Klebsiella pneumoniae, an opportunistic gram-negative bacterium causing community-acquired diseases, is mostly encapsulated by a considerable thick polysaccharidic capsule acting to protect the bacterium from phagocytosis and prevent from damage by serum factors. Our previous study has demonstrated that deletion of yco6, coding for a protein-tyrosine kinase, in K. pneumoniae CG43 reduced significantly the bacterial mucoidy and content of capsular polysaccharide. KpUgd, Gnd and ManC, required for regulation and synthesis of CPS sugar nucleotide precursors, were able to phosphorylated by KpWzcE23. In this study, we want to demonstrate that phosphorylation affects enzyme activity of ManC and Gnd. ManC is a bifunctional protein, with N-terminal GDP-mannose pyrophosphorylase and C-terminal phosphomannose isomerase activity and Gnd is a 6-phosphogluconate dehydrogenase involved in pentose phosphate pathway. Recombinant ManC and Gnd were purified and treated with calf intestine alkaline phosphatase, and we tested their specific enzyme activities after incubation with or without KpWzcE23. The results showed that the VMAX of Gnd and both PMI domain and GMP domain of ManC were elevated after phosphorylated. We also constructed four recombinant truncated KpUgd fragments, each containing 2 to 7 tyrosine residues, to investigate the phosphotyrosine sites. The results showed all four fragments showed phosphorylation signals by isotope autoradiography. We also tried to search other phophorylation proteins in a phosphoproteomic way, but did not find potential targets. In conclusion, this study have suggested KpUgd, ManC and Gnd are involved in KpWzc-dependent CPS synthesis.

克雷白氏肺炎桿菌為一株伺機性引起院內感染疾病的格蘭氏陰性菌，其外部包覆著由多醣體所組成的厚莢膜。此一莢膜可以讓細菌逃避細胞的吞噬作用以及避免被血清因子所毒殺。在實驗室之前的研究中，證明了剔除酪胺酸激酶的基因KpWzc會明顯地減少其原本所具有的黏性和莢膜多醣體的量。證實了克雷白氏肺炎桿菌蛋白質酪胺酸激酶KpWzc夠將克雷白氏肺炎桿菌中的KpUgd、Gnd和ManC這三個酵素磷酸化。而磷酸化對KpUgd酵素活性有顯著提升。本實驗目的在驗證蛋白質酪胺酸激酶KpWzc對其下游磷酸化蛋白質Gnd, ManC之酵素活性影響以及尋找KpUgd被磷酸化之酪胺酸殘基。ManC 是一個雙功能酵素，前半段具有磷酸甘露糖異位轉化酶活性，後半段則具有磷酸甘露糖異構酶活性，而Gnd 具有磷酸葡萄糖酸去氫酶活性，過量表現與被純化的兩個酵素先經由牛小腸鹼性去磷酸酶處理後，與蛋白質酪胺酸激酶KpWzcE23進行磷酸化反應，並測試期專有酵素活性，結果顯示兩個酵素的三個酵素最大反應速率皆有上升。在此研究之中，我們亦構築了四段截短的KpUgd片段，用以探討磷酸酪胺酸殘基位置，同位素放射能照像結果顯示，四段皆有被偵測到磷酸化訊號，證明蛋白質酪胺酸激酶KpWzcE23可同時對KpUgd上多個酪胺酸殘基進行磷酸化。另外，我們也用磷酸化蛋白質體學技術來尋找細菌內其他可能被磷酸化的蛋白質，但是並沒有找到可能的磷酸化蛋白質。以上實驗結果可推測KpUgd, ManC與Gnd三個酵素皆參與由蛋白質酪胺酸激酶KpWzc調控之莢膜多醣體合成。

Alvarez, D., S. Merino, et al. (2000). "Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates." Infect Immun 68(2): 953-5.
Arakawa, Y., R. Wacharotayankun, et al. (1995). "Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid." J Bacteriol 177(7): 1788-96.
Brown, C. and R. J. Seidler (1973). "Potential pathogens in the environment: Klebsiella pneumoniae, a taxonomic and ecological enigma." Appl Microbiol 25(6): 900-4.
Bugert, P. and K. Geider (1997). "Characterization of the amsI gene product as a low molecular weight acid phosphatase controlling exopolysaccharide synthesis of Erwinia amylovora." FEBS Lett 400(2): 252-6.
Chang, H. Y., J. H. Lee, et al. (1996). "Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43." Microb Pathog 20(5): 255-61.
Crater, D. L. and I. van de Rijn (1995). "Hyaluronic acid synthesis operon (has) expression in group A streptococci." J Biol Chem 270(31): 18452-8.
