The Flagellar World The Flagellar World Electron microscopic images of bacterial flagella and related surface structures from more than 30 species Shin-Ichi Aizawa Prefectural University of Hiroshima AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK 225 Wyman Street, Waltham, MA 02451, USA First published 2014 Copyright © 2014 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. 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To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-417234-0 For information on all Academic Press publications visit our website at store.elsevier.com PREFACE I graduated from the physics department of Tohoku University in 1974 and moved into a brand-new (at the time) science, Biophysics, at Nagoya University, where I met bac- terial flagella as one of the rotation programs for graduates (under Prof. Sho Asakura). As a physics student, I was very much interested in the flagellar motor. I attempted to purify the motor and failed, which gave me an opportunity to quickly learn biology, biochemistry, genetics, and molecular biology. As a postdoc (1980–1984, under the late Prof. Robert Macnab) at Yale University, I successfully developed a new method for puri- fication of flagella with the motor still attached. Since then, flagella has been the main theme of my research. In the early years of research, I worked with Salmonella typhimurium only, using abundant mutants of SJW strains (see Topic: History of Salmonella SJW (Salmonella Japan Waseda) strains). After 10 years of constant publications on Salmonella flagella, people from around the world started sending their “bugs” for me to take a look at with the electron microscope (EM). I accepted almost all bugs. I was very happy to see their flagella, even though some of them were human pathogens. Now I am bewildered by the piles of electron microscope negatives, as my retire- ment is getting close. I took thousands of EM pictures of flagella from various species. Some of them were published, but many were left unused. What should I do with them: burn them or shred them? Rather than terminating them forever, I chose a way to leave them in a book. This book, then, is naturally not a review of flagella research of the world, but a collection of my work on flagella with my view of this small world. This book contains 35 strains of eubacteria and archaeal species, aligned one by one in alphabetical order. Each chapter contains one species on two facing pages. On the first page, I show pictures of a whole cell, then isolated flagella, and then other cell surface appendages, if any, and on the second page is the genetic map regarding flagellar genes and the analysis of genes. Five species were placed into an exceptions section, because they are not really flagella. In the introduction, I summarize the flagellar structures and the flagellar genetics for those who are not familiar with this world. In the appendix, I describe the flagellar family, protocols for purification of flagella, and microscopic tech- niques for the observation of flagella. Some lines might (unintentionally) resemble lines in “Flagella” chapters in the Encyclopedia of Microbiology and in the Encyclopedia of Genetics. It is not easy to write about flagella differently from previous writing. I have to warn readers, again, that this analysis of the flagellar world is totally from my own perspective of the world. My knowledge about flagella was obtained mainly from S. typhimurium. Today, the Salmonella flagellum is the most extensively studied one, and I regard them as representative of eubacteria flagella in this book. I chose numerous papers on flagella and placed them in the References section, in which they were grouped according to each species. When the text in each chapter is too short to mention all papers involved or when there are not many papers to cite, the references are presented in order of the published years. At the end of each chapter, I mention names of those who provided their strains to us, and the institutes they belong or belonged to (as indicated by “as of”). Their titles are omitted. Many of them were my good collaborators for years and then also gave me kind and encouraging comments for a draft of the chapter they were involved in for this viii Preface book. I thank all the students who worked in my lab. Most of them were undergradu- ates and there are more than 100 altogether by now. I have been lucky to have so many students, which gave me an opportunity to think about as many projects as possible and thus turned an otherwise monotonous world into a colorful one. In case their