ENDOGENOUS LIPID CHEMOATTRACTANTS AND EXTRACELLULAR MATRIX PROTEINS INVOLVED IN DEVELOPMENT OF MYXOCOCCUS XANTHUS by PATRICK DAVID CURTIS (Under the Direction of Lawrence J. Shimkets) ABSTRACT The soil bacterium Myxococcus xanthus is a model organism to study multicellular development and biofilm formation. When starved, swarms of M. xanthus cells aggregate into a multicellular architecture called a fruiting body, wherein cells differentiate into metabolically dormant myxospores. Fruiting body formation requires directed cell movement and production of an extracellular matrix (ECM) to facilitate cell-contact dependent motility (Social motility), and biofilm formation. M. xanthus displays chemotaxis towards phospholipids derived from its membrane containing the rare fatty acid 16:1ω5c. This study demonstrates that 16:1ω5c is primarily found at the sn-1 position within the major membrane phospholipid, phosphatidylethanolamine (PE), which is contrary to the established dogma of fatty acid localization in Gram-negative bacteria. Additionally, 16:1ω5c at the sn-1 position stimulates chemotaxis stronger than 16:1ω5c located at the sn-2 position. These results suggest that the endogenous lipid chemoattractants may serve as a self-recognition system. Chemotaxis towards a self-recognition marker could facilitate movement of cells into aggregation centers. Lipid chemotaxis is dependent on the ECM-associated zinc metalloprotease FibA, suggesting that the ECM may harbor protein components of extracellular signaling pathways. Protein components of prokaryotic biofilms are largely unexplored. Twenty one putative ECM-associated proteins were identified, including FibA. Many are novel proteins. A large portion of the putative ECM proteins have lipoprotein secretion signals, unusual for extracellular proteins. An MXAN4860 pilA mutant displays a 24 hour delay in fruiting body formation and sporulation compared to the pilA parent, indicating that MXAN4860 functions in the FibA-mediated developmental pathway previously described. The ECM provides the main connective network between cells in fruiting bodies and biofilms, and the proteins identified here may be components of novel signaling pathways controlling communal cellular behavior. INDEX WORDS: Myxococcus xanthus, Lipid Chemotaxis, Biofilm, Extracellular Matrix, Hypothetical Proteins, Development ENDOGENOUS LIPID CHEMOATTRACTANTS AND EXTRACELLULAR MATRIX PROTEINS INVOLVED IN DEVELOPMENT OF MYXOCOCCUS XANTHUS by PATRICK DAVID CURTIS B.S., Purdue University, 2001 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2007 © 2007 Patrick David Curtis All Rights Reserved ENDOGENOUS LIPID CHEMOATTRACTANTS AND EXTRACELLULAR MATRIX PROTEINS INVOLVED IN DEVELOPMENT OF MYXOCOCCUS XANTHUS by PATRICK DAVID CURTIS Major Professor: Lawrence J. Shimkets Committee: Russell W. Carlson Harry A. Dailey Timothy R. Hoover Eric V. Stabb Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2007 DEDICATION I dedicate this work to my mother, Diane M. Curtis. She has been a source of encouragement, inspiration and support my entire life, no matter the field or endeavor I put myself to. This is for you, Mom. iv ACKNOWLEDGEMENTS First off I must thank my P.I. Larry Shimkets. His guidance and patience has helped me become to the scientist I am today. I can see his stamp on my thought processes and approach to the scientific method, and I understand why he is well respected not just in the myxobacterial community, but the scientific community at large. I also must thank him for putting up with me during the middle when things got rough, for sticking with me until I emerged on the other side. I would also like to thank my committee members Russ Carlson, Harry Dailey, Tim Hoover and Eric Stabb, who helped me to write better, helped me to apply for postdoctoral positions, helped to refocus and reapply my research each year, and provided moral support, often in the form of Packer games. I also thank the past and present members of the Shimkets lab for their training and support from day 1 in the lab. In particular I must acknowledge Pam Bonner, who was my compatriot, sympathizer, empathizer, psychologist, shoulder-to-lean-on, sister in practice if not blood, and friend for 5 years. Without the people of the lab, I would not have made it as far as I did. To my friends in and out of the department, thank you for making my time here special and full of fond memories. Thank you to the members of my class, in particular James Henriksen who has been my brew partner, philosophical foil, person to argue with like the 80 year-old grumpy men we were born as, and my best friend. Thanks to Turner Classic movies for keeping me sane the first three years, Blizzard Entertainment for keeping me sane the last three years, and ABC Package and The Shroom for keeping me sane at sporadic points in between. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW: METABOLIC PATHWAYS RELEVANT TO PREDATION, SIGNALING, AND DEVELOPMENT...............1 Introduction ...............................................................................................................2 Amino Acid Catabolism............................................................................................4 Purine and Pyrimidine Salvage ...............................................................................15 Lipid Catabolism .....................................................................................................16 Carbohydrate Utilization .........................................................................................21 Lipid Biogenesis......................................................................................................25 Spore-Specific Products ..........................................................................................36 Summary .................................................................................................................40 Purpose ....................................................................................................................41 2 NOVEL LIPIDS IN MYXOCOCCUS XANTHUS AND THEIR ROLE IN CHEMOTAXIS.......................................................................................................54 Introduction .............................................................................................................56 Experimental Procedures.........................................................................................57 vi Results .....................................................................................................................66 Discussion ...............................................................................................................83 3 PROTEINS ASSOCIATED WITH THE MYXOCOCCUS XANTHUS EXTRACELLULAR MATRIX..............................................................................98 Introduction .............................................................................................................99 Experimental Procedures.......................................................................................102 Results ...................................................................................................................111 Discussion .............................................................................................................120 4 CONCLUSION..........................................................................................................135 Lipid Chemotaxis as a Self-Recognition Marker..................................................137 ECM-Mediated Signaling Networks.....................................................................141 Lipoprotein Secretion............................................................................................144 Concluding Thoughts ............................................................................................149 APPENDICES .............................................................................................................................156 A SPATIAL ORGANIZATION OF MYXOCOCCUS XANTHUS DURING FRUITING BODY FORMATION...........................................................................................156 B LIPASES IN MYXOCOCCUS XANTHUS ................................................................169 vii LIST OF TABLES Page Table 2-1: Primers used in construction and examination of plsB and plsC mutants....................59 Table 2-2: Mass Spectrometry – Collision Induced Dissociation analysis of PE in M. xanthus strains.............................................................................................................................69 Table 2-3: PE enriched in sn-1 16:1 stimulates chemotaxis..........................................................80 Table 2-4: 16:1ω5 and 16:1ω7/16:1ω6 content in cultured bacteria and environment samples...82 Table 3-1: Strains Used In This Study.........................................................................................103