Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2013 Study of Genes Relating To Degradation of Aromatic Compounds and Carbon Metabolism in Mycobacterium Sp. Strain KMS Chun Zhang Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biology Commons, Environmental Sciences Commons, and the Microbiology Commons Recommended Citation Zhang, Chun, "Study of Genes Relating To Degradation of Aromatic Compounds and Carbon Metabolism in Mycobacterium Sp. Strain KMS" (2013). All Graduate Theses and Dissertations. 1532. https://digitalcommons.usu.edu/etd/1532 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. STUDY OF GENES RELATING TO DEGRADATION OF AROMATIC COMPOUNDS AND CARBON METABOLISM IN MYCOBACTERIUM SP . STRAIN KMS by Chun Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biology Approved: ____________________________ ____________________________ Anne J. Anderson, Ph.D. Dennis L. Welker, Ph.D. Major Professor Committee Member ____________________________ ____________________________ Jeanette M. Norton, Ph.D. Ronald C. Sims, Ph.D. Committee Member Committee Member ____________________________ ____________________________ Charles D. Miller, Ph.D. Mark R. McLellan, Ph.D. Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2013 ii Copyright © Chun Zhang 2013 All Rights Reserved iii ABSTRACT Study of Genes Relating to Degradation of Aromatic Compounds and Carbon Metabolism in Mycobacterium sp . Strain KMS by Chun Zhang, Doctor of Philosophy Utah State University, 2013 Major Professor: Dr. Anne J. Anderson Department: Biology Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp . strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene- degrading gene islands that also were found with high conservation in five other pyrene- degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. iv KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA , encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA . Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization. (285 pages) v PUBLIC ABSTRACT Polycyclic aromatic hydrocarbons are produced from incomplete combustion of organic materials by human or natural activities. These polycyclic aromatic hydrocarbons are classified as pollutants because of their toxic, mutagenic, and carcinogenic characteristics. Mycobacterium sp . strain KMS, isolated from a contaminated soil, grows on the model polycyclic aromatic hydrocarbon, pyrene, with its degradation to water and carbon dioxide. This study locates genes on the chromosome and plasmids of isolate KMS relating to pyrene degradation, elucidates the influence of other carbon sources available in the habitats of isolate KMS on degradation of pyrene, and deduces possible metabolic pathways used by isolate KMS for its survival. Pyrene-degrading genes are clustered on the KMS chromosome. Duplication of some of the genes may help the degradation of pyrene by KMS in contaminated soils. Pyrene- degrading genes are clustered and are in similar positions in the genome of six pyrene- degrading Mycobacterium isolates. Those genes were possibly acquired from a common ancestor. Isolate KMS also grows on a simple aromatic compound, benzoate. The presence of benzoate does not help pyrene degradation by inducing the pyrene-degrading genes. Also the presence of carbon sources typical of those in the rhizosphere, such as sugars, organic acids, and benzoate, delayed utilization of pyrene until these carbon sources were utilized. Benzoate did not repress growth on the sugars or organic acids. Analysis of gene transcription and enzyme activities revealed that isolate KMS had novel pathways for interconversion of carbon compounds required for effective cell growth, especially for the structures required for the thick cell wall of the mycobacteria. vi ACKNOWLEDGMENTS I would like to appreciate my major advisor, Dr. Anne J. Anderson, for your guidance, help, support, and inspiration on my study. When I began my study in Utah State University, I had few experience on how to make presentation and how to write a logical paper in English. Spending lots of efforts and time on my study, Dr. Anderson trained me how to think, how to organize the sentences in the writing and how to prove my ideas in scientific researches. I’ll memorize your kindly help. I am appreciative to my committee members, Dr. Charles D. Miller, Dr. Dennis L. Welker, Dr. Jeanette M. Norton, and Dr. Ronald C. Sims, for discussing experiments, reviewing papers, enlightening ideas, supporting my study, and using the machines. I will remember your kindly help. I will thank Dr. Jon Takemoto, Dr. Brad Kropp, Dr. Joseph Li, Dr. John Stark, Dr. Andy Anderson, Dr. Paul Cliften, Dr. Dane Hansen, Dr. Alan Savitzky, Dr. Edmund Brodie, Dr. Marie Walsh, Dr. Jeff Broadbent, Dr. Yajun Wu, Dr. Judith Sims, Dr. David Britt, Dr. Ningling Yin, Nancy Kay Harrison, Kami Mcneil, and Brian Joy for using the machines in my research and for your advice and help in my research. I will also thank the people in my lab and students from the Department of Biology and Biological Engineering: Dr. Young Choel Kim, Dr. Christian Dimkpa, Dr. Songhee Han, Brian Pettee, Dr. Tian Yu, Dr. Sitaram Harihar, Dr. Yun Peng, Dr. Pin Liu, Dr. Junling Huo, Yukie Kawasaki, Sanjib Shrestha, Angela Cefalo, Han Xu, and Alyssa Calder for your help in my research. Thanks are also given to the Department of Biology, Utah State University, and the nice people in Logan, Utah. vii I acknowledge the Utah Water Research Laboratory for partial financial support for my research. Finally, I would like to thank my parents, Qingwei Zhang and Mengling Gao, for your encouragement and love as well as my friends Han Feng, Dong Wang, Hong Lv, Yi Ren, Dongliang Wang, Danning Huang, Jiahua Cao, Hui Wang, Yongjia Wang, Lei Quan, Wang Tian, Dongli Wang, Ji Qi, Hao Yu, Hao Feng, Yue Li, Lin Yang, Gary Chiu, Frankie Chiu, Fluorence Chiu, Wenbin Yu, Mingbo Li, Guanchiang Chen, Xin Dai, Chet Lu, Siewsun Wong, Mai Li, Lei Ye, Yongjun Tang, Dong Chen, Zhonghua Xu, and Ce Wang for your concern and friendship. Chun Zhang viii CONTENTS Page ABSTRACT………………………………………………………………………….......iii PUBLIC ABSTRACT…………………………………………………………………….v ACKNOWLEDGMENTS……………………………………………...…….…….......vi LIST OF TABLES……………………………………………………………………….xii LIST OF FIGURES………………………………………………………….…..…...…xiv LIST OF ABBREVIATIONS……………………………….………………..………..xvii CHAPTER 1 INTRODUCTION……………………………………………………….….…….1 Outline of dissertation…………….………………………..…..………..…..1 Chemical structures and origins of aromatic hydrocarbons…….………...….4 Biological risks from aromatic hydrocarbons……………...………………..7 Biodegradation of PAHs by microbes in soils………..……..……..….…8 Mycobacterium sp . strain KMS…………………………………………..….12 Pyrene-degradation mechanisms in mycobacteria………..……..…..…….....15 Summary of pyrene-degradation pathways in isolates KMS and PYR-1………………………………….…………………………15 Components of ring-hydroxylating dioxygenases…………..………..…20 Organization of pyrene-degrading genes in Mycobacterium