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Functional Characterization of the Arginine Transaminase Pathway in Pseudomonas Aeruginosa PAO1

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Functional Characterization of the Arginine Transaminase Pathway in Pseudomonas Aeruginosa PAO1 Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology 11-27-2007 Functional Characterization of the Arginine Transaminase Pathway in Pseudomonas aeruginosa PAO1 Zhe Yang Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Part of the Biology Commons Recommended Citation Yang, Zhe, "Functional Characterization of the Arginine Transaminase Pathway in Pseudomonas aeruginosa PAO1." Dissertation, Georgia State University, 2007. https://scholarworks.gsu.edu/biology_diss/29 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. FUNCTIONAL CHARACTERIZATION OF THE ARGININE TRANSAMINASE PATHWAY IN PSEUDOMONAS AERUGINOSA PAO1 by ZHE YANG Under the Direction of Chung-Dar Lu ABSTRACT Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR. The potential physiological functions of those candidate genes in L-arginine utilization were studied by growth phenotype analysis in knockout mutants. The insertion mutation of aruH encoding an L-arginine:pyruvate transaminase abolished the capability to grow on L-arginine of an aruF mutant devoid of a functional arginine succinyltransferase (AST) pathway, the major route of arginine utilization. The aruH gene was cloned and over-expressed in E. coli. Taking L-arginine and pyruvate as the substrates, the reaction products of recombinant enzyme were identified by MS and HPLC as 2-ketoarginine and L-alanine. Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of ping-pong kinetics mechanism, and the apparent Km and catalytic efficiency (Kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM-1 s-1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM-1 s-1 for L-arginine. Recombinant AruH showed an optimal pH at 9.0 and substrate specificity with an order of preference being Arg > Lys > Met > Leu > Orn > Gln. These data led us to propose the arginine transaminase (ATA) pathway that removes the α-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the ATA pathway. Our latest DNA microarray experiments under D-arginine conditions also revealed PA3863 as the candidate gene encoding D-arginine dehydrogenase which might lead to the recognition of a wider network of arginine metabolism than we previously recognized. INDEX WORDS: arginine catabolism, Pseudomonas aeruginosa, ATA pathway, two- component system, ArgR, DNA microarray, transaminase, kinetics. FUNCTIONAL CHARACTERIZATION OF THE ARGININE TRANSAMINASE PATHWAY IN PSEUDOMONAS AERUGINOSA PAO1 by ZHE YANG A Dissertation Submitted in Partial Fulfillment of Requirements for the Degree of Doctor of Philosophy In the College of Arts and Sciences Georgia State University 2007 Copyright by Zhe Yang and Chung-Dar Lu 2007 FUNCTIONAL CHARACTERIZATION OF THE ARGININE TRANSAMINASE PATHWAY IN PSEUDOMONAS AERUGINOSA PAO1 by ZHE YANG Major Professor: Chung-Dar Lu Committee: Phang C. Tai Jenny J. Yang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2007 iv ACKNOWLEDGEMENTS I would like to first thank my advisor Dr. Chung-Dar Lu for all of his patience and guidance. You spent much time not only sharing your knowledge and discussing the science with me, but also giving me suggestions and directions for my career. I have learned so much from you, which can be of benefit for my career as well as for my whole life. I am also very thankful to my other two committee members, Dr. Phang C. Tai and Dr. Jenny J. Yang, for your interest, advice and critical review of my dissertation. I thank Dr. Dieter Haas for the detailed method of 2-ketoarginine synthesis and Dr. Yoshifumi Itoh for providing the PAO4558 and PAO4566 strains used in this study. I also thank Dr. Michiya Kamio for assistance with HPLC analysis and Dr. Siming Wang for ESI-MS analysis. A special note of thanks goes to Dr. Giovanni Gadda for generously sharing his knowledge and experience in enzyme kinetics. I am grateful to all my labmates and friends, especially Drs. Hosam Ewis, Hassan Wally, Mohamed Hegazy, Shehab Hashim, Dong-Hyeon Kwon, Hsiuchin Yang, Yunfeng Tie, Hao Wang, Bin Na, Congran Li, Xiaozhou Zhang, Han-Ting Chou, Wei Li, Ying-Ju