University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2013 Characterization of uncultured, human oral microbiota using a targeted, single-cell genomics approach Alisha Gail Campbell [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Other Microbiology Commons Recommended Citation Campbell, Alisha Gail, "Characterization of uncultured, human oral microbiota using a targeted, single-cell genomics approach. " PhD diss., University of Tennessee, 2013. https://trace.tennessee.edu/utk_graddiss/1703 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Alisha Gail Campbell entitled "Characterization of uncultured, human oral microbiota using a targeted, single-cell genomics approach." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Mircea Podar, Major Professor We have read this dissertation and recommend its acceptance: Alison Buchan, Robert Hettich, Chris Schadt, John Biggerstaff Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Characterization of uncultured, human oral microbiota using a targeted, single-cell genomics approach A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Alisha Gail Campbell May 2013 Copyright © 2013 by Alisha Gail Campbell All rights reserved. ! ""! DEDICATION I dedicate this dissertation to my husband Jim for his patience and unwavering support during my graduate career. In addition, I dedicate this work to my parents, Scott and Linda Jo and Jeff and Marie, who have encouraged me throughout the pursuit of my education. ! """! ACKNOWLEDGEMENTS I would like to acknowledge my advisor Mircea Podar for giving me the opportunity to work on such an exciting project. I would also like to acknowledge my committee members: Drs. Alison Buchan, Robert Hettich, Chris Schadt and John Biggerstaff for the time, comments and suggestions they provided during each of our meetings. I would like to thank several past and present members of the Biosciences Division at ORNL for their friendship, advice and assistance throughout my dissertation work including Migun Shakya, Scott Hamilton-Brehm, Jennifer Mosher, Donna Kridelbaugh, Sarah Kauffman, Zhiwu Wang, Taniya Roy Chowdhury, Steve Allman, Marilyn Kerley, Dawn Klingeman, Sue Carroll and Kim Drew. In addition, I would like to thank the Genome Science and Technology program and its staff including Kay Gardner, Terrie Yeatts and Dr. Albrecht Von Arnim for all of their support during my graduate career. In addition, I would like to thank Dr. Cynthia Peterson for her guidance as I began my graduate experience. I would like to acknowledge Joe Mays at the UT sequencing facility for his great work and helpful discussions. I would also like to thank all of my friends within the GST program that commiserated with me about all the shared experiences of graduate school. I would like to thank my many collaborators including Drs. Jeff Becker and Melinda Hauser at UT, as well as the Tanja Woyke lab at the Joint Genome Institute and collaborators at Ohio State University for the insight and expertise they were able to provide for many aspects of my project. The research presented within this dissertation was supported by grant R01 HG004857 from the National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH) and by grant 1R56DE021567 from the National Institute for Dental and Cranial Research (NIDCR) of the NIH to Mircea Podar. ! "#! Lastly, I would like to thank my entire family for all of their love and support throughout my education, even when they thought I might continue to go to school forever. A special thank you goes to my husband Jim, who believed in me and continued to encourage me despite my occasional anxiety and bad temper. ! #! ABSTRACT Microbial communities associated with the human oral cavity are complex, and many oral microbes have yet to be cultured. These uncultured community members are of interest ecologically and phylogenetically, and a number of uncultured species have been positively correlated with oral diseases such as periodontitis. Thus, an approach was adapted to selectively separate single cells from mixed populations of oral bacteria and obtain genomic information for uncultured community members. A combination of fluorescent labeling, cell sorting with flow cytometry and multiple displacement amplification was used to obtain sufficient genomic material for whole-genome pyrosequencing. The first targets were from uncultured oral lineages within Deltaproteobacteria groups Desulfobulbus