綠膿桿菌PAO1中兩個雙功能酵素PslB和PA3346功能特性之研究與克雷白氏肺炎桿菌CG43 galU突變株在半乳糖壓力下其轉錄體之分析

Abstract

本論文共分為三個章節，第一章和第二章主要是探討綠膿桿菌PAO1中兩個雙功能酵素PslB與PA3346之酵素特性，第三章則是分析克雷白氏肺炎桿菌CG43尿嘧啶雙磷酸葡萄糖焦磷酸化酶(GalU)突變株在半乳糖存在下其基因轉錄之變化。 綠膿桿菌中PslB是具有磷酸甘露糖異構酶(PMI)與鳥苷雙磷酸甘露糖焦磷酸化酶(GDP-man PPase)的雙功能酵素，分別參與鳥苷雙磷酸甘露糖(GDP-man)合成的第一步和第三步催化反應。而目前為止，PslB的酵素活性及參與催化反應的重要胺基酸位置尚未被報導。在第一章的研究裡，我們證實了PslB的確具有PMI與GDP-man PPase的酵素功能。GDP-man PPase 酵素反應需要正二價鎂離子的幫助；PMI酵素反應則是需要正二價鈷離子，而且其活性會被GDP-man所抑制。另外，2, 3-丁二酮(2,3-butanedione)能將PslB的PMI活性去活化，顯示PslB上某個位置的精胺酸(Arg)可能在催化過程中扮演重要的角色。我們進一步利用點突變的實驗技術，發現將PslB第408號位置的精胺酸更改為賴胺酸(Lys)或者丙胺酸(Ala)後，其PMI的活性則是完全消失。經由圓二色光譜的分析，野生株及定點突變株的PslB在蛋白質二級結構上未有改變。此結果證實PslB Arg408的確是參與催化反應的重要胺基酸，亦可做為未來了解PMI催化機制的初步依據。 第二章主要研究綠膿桿菌另一雙功能酵素PA3346。先前的研究發現，HptB訊息傳遞路徑在綠膿桿菌PAO1細菌群體移動與生物膜形成中扮演重要的調控角色。當細菌接受到環境的刺激，感應激酶蛋白(PA1611, PA1976, PA2824)會自我磷酸化，其磷酸根再經由HptB傳遞給下游的反應調控子PA3346。PA3346 N端磷酸化後，使得其絲胺酸蛋白磷酸酶活性增加，並將anti-sigma factor拮抗子PA3347 Ser56上之磷酸根去除。然而，能將PA3347 Ser56位置磷酸化的絲胺酸蛋白激酶仍然未知。經由蛋白質結構域分析，我們發現PA3346C端(PA3346-L408-A571)的結構域與枯草桿菌的SpoIIAB(絲胺酸蛋白激酶/anti-sigma factor)極為相似，因此推測PA3346(L408-A571)具有絲胺酸蛋白激酶的活性；而且，PA3346-PA3347可能與枯草桿菌SpoIIE-SpoIIAB-SpoIIAA的調控系統類似，形成所謂的partner-switching調控模組。本論文建構PA3346(L408-A571)與PA3347重組蛋白質，以進行試管內磷酸化反應。結果證實PA3346(L408-A571)能將PA3347 Ser56位置磷酸化，其絲胺酸蛋白激酶的活性需要Mg2+, Ca2+與Mn2+等正二價金屬離子的幫助。在進行GST pull-down assay與GFP assembly assay後，我們發現PA3346(L408-A571)與PA3347蛋白質不管是在試管內或是在細菌體內，都能夠互相結合。這些結果證實了PA3346(L408-A571)的確具有絲胺酸蛋白激酶/anti-sigma factor的功能。為了尋找參與partner-switching調控系統的sigma factor，我們亦進行了雙分子螢光互補實驗，而詳細的分子調控機制需要進一步的探討研究。 本論文的第三章，主要對克雷白氏肺炎桿菌galU突變株進行研究。當使用galU突變株進行半乳糖紙錠擴散實驗時，紙錠周圍會出現清晰的抑菌圈；將細菌培養在以2%甘油為主要碳源的M9培養液時，加入半乳糖會使得細菌生長緩慢並且有死亡的現象。半乳糖對克雷白氏肺炎桿菌galU突變株的毒性，主要是因為尿嘧啶雙磷酸葡萄糖焦磷酸化酶(UDP-glc PPase)缺損，導致了有毒的半乳糖代謝產物累積。然而，這些代謝產物如何影響細菌基因表現及造成細菌死亡的原因仍然未知。因此，我們利用RNA-seq技術研究在半乳糖存在的狀況下，克雷白氏肺炎桿菌galU突變株的基因表現變化。我們發現在半乳糖的存在下，galETKM、galP、lacYZ以及胺基酸合成相關的基因表現有上升的趨勢；而甘油代謝(pduCDE)、鐵離子攝取系統(sitABCD、feoABC)與12個調控因子(hns、csrA與10個轉錄調控子)的基因表現則被抑制。廣泛型調控因子H-NS與CsrA控制許多細菌的重要生理作用，如碳源代謝、毒性、移動與應激反應系統。我們推測本研究使用的培養條件使得有毒的半乳糖代謝產物累積，並間接地抑制H-NS、CsrA與其它轉錄調控子的基因表現，影響細菌的轉譯與轉錄作用，最後因為生理系統紊亂導致死亡。

