Bacterial and Archaeal Motility

Preface
Contents
Contributors
Part I: Bacterial Flagellar Protein Export and Assembly
	Chapter 1: Purification of the Transmembrane Polypeptide Channel Complex of the Salmonella Flagellar Type III Secretion System
		1 Introduction
		2 Materials
			2.1 Salmonella Strains and Plasmids
			2.2 Culture Media
			2.3 Cell Growth and Harvest
			2.4 Transformation
			2.5 Preparation and Solubilization of Cellular Membranes
			2.6 Measurement of Protein Concentration in Crude Membrane Fractions
			2.7 Purification of the FliP/FliQ/FliR Complex
			2.8 Identification of Protein Expression and Purified Protein Sample
			2.9 Observation of Purified FliP/FliQ/FliR Complex by Electron Microscopy
		3 Methods
			3.1 Transformation
			3.2 Cell Culture and Harvest
			3.3 Preparation of Membrane Fractions
			3.4 Measurement of Protein Concentration by the Lowry Method
			3.5 Solubilization of Membrane Fractions by LMNG
			3.6 Purification of the FliP/FliQ/FliR Complex
			3.7 Observation of Negatively Stained FliP/FliQ/FliR Complex by Electron Microscopy
		4 Notes
		References
	Chapter 2: In Vitro Flagellar Type III Protein Transport Assay Using Inverted Membrane Vesicles
		1 Introduction
			1.1 Background
			1.2 Overview of the Method
		2 Materials
			2.1 Salmonella enterica Strain and Plasmid
			2.2 Culture
			2.3 Preparation of IMV Stock
			2.4 Preparation of IMV
			2.5 In Vitro Transport Assay
		3 Methods
			3.1 Cell Culture
			3.2 Preparation of Spheroplast
			3.3 Preparation of IMV Stock
			3.4 Preparation of the IMV Solution for Transport Assay
			3.5 Transport Assay (500 μL)
			3.6 Detection of the Transported Proteins
		4 Notes
		References
	Chapter 3: Molecular Simulation to Investigate Open-Close Motion of a Flagellar Export Apparatus Protein FlhAC
		1 Introduction
		2 Materials
			2.1 Initial Structure for the Wild-Type Simulation
			2.2 Initial Structure for the G368C Mutant Simulation
			2.3 Force Field for the Simulated Systems
			2.4 Periodic Boundary Condition
		3 Methods
			3.1 Molecular Dynamics Simulation for Equilibration
			3.2 Molecular Dynamics Simulation for Production
			3.3 Analysis of MD Trajectories
			3.4 PaCS-MD Simulation of Open-Close Movements
			3.5 Calculation of Free Energy Profile for Open-Close Motion
		4 Notes
		References
	Chapter 4: Live-Cell Imaging of the Assembly and Ejection Processes of the Bacterial Flagella by Fluorescence Microscopy
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Channel Slide
			2.3 Flagellar Filament Labeling
			2.4 Microscope Configuration
			2.5 Image Analysis
			2.6 V. alginolyticus Strains
		3 Methods
			3.1 Making Tunnel Slide
			3.2 Flagella Growth Measurements
			3.3 Flagellar Ejection Observation
		4 Notes
		References
	Chapter 5: Purification and CryoEM Image Analysis of the Bacterial Flagellar Filament
		1 Introduction
		2 Materials
			2.1 Bacterial Strain and Plasmid
			2.2 Cell Culture and Solution
			2.3 Equipment and Electron Microscope
			2.4 Program for Data Collection and Structural Analysis by Single-Particle Image Analysis
		3 Methods
			3.1 Purification of FljB
			3.2 Negative Staining and Sample Observation
			3.3 CryoEM Sample Preparation and Data Collection
			3.4 Image Processing and Model Building
		4 Notes
		References
Part II: Flagella-Driven Motility of Bacteria
	Chapter 6: Site-Specific Isotope Labeling of FliG for Studying Structural Dynamics Using Nuclear Magnetic Resonance Spectrosco...
