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Molecular Analysis of Ftsz-Ring Assembly in <I>E. Coli</I> Cytokinesis

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City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 9-2016 Molecular Analysis of FtsZ-Ring Assembly in E. coli cytokinesis Kuo-Hsiang Huang The Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/1530 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] MOLECULAR ANALYSIS OF FTSZ-RING ASSEMBLY IN E. COLI CYTOKINESIS by Kuo-Hsiang Huang A dissertation submitted to the Graduate Faculty in Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2016 i © 2016 Kuo-Hsiang Huang All rights Reserved ii This manuscript has been read and accepted for the Graduate Faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy _______________________ _____________________________________________ Date Chair of Examining Committee Dr. Anuradha Janakiraman, City College of New York _______________________ _____________________________________________ Date Executive Officer Dr. Laurel A. Eckhardt _____________________________________________ Dr. David Jeruzalmi, City College of New York _____________________________________________ Dr. Tadmira Venkatesh, City College of New York _____________________________________________ Dr. David Dubnau, Rutgers University _____________________________________________ Dr. Heran Darwin, New York University Supervising Committee The City University of New York iii Abstract of the Dissertation Molecular Analysis of FtsZ-ring Assembly in E. coli Cytokinesis By Kuo-Hsiang Huang Advisor: Dr. Anuradha Janakiraman An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions of FtsZ polymers and their impacts on Z-ring assembly in E. coli, and (ii) the structure-function relationship of FtsZ with two regulators, ZapC and ZapD, which have no primary sequence homology but have overlapping functions in Z-ring assembly and stability in E. coli. This work is expected to provide significant insight into the roles of different FtsZ domain sequences on the assembly of dynamic FtsZ polymers as well as the roles of modulatory proteins in mediating the assembly of an efficient cytokinetic ring during the early stage of E. coli cytokinesis. iv Acknowledgement I would like to first thank Dr. Janakiraman for her advisement of this dissertation. Dr. Janakiraman allowed me to be an independent researcher while working on the topics of this dissertation. She has enhanced my ability to design experiments to address specific scientific questions and also allowed me the freedom to answer the questions that I was interested in. This will help my future research since independent thinking is an important skill for being a competent scientist. In addition, she taught me to be an effective communicator, both writing about and presenting my research and assisted me to improve greatly in those areas. Therefore, I would like to show my great appreciation to her. I would also like to thank the members of my committee for providing helpful advice and criticism and improving this work. Secondly, I would like to thank all the present and previous members of the Janakiraman lab and the Ghose lab for numerous discussions and suggestions since I joined Dr. Janakiraman’s lab. All of them make the lab an awesome place to work. Especially, I appreciate that Aaron Mychacke and Lukasz Tchorzewski worked for, repeated or analyzed some experiments in this thesis. I would also like to thank Dr. Jorge Morales at the Microscopy Facilities of CCNY because he assisted and trained me to collect excellent TEM images from the transmission electron microscope. Finally, I really appreciate all of my family, friends and previous mentors who have ever encouraged or supported me in my work towards a PhD degree at CUNY. v All published works are also listed below: Some material in Chapter 1 was previously published in Huang KH, Durand-Heredia J, Janakiraman A (2013) FtsZ ring stability: of bundles, tubules, crosslinks, and curves. J Bacteriol 195: 1859-1868. Most material in Chapter 2 was previously published in Schumacher, M.A., Zeng, W., Huang, K.H., Tchorzewski, L., and Janakiraman A. (2015) Structural and functional analyses reveal insights into the molecular properties of the E. coli Z ring stabilizing protein, ZapC. The Journal of Biological Chemistry. M115.697037 Most material in Chapter 3 was previously published in Huang, K.H., Mychack, A., Tchorzewski, L., and Janakiraman, A. “Characterization of the FtsZ C-Terminal Variable (CTV) Region in Z- Ring Assembly and Interaction with the Z-Ring Stabilizer ZapD in E. coli Cytokinesis” (2016) PlosOne 11(4): e0153337 All abovementioned journal publishers allow authors to reuse published materials or articles in dissertation. The copyright permission policies of three publishers can be found below: 1. Journal of Bacteriology http://journals.asm.org/site/misc/ASM_Author_Statement.xhtml 2. The Journal of Biological Chemistry http://www.jbc.org/site/home/about/permissions.xhtml 3. PlosOne http://journals.plos.org/plosone/s/content-license#loc-submitting-copyrighted-or-proprietary- content vi Table of Contents Chapter 1: Introduction and background…………………………..….……..……….1 1.1. Overview of bacterial cytokinesis……………………………………….....….....…....2 1.2. Bacterial cytokinesis is initiated by the assembly of FtsZ polymers at midcell…………………………………………………………….….....……...………5 1.2.1. The structure and function of FtsZ…...……………………….……………….....…5 1.2.2. The architecture of the Z-ring: FtsZ polymers………………………………….…..9 1.2.3. FtsZ positive regulators mediate the Z-ring assembly during the early cytokinesis………………………………………………….………………….…..10 1.3. FtsA and ZipA function in mediating the divisome assembly via different mechanisms during E. coli cytokinesis………………………………………….…….13 1.3.1. The role of FtsA in FtsZ assembly………………………..……………………..…15 1.3.2. The role of ZipA in FtsZ assembly………………………………………………...18 1.4. The role/s of Z-ring associated proteins (Zaps) in FtsZ assembly ……..…........…..21 1.4.1. ZapA……………………………………………………….…………………….....21 1.4.2. ZapB……………………………………………..………………………………....26 1.4.3. ZapC and ZapD……………………………………..………………………….…..29 1.5. Summary………………………………………………………………….……………32 1.6. References…………………………………………………………………….………..34 Chapter 2: Identification and characterization of an FtsZ-interacting domain on ZapC………………………………………………………............41 2.1. Abstract…………………………………………………………..………........……….42 2.2. Introduction…………………………………………………..……………….……….43 2.2.1. FtsZ: an essential cell division protein……………………………………….…….43 2.2.2. FtsZ positive regulators mediate the Z-ring assembly and stability……….……….43 2.2.3. ZapC…………………………………………………………………..…………....44 2.2.4. The structural and functional analysis of ZapC in this study…………...……..…...45 2.3. Methods and Materials……………………...…………………….……….…………...47 2.3.1. E. coli or Yeast Strains and growth conditions……………………….……...…….47 2.3.2. Plasmid construction…………………………………………………...……..……47 2.3.3. Site-directed mutagenesis………………………………………………………….48 2.3.4. Protein expression and purification………………………………………………..49 2.3.5. Yeast-two-hybrid (Y2H) β-galactosidase assays……………………..…….….…..49 2.3.6. Yeast-two-hybrid(Y2H) cell growth assays………………….………...…………..50 vii 2.3.7. Spot-viability assays………………………………………………….………….…51 2.3.8. Fluorescence microscopy………...…………………..……………............…….…51 2.3.9. Western blotting analyses……………………………………………….………….52 2.3.10. FtsZ Sedimentation assays……………………………………………….……….52 2.3.11. Electron microscopy…………...……………………………………….…………53 2.4. Results…………………...…………………………………………………………..…...54 2.4.1. Overall structure of E. coli ZapC…………………………………………..……….54 2.4.2. Monomeric ZapC likely mediates FtsZ bundling in vitro………………….………56 2.4.3. ZapC structure reveals pockets lined by residues involved in FtsZ binding…….…61 2.4.4. Residues in ZapC pockets contribute to interaction with FtsZ in a Y2H assay…….67 2.4.5. Overexpression of ZapC (K89D) or ZapC (K94D) in E. coli does not lead to lethal filamentation of the cells……………..……………………………………………..72 2.4.6. ZapC (K89D) and ZapC (K94) are not recruited to Z-ring at midcell………..…….77 2.4.7. ZapC (K94D) fails to interact with FtsZ in vitro…………………….……………..82 2.4.8. ZapC does not require the FtsZ conserved C-terminal peptide (CCTP) domain for interaction…………..…………………………………………………...………….85 2.5. Discussion……………………………………………………..……………….………...91 2.6. Supplemental Information…………………………………….....…………..…………95 2.7. References……………………………………………...……...…..…………..………..108 Chapter 3: Role(s) of the FtsZ CTV region in E.coli cytokinesis: assembly of the Z-ring and interaction with ZapD………………………………..…..…..……….112 3.1. Abstract…………………………………………………………….……………..…….113 3.2. Introduction……………………………………………………….…………..………..115
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  • The Microbiome of the Footrot Lesion in Merino Sheep Is Characterized by a Persistent Bacterial Dysbiosis T ⁎ Andrew S