Huang, Chun-Kai Yang, Jinshan Jin and Chun-Ko Ko. You make the lab such an exciting place to stay and I have had a great time with all of you. I am also very grateful to all faculty and staff in the Department of Biology at Georgia State University for their help, support and encouragement. The love and support from my entire family are greatly appreciated. Most importantly, I am deeply indebted to my wife, Jing Song, for her unwavering love, great understanding and endless support, and for bringing our lovely daughter Elaine Yang into v my life. Without her, this dissertation would not have been possible. I am also like to show my whole-heart thankfulness to my parents and my little brother, Yi Yang, Fengxian Zhang and Xinrui Yang, for their unconditional love and kind encouragement. Last but certainly not the least, this dissertation is dedicated to my grandmother, Zhiping Duan, who kindly helped to raise me up and shared me with her positive attitude toward life. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………. iv LIST OF TABLES……………………………………………………………... viii LIST OF FIGURES……………………………………………………………. ix LIST OF ABBREVIATIONS………………………………………………….. xi GENERAL INTRODUCTION………………………………………………… 0.1 Arginine catabolism by microorganisms……………………………. 1 0.2 P. aeruginosa is a good model microorganism to study arginine 2 catabolism……………………………………………………………….. 0.3 ArgR and arginine catabolism in P. aeruginosa……………………. 3 0.4 Proposal of the ADH pathway……………………………………… 4 0.5 DNA microarray and cell metabolism………………………………. 6 CHAPTER ONE: Functional Genomics Approach Enables the Identification of Genes of the Arginine Transaminase Pathway in Pseudomonas aeruginosa 1.1 Introduction…………………………………………………………. 11 1.2 Materials and Methods……………………………………………… 13 1.3 Results………………………………………………………………. 21 1.4 Discussion…………………………………………………………... 31 CHAPTER TWO: Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1………………….. 2.1 Introduction…………………………………………………………. 49 2.2 Materials and Methods……………………………………………… 50 vii 2.3 Results………………………………………………………………. 56 2.4 Discussion…………………………………………………………... 60 CHAPTER THREE: Identification of Candidate Genes for D-arginine Catabolism in Pseudomonas aeruginosa PAO1 Using DNA Microarray……... 3.1 Introduction…………………………………………………………. 75 3.2 Materials and Methods……………………………………………… 76 3.3 Results and Discussion…………………………...…………………. 79 OVERALL SUMMARY………………………………………………………. 88 REFERENCES………………………………………………………………… 92 viii LIST OF TABLES Table 1.1 Bacterial strains and plasmids used in the study. 37 Table 1.2 Microarray analysis of genes induced by L-arginine in the 38 absence of ArgR in P. aeruginosa. Table 1.3 Verification of microarray data by promoter-lacZ fusions. 40 Table 1.4 Measurements of L-arginine transaminase activities in 41 P. aeruginosa PAO1 and its mutant strains. Table 1.5 Growth of P. aeruginosa PAO1 and its mutant strains on 42 L-arginine. Table 1.6 Measurements of β-galactosidase activity in P. aeruginosa strains PAO1 harboring PA4980::lacZ translational fusion 43 plasmid pZY5. Table 1.7 Measurements of β-galactosidase activity in P. aeruginosa strains PAO1 and PAO4558 harboring PA4980::lacZ 44 translational fusion plasmid pZY5. Table 2.1 Substrate specificity of AruH. 63 Table 3.1 Microarray analysis of genes induced by D-arginine in P. 84 aeruginosa PAO1. Table 3.2 Measurements of D-arginine dehydrogenase activities in 85 P. aeruginosa PAO1 and its mutant strains. Table 3.3 Verification of microarray data by promoter-lacZ fusions. 86 ix LIST OF FIGURES Figure 0.1 Diagram of arginine structure and known catabolic reactions. 9 Figure 0.2 Predicted gene expression patterns of the arginine metabolic 10 genes identified by DNA microarray. Figure 1.1 Arginine catabolic pathways in P. aeruginosa PAO1. 45 Figure 1.2 HPLC analysis of the AruI reaction products. 46 Figure 1.3 Conserved gene organization of the aruRS-aruIH locus in 47 pseudomonads. Figure 1.4 Microarray analysis of genes induced by L-arginine in the 48 absence of ArgR in P. aeruginosa Figure 2.1 Purification of the recombinant AruH. 64 Figure 2.2 Dimeric state of recombinant AruH. 65 Figure 2.3 HPLC analysis of the AruH reaction products. 66 Figure 2.4 ESIMS analysis of the AruH reaction products using L- 67 arginine
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