		1 Introduction
			1.1 Background
			1.2 Overview of the Method
		2 Materials
			2.1 E. coli Cultivation Using a Deuterated M9 Medium
			2.2 Protein Purification
			2.3 NMR Spectroscopy
		3 Methods
			3.1 Phe and Ile Residue-Specific Isotope Labeling
			3.2 Protein Purification (See Note 10)
			3.3 NMR Signal Assignment of Ile δ1 Methyl in FliGM-FliGC
			3.4 Sequence-Specific Signal Assignment of SAIL-Phe in FliGM-FliGC
		4 Notes
		References
	Chapter 7: Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo
		1 Introduction
			1.1 Background
			1.2 Overview of Methods
		2 Materials
			2.1 Site-Directed In Vivo Photo-Cross-Linking
			2.2 Site-Directed In Vivo Cysteine Disulfide Cross-Linking
			2.3 Sample Preparation for SDS-PAGE and Immunoblotting
		3 Methods
			3.1 Site-Directed In Vivo Photo-Cross-Linking
			3.2 Site-Directed In Vivo Cysteine Disulfide Cross-Linking
			3.3 SDS-PAGE and Immunoblot
		4 Notes
		References
	Chapter 8: Measurements of the Ion Channel Activity of the Transmembrane Stator Complex in the Bacterial Flagellar Motor
		1 Introduction
		2 Materials
			2.1 Bacteria Strain and Plasmids
			2.2 Culture Media
			2.3 Fluorescence Spectrophotometry
			2.4 Fluorescence Microscopy
		3 Methods
			3.1 Preparation of Bacterial Samples
			3.2 Acquisition of Excitation Spectrum of pHluorin(M153R) Expressing in E. coli Cells
			3.3 pH Calibration
			3.4 Calculation of the Cytoplasmic pH
			3.5 Acquisition of Fluorescence Images of E. coli Cells Stained with CoroNa Green
			3.6 Estimation of the Cytoplasmic Na+ Concentrations
		4 Notes
		References
	Chapter 9: Purification of the Na+-Driven PomAB Stator Complex and Its Analysis Using ATR-FTIR Spectroscopy
		1 Introduction
			1.1 Background
			1.2 Overview of the Methods
		2 Materials
			2.1 Purification of the PomAB Stator Complex
			2.2 ATR-FTIR Analysis of the PomAB Stator Complex
		3 Methods
			3.1 Purification of the PomAB Stator Complex
			3.2 ATR-FTIR Analysis of the PomA/PomB Stator Complex
		4 Notes
		References
	Chapter 10: Purification of Na+-Driven MotPS Stator Complexes and Single-Molecule Imaging by High-Speed Atomic Force Microscopy
		1 Introduction
		2 Materials
			2.1 Reagents and Buffers Used for Preparation of Membrane Fractions
			2.2 Reagents and Buffers Used for Purification of MotPS Complexes
			2.3 HS-AFM Imaging
		3 Methods
			3.1 Preparation of Membrane Fractions Containing His6-Tagged MotPS Complexes
			3.2 Purification of His6-Tagged MotPS Complexes
			3.3 HS-AFM Imaging
			3.4 Buffer Exchanging System
			3.5 Image Analysis
		4 Notes
		References
	Chapter 11: High-Resolution Rotation Assay of the Bacterial Flagellar Motor Near Zero Loads Using a Mutant Having a Rod-Like S...
