeau, and R. Villemur. 2001. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with Emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J. Bacteriol. 184:1195-1204.

Egger, L. A., H. Park, and M. Inouye. 1997. Signal transduction via the histidyl-aspartyl phosphorelay. Genes Cells. 2:167-184.

Essar D. W., L. Eberly, A. Hadero, and I. P. Crawford. 1990. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolution implications. J. Bacterial. 172: 884-900.

Ferrandez, A., A. C. Hawkins, D. T. Summerfield, and C. S. Harwood. 2002. Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response. J. Bacteriol. 184:4374-4383.

Fields, S. 1993. The two-hybrid system to detect protein-protein interactions. METHODS: A Companion to Meth. Enzymol. 5:116–124.

Finelli, A., C. V. Gallant, K. Jarvi, and L. L. Burrows. 2003. Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development. J. Bacteriol. 185:2700-2710.

Fletcher, M. 1977. The effects of culture concentration and age, time, and temperature on bacterial attachment to polystyrene. Can. J. Microbiol. 23:1-6.

Freeman, J. A.,and Bassler, B. L. 1999. Sequence and fuction of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. J. bacterial. 181:899-906.

Fujita, M., K. Tanaka, H. Takahashi, and A. Amemura. 1994. Transcription of the principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase. Mol. Microbiol. 13:1071-1077.

Furste, J. P., and W. Pansegrau, R. Frank, H. Blocker, P. Scholz, M. Bagdasarian, and E. Lanka. 1986. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 48:119-131.

Garrett, D. S., Y. J. Seok, A. Peterkofsky, A. M. Gronenborn, and G. M. Clore. 1999. Solution structure of the 40,000 Mr phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr. Nat. Struct. Biol. 6:166-173.

Georgelli D., A. S. Lynch, and E. C. Lin. 1997. In vitro phosphorylation study of the Arc two component signal transduction system of Escherichia coli. J. Bacteriol. 179:5429-5435

Han, C. Y., I. P. Crawford, and C. S. Harwood. 1991. Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa. J. Bacteriol. 173:3756-3762.

Hancock, R. E., and H. Nikaido. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136:381-90.

Harper, J. W., G. R. Adami, N.Wei, K. Keyomarsi, and S. J. Elledge. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 75:805–816.

Hoch, J. A. 2000. Two-component and phosphorelay signal transduction. Curr. Opin. Microbiol. 3:165-170.

Iuchi, S., and E. C. Lin. 1988. ArcA, a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways Proc. Natl. Acad. Sci. USA. 85:1888-1892.

Ishige, K., S. Nagassawa, S. Tokishita, and T. Mizuno. T. 1994. A novel device of bacterial signal transducer. EMBO J. 13:5195-5202.

James, P., J. Halladay, and E. A. Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 144:1425–1436.

Kang, C. M., M. S. Brody, S. Akbar, X. Yang, and C. W. Price. 1996. Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor sigma(b) in response to environmental stress. J. Bacteriol. 178:3846-3853.

Kato J., T. Nakamura, A. Kuroda, and H. Ohtake. 1999. Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa. Biosci. Biotechnol. Biochem. 63:155-161.

Knobloch, J. K, K. Bartscht, A. Sabottke, H. Rohde, H. H. Feucht, and D. Mack. 2001. Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress. J Bacteriol. 183:2624-2633.

Kohler, T., L. K. Curty, F. Barja, C. Van Delden, and J. C. Pechere. 2000. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J. Bacteriol. 182:5990–5996.

Li, L., S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski. 1994. Specific association between the human DNA repair proteins XPA and ERCC1. Proc. Natl. Acad. Sci. USA 91:5012–5016.

Li, S., A. Ault, C. L. Malone, D. Raitt, S. Dean, L. H. Johnston, R. J. Deschenes, and J. S. Fassler. 1998. The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators Ssk1p and Skn7p. EMBO J. 17:6952–6962.

Manch, J. N. and I. P. Crawford. 1982. Genetic evidence for a positive-acting regulatory factor mediating induction in the tryptophan pathway of Pseudomonas aeruginosa. J. Mol. Biol. 156:67-77

Martinez-Argudo, I., P. Salinas, R. Maldonado, and A. Contreras. 2002. Domain interaction on ntr signal transduction pathway: two-hybrid analysis of mutant and truncated derivatives of histidine kinase NtrB. J. Bacteriol. 184:200-206.

Matsushika, A., and T. Mizuno. 1998. A dual- signaling mechanism mediated by the ArcB hybrid sensor kinase containing the histidine-containing phosphotransfer domain in Escherichia coli. J. Bacteriol. 180:3973-3977.

Mavrodi D. V., R. F. Bonsall, S. M. Delaney, M. J. Soule, G. Phillips, and L. S. Thomashow. 2001. Functional Analysis of Genes for Biosynthesis of Pyocyanin and Phenazine-1-Carboxamide from Pseudomonas aeruginosa PAO1. J. Bacteriol. 183:6454-6465.

