THE ROLE OF A PORIN-CYTOCHROME FUSION IN NEUTROPHILIC FE OXIDATION: INSIGHTS FROM FUNCTIONAL CHARACTARIZATION AND METATRANSCRIPTOMICS by Arkadiy Garber A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Geology Summer 2018 © 2018 Arkadiy Garber All Rights Reserved THE ROLE OF A PORIN-CYTOCHROME FUSION IN NEUTROPHILIC FE OXIDATION: INSIGHTS FROM FUNCTIONAL CHARACTARIZATION AND METATRANSCRIPTOMICS by Arkadiy Garber Approved: __________________________________________________________ Clara Chan, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Neil Sturchio, Ph.D. Chair of the Department of Geological Sciences Approved: __________________________________________________________ Estella Atekwana, Ph.D. Dean of the College of Earth, Ocean, and Environment Approved: __________________________________________________________ Ann L. Ardis, Ph.D. Senior Vice Provost for the Office of Graduate and Professional Education ACKNOWLEDGMENTS I would like to thank my advisor, Clara Chan, for all the support and guidance over the last years. In addition to learning many valuable skills, my growth as a scientist would not have been possible without the direction and focus that Clara’s mentorship has instilled in me. Additionally, I would like to thank Sean McAllister for all the guidance and feedback; my understanding of bioinformatics and phylogenetics has benefited greatly from our many conversations. I would also like to thank other Chan lab members, past and present, for always being available and willing to help out and provide feedback, and for providing a great atmosphere to do science in. Thank you to my committee members, Dr. Thomas Hanson and Dr. Shawn Polson, for guidance and feedback on my research. Dr. Hanson’s extensive understanding of microbial physiology and Dr. Polson’s intimate knowledge of bioinformatics have been limitless resources during my time at the University of Delaware. Thank you to Dr. Sharon Rozovsky for kindly providing the resources for me to learn about protein expression and purification, as well as for advice about project design. This thesis is dedicated to my parents and sister, who have always supported me in my research endeavors, even without much understanding of what it is that I actually study. iii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vii LIST OF FIGURES .......................................................................................................... viii ABSTRACT ..................................................................................................................... xii Chapter 1 INTRODUCTION ................................................................................................. 1 1.1 Motivation to understand Fe-oxidation mechanisms .............................. 1 1.2 Fe oxidation in acidophilic FeOB. ............................................................. 2 1.3 Fe oxidation in neutrophilic FeOB. ........................................................... 4 1.4 Phylogenetic distribution of cyc2 ............................................................. 8 1.5 Hypotheses and overall approach ............................................................ 9 2 FUNCTIONAL CHARACTERIZATION OF CYCPV-1 ................................................. 11 2.1 Section Introduction ............................................................................... 11 2.2 2.2 Materials and Methods .................................................................... 12 2.2.1 Design of Escherichia coli expression system ............................. 12 2.2.2 Expression of Cyc2 and control vectors ...................................... 14 2.2.3 Western blot and heme stain ..................................................... 15 2.2.4 Whole-cell Fe oxidation assay .................................................... 16 2.2.5 Cyc2 purification ......................................................................... 17 2.3 2.3 Results and Discussion ...................................................................... 18 2.3.1 Expression of Cyc2 and controls in E. coli ................................... 18 2.3.2 Western blot and heme stain identification of Cyc2 and Cyc2 porin ........................................................................................... 21 2.3.3 Fe oxidation by Cyc2-expressing cells ......................................... 23 2.3.4 Cyc2 expression without successful heme insertion .................. 25 2.3.5 Inhibition of Fe oxidation activity using sodium azide ............... 27 2.3.6 E. coli growth anaerobically with nitrate .................................... 28 2.3.7 Preliminary attempts for Cyc2 purification: ............................... 30 iv 2.4 Conclusions ............................................................................................. 32 3 CYC2 EXPRESSION IN THE ENVIRONMENT ...................................................... 34 3.1 Section Introduction ............................................................................... 34 3.2 Materials and Methods .......................................................................... 34 3.2.1 Development and Use of HMM Profiles ..................................... 34 3.2.2 Re-analysis of the metagenome/metatranscriptome from the Rifle aquifer ................................................................................ 35 3.2.3 Re-analysis of the metatranscriptome from Brocéliande stream ......................................................................................... 37 3.3 Results and Discussion ........................................................................... 38 3.3.1 Expression of cyc2 in the Rifle Aquifer ....................................... 38 3.3.2 Re-analysis of the metatranscriptome from Brocéliande stream ......................................................................................... 40 3.3.3 Genomic Co-occurrence of cyc2 and other EET enzymes ........... 47 3.4 Conclusions ............................................................................................. 50 4 INSIGHTS INTO DIVERSITY OF CYC2 ................................................................. 51 4.1 Introduction ............................................................................................ 51 4.2 Materials and Methods .......................................................................... 51 4.2.1 Identification of Cyc2 and MtrCAB Homologs Within Sequence Databases ................................................................................... 51 4.2.2 Multiple Sequence Alignments of Cyc2 and MtrA homologs ..... 52 4.2.3 Cyc2 and MtrA Amino Acid Identity Analysis ............................. 52 4.2.4 Hidden Markov Model (HMM) Validation .................................. 53 4.3 Results and Discussion ........................................................................... 54 4.3.1 Cyc2 lateral gene transfer ........................................................... 54 4.3.2 Cyc2 tertiary structure ................................................................ 55 4.3.3 HMM Validation and Identification of Cyc2-related “standalone” cytochromes ......................................................... 58 4.4 Conclusions ............................................................................................. 65 v 5 PRELIMINARY WORK WITH SIDEROXYDANS LITHOTROPHICUS ES-1: CYC2 EXPRESSION UNDER GROWTH ON THIOSULFATE AND FE(II) .......................... 67 5.1 Introduction ............................................................................................ 67 5.2 Methods ................................................................................................. 68 5.2.1 Culturing of Sideroxydans lithotrophicus ES-1: .......................... 68 5.2.2 Ferrozine measurements ............................................................ 68 5.2.3 Culture harvesting, DNA/RNA extraction, and molecular analyses: ..................................................................................... 69 5.3 Results .................................................................................................... 70 5.3.1 Growth of Sideroxydans lithotrophicus ES-1: ............................. 70 5.3.2 Amplification of cyc2 from ES-1 DNA: ........................................ 72 5.3.3 Amplification of cyc2 from ES-1 RNA: ......................................... 73 5.4 Conclusions ............................................................................................. 74 REFERENCES ................................................................................................................ 75 Appendix SUPPLEMENTARY FIGURES FOR CYC2 EXPRESSION IN THE ENVIRONMENT .. 82 vi LIST OF TABLES Table 2.1: Final gene construct sequences, including the OmpA signal sequence (italicized), TEV site (underlined), and Strep Tag (bolded). .................... 14 Table 2.2: Oxygen concentrations within sterile LB and suspensions of E. coli cells expressing Cyc2 and controls. Cells were at incubated at an optical density of 2 (109 cells/ml) for 5 minutes with vigorous stirring (300 RPM) during measurement of oxygen concentrations (error intervals reflect the fluctuation during those five minutes). Measurement of oxygen in sterile LB represents