		1 Introduction
		2 Materials
			2.1 Bacterial Strain
			2.2 Media
			2.3 Cell Growth and Harvest
			2.4 Probe
			2.5 Dark-Field Microscope
			2.6 Flow Chamber
		3 Methods
			3.1 Preparation of a Flow Chamber
			3.2 Preparation of Gold Nanoparticles
			3.3 Sample Preparation
			3.4 Rotation Measurements
		4 Notes
		References
	Chapter 12: Live-Cell Fluorescence Imaging of Magnetosome Organelle for Magnetotaxis Motility
		1 Introduction
		2 Materials
			2.1 Preparation for AMB-1 Cells Expressing GFP-Fused Magnetosome Membrane Proteins
			2.2 Live-Cell Fluorescence Imaging of Magnetosome Positioning
			2.3 Live-Cell pH Measurements in Magnetosome Lumen
		3 Methods
			3.1 Preparation for AMB-1 Cells Expressing GFP-Fused Magnetosome Membrane Proteins
			3.2 Live-Cell Imaging of Magnetosome Positioning
			3.3 Live-Cell pH Measurements in Magnetosome Lumen
		4 Notes
		References
	Chapter 13: Swarming Motility Assays in Salmonella
		1 Introduction
		2 Materials
			2.1 Swimming Motility Assay
			2.2 Swarming Motility Assay
			2.3 The Border-Crossing Swarming Motility Assay
		3 Methods
			3.1 Swimming Motility Assay
			3.2 Swarming Motility Assay
			3.3 The Border-Crossing Swarming Motility Assay
		4 Notes
		References
	Chapter 14: Analysis of Adhesion and Surface Motility of a Spirochete Bacterium
		1 Introduction
			1.1 Spirochete
			1.2 Two-Type Motility of the Spirochete Leptospira: Swimming and Crawling
			1.3 Principle 1: Steady-State Adhesion
			1.4 Principle 2: Diffusion by Crawling
		2 Materials
			2.1 Bacterial Strain
			2.2 Media for Leptospira
			2.3 Bacterial Growth and Harvest
			2.4 Microscope Setup
			2.5 Glass-Made Flow Chamber
			2.6 Slide Chamber Cultivating Kidney Cells
			2.7 Software for Data Analysis
		3 Methods
			3.1 Adhesion Assay
			3.2 Crawling Assay Using a Glass-Made Flow Chamber
			3.3 Crawling Assay on Kidney Cells Cultivated in a Chamber Slide
		4 Notes
		References
	Chapter 15: Force Measurement of Bacterial Swimming Using Optical Tweezers
		1 Introduction
			1.1 Force of Bacterial Motion
			1.2 Force Measurement Using Optical Tweezers
			1.3 Motility and Pathogenicity
			1.4 Force Balance in Trapped Bacterium
			1.5 Principle for the Determination of the Spring Constant of Optical Tweezers
		2 Materials
			2.1 Materials for Leptospira Strains
			2.2 Microscope Setup for Optical Tweezers
			2.3 Materials for Bead Labeling
			2.4 Flow Chamber
		3 Methods
			3.1 Preparation of Bacterial Sample
			3.2 Measurement of Spring Constant
			3.3 Δx-F Calibration
		4 Notes
		References
Part III: Archaella-Driven Motility of Archaea
	Chapter 16: Archaella Isolation
		1 Introduction
		2 Materials
			2.1 Isolation of Archaella from Sulfolobus acidocaldarius by Shearing with a Syringe Needle
			2.2 Shearing Archaella from Methanogens with a Waring Blender
			2.3 Isolation of Archaella by Detergent Extraction of Whole Cells of Methanogens
			2.4 Observation of Archaella by Transmission Electron Microscopy
		3 Methods
			3.1 Isolation of Archaella by Shearing with a Syringe Needle (See Note 1)
			3.2 Shearing Archaella from Methanogens with a Waring Blender (See Note 3)
			3.3 Isolation of Archaella by Detergent Extraction of Whole Cells of Methanogens (See Note 8)
			3.4 Imaging of Negative Stained Archaella by Transmission Electron Microscopy
		4 Notes
		References
	Chapter 17: Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques
		1 Introduction
		2 Materials
			2.1 Archaea Strains
			2.2 Chemicals
			2.3 Stock Solution
			2.4 Fluorescence Microscope
			2.5 Phase-Contrast Microscope
			2.6 Tunnel Slide
			2.7 Software
		3 Methods
			3.1 Cultivation of Hbt. salinarum
			3.2 Cultivation of Hfx. volcanii
			3.3 Preparation of Biotinylated Cells
			3.4 Preparation of Fluorescent Dye-Labeled Cells
			3.5 Streptavidin-Bead Preparation
			3.6 Visualization of Swimming Motility of Fluorescent-Labeled Cells
			3.7 Simultaneous Observation of the Architecture and Function of Helical Filaments Under Total Internal Reflection Fluorescenc...
