University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2012 Characterization of the Function of the Azospirillum brasilense Che1 Chemotaxis Pathway in the Regulation of Chemotaxis, Cell Length and Clumping Amber Nicole Bible [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Cell Biology Commons Recommended Citation Bible, Amber Nicole, "Characterization of the Function of the Azospirillum brasilense Che1 Chemotaxis Pathway in the Regulation of Chemotaxis, Cell Length and Clumping. " PhD diss., University of Tennessee, 2012. https://trace.tennessee.edu/utk_graddiss/1269 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 Amber Nicole Bible entitled "Characterization of the Function of the Azospirillum brasilense Che1 Chemotaxis Pathway in the Regulation of Chemotaxis, Cell Length and Clumping." 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 requirements for the degree of Doctor of Philosophy, with a major in Biochemistry and Cellular and Molecular Biology. Gladys Alexandre, Major Professor We have read this dissertation and recommend its acceptance: Beth Mullin, Barry Bruce, Alison Buchan, Elena Shpak 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 Function of the Azospirillum brasilense Che1 Chemotaxis Pathway in the Regulation of Chemotaxis, Cell Length and Clumping A dissertation presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Amber Nicole Bible May 2012 Abstract Azospirillum brasilense is a gram-negative alphaproteobacterium that lives in the soil where it colonizes the root surfaces of cereals and grasses. The genome of A. brasilense has recently been sequenced and shown to possess four different chemotaxis-like operons. This dissertation project focused on characterizing the Che1 chemotaxis-like signal transduction pathway, which was initially implicated in regulation of the chemotaxis behavior. Deletions of individual genes within the Che1 pathway did not exhibit a null chemotaxis phenotype, leading us to investigate the role of this pathway in the lifestyle of A. brasilense and the mechanism(s) by which it functions. We have used a combination of microbiology and molecular genetics methods, including construction and characterization of several mutant strains lacking che1 genes, as well as molecular biology and microscopy. The data obtained suggest that Che1 is involved in regulating multiple cellular behaviors such as cell length and swimming speed, as well as having indirect effects on cell-to-cell clumping behavior, cell surface properties, and a minor role in regulating the motility bias. The data obtained also shed light on the function of the N-terminal HlyIII-like domain of CheA1 in A. brasilense which is also found in other bacterial species, as a single domain protein. Using A. brasilense, Escherichia coli and Bacillus subtilis, evidence is provided that HlyIII-like domains function to modulate membrane properties with effects on fatty acid composition that appear to also impair protein localization and function, including the control of cell length at division and chemotaxis. ii Table of Contents Introduction………………………………………………………………………………………1 References……………………………………………………………………………………….21 Chapter 1. Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense………………………………………………………………………………………...28 Section A: Introduction………………………………………………………………….31 Section B: Materials and Methods………………………………………………………33 Section C: Results……………………………………………………………………….43 Part 1. CheA1 and CheY1 play a minor role in chemotaxis…………….………43 Part 2. Che1 mutants have different cell lengths under specific growth conditions………………………………………………………………………...51 Part 3. Changes in cell length do not affect growth rate………………….….….53 Part 4. The changes in cell length are predicted to affect nutrient uptake in shorter but not in longer cells……………………………………………………………54 Part 5. Che1 mutants have a different ability to clump under certain growth conditions………………………………………………………………………...57 Part 6. Che1 mutants are affected in the production of EPS………………….…60 Section D: Discussion………………………………………………………………...…62 Section E: References…………………………………………………………………...72 Chapter 2. The Azospirillum brasilense Che1 chemotaxis pathway controls the swimming speed which affects transient cell-to-cell clumping…………………………………...............78 Section A: Introduction………………………………………………………...………..81 Section B: Materials and Methods……………………………………………...