This thesis consists of three chapters. Chapter 1 and chapter 2 describe the functional studies of two bifunctional enzymes, PslB and PA3346, in Pseudomonas aeruginosa PAO1. Chapter 3 describes the results of transcriptome analysis of Klebsiella pneumoniae CG43S3 ∆galU mutant under galactose stress. Pseudomonas aeruginosa pslB gene encodes a bifunctional enzyme phosphomannose isomerase/GDP-D-mannose pyrophosphorylase (PMI-GDP-man PPase). The enzyme catalyzes the first and third steps in the GDP-D-mannose biosynthetic pathway, an important precursor of many polysaccharides. So far, very little is known about PslB. In Chapter 1, we demonstrate that Pseudomonas aeruginosa pslB encodes a protein with GDP-man PPase/PMI dual activities. The GDP-man PPase activity is Mg2+dependent, whereas the PMI activity is Co2+dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the PMI activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8- to 20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. The CD spectra of R408A, R408K and wild type PslB are nearly identical, indicating that there is nearly no alterations of their secondary structures. Overall, these results provide a basis for understanding the catalytic mechanism of PMI. In chapter 2, we focus on the other bifunctional enzyme PA3346. We have previously observed that the HptB-mediated phosphorelay pathway plays an important role in swarming phenotype and biofilm formation in P. aeruginosa PAO1. Upon activation by an environmental stress, sensor kinase (PA1611, PA1976 and PA2824) autophosphorylates itself and then transfers a phosphoryl group to HptB, which then relays the signal to response regulator PA3346. The phosphorylation on PA3346 N-terminal receiver domain results in an increase in its Ser protein phosphatase activity leading to dephosphorylation of the putative anti-sigma factor antagonist PA3347. While the target phosphorylation site on PA3347 has been shown to be located at Ser-56, the corresponding serine protein kinase and anti-sigma factor remain elusive. Protein domain analysis revealed that the C-terminal region of PA3346 (PA3346-L408-A571) contains conserved domains similar to the Ser protein kinase/anti-sigma factor SpoIIAB in Bacillus subtilis. Thus, we proposed that PA3346-PA3347 forms a partner-switching regulatory module as observed for SpoIIE-SpoIIAB-SpoIIAA in B. subtilis. This thesis has cloned and purified PA3346(L408-A571) and PA3347-GST for in vitro phosphorylation and GST pull-down assays. The results clearly demonstrate that PA3346(L408-A571) possesses a kinase activity toward PA3347 and the activity is divalent cation, especially Mg2+, Ca2+ and Mn2+ dependent. The PA3347-S56A mutant protein could not serve as a substrate for the kinase. In the GST pull-down assay, PA3346(L408-A571) could be co-eluted with PA3347-GST in the presence of ADP. As for the GFP fragment reassembly assay, the bacterial cells harboring the plasmids of PA3346(L408-A571)-CGFP and PA3347-NGFP display the green fluorescence signals. These data indicate that PA3346(L408-A571) is a serine protein kinase/anti-sigma factor playing a key role in the partner-switching regulatory module. In order to identify the specific sigma factor participated in this partner-switching system, in vivo bimolecular fluorescence complementation assay are performed and the detailed molecular regulation mechanism is under investigation. Chapter 3 focuses on the response of K. pneumoniae CG43 ΔgalU mutant to the galactose stress. K. pneumoniae ΔgalU mutant, which is defective in UDP-Glc PPase, exhibited a growth-inhibition zone in the galactose disk diffusion assay. Galactose can increase the mortality when the ΔgalU mutant was cultivated in M9 minimal medium with 2% glycerol as the sole carbon source. The toxic galatose metabolite affecting the gene expression and causing the cell death remains unclear. Therefore, we investigate the gene expression profiles of the K. pneumoniae ∆galU mutant under the galactose stress by RNA-seq. The results indicate that galETKM, galP, lacYZ and genes responsible for biosynthesis of certain amino acids are upregulated in the presence of galactose. The gene expressions of glycerol metabolism (pduCDE), iron-acquisition systems (sitABCD and feoABC) and 12 regulatory factors (hns, csrA and 10 transcriptional regulators) are repressed. Notably, H-NS and CsrA are global regulatory proteins controlling a variety of physiological processes. Besides, E. coli csrA gene was shown to be essential for bacterial growth in both LB and minimal medium. The toxic galactose metabolites might indirectly repress the gene transcription of H-NS, CsrA and several other transcriptional regulators and lead to the bacterial growth arrest and death.