			3.8 Bead Assay (Fig. 3a)
			3.9 Ghost Preparation
		4 Notes
		References
Part IV: Type IV-Driven Twitching Motility of Bacteria
	Chapter 18: In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Instruments
			2.3 Software
		3 Methods
			3.1 Bacterial Culture and Grid Preparation
			3.2 Assembly of AutoGrids
			3.3 Initial Screening of Grids by Cryo-Light Microscope
			3.4 Loading of AutoGrids onto Cryo-TEM
			3.5 Microscope and Camera Tuning
			3.6 Prepare Imaging Settings in SerialEM
			3.7 Collection of a Whole Grid Montage Map at Low Magnification
			3.8 Grid Square Exploration to Identify Regions of Interest
			3.9 Set Image Shift Offset Between 470x and the ``View´´ Magnification (4800x)
			3.10 Target Region Exploration at Medium Magnification
			3.11 Target Identification at High Magnification
			3.12 Determine the Electron Dose and Exposure Time for Data Collection
			3.13 Set Record Camera Parameters and File Options for Data Collection
			3.14 Set the Tilting Scheme and Other Data Collection Parameters
			3.15 Define the Focus Position and Set Periodic Energy Filter Centering
			3.16 Start the Batch Tilt Series Acquisition
			3.17 Automatic Data Processing and Tomogram Reconstruction
			3.18 Manual Tomogram Reconstruction
			3.19 Particle Picking Using IMOD
			3.20 Subtomogram Averaging Using Dynamo
			3.21 Resolution Estimation and Structural Analysis
		4 Notes
		References
	Chapter 19: Twitching Motility Assays of Lysobacter enzymogenes OH11 Under a Light Microscope
		1 Introduction
		2 Materials
		3 Methods
			3.1 Activation of Culture
			3.2 Preparation of Solid Medium
			3.3 Microscope Slide Preparation
			3.4 Microscope Observation
		4 Notes
		References
	Chapter 20: Live Cell Imaging of the Twitching Motility of Cyanobacteria by High-Resolution Microscopy
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Growth Medium
			2.2 Glass Chamber Assembly
			2.3 Optical Microscopy
			2.4 Bead Assay
		3 Methods
			3.1 Phototaxis on a Short Time Scale Under Optical Microscopy
			3.2 Visualizing T4P Dynamics with Beads
		4 Notes
		References
Part V: Adhesion-Based Gliding Motility of Bacteria
	Chapter 21: Isolation and Visualization of Gliding Motility Machinery in Bacteroidota
		1 Introduction
		2 Materials
			2.1 Cell Preparation
			2.2 Isolation of SprB Filaments from F. johnsoniae
			2.3 Osmotically Shocked Cells
			2.4 TEM
		3 Methods
			3.1 Cell Preparation
			3.2 Observation of SprB Filaments on the Cell Surface of F. johnsoniae by TEM
			3.3 Isolation of SprB Filament from F. johnsoniae
			3.4 Observation of Isolated SprB Filaments by TEM
			3.5 Preparation of Osmotically Shocked F. johnsoniae Cells
			3.6 Preparation of Osmotically Shocked S. grandis Cells
			3.7 Observation of the Gliding Machinery of Osmotically Shocked Cells by TEM
		4 Notes
		References
	Chapter 22: Live Cell Imaging of Gliding Motility of Flavobacterium johnsoniae Under High-Resolution Microscopy
		1 Introduction
		2 Materials
			2.1 Strain Preparation
			2.2 Optical Microscopy
			2.3 Glass Chamber Assembly
			2.4 Immunofluorescent Labeling of SprB
			2.5 Inhibitor and Probe for Cell Surface Movement