work has never been published in some other form, I mention names of students who worked on the project. I offer a special thanks to Kaoru Uchida, my last Ph.D. student, who learned all the EM techniques I have used and took several pictures anew for this book. I also thank Tatsuya Yamasaki for his drawings that have been inserted to fill the page. “It is very important to have a patron who loves your work,” facetiously said Hirokazu (Q-chan) Hotani—who was actually my patron—in his memoir. He organized a big proj- ect twice in his time and allowed me to join both. I learned a lot about science and life from Bob Macnab; one of his lessons was “Write papers every single year, but not just for Science or Nature.” Thanks to this lesson, I could publish papers every single year, includ- ing a couple of Science papers (I’m sorry, Bob. Now I know what you meant). I have been writing Haiku (the shortest style of poem) for the last 30 years. It may be out of character for me not to make one for this occasion. 振り返る勇気を得たり夏の果 (Furikaeru/yuuki wo etari/Natsu no hate) Finally got a courage/to look back/in the end of the summer August 2013, the hottest summer ever (Depicted by Michiko Kobayashi, a mushroom painter) INTRODUCTION 1. BASIC KNOWLEDGE ABOUT FLAGELLA The flagellum is an organelle of bacterial motility. It is a gigantic protein complex, consisting of three major substructures: the filament, the hook, and the basal body. The basal body includes an actively rotating part of the flagellum, the flagellar motor, which can generate torque from an electrochemical potential of proton gradient across the membrane, called proton motive force. Before going into chapters, I explain the basic knowledge and some unique terminologies only used for flagella, and introduce related Chapters and Topics in which you may find more references. (A) Flagella Arrangement Many (more than 70%) bacteria carry one or more flagella per cell and swim in water. Depending on the position of a flagellum growing on a cell, flagella are called with dif- ferent names: polar (at one or both poles), subpolar (near the pole), lateral (from the middle half of the cell body), and peritrichous (randomly arranged on the cell body) flagella.1 Figure I.1 Flagella position. (From left to right: Polar, Subpolar, Lateral, and Peritrichous flagella) In recent studies, peritrichous flagella and lateral flagella are often confused for his- torical reasons (see Topic: Flagella position and shape). Flagellar shape is not defined by its growing position, but is defined by the intrinsic properties of the flagellin (see Appendix: Flagellar family). Strictly speaking, the only example of lateral flagella included in this book is those of Selenomonas ruminantium (see Chapter 25). However, it is not easy to change history, and I, myself, sometimes use those names (pof for polar flagella and laf for lateral flagella) to distinguish two types of flagellins in one strain. I do not use some more conventional names: monotrichous (single flagellum), mul- titrichous (more than two flagella), and lophotrichous (tuft at polar end of flagella) in this book. Instead, I use only the four positions mentioned above and the number of flagella: single, few, or numerous. (B) Gram Staining Gram staining of bacterial cells is neither an accurate nor elaborate technique, but nevertheless it is practically useful to distinguish two big domains of eubacteria spe- cies: Gram-positive bacteria that include most of the Firmicutes, and Gram-negative bacteria that include the rest. The structure of the flagellar basal body differs between the two types due to the difference of membrane structures; Gram-positives have two rings, while Gram-negatives have four rings (see next section). Accordingly, the genes 2 The Flagellar World encoding the two extra rings in Gram-negatives are missing in Gram-positives, as seen in Bacillus subtilis (see Chapter 4). (C) Salmonella enterica Serovar Typhimurium Salmonella enterica serovar Typhimurium had been just called Salmonella typhimurium until 2005 when the present name was proposed to classify members in a big Salmonellae family.2 However, I will be talking about serovar Typhimurium only—and no other strains—among the Salmonellae family in this book. Therefore, hereafter, I will call this particular strain with the old shortened name, Salmonella typhimurium. When S. typhimurium and E.coli are dealt with to the same extent, I refer to them as E.coli/Salmonella. 