STUDIES ON ENVIRONMENTAL RELEVANCE OF QUORUM SENSING SIGNAL DECAY Thesis by Ya-Juan Wang In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2007 (Defended December 8, 2006) ii © 2007 Ya-Juan Wang All Rights Reserved iii Acknowledgements The science of today is the technology of tomorrow. Cultivated by the outstanding scientific soil and atmosphere of Caltech, in rewards I am involved in exploring environmental science striving to contribute to the future technology development and benefit the society at large. In retrospect, my past five-year graduate student life at Caltech could not be this wonderful without so many people to whom I own my greatest gratefulness. First of all, I would like to thank my advisor, Professor Jared Leadbetter. Jared started his lab at Caltech in 2000 and I was lucky enough to be among his first graduate students. Jared impressed me by his passion for science and keen insight into key points on research. Not only did he direct me into this incredible field of quorum sensing, but also he taught me many hands-on techniques. Jared provided supportive and creative research environment and many training opportunities such as conferences, and I cherish all of them. Besides research, I had many wonderful times at the Leadbetters’ with his lovely wife JoEllyn and daughters Chloe and Olivia. I am also grateful for his attendance and very kind toast at my wedding. Jared is a perfect mentor for me. I would like to thank all my other committee members: Professor Janet Hering, Professor Michael Hoffmann, and Professor Dianne Newman, for their advice and help during these years. I did one project in Professor Janet Hering’s lab and learned trace iv metal specification in natural waters. Professor Michael Hoffmann gave me lots of help and encouraged me during my graduate life. I am grateful for the usage of the scintillation counter from Professor Dianne Newman and her critical comments on my manuscript. I also want to thank Professor Paul Wennberg and Professor Jess Adkins who served as committee members on my qualifying exam. I am grateful for many friendly and smart colleagues in the Leadbetter group. In the lab I spent most of my graduate life. I would like to thank Jean Huang, with whom I struggled to understand quorum sensing. Her enthusiasm for science amazes me a lot. Liz Ottesen is a good source of knowledge and has many scientific ideas. Jian Yuan Thum is always ready to help even though she is not in the lab now. Wanwan Yang is an outgoing girl who is good at dancing. Cynthia Collins familiarized us with directed enzyme evolution. Amy Vu is good at finding great deals and we benefited a lot from her information. Suvi Flagan is a smart girl and I hope all the best for her graduate study at University of Washington. Xinning Zhang is the new comer of the lab and she is working on her very cool project on termites. The termite-hunting trips were of a lot of fun. The Leadbetter group is a wonderful place to do science. I would also like to thank current and former postdoctoral fellows. Dr. Eric Matson is experienced in microbiology and molecular biology and I believe that his future career will be successful. Dr. Andrew Hawkins was my officemate and he owned great passion for research and his current industry career must be going on great. Dr. Jong-In Han is now an assistant professor at Rensselaer Polytechnic Institute and I wish him all the best to his research and family. I v also want to thank Dr. Paul Orwin, Dr. Shana Goffredi, Dr. Weng-Ki Ching and Dr. Tina Salmassi for their companion and help. I also want to thank many members in the Environmental Science and Engineering Program. I appreciate all the help from Nathan Dalleska for trouble-shooting the LC/MS problems and Mike Vondrus for making the heat-fitting appliance for my biofilm reactor. I want to thank Linda Scott for her kindest assistance. I thank Fran Matzen and Cecilia Gamboa who made things flow smoothly. My final thanks are reserved for my family. I thank my parents and my younger brother for their love. I want to thank my dearest husband Tingwei Mu who celebrated my triumphs and helped me overcome difficult moments. He has been the most supportive and thoughtful and kindest husband I could wish for. I never thought I could be this happy. With him, life becomes meaningful. vi Abstract Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show for the first time that acyl-HSLs are biodegraded in soils sampled from diverse US sites and by termite hindgut contents. Furthermore, high-affinity acyl-HSL degraders were enriched in oligotrophic biofilm reactors that were inoculated with a typical turf soil. When amended to soil samples at physiologically relevant concentrations, 14C- 14 labeled acyl-HSLs were mineralized to CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 µM acyl-HSL, ca. 1000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol ⋅ h-1 ⋅ g of fresh weight soil-1. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2, but that the two processes were well coupled kinetically. An MPN of 4.6 × 105 cells ⋅ g of turf soil-1 degraded physiologically relevant 14 14 amounts of hexanoyl-[1- C]HSL to CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. The results implicate a real-world challenge for acyl-HSL-producing bacteria to outpace vii biological acyl-HSL degradation and thus to successfully engage in cell-cell communications in soils and other environments. Furthermore, high-affinity acyl-HSL degraders were enriched in oligotrophic biofilm reactors that were inoculated with the turf soil. When supplied at physiological concentration, acyl-HSL was observed to be >95% consumed at certain stage. Greater species diversity was formed in the acyl-HSL-supplied reactor than the otherwise acyl- HSL-absent reactor. The most abundant bacterial species belonged to six different phyla based on 16S rDNA, including Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria, Planctomycetes and Bacteroidetes. Six bacterial strains were successfully isolated from the biofilm reactors and designated Soil Oligotrophic Degraders which are Variovorax strains SOD31 and SOD32, Mesorhizobium strains SOD33 and SOD35, Bradyrhizobium strain SOD34 and Labrys strain SOD36. Unambiguous growth of the six isolates on physiological amount of acyl-HSLs under oligotrophic condition has been demonstrated. In further investigation of Variovorax strain SOD31, the bacterium exhibited an acyl-HSL-limiting growth kinetics with a half-saturation constant of 1.7 µM, which is in high agreement with what we observed from the turf soil. The results suggests that strain SOD31 may be an active contributor to the soil’s activity of degrading acyl- HSL. Rapid signal decay might serve either to quiet signal crosstalk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate viii cellular communications and the accuracy of population density based controls on gene expression in species-rich ecosystems. The successful isolation and cultivation of high- affinity acyl-HSL degraders capable of utilizing physiological concentration of acyl-HSL under oligotrophic conditions will allow determination of the distribution of signal- producing and –degrading microbes in soils and other environment. This advance will lead to improved understanding of signal decay influences quorum sensing and community function and structure in naturally occurring microbial communities. ix Table of Contents Acknowledgements ......................................................................................................iii Abstract........................................................................................................................ vi List of Figures ............................................................................................................xiii List of Tables............................................................................................................... xv Chapter 1 Introduction and Overview........................................................................ 1 1.1 Introduction............................................................................................................ 2 1.1.1 Quorum sensing and signaling molecules of acyl-homoserine lactones ........ 2 1.1.2 Chemical and biologically degradation of acyl-HSLs................................... 3 1.2 Overview of the thesis............................................................................................ 4 1.3 References ............................................................................................................. 7 Chapter 2 Acyl-HSL Signal Decay: Intrinsic to Bacterial Cell-cell