		3 Methods
			3.1 Culture Conditions
			3.2 Gliding Motility Observed Under Phase-Contrast Microscopy
			3.3 Visualization of SprB Dynamics with Immunofluorescent Labeling
			3.4 Inhibition of Gliding Motility by CCCP
			3.5 Visualization of Helical Movement on the Cell Surface Through Fluorescent Beads
		4 Notes
		References
	Chapter 23: Social Motility Assays of Flavobacterium johnsoniae
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Plasmids
			2.2 Bacterial Culture Media
			2.3 Bacterial Media for Colony Morphology Observation
			2.4 Construction of gld or spr Deletion Mutant
			2.5 Carbon-Grid Stamp-Peel Method [17]
			2.6 Atmospheric Scanning Electron Microscopy (ASEM) [16]
			2.7 TEM of F. johnsoniae Spreading Colonies (Biofilms) [15-17]
		3 Methods
			3.1 Construction of Targeting Vector Plasmid
			3.2 Introduction of the Targeting Vector Plasmid into E. coli
			3.3 Construction of a F. johnsoniae Deletion Mutant Strain
			3.4 Construction of the Shuttle Vector to Produce a Complemented Strain of F. johnsoniae
			3.5 Transformation of E. coli S17-1λ with the Shuttle Vector Plasmid
			3.6 Conjugative Transfer to Produce a Complemented Strain of F. johnsoniae
			3.7 Construction of Green Fluorescent Protein (GFP)-Marked Strains of Flavobacterium sp.
			3.8 Colony Spreading (See Note 4)
			3.9 Carbon-Grid Stamp-Peel Method [17]
			3.10 Atmospheric Scanning Electron Microscopy (ASEM) [16]
			3.11 NCMIR Staining Method for ASEM [16]
			3.12 Labeling with Charged Nanogold [16]
			3.13 ASEM Imaging [16]
			3.14 TEM Imaging of F. johnsoniae Spreading Colonies (Biofilms) [15-17]
		4 Notes
		References
	Chapter 24: Visualization of Peptidoglycan Structures of Escherichia coli by Quick-Freeze Deep-Etch Electron Microscopy
		1 Introduction
		2 Materials
			2.1 Bacterial Strain
			2.2 Cell Culture
			2.3 Cell Suspension
			2.4 Quick-Freezing (See Fig. 1)
			2.5 Platinum Replica (See Fig. 2)
		3 Methods
			3.1 Cell Culture
			3.2 SDS Treatment
			3.3 Negative Staining EM
			3.4 Quick-Freezing
			3.5 Platinum Shadowing and Carbon Backing
			3.6 Recovery and Observation of the Platinum Replica
		4 Notes
		References
Part VI: Unique Motility System in Bacteria
	Chapter 25: Purification and Structural Analysis of the Gliding Motility Machinery in Mycoplasma mobile
		1 Introduction
		2 Materials
			2.1 Strains
			2.2 Aluotto Medium
			2.3 Cell Growth and Harvest
			2.4 Purification of Motor and Motor Chain
			2.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.6 Electron Microscopy
			2.7 Image Collection and Structural Analysis
		3 Methods
			3.1 Cultivation of M. mobile
			3.2 Purification of Motor for Gliding
			3.3 Purification of the Gliding Motor Chain
			3.4 Specimen Preparation for Negative-Staining EM and Data Collection
			3.5 Image Processing of the Motor Proteins
			3.6 Image Processing of the Motor Chain
		4 Notes
		References
	Chapter 26: Motility Assays of Mycoplasma mobile Under Light Microscopy
		1 Introduction
		2 Materials
			2.1 Cultured Cell
			2.2 Tunnel Chamber
			2.3 Microscopic Observation and Image Analysis
		3 Methods