Dalessandro, G. and D. H. Northcote (1977). "Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir." Biochem J 162(2): 281-8.
Drummelsmith, J. and C. Whitfield (1999). "Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30)." Mol Microbiol 31(5): 1321-32.
Duclos, B., C. Grangeasse, et al. (1996). "Autophosphorylation of a bacterial protein at tyrosine." J Mol Biol 259(5): 891-5.
Grangeasse, C., A. J. Cozzone, et al. (2007). "Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology." Trends Biochem Sci 32(2): 86-94.
Grangeasse, C., P. Doublet, et al. (1997). "Characterization of a bacterial gene encoding an autophosphorylating protein tyrosine kinase." Gene 204(1-2): 259-65.
Grangeasse, C., B. Obadia, et al. (2003). "Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase." J Biol Chem 278(41): 39323-9.
Griffith, C. L., J. S. Klutts, et al. (2004). "UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans." J Biol Chem 279(49): 51669-76.
Hauser, S. C., J. C. Ziurys, et al. (1984). "Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase." J Biol Chem 259(7): 4527-33.
Huang, J. and M. Schell (1995). "Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter." Mol Microbiol 16(5): 977-89.
Jayaratne, P., D. Bronner, et al. (1994). "Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide." J Bacteriol 176(11): 3126-39.
Klein, G., C. Dartigalongue, et al. (2003). "Phosphorylation-mediated regulation of heat shock response in Escherichia coli." Mol Microbiol 48(1): 269-85.
Lin, W. S., T. Cunneen, et al. (1994). "Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus." J Bacteriol 176(22): 7005-16.
Mijakovic, I., D. Petranovic, et al. (2006). "Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine." Nucleic Acids Res 34(5): 1588-96.
Mijakovic, I., S. Poncet, et al. (2003). "Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases." Embo J 22(18): 4709-18.
Morona, J. K., R. Morona, et al. (2002). "Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase." J Bacteriol 184(2): 577-83.
Morona, J. K., J. C. Paton, et al. (2000). "Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in streptococcus pneumoniae." Mol Microbiol 35(6): 1431-42.
Paulsen, I. T., A. M. Beness, et al. (1997). "Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria." Microbiology 143 ( Pt 8): 2685-99.
Podschun, R. and U. Ullmann (1998). "Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors." Clin Microbiol Rev 11(4): 589-603.
Preneta, R., S. Jarraud, et al. (2002). "Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae." Comp Biochem Physiol B Biochem Mol Biol 131(1): 103-12.
Princivalle, M. and A. de Agostini (2002). "Developmental roles of heparan sulfate proteoglycans: a comparative review in Drosophila, mouse and human." Int J Dev Biol 46(3): 267-78.
Rahn, A., K. Beis, et al. (2003). "A novel outer membrane protein, Wzi, is involved in surface assembly of the Escherichia coli K30 group 1 capsule." J Bacteriol 185(19): 5882-90.
Rahn, A., J. Drummelsmith, et al. (1999). "Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae." J Bacteriol 181(7): 2307-13.
Seidler, R. J., M. D. Knittel, et al. (1975). "Potential pathogens in the environment: cultural reactions and nucleic acid studies on Klebsiella pneumoniae from clinical and environmental sources." Appl Microbiol 29(6): 819-25.
Sieberth, V., G. P. Rigg, et al. (1995). "Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes." J Bacteriol 177(15): 4562-5.
Simoons-Smit, A. M., A. M. Verweij-van Vught, et al. (1986). "The role of K antigens as virulence factors in Klebsiella." J Med Microbiol 21(2): 133-7.
Sprenger, G. A. (1995). "Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12." Arch Microbiol 164(5): 324-30.
Warit, S., N. Zhang, et al. (2000). "Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species." Mol Microbiol 36(5): 1156-66.
Whitfield, C. (2006). "Biosynthesis and assembly of capsular polysaccharides in Escherichia coli." Annu Rev Biochem 75: 39-68.
Whitfield, C. and I. S. Roberts (1999). "Structure, assembly and regulation of expression of capsules in Escherichia coli." Mol Microbiol 31(5): 1307-19.
白平輝(2004). "Capsular polysaccharide production and protein-tyrosine phosphorylation in Klebsiella pneumoniae" CG43. NCTU 生物科技研究所 碩士論文
Li Zhi-Kai (2006). "Functional analysis of the core elements, Wza, Yor5, Yco6 and Wzx, involved in capsular polysaccharide biosynthesis in Klebsiella pneumoniae CG43." NCTU 生物科技研究所 碩士論文
Li Mei-Ju (2008). "Identification of phosphotyrosine residues in UDP-glucose dehydrogenase of Klebsiella pneuminae CG43." NCTU 生化工程研究所 碩士論文