2. FLAGELLAR STRUCTURE (A) The Structure–Function–Gene Relationship in the Flagellum The modern research on the bacterial flagellum started in 1974, when DePamphilis and Adler biochemically isolated the flagellar basal body and showed the electron micro- scopic images of the structure in a series of papers.3–5 In 1985, I refined the purification method and established a protocol on how to purify the protein components of the basal body.6 By the year 2000, most components of the flagellum had been identified, the pathway of flagellar assembly had been revealed, and roles of ca. 40 flagellar genes in the assembly process were now known.7–9 The figure shows the structure–function– gene relationship in the flagellum. Figure I.2 Structure of the flagellum. The name of a gene product is followed by the name of corresponding structure in parenthesis. Introduction 3 The largest part of the flagellum is the filament, which is composed of thousands of subunits of one or multiple kind(s) of protein called flagellin or FliC. The reasons why a filament uses more than two flagellins are not completely understood (see Topic: Multiple flagellins). The filament is a helical tube. The question why one kind of chem- ically-identical protein can form a helix has not been fully answered and thus solving explains one of the mysteries about the flagellum (see Appendix: Flagellar family). The filament is connected to the hook, which is hooked or sharply curved. Unlike the filament, the hook is always composed of a single kind of protein called hook protein or FlgE. The length of the hook is regulated to be short by secretion of a soluble protein called FliK (see Topic: Hook length). The mechanism of length control is still controver- sial.10–13 FliK does not just regulate the hook length, but also indirectly regulates the gene expression of the late genes (fliC, flgK, flgL, and che genes) (see Topic: Gene regulation). Between the filament and the hook, there is a small spacer called the HAP (hook- associated protein) region, which contains HAP1 (or FlgK) and HAP3 (FlgL). The HAP region is necessary for assembly of filaments and for stabilizing the filament shape as a helix.14 Judging from the fact that HAP1 and HAP3 exist in any type of flagella, I suspect that the HAP region may play an important roles in conveying torque from the flexible hook (changing its conformation continuously during rotation) to the rigid filament. The flagellum originates at the inner membrane and passes through the outer mem- brane or the cell wall. The structural entity for the anchoring in the membrane is called the basal structure or basal body. The basal body does not contain just those compo- nents necessary for motor function. In addition to the stator components (MotA and MotB), some fragile components have been detached from the basal body during puri- fication. In 1985, one such fragile structure was found attached to basal bodies puri- fied by a modified method; it was named the C (cytoplasmic) ring.15,16 In 1990, another rod-like structure was found in the center of the C ring and named the C rod.17 In 2006, flagellar export ATPase (FliI) was found at the periphery of the C ring as a complex with the supporter protein FliH.18 Therefore, the basal structure (as of 2007) consists of the basal body, the C ring, the C rod, the export ATPase, and their regulators. There remain some genes with unknown functions even in S. typhimurium; they are flhE, fliY (not the same as fliY of Bacillus family, which is fliM + fliN), and flk.19 (B) Assembly Process of the Flagellum The order of the steps toward the completion of a flagellum (the morphology pathway) has been analyzed in the same way as that which was used for bacteriophages: iden- tifying intermediate structures in various flagellar mutants and aligning them in size from small to large ones.20 Flagellar construction starts from the cytoplasm, progresses through the periplasmic space, and finally extends to the outside of the cell. Figure I.3 Flagellar assembly process. (Assembly proceeds from left to right, from small to large substructures.) 