			3.1 Preparation of Tunnel Chambers
			3.2 Preparation of Cells
			3.3 Observation and Analysis of Mycoplasma Motility
		4 Notes
		References
	Chapter 27: Detection of Steps and Rotation in the Gliding Motility of Mycoplasma mobile
		1 Introduction
		2 Materials
			2.1 Strains
			2.2 Chemicals
			2.3 Stock Solution
			2.4 Fluorescence Microscopy (Fig. 1)
			2.5 Phase-Contrast Microscopy
			2.6 Tunnel Slide
			2.7 Software
		3 Methods
			3.1 Cultivation
			3.2 Preparation of Fluorescently Labeled Cells
			3.3 Construction of Tunnel Slide
			3.4 Detection of Stepwise Movements
			3.5 Detection of Rotation
		4 Notes
		References
	Chapter 28: Direct Measurement of Kinetic Force Generated by Mycoplasma
		1 Introduction
		2 Materials
			2.1 Optical Tweezers
			2.2 Bead
			2.3 Tunnel Chamber
			2.4 Cells and Cultivation
			2.5 Aluotto Medium
			2.6 SP-4 Medium
			2.7 Surface Modification and Preparation of Cells
		3 Methods
			3.1 Cultivation of Mycoplasma mobile
			3.2 Cultivation of Mycoplasma pneumoniae
			3.3 Biotin Conjugation to Mycoplasma mobile Cell Surface
			3.4 Biotin Conjugation to Mycoplasma pneumoniae Cell Surface
			3.5 Avidin Conjugation to Polystyrene Beads
			3.6 Construction and Surface Coating of Tunnel Chamber
			3.7 Measurements of Spring Constant of Optical Tweezers
			3.8 Data Analysis for Spring Constant Measurement
			3.9 Measuring Force Using Optical Tweezers
			3.10 Data Analysis for Force Measurement
		4 Notes
		References
	Chapter 29: Genetic Manipulation of Mycoplasma pneumoniae
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Plasmids
			2.2 Media
			2.3 Enzymes and Kits
			2.4 Electroporation
			2.5 Oligo DNA Primers
		3 Methods
			3.1 Construction of Tn4001 Vector Plasmid Using Gateway Cloning (See Note 2)
			3.2 Transformation of M. pneumoniae
			3.3 Analysis of Tn4001 Insertion Site (Inverse PCR Method)
		4 Notes
		References
	Chapter 30: Purification and ATPase Activity Measurement of Spiroplasma MreB
		1 Introduction
		2 Materials
			2.1 E. coli Strains
			2.2 Plasmid
			2.3 Cell Cultivation and Harvesting
			2.4 MreB Purification
			2.5 Pi Release and Pi Standard Measurements
		3 Methods
			3.1 Cell Cultivation and Harvesting
			3.2 Purification of MreB
			3.3 Pi Release Measurement of MreB
			3.4 Acquisition of the Pi Standard
			3.5 Construction of Pi Standard Curve
			3.6 Construction of Pi Release Curve of MreB
		4 Notes
		References
	Chapter 31: Swimming Motility Assays of Spiroplasma
		1 Introduction
		2 Materials
			2.1 Growth Medium and Bacterial Strains
			2.2 Optical Microscopy and Data Analysis
			2.3 Glass Chamber Assembly
			2.4 Motility Assay
			2.5 Stopping Cell Motility
		3 Methods
			3.1 Cell Preparation
			3.2 Setting for Cell Observation
			3.3 Cell Observations with Phase-Contrast Microscopy
			3.4 Measuring the Kink Position After Recording
			3.5 Inhibition of Cell Motility with CCCP
			3.6 Inhibition of Cell Motility with Light Irradiation
		4 Notes
		References
	Chapter 32: Motility Assays of Chloroflexus
		1 Introduction
		2 Materials
			2.1 Cultivation of Chloroflexus
			2.2 Microscopy
			2.3 Spectroscopy