Structure Elucidation and Biosynthetic Enzyme Characterization of Bacteriocins by Jeella Zara Acedo A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Alberta © Jeella Zara Acedo, 2017 Abstract Acidocin B (AcdB), a bacteriocin from Lactobacillus acidophilus M46 that was initially reported to be a linear peptide, was purified and shown to be circular based on MALDI-TOF MS and MS/MS sequencing. MS analysis further revealed that AcdB is comprised of 58 amino acid residues, instead of 59 residues as initially reported. The NMR solution structure of AcdB in sodium dodecyl sulfate micelles was solved, revealing that AcdB consists of four !-helices that are folded to form a globular bundle with a central pore. This is the first reported three-dimensional structure (3D) for a subgroup II circular bacteriocin. Comparison of the structure of AcdB to that of carnocyclin A, a subgroup I circular bacteriocin, highlighted the differences between the two subgroups. At least seven putative subgroup II circular bacteriocins were identified using BLAST, and sequence analysis revealed a highly conserved asparagine residue at the leader peptide cleavage sites, suggesting that an asparagine endopeptidase might be involved in their biosynthesis. Lastly, the biosynthetic gene cluster of AcdB was sequenced and characterized. Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins (class IIc) from Lactococcus lactis QU 5 and Staphylococcus aureus A53, respectively. Their 3D NMR solution structures were determined, revealing that both peptides are composed of four !-helices that assume a saposin-like fold with a highly cationic surface and a hydrophobic core. The observed structural motif is remarkably similar to the overall fold of the two-component leaderless bacteriocins, enterocin 7A and 7B. Homology modeling showed that the aforementioned motif may be shared among broad-spectrum leaderless bacteriocins despite the variations in their sequence identities and lengths. The structures ii of LnqQ and AucA were also demonstrated to exhibit certain similarities to those of the circular bacteriocins. Activity assays showed that the two peptides, LnqQ and AucA, combined do not act synergistically and have different antimicrobial spectra and potency, suggesting that sequence disparities play a vital role in their modes of action. Carnobacteriocin X (CbnX) was originally reported as a single-peptide bacteriocin (class IId). However, sequence analysis and synergy assays revealed that CbnX belongs to a two-peptide bacteriocin (class IIb), with CbnY as its partner. CbnXY is the first two- peptide bacteriocin reported in carnobacteria. CbnX and CbnY are inactive individually, but elicit synergistic activity against closely related strains when combined. The NMR solution structures of CbnX and CbnY were elucidated and shown to strongly resemble the structures of other class IIb bacteriocins (i.e. LcnG, PlnEF, PlnJK). CbnX consists of an extended, amphipathic !-helix and a flexible C-terminus. CbnY has two !-helices (one hydrophobic, one amphipathic) connected by a short loop, and a cationic C-terminus. Binding studies showed that CbnX and CbnY do not interact directly, suggesting that a membrane-bound receptor may be required to mediate the formation of the CbnXY complex. Pneumococcin is a two-component lantibiotic, comprised of PneA1 and PneA2, from Streptococcus pneumoniae R6. Its biosynthetic machinery encodes a putative flavin- dependent reductase, named PneJB, which is likely involved in the formation of D-Ala and D-Abu. The activity of PneJB was investigated through the heterologous expression of pneumococcin biosynthetic proteins in Escherichia coli. Coexpression of the precursor peptides (PneA1 and PneA2) and the lantibiotic synthetase (PneM) with and without PneJB produced a mixture of partially modified substrates that could not be separated by iii RP-HPLC. To potentially address this issue, truncated precursor peptides were designed and cloned. The truncated peptides, however, could not be successfully expressed in E. coli. Hence, chemical synthesis of substrate analogues is currently being pursued. Other approaches to obtain the precursor peptides are presented herein. The PneJB enzyme was expressed and purified as a SUMO fusion protein, wherein the SUMO tag could be readily cleaved. The substrate analogues and the PneJB enzyme will consequently be used for in vitro PneJB activity assays and future crystallization trials. iv Preface The content of Chapter 2 was published as Acedo et al. Appl. Environ. Microbiol. 2015, 81, 2910-2918. I performed all the experiments with assistance from the other authors, specifically in the sequencing of the AcdB biosynthetic gene cluster and acquisition of NMR spectroscopic data. I wrote the manuscript except for the section on the gene cluster. Studies described in Chapter 3 were published as Acedo et al. Biochemistry. 