    The Microbiome of the Footrot Lesion in Merino Sheep Is Characterized by a Persistent Bacterial Dysbiosis T ⁎ Andrew S

    Veterinary Microbiology 236 (2019) 108378 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic The microbiome of the footrot lesion in Merino sheep is characterized by a persistent bacterial dysbiosis T ⁎ Andrew S. McPherson, Om P. Dhungyel, Richard J. Whittington Farm Animal Health, Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, 425 Werombi Rd, Camden, New South Wales, 2570, Australia ARTICLE INFO ABSTRACT Keywords: Footrot is prevalent in most sheep-producing countries; the disease compromises sheep health and welfare and Footrot has a considerable economic impact. The disease is the result of interactions between the essential causative Merino agent, Dichelobacter nodosus, and the bacterial community of the foot, with the pasture environment and host Sheep resistance influencing disease expression. The Merino, which is the main wool sheep breed in Australia, is Microbiome particularly susceptible to footrot. We characterised the bacterial communities on the feet of healthy and footrot- Dichelobacter nodosus affected Merino sheep across a 10-month period via sequencing and analysis of the V3-V4 regions of the bacterial 16S ribosomal RNA gene. Distinct bacterial communities were associated with the feet of healthy and footrot- affected sheep. Infection with D. nodosus appeared to trigger a shift in the composition of the bacterial com- munity from predominantly Gram-positive, aerobic taxa to predominantly Gram-negative, anaerobic taxa. A total of 15 bacterial genera were preferentially abundant on the feet of footrot-affected sheep, several of which have previously been implicated in footrot and other mixed bacterial diseases of the epidermis of ruminants.