……….84 Section C: Results………………………………………………………….……………92 Part 1. Che1 affects clumping but not flocculation……………….……..………92 iii Part 2. Stable, but not reversible (transient) clumping involves changes in the extracellular EPS matrix…………………………………………….………..…99 Part 3. Clumping is modulated by temporal changes in aeration……..……….102 Part 4. Clumping is not correlated with changes in reversal frequency……………………………………………………………………….106 Part 5. Clumping and swimming velocity correlate…………………………...106 Part 6. Control of the swimming speed is a signaling output of Che1….……..109 Part 7. A taxis receptor modulates Che1-dependent effects on clumping……..115 Section D: Discussion…………………………………………………………………117 Section E: References………………………………………………………………....125 Chapter 3. Functional analysis of CheA1 domains in behaviors regulated by the Che1 pathway……………………………………….………………………………………….……130 Section A: Introduction……………………………………………………………….132 Section B: Materials and Methods……………………………………………………136 Section C: Results………………………………………………………………...…..144 Part 1. Domain architecture of CheA1………………………………………..144 Part 2. HlyIII-like domain of CheA1 is required for polar localization…........145 Part 3. Complementation of ΔcheA1 (AB101) for functional analysis……..…149 Part 4. TM, P5B, and Rec domains are essential for cell length regulation…...149 Part 5. The A. brasilense Sp245 cheA1 mutant is affected in chemotaxis, but not cell length………………………………………………………………….…..153 Part 6. Contribution of CheA1 domains to chemotaxis and aerotaxis behavior………………………………………………………………………..153 Part 7. Swimming motility bias………………………………………….....…158 Part 8. CheA1 and swimming speed, the output of Che1……………………..158 Part 9. Role of CheA1 domains in clumping and flocculation behavior……....162 iv Section D: Discussion…………………………………………………………………167 Section E: References…………………………………………………………………175 Chapter 4. Characterization of HlyIII-like proteins in A. brasilense, E. coli, and B. subtilis…………………….…………………………………………………………………….181 Section A: Introduction………………………………………………………………..183 Section B: Materials and Methods………………………………………………….…185 Section C: Results……………………………………………………………………..192 Part 1. Loss of HlyIII-like proteins results in defective/altered membrane lipid staining…………………………………………………………………………192 Part 2. Loss of HlyIII-like proteins affects protein localization…………….....195 Part 3. HlyIII-like proteins are involved in cell length regulation…………..…197 Part 4. HlyIII-like proteins do not directly affect cell division machinery or processes…………………………………………………………………….…198 Part 5. Perturbations in Min localization in HlyIII mutants……………….…..201 Part 6. HlyIII-like proteins affect fatty acid profiles……………………......…203 Part 7. HlyIII-like proteins affect behavior in E. coli and B. subtilis……….…207 Section D: Discussion………………………………………………………………....209 Section E: References………………………………………………………….………215 Conclusions………………………………………………………………………………….…221 Vita……………………………………………………………………………………………..225 v List of Tables Chapter 1: Table 1. Complementation of che1 mutant strains for cell length………………….…...55 Table 2. Effect of mutations in genes of the che1 operon of A. brasilense on the doubling times of cells grown with different substrates…………………………………58 Chapter 2: Table 3. Strains and plasmids used in this study………………………………………..85 Table 4. Time course of clumping and flocculation in wild type and mutant derivatives of A. brasilense………………………………………………………………….….……96 Table 5. Fraction of clumping cells in wild type (Sp7) A. brasilense and its che1 mutant derivatives during growth under flocculation permissive conditions………………..….98 Table 6. Timing of clumping behavior in wild type A. brasilense and its che1 mutant strain derivatives complemented with wild type or mutant alleles of cheA1, cheB1, or cheY1 (in hours)……………………………………………………………..…………110 Chapter 3: Table 7. Strains and plasmids used in this study……………………………………....137 Table 8. List of primers used in this study………………………………………..…....141 Table 9. Motility bias of wild type A. brasilense, ΔcheA1 (AB101), and ΔcheA1 (AB101) complemented with wild type and mutant derivatives of cheA1…………..…159 Chapter 4: Table 10. Strains and plasmids used in this study………………………...……….….187 vi List of Figures Introduction: Figure 1. Polar and lateral flagella in Azospirillum brasilense………………......……..3 Figure 2. Chemotaxis