Miller, J. H. 1972. Experiments in molecular genetics. Cold spring Harbor Laboratory, Cold Spring Harbor, New York.

Ohta N., and A. Newton. 2003. The core dimmerization domains of histidine kinase contain recognition specificity for the cognate response regulator. J. Bacteriol. 185:4424-4431.

O’Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30:295-304.

O’Toole, G. A., K. A. Gibbs, P. W. Hager, P.V. Phibbs, and R. Kolter. 2000. The global carbon metabolism regulator Crc is a component of a signal trasduction pathway required for biofilm development by Pseudomonas aeruginosa. J. Bacteriol. 182:425-431.

Parkins, M. D., H. Ceri, and D. G. Storey. 2001. Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation. Mol. Microbiol. 40:1215-1226.

Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26:71-112.

Parkinson, J. S. 1993. Signal transduction schemes of bacteria. Cell. 73:857-871

Perraud A. L., V. Weiss, and R, Gross. 1999. Signaling pathway in two-component phosphorelay systems. Trends Microbiol. 7:115-120.

Perraud A. L., B. kimmel, V. Weiss, and R. Gross. 1998. Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal Hpt domains of the sensor proteins. Mol. Microbiol. 27:875-887.

Rangaswamy, V., and C. L. Bender. 1992. Phosphorylation of CorS and CorR, regulatory proteins that modulate production of phytotoxin coronatine in Pseudomonas syringae. FEMS Microbiol. Lett. 193:13-18.

Rashid, M. H., and A. Kornberg. 2000. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA. 97:4885–4890.

Reimmann, C., M. Beyeler, A. Latifi, H. winteler, M. Foglino, A. Lazdunski, and D. Haas. 1997. The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, canide, and lipase. Mol. Microbiol. 24:309-319.

Rodrigue A., Y. Quentin, A. Lazdunski, V. Mejean, and M. Foglino. 2000. Two-component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol. 8:498-504.

Sambrook J., and D. W. Russell. 2001. Molecular Cloning: a laboratory manual—3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.


Song H. K., J. Y. Lee, M. G. Lee, J. Moon, K. Min, J. K. Yang, and S. W. Suh. 1999. Insights into eukaryotic multistep phosphorelay signal transduction revealed by the crystal structure of Ypd1p from Saccharomyces cerevisiae. J. Mol. Biol. 293:753-761.

Stock A. M., V. L. Robinson, and P. N. Goudreau. 2000. Two-component signal transduction. Annu. Rev. Biochem. 69:183-215.

Stout, V. 1994. Regulation of capsule synthesis includes interactions of the RcsC/RcsB regulatory pair. Res. Microbiol. 145:389-392.

Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature. 406:959-964.

Suh, S. J., L. Silo-Suh, D. E. Woods, D. J. Hassett, S. E. West, and D. E. Ohman. 1999. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol. 181:3890-3897.

Tsuzuki, M., K. Ishige, and T. Mizuno. 1995. Phosphotransfer circuitry of the putative multi-signal transducer, ArcB, of Escherichia coli: in vitro studies with mutants. Mol. Microbiol 18:953-962.

Takeda, S., Y. Fujisawa, M. matsubara., H. Aiba., and T. Mizuno. 2001. A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC-YojN- RcsB signaling pathway implicated in capsular synthesis and swarming behaviour. Mol. Microbiol. 40: 440-450.

Vallet I., J. W. Olson, S. Lory, A. Lazdunski, and A. Filloux. 2001. The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. USA. 98:6911-6916.

West A. H., and A. M. Stock. 2001. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 26:369-376

Wimpenny, J. W. T., and R. Colasanti. 1997. A unifying hypothesis for structure of microbial biofilms based on cellular automaton models. FEMS Microbiol Ecol. 22:1-16.

Wise, A. A., and C. W. Prise. 1995. Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor �禒 in response to environmental signals. J. Bacteriol. 177:123-133.

Wurgler-Murphy, S. M., and H. Saito. 1997. Two-component signal transducers and MAPK cascades. Trends Biochem. Sci. 22:172-176.

Xu, K. D., M. J. Franklin, C. H. Park, G. A. McFeters, and P. S. Stewart. 2001. Gene expression and protein levels of the stationary phase sigma factors, RpoS, in continuously-fed Pseudomonas aeruginosa biofilm. FEMS Microbiol. Lett. 199:67-71.

Xu Q., and H. A. West. 1999. conservation of structure and function among histidine-containing phosphotransfer (Hpt) domains as revealed by the crystal structure of YPD1. J. Mol. Biol. 292: 1039-1050.

Zhou, L., X. H. Lei, B. R. Bochner, and B. L. Wanner. 2003. Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two- component systems. J. Bacteriol. 185:4956-4972.