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UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Bioinformatic functional characterization of the prokaryotic FUPAs through co-localization and co- regulatory analysis : taking the FU out of the FUPAs Permalink https://escholarship.org/uc/item/9h99s37m Author De La Mare, Russell Wade Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Bioinformatic Functional Characterization of the Prokaryotic FUPAs through Co-localization and Co-regulatory Analysis: Taking the FU out of the FUPAs A thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Russell Wade De La Mare Committee in charge: Milton H. Saier, Jr., Chair Kathleen French Eric Allen 2012 The Thesis of Russell Wade De La Mare is approved and it is acceptable in quality and form for publication on microfilm and electronically: _________________________________________________________________ _________________________________________________________________ _________________________________________________________________ Chair University of California, San Diego 2012 iii DEDICATION I dedicate this thesis to my family, friends, Dr. Saier and all of the members of Saier Lab with whom I have had the pleasure of working and those whose work made this project possible. This thesis is also dedicated to all of the professors who have prepared me for this and future undertakings, especially Dr. Kathy French, for providing much of the mentorship I sorely needed. A huge “thank you” is owed to Rostislav Castillo, Dorjee Tamang, Andrei Osterman, Dmitry Rodionov and Pavel Novichkov for their support and guidance. Naturally, I would like to thank Dr. Milton H. Saier, Jr., for his guidance, support and friendship, as well as his passion and enthusiasm for scientific research, which he has inspired in many students, me included. In the four years that I have known Dr. Saier, I have learned a lot about science and life in general. From times when we were alone in the lab to those when it was so crowded one couldn’t hear themselves think to that time when no one was allowed in at all for all the wrong reasons, he has been a fine friend and a mentor, as well as inspiring us to work hard by setting the example. As Dr. Saier once told me, he made it his responsibility to “raise the kids in the lab”. Dr. Saier’s work beyond academia has also inspired me and expanded my worldview in ways I am glad I will never part from. I sincerely thank Dr. Saier and all of the members of Saier Lab. iv EPIGRAPH Insert clever quote here Anonymous v TABLE OF CONTENTS Signature Page …………………………………………………...……………… iii Dedication ……………………………………………………….……………… iv Epigraph ………………………………………………………………………… v Table of Contents ……………...………………………………………………… vi Acknowledgements ……………………………………………………………… viii Abstract of the Thesis …………………………………………………………… ix Introduction ……………………………………………………………………… 1 Methods …………………………………………………………………………. 4 FUPA23 ATPase Family .………………………..………….......……………… 6 FUPA24 ATPase Family ………………………………………..……………… 32 FUPA25 ATPase Family …………………………………..…………………… 59 FUPA26 ATPase Family ………………………………..……………………… 103 FUPA27 ATPase Family .………………………………….…………………… 113 FUPA28 ATPase Family ………………………………………..……….……… 166 FUPA29 ATPase Family ………………………………………………………………169 FUPA30 ATPase Family ………………………………..……………………… 205 vi FUPA31 ATPase Family .………………………………….…………………… 243 FUPA32 ATPase Family ………………………………………..……….……… 253 Discussion…..………………………………………………………………….. 303 Table 1: Summary of functional predictions made for FUPA23-32 .…………… 310 References ……………………………..………………………………………… 311 vii ACKNOWLEDGEMENTS I would like to acknowledge Dr. Milton H. Saier, Jr., for his support and for serving as the chair of my committee. Similarly, I wish to acknowledge Dr. Kathleen French and Dr. Eric Allen for taking the time and making the effort to serve on my committee. Having enjoyed taking one engaging course and assisting with teaching thrice more with Dr. French, it is an honor to have her evaluate my performance once again, in the context of a thesis defense. Dr. Allen has been especially generous and accommodating in a pinch with his time and advice. Aside from Dr. Saier, Rostislav Castillo and Andrei Osterman were my primary sources of mentorship and were invaluable in helping me get over the learning curve for the research performed in our lab. I am grateful to both them both and they most certainly deserve honorary mentions. I would like to acknowledge Dorjee Tamang, Robert Olson, Dmitry Rodionov and Pavel Novichkov for all of their help on the technical side of this project. I thank Andrew Lukosus for all of his help on the administrative side and Mark Whelan for his work as a TA coordinator, which allowed me to experience