2016, 55, 4798-4806. I performed all the experiments with assistance from the other authors, specifically in the cloning of the expression plasmids, acquisition of NMR spectroscopic data, and activity assays. I wrote the manuscript. The studies in Chapter 4 were published as Acedo et al. FEBS Lett. 2017, 591, 1349- 1459. I performed the circular dichroism experiments, expression and purification of [13C,15N]-CbnX and CbnY, and the elucidation of their NMR solution structures. The other authors performed the activity assays, cloning of expression plasmids, binding studies, and assisted with the acquisition of NMR spectroscopic data. I performed approximately 75% of the work and assisted in writing the manuscript. Chapter 5 is an unpublished work. I performed all the experiments described in this chapter. v Acknowledgements This thesis would not have been possible without the help, guidance, and support of several individuals. First and foremost, I am beyond grateful to my supervisor, Prof. John C. Vederas, for giving me the opportunity to do research in his lab. His insights, constant support, and encouragement kept me moving forward and honed my skills as a researcher. The lab environment and resources provided in the Vederas group have been ideal for learning and professional growth. I am thankful to all the people I worked with in the various projects presented in this thesis. I am also very grateful to the exceptional support staff members in the Department of Chemistry who are among the kindest and most approachable people I have worked with. In particular, I would like to thank Mark Miskolzie and Dr. Ryan McKay of the NMR facility, Jing Zheng, Dr. Randy Whittal and Bela Reiz of the MS facility, Wayne Moffat of the Analytical Instrumentation laboratory, and Gareth Lambkin of the Biological Services laboratory. I am also grateful to Pascal Mercier of NANUC for his help in CYANA. I am thankful to Dr. Marco van Belkum, Dr. Conrad Fischer, and Sorina Chiorean for proofreading this thesis, and all their inputs in improving this manuscript. I am thankful to the Alberta Innovates Health Solutions and the University of Alberta for funding my graduate studies. I also would like to acknowledge special people who have been instrumental not only in the completion of this thesis, but also in the success of my graduate studies as a whole. On top of my thank you list is Dr. Marco van Belkum, who has been my mentor since day one. I am extremely grateful for all the things that you taught me, and it was much pleasure working with you. I am thankful to Dr. Christopher Lohans, who also helped me start up in the lab, and has since been a constant source of advice. Thank you vi Chris for unselfishly sharing your time and ideas. To Dr. Shaun McKinnie, thank you for being supportive throughout my graduate studies. I found your inputs in my seminars, scholarship applications, and random research questions very helpful. You and Chris have been inspirational and I look up to both of you for your character and brilliance. To Dr. Kaitlyn Towle, thank you for always being willing to help and for all our fruitful discussions. To Dr. Leah Martin-Visscher, thank you for always being reachable by e-mail to answer my NMR questions, and for dropping by the lab whenever we need help. You have been a very good teacher. To Albert Remus, thank you for assisting with genome work and answering all my genome-related questions. It was wonderful having to work with you. To Randy Sanichar, thank you for being my go-to person for my organic chemistry questions and for being a good friend. To everyone in the Vederas group who offered me advice and assistance at certain points during the last five years, and with whom I have shared some good times with, thank you very much! I am also extremely grateful to my family and friends for the encouragement and support. To my parents, thank you for all your sacrifices, love, and support that paved the way to several amazing opportunities for me, and led me to where I am today. To my husband, Kristian, words are not enough to say how grateful I am to have gone through grad school with you. As a research-partner, you have been an endless source of ideas and your passion for research is inspiring. As a life-partner, your love and support are definitely unmatched. Above all, I am grateful to God for the gift of life and for bringing good people along the way throughout this journey. To God be the glory! vii Table of Contents Chapter 1 ......................................................................................................