4 The Flagellar World In the Cytoplasm In the assembly process, the very first flagellar structure recognizable by electron microscopy is the MS ring complex21; other components assemble on the MS ring com- plex one by one. Therefore, the MS ring complex is regarded as the construction base for the flagellum. When two other flagellar substructures, the C ring and the C rod, are constructed on the cytoplasmic side of the M ring, the gigantic complex starts secreting other flagellar proteins to construct the extracellular structures of the flagellum. In the Periplasmic Space The first extracellular structures constructed on the MS ring complex are the proximal rod and then the distal rod. When the rod has grown large enough to reach the outer membrane, the hook starts growing. However, the outer membrane physically ham- pers the hook growth until the outer ring complex makes a hole in it. Among flagellar proteins, FlgH and FlgI, the component proteins of the outer ring complex, are excep- tional in terms of the manner of secretion: these two proteins have cleavable signal pep- tides and are exported through the general secretion pathway.22 However, under special conditions, filaments grow in the absence of the outer rings but stay in the periplasm to form the periplasmic flagella as seen in spirochetes (see Chapter 6). Outside the Cell Once the physical block by the outer membrane has been removed by the outer PL rings, the hook resumes growth with the aid of FlgD until the length reaches ca. 55 nm (see Topic: Hook length). Then, FlgD is replaced by HAPs, which is followed by the filament growth. The filament growth proceeds only in the presence of FliD (HAP2 or filament cap protein); without this cap, exported flagellin molecules are lost to the medium. In conclusion, a flagellum grows from bottom to tip.23 The component proteins of the axial structures (the rod, hook, and filament) do not retain the signal peptides, but are secreted without cleavage through a special secretion system of the flagellum. Today it is called the type III secretion system (T3SS), which was originally used for one type of virulence secretion system in invasive pathogenic species and adopted for the flagellar secretion system due to the structural and functional similarities between the two.24 The axial structures do not self-assemble in vivo, but require helper proteins or cap proteins: FlgJ for the rod, FlgD for the hook, and FliD for the filament (find them in the Figure above). The other type of helper proteins are chaperones: FlgA is a chaperone for P-ring formation, FliS for flagellin, FliT for FliD, and FlgN for FlgK and FlgL. Flagellar assembly is not a simple aggregation of proteins, but a sophisti- cated assembly system with self-controlled devices far more developed than any other cell-surface structures. 3. FLAGELLAR GENETICS (A) Unified Gene Names for E. coli/ Salmonella Names of the flagellar genes were originally specific for each of these two species. But when the number of genes identified year after year increased over the number of alphabets, it was necessary to rename the genes. By then, people noticed the similarity of gene function between E.coli and S. typhimurium. Bob Macnab proposed that flagel- lar geneticists use unified nomenclature for the flagellar genes for both species. After tough negotiation, the proposal was successfully accepted.25 I summarize the unified genes together with old names, and their functions on a list, which will serve as a good guide when you have to enter the forest of the classic papers written before 1985. Introduction 5 Table I.1 Unified Flagellar Gene Names for E. coli and S. typhimurium Old Symbols New Symbols Structure or Function E. coli S. typhimurium flaU flaFI flgA Periplasmic chaperone for P-ring formation flbA flaFII flgB Proximal rod protein (Rod 1) flaW flaFIII flgC Proximal rod protein (Rod 1) flaV flaFIV flgD Cap protein for hook growth flaK flaFV flgE Hook protein flaX flaFVI flgF Proximal rod protein (Rod 1) flaL flaFVII flgG Distal rod protein (Rod 2) flaY flaFVIII flgH L-ring protein flaM flaFIX flgI P-ring protein flaZ flaFX flgJ Cap protein for rod growth, muramidase flaS flaW flgK HAP 1 flaT flaU flgL HAP 3 * * flgM Anti-sigma factor * * flgN Chaperone for FlgK, FlgN flaH flaC flhA Secretion gate keeper flaG fiaM flhB Secretion gate keeper flaI flaE flhC++ Regulation of gene expression flbB flaK flhD++ Regulation of gene expression * * flhE++ Required for swarming motility flaD flaL fliA Sigma factor 28 – nml fliB++ N-methylation of lysine residues in flagellin hag HI fliC Flagellin flbC flaV fliD HAP 2 or cap protein for filament growth flaN flaAI fliE Proximal rod protein (Rod 1) flaBI flaAII.1 fliF MS-ring protein flaBII flaAII.2 fliG C ring, torque generation by interacting with MotA flaBIII flaAII.3 fliH Anchoring FliI to FliN flaC flaAIII fliI ATPase for T3SS flaO flaS fliJ Interact with FlhA flaE flaR fliK Hook length control, switching of secretion substrate specificity flaAI flaQI fliL Basal body-associated membrane protein flaAII flaQII fliM C ring. Motor switching by interacting with CheY-Phosphate motD flaN fliN C ring flbD flaP fliO++ FliP integrity flaR flaB fliP Cytoplasmic (C) rod, Secretion gate (Continued)