University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2012 Characterization of Chemosensing in the Alphaproteobacterium Azospirillum brasilense Matthew Hamilton Russell University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Organismal Biological Physiology Commons Recommended Citation Russell, Matthew Hamilton, "Characterization of Chemosensing in the Alphaproteobacterium Azospirillum brasilense . " PhD diss., University of Tennessee, 2012. https://trace.tennessee.edu/utk_graddiss/1469 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 Matthew Hamilton Russell entitled "Characterization of Chemosensing in the Alphaproteobacterium Azospirillum brasilense ." 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 Biochemistry and Cellular and Molecular Biology. Gladys M. Alexandre, Major Professor We have read this dissertation and recommend its acceptance: Dan Roberts, Andreas Nebenfuehr, Erik Zinser 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 the chemosensory abilities of the alphaproteobacterium Azospirillum brasilense A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Matthew Hamilton Russell August 2012 ii Copyright © 2012 by Matthew Russell All rights reserved. iii Dedication This body of work is dedicated to my loving, patient wife. Without her patience and support I would not have been able to pursue and complete this endeavor. She serves as an example in life and work for me to follow and I thank her for all she does. I also dedicate this work to our daughter who reminds me daily why all of this is worth it. Thank you both. I love you. iv Acknowledgements I would like to thank Dr. Gladys Alexandre for ALL of her patience and help as I have pursued my dream. Without her passion and guidance to help me become a good scientist, I would have not have become the person I am. All my career endeavors will be a consequence of her passion to answer the basic questions or how biology works. I thank you. I would also like to thank my committee for helpful advice and the opportunity to pursue this Ph.D. degree. I would like to thank Dr. Dan Roberts and Dr. John Koontz for the inspiration to turn my curiosity of how cells work into a career path. I would also like to thank all current and previous lab members for making daily lab life interesting and enjoyable (usually). v Abstract Motile bacteria must navigate their environment in constant search of nutrients to sustain life. Thus they have evolved precise and adaptable sensory systems to achieve this goal, making the navigation system of the model bacterium Escherichia coli the best characterized signal transduction pathway in Biology. However, many bacteria have evolved more sophisticated arsenals for sensing and responding to their environment including chemoreceptors to identify novel attractants in the microenvironment. The diazotrophic alphaproteobacterium Azospirillum brasilense inhabits the soil and colonizes the roots of cereals like rice, corn, and wheat. Like most proteobacterial, A. brasilense encodes multiple chemotaxis-like pathways, 4, of which only Che1 has been characterized in detail. Also, of the approximately 50 chemoreceptors encoded within the genome, only the function of AerC and Tlp1 have been determine and their role in energy taxis, the dominant behavior of A. brasilense. In this dissertation, I will describe the characterization of another chemoreceptor, Tlp2, with a sensing domain of unknown function and the role it plays in A. brasilense behavior. I will also describe my work in expanding knowledge of the chemotaxis-like pathway of Che1. Also, the role of Tlp1 in root colonization, chemotaxis, and aerotaxis, the ability to navigate oxygen gradients, has been published. My work will detail the role of the C-terminal PilZ domain, a domain shown to bind the ubiquitous bacterial second messenger cyclic-di-GMP. I will characterize the necessity of c-di-GMP binding to Tlp1 for cells to maintain the ability to remain sensitive to temporal changes in aeration. I will also discuss the novel role c-di-GMP plays in modulating the cell’s ability to remain motile and remain sensitive to addition changes in oxygen availability. vi Table of Contents Chapter 1 Introduction..................................................................................................................1 Chemotaxis…………………………………………………………...