Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1997 Physiological Role of the Short Form of the Histidine Protein Kinase, Cheas, in Bacterial Chemotaxis Barry Patrick McNamara Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Microbiology Commons Recommended Citation McNamara, Barry Patrick, "Physiological Role of the Short Form of the Histidine Protein Kinase, Cheas, in Bacterial Chemotaxis" (1997). Dissertations. 3422. https://ecommons.luc.edu/luc_diss/3422 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1997 Barry Patrick McNamara LOYOLA UNIVERSITY CHICAGO PHYSIOLOGICAL ROLE OF THE SHORT FORM OF THE HISTIDINE PROTEIN KINASE, CheA8 , IN BACTERIAL CHEMOTAXIS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY BY BARRY PATRICK MCNAMARA CHICAGO, ILLINOIS MAY 1997 Copyright by Barry P. McNamara, 1997 All rights reserved. ii ACKNOWLEDGMENTS The culmination of this work would not have been possible were it not for the encouragement and generous support provided to me by my parents Justina and Eugene McNamara during the course of all my studies. I also wish to recognize my immediate co-workers, with whom I have had the honor and privilege of working with for the past several years. Specifically, I thank them for their help in navigating me through the computer world, for their generosity in sharing materials, and for their spirited guidance and technical support. Without question, I must express my gratitude to each of my committee members, Drs. Thomas Gallagher, John Lopes, Mike Fasullo, Charles Lange for their critical evaluation of my experimental data at each step of the way. and to Dr. David Hecht for the leadership he provided during each committee meeting. I especially thank my director, Dr. Alan J. Wolfe, for his persistence and belief in my abilities in light of the occasional setback. Finally, I will and do thank God for the special opportunity He has given me in being able to take but a small peek into the wonderment of rii s awesome creation and, especially, for the greatest creation and blessing of all, my new wife Ulrike, of whom I do adore. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................... iii LIST OF ILLUSTRATIONS ........................................ vii LIST OF TABLES ................................................ ix Chapter I. INTRODUCTION . 1 II. LITERATURE REVIEW ...................................... 4 2.1. Introduction . 4 2.2. Chemosensing and behavioral responses of motile bacteria . 5 2.3. Bacterial chemoreceptors ........................... 7 2.3.1. Structure and ligand interactions of chemoreceptors 9 .2. Transmembrane signaling ................... 11 .3. Cellular distribution of chemoreceptors ......... 12 2.4. Biochemistry of the chemotaxis signaling pathway . 13 2. 5. Adaptation responses . 17 2.6. Genetics of the chemotaxis signaling pathway .......... 19 2. 7. Gene products of the cheA locus . 21 Ill. MATERIALS AND EXPERIMENTAL METHODS .................. 25 3. 1. Chemicals and biological reagents. 25 3.2. Construction of plasmid-borne cheA mutant al lei es . _ . 25 3.3. Bacterial strains ............................ _ . 28 3.3.1. Isolation of cheA deletion and truncation mutants . 29 .2. Isolation of chromosomal cheA start(S) muian1s . 32 3.4. Bacterial media ............................ _ . 35 3.5. Growth conditions .......................... _ ..... 36 3.5.1. Measurement of microbial growth ....... _ ..... 36 .2. Proteus mirabilis swarmer cell isolation ... _ ..... 36 3.6. Genetic manipulations ....................... _ . 37 3.6.1. Transformation of competent E. coli ...... _ . 37 .2. Generalized transduction . _ . 38 .3. Replica plating for mutant selection ...... _ . 39 .4. Complementation analysis ............. _ . 39 3.7. Methods to assess motility and chemotactic beha\Jior ..... 40 iv 3. 7 .1. Swim plate analysis . 41 .2. Anaerobic swim assay conditions . 42 .3. Competitive swim assay analysis .............. 42 .4. Swimming speed and tumble frequency determinations . 43 3.8. Protein methods .................................. 44 3.8.1. CheA overexpression and purification .......... 44 .2. SOS-PAGE and western blot analysis .......... 45 .3. CheA in vitro autophosphorylation assays ....... 46 .4. Generation of affinity purified anti-E. coJ; CheA polyclonal antibodies ...................... 47 3.9. Molecular biology methods ................... _ ..... 48 3.9.1. Plasmid and chromosomal DNA preparation ..... 48 .2. DNA cloning and restriction endonuclease analysis . _ . 