teaching a myriad of lecture courses. viii ABSTRACT OF THE THESIS Bioinformatic Functional Characterization of the Prokaryotic FUPAs through Co- localization and Co-regulatory Analysis: Taking the FU out of the FUPAs by Russell Wade De La Mare Master of Science in Biology University of California, San Diego, 2012 Professor Milton H. Saier, Jr., Chair P-type ATPases are ubiquitous in all domains of life. Chan, et al. (2010) analyzed P-type ATPases in all the major prokaryotic phyla for which complete genome sequence data were available. The P-type ATPase superfamily consists of thirty-two recognized families, 17 of which are strictly found in prokaryotes. The first seven of these are Families 1-7, which are generally well characterized. Ten functionally uncharacterized ix P-type ATPase (FUPA) families were identified in as well, FUPA23 through FUPA32. Here, these families are analyzed individually rather than by phyla using the genomic context program SEED (Overbeek et al., 2005) and the co-regulation prediction program RegPredict (Novichkov PS, et al., 2010). By examining the known function of genes co- localized near those encoding FUPAs and using co-localization as a proxy for co- regulation, and finding other genes regulated by similar regulatory sequences, we are able to make highly robust predictions about the putative functions of these ATPases and functionally characterize them. In the process of doing this, unique characteristics of the proteins are illuminated and discussed in the context of phylogeny, environmental context and horizontal gene transfer and how each contributes to the functionality of these ATPases in genomes in which they are found. x Introduction P-type ATPases are found in all domains of life. According to the Transporter Classification Database (http://www.tcdb.org), there are 32 families of P-type ATPases. Nine families are characterized, all as cation transporters, except for Family 8, found only in eukaryotes, which transports phospholipids from the outer leaflet to the inner leaflet of the membrane. Families 23-32 are functionally uncharacterized P-type ATPases, or FUPAs. The use of functional, phylogenetic, and membrane topology information extracted from over 10,000 publications on functional data and novel transport systems has allowed our lab to classify over 5,000 transport proteins into over 600 families. The fruits of this work can be found in the IUBMB approved Transporter Classification Database (http://www.tcdb.org), a curated database which employs the TC system, and which is analogous to the function-only based Enzyme Commission (EC) system (http://www.chem.qmul.ac.uk/iubmb/enzyme/; Saier et al. 2005; 2009). This research emphasizes the synergy of database collaboration as the SEED database (http://pubseed.theseed.org/) and TCDB have been used to augment the content found in both. My study focuses on the functionally uncharacterized P-type ATPases (FUPA) subfamilies (TCID 3.A.3.23-32) of transmembrane proteins such that the gaps of knowledge for these protein subfamilies are bridged, leading to more directed future experiments, and improved resolution and clarity regarding the functions of the FUPAs, so that SEED and TCDB can be updated and expanded. Transport systems play a crucial role in every process of life. Some examples include nutrient transport, metabolite excretion, essential cation acquisition and 1 2 allocation, drug/toxin secretion, establishing electrochemical gradients, macromolecular export, stress response-related transport and transport of signaling molecules (Busch et al. 2002). These functions have all been suggested, to varying degrees, for various P-type ATPases, necessitating consideration for any evidence of each. Stress response-related transport is of special interest here, as many of the FUPA proteins have been implicated in this type of transport (Chan, et al., 2010). UspA expression rises when the organism is exposed to stress conditions. UspA enhances cell survival during prolonged exposure these conditions. The occurrence of multiple universal stress protein-encoding genes amongst many of those genes that encode FUPAs further supports the hypothesis that many of the FUPAs participate in stress- condition response and endurance. The functionally uncharacterized P-type ATPases (FUPA) subfamilies (TCID 3.A.3.23-32), are comprised of over 1000 members spanning the prokaryotic and archaeal domains. Within the prokaryotic domain, these proteins have been identified in both Gram-positive and Gram-negative bacteria. Various genomic sources are found for most phylogenetic clusters, which suggests horizontal gene transfer (HGT) is responsible for some
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