…………………...1 Model Organisms for Chemotaxis……………………………………………………...…2 Diversity in Bacterial Chemotaxis……………………………………………………...…6 Chemoreceptor Architecture and Function………………………………………...….....10 Role of the Signaling Complex for Sensitivity and Amplification…………………...….12 Chemoreceptor Diversity…………………………………………………………...……14 Azospirillum brasilense Chemotaxis Systems from a Genomic Perspective…………….16 A. brasilense sensing, Locomotor Behavior, and Chemoreceptors……………...……....16 Concluding Remarks………………………...……………….…………………………..20 List of References……………………………………………...…………...……………21 Chapter 2 Characterization of a Nitrogen Compound Transducer in Azospirillum brasilense………………………………………………………………………………………....43 Disclosure...…………………………………………………………………..………….44 Abstract……………………………………………………………………...…………...44 Introduction…………………………………………………………………...………….45 Experimental Procedures………………………………………………………...………47 Results…………………………………………………………………………...……….56 Discussion………………………………………………………………………...……...82 List of References……………………………………………………………………......87 Chapter 3 The Azospirillum brasilense Che1 Chemotaxis Pathway Controls the Swimming Speed Which Affects Transient Cell-to-Cell Clumping………………………………….…...96 vii Table of Contents, continued Disclosure…………………………………………...…………………………………...97 Abstract………………………………………………………………...…………….…..97 Introduction……………………………………………………………...…………….....98 Experimental Procedures………………………………………………………...……..101 Results…………………………………………………………...……………………...109 Discussion………………………………………………………...…………………….128 List of References………………………………………………………………...…….137 Chapter 4 Characterization of the PilZ Domain of Azospirillum brasilense Tlp1 and its Role in Modulating Cellular Response During Aerotaxis……………………………………...….143 Disclosure……………………………………...……………………………………….144 Abstract…………………………………………...…………………………………….144 Introduction……………………………………………...……………………………...145 Experimental Procedures…………………………………………………………...…..147 Results……………………………………………………...…………………………...154 Discussion…………………………………………………………...………………….180 List of References…………………………………………………………………...….188 Chapter 5 Concluding Remarks and Future Research Directions…………………..…….196 C-di-GMP Signaling in A. brasilense………………………………………...………...197 Possible GGDEFs that synthesize c-di-GMP which binds to PilZ-containing receptors in A. brasilense……………………………………………………...……………………..198 Possible signals for c-di-GMP synthesis and degradation in A. brasilense………...…..200 Potential c-di-GMP binding proteins encoded within the A. brasilense genome…...….201 Chemotaxis pathway crosstalk in A. brasilense…………………………………...……208 viii Table of Contents, continued List of References……………………………………………………...……………….218 Appendix…………………………………………………………...…………………………...226 Diversity in Bacterial Chemotactic Responses and Niche Adaptation…………...…….227 Vita…………………...…………………………………………………………………………267 ix List of Tables Table 2.1: Bacteria strains and plasmids used in this study…………………………...………….50 Table 3.1: Strains and plasmids used in this study………………………………………...…...105 Table 3.2: Time course of clumping and flocculation in wild type and mutant derivatives of A. brasilense…………………………………………………………….115 Table 4.1: Swimming reversal frequency of A. brasilense Sp7, Δche1, and Δche1Δtlp1 double mutant derivatives upon temporal changes in aeration…………………………..……………………………………...............….168 Table 4.2: Swimming reversal frequency of the chsA::Tn5 mutant derivative of A. brasilense upon temporal changes in aeration conditions.……………….……….…172 Table 4.3: Swimming velocity of a free-swimming population of chsA::Tn5 mutant derivatives of A. brasilense in the gas perfusion chamber assay…………………….....173 Table 4.4: Clump fractions of A. brasilense Sp7 and Δtlp1 mutant derivatives under conditions of growth with high aeration………………………………………………..179 Table 5.1: PSI-BLAST hits using the N-terminal domain of E. coli YcgR as the query sequence within the genome of A. brasilense and related bacteria C. crescentus and R. centenum……………… …………………………………………...…………...210 Table 5.2: Relevant PSI-BLAST hits using the PilZ domain of E. coli YcgR as the query sequence within the genome of A. brasilense and Rhodospirillum centenum………………………………………………………………………………..214 Table 5.3: Relevant PSI-BLAST hits using the PilZ domain of P. aeruginosa Alg44 as the query sequence within the genome of P. aeruginosa PAO1……………..215 x List