49 .3. Site-directed mutagenesis ............. _ . 50 .4. Polymerase chain reaction ............. _ ..... 51 .5. DNA hybridizations ......................... 52 .6. DNA sequence analysis . 53 IV. EXPERIMENTAL RESULTS ........................... _ . 55 4.1. In vivo complementation of CheA C-terminal truncation mutants by CheAs . .. _ . 55 4.2. CheAs overexpression studies ............ _ .......... 58 4.3. In vitro transducer-mediated transphosphorylation by CheA5 ................................. _ ... 62 4.4. Chemotactic behavior among clinical isolates of Enterobacteriaceae . _ . _ . 63 4.5. CheA expression in the Enterobacteriaceae ... _ ........ 67 4.6. CheZ expression in the Enterobacteriaceae ... _ .... _ ... 73 4. 7. Analysis of the 5' region of enteric cheA genes . _ . _ . 73 4.8. Behavioral studies with strains of E. coli that differ only in their ability to synthesize CheA5 ......•.... _ ....•... 80 4.8.1. Design and construction of cheA start(S~ muianis . 81 .2. CheA expression from cheA start(S) mutants .... 83 4.9. Chemotactic behavior of cheA start(S) mutant strains ..... 85 4.10. CheA5 complementation analysis . _ . 89 4. 11. Competitive swim assay analysis . _ . 92 4.12. In vitro phosphorylation of Met-98 CheA._ variants ....... 94 4.13. Relative expression levels of CheA._ and C heAs . _ . 96 V. DISCUSSION .................................... - . _ ..... 101 v 5.1. Chemotaxis-associated activities exhibited by CheA$. 101 5.2. Coexpression of CheAt_ and CheA5 by members of the Enterobacteriaceae. 105 5.3. Putative physiological role of CheA5 in chemotaxis ...... 109 5.4. Putative signaling checkpoints affected by CheAs . 113 APPENDIX A List of common abbreviations used in this study . 117 WORKS CITED . 118 VITA ........................................................ 132 vi LIST OF ILLUSTRATIONS Figure Page 1. Membrane topology and domain organization of MCP molecules . 1O 2. Circuitry of the chemotactic signal transduction pathway . 16 3. Genomic organization of the mocha and meche operons in E. coli . 20 4. Organization of structural domains and associated functions shared among both forms of CheA . 22 5. CheAs complementation studies with strains that express various CheA truncation mutants . 56 6. Restoration of swimming ability by CheA8 to cells expressing CheA616K(Am) ........................................... 57 7. Effect of CheA8 concentration on the swimming ability of cells expressing CheA616K(Am) .................................. 60 8. Effect of CheA8 overexpression on swimming ability in a wild-type strain ................................................... 61 9. Swim colony morphologies exhibited by various clinical isolates of Enterobacteriaceae . 65 1O. CheA expression among motile Enterobacteriaceae . 70 11. CheA expression in isolates of Klebsiella ssp. and Yersin;a s sp. 72 12. CheZ expression in motile and non-motile enteric bacteria . 74 13. Comparison of nucleotide sequences spanning the '3' motB-5' cfleA region of the mocha operon . 77 vii 14. Predicted amino acid sequence alignments of the amino-terminal portion of CheA . 79 15. Schematic of the mocha operon of E. coli and mutant cheA start(S) alleles used in this study . 82 16. lmmunoblot analysis on recombinant strains of E. coli harboring cheA start(S) alleles ............................................ 84 17. Swimming ability of cells that carry indicated cheA start(S) mutations under aerobic (A) or anaerobic (B) assay conditions .............. 86 18. Influence oh-serine upon the chemotactic ability of cells wild-1ype for chemotaxis when grown under anaerobic conditions . 88 19. CheAs induction as a function of IPTG inducer concentration . 90 20. Effect of CheAs concentration on the swimming ability of cells harboring cheA start(S) mutations ............................. 91 21. Growth characteristics of M98L mutant strains grown in tryptone broth at 31EC ................................................. 95 22. Purified CheA variant proteins isolated from strain BL21(DE3) transformants harboring CheA expression vectors . 97 23. Relative in vitro phosphorylation activities of HisCTag purified CheAL variants . 98 24. In vivo steady state levels of CheAt_ and CheAs as a func1ion oi the growth phase of E. coli grown in tryptone broth . 100 viii LIST OF TABLES Table Page 1. Components of the chemotaxis signal transduction system . 19 2. Plasmids used in complementation studies ...................... 26 3. Plasmids used in strain construction . 28 4. Bacterial strains