and Desulfovibrio, and cells were selectively isolated with group-specific, fluorescent in situ hybridization probes. Deltaproteobacteria were targeted to better understand their increased abundance in periodontitis patients. The resulting oral Deltaproteobacteria genomes encoded several unique proteins that appear to function in survival in a host environment, including proteins similar to virulence factors of known human pathogens. Thus, it is likely that the oral Deltaproteobacteria are not just opportunistic community members of periodontitis sites but also have the potential to play a role in disease etiology. A second target was a low abundance community member belonging to the class Anaerolineae, within Chloroflexi subphylum I. This diverse group of organisms has only a few cultured isolates, and limited genomic information is ! #"! available, making it difficult to discern their ecological significance. Genomic data suggest that oral Anaerolineae are anaerobic heterotrophs with an abundance of carbohydrate transport and metabolism genes. The presence of a unique phosphotransferase system and other genes necessary for N-acetylglucosamine metabolism suggests this organism grows by scavenging material from surrounding lysed bacterial cells. These single-cell genomic studies have provided insight into the ecological niches of two uncultured groups within the oral environment, revealed differences between environmental and host-associated organisms within this groups and provided possible gene targets for future studies. Ultimately, methods employed in these studies could be readily applied to gain information on other uncultured microbes from any environment. ! #""! TABLE OF CONTENTS Chapter 1. The importance of uncultured members of human-associated, oral microbial communities!!!!!!!!!!!!!!!!!!!!!!.1 Chapter 2. Methods for isolation and analysis of single amplified genomes of oral microbiota!!!!!!!!!!!!!!!!...!!!!..!!!!!15 Chapter 3. Multiple single-cell genomes provide insight into functions of uncultured Deltaproteobacteria in the human oral cavity!!!!!!!!.49 Chapter 4. Single-cell genomic analyses of an uncultured member of phylum Chloroflexi found in the human oral cavity!!!!!!!!!!!94 Chapter 5. Conclusions and future directions of the studies of uncultured microbiota!!!!!!!!!!!!!!!!!!!!!!!!!!!!!118 References!!!!!!!!!!!!!!!!!!!!!!!!!!!!...123 Vita!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.136 ! #"""! LIST OF TABLES Table 2.1. Composition of lysis, neutralization and amplification buffers used for single-cell lysis and amplification procedures!!!!!!!!!!!!!..37 Table 2.2. Reagents for the setup of one polymerase chain reaction (50 µL)!.39 Table 2.3. Commands used for genome normalization, assembly and gene prediction!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.43 Table 2.4. Optimization of resulting Velvet assemblies over a range of hash lengths for SAG Dsb3 discussed in Chapter 3!!!!!!!!!!!!!!..44 Table 3.1. Metadata and genome statistics for single and multi-cell oral amplicons!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.59 Table 3.2. Similarity matrix of Desulfobulbus genomes based upon average nucleotide identities!!!!!!!!!!!!!!!!!!!!!!!!!!63 Table 3.3. Similarity matrix of Desulfobulbus genomes based upon tetranucleotide frequencies!!!!!!!!!!!!!!!!!!!!!!...64 Table 3.4. Categories of putative virulence factors found in Dsb1-5 and Dsv1!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.......74-75 Table 3.5. Dsb1-5 contigs that contain large numbers of horizontally acquired polysaccharide metabolism genes!!!!!!!!!!!!!!!!!!.78-79 Table 3.6. COGs unique to either human-associated or environmental Desulfovibrio genomes!!!!!!!!!!!!!!!!!!!!!!!84-85 Table 4.1. Assembly statistics and metadata for combined Chl1-2 genome!.103 ! "$! Table 4.2. COGs present in Chl1-2 and absent in other available Chloroflexi genomes!!!!!!!!!!!!!!!!!!!!!!!!!!!!108-109 ! $! LIST OF FIGURES Figure 1.1. A comparison of a healthy tooth (left) versus the effects of periodontitis (right)!!!!!!!!!!!!!!!!!!!!!!!!..!!..8 Figure 2.1. An overview of single-cell fluorescent labeling and isolation and subsequent genome amplification, sequencing and data processing!!!!!16 Figure 2.2. Output from the flow cytometer showing the selection of a specific group of cells based upon forward scatter (FSC) and fluorescent emission (528-538)!..!!!!!!!!!!!!!!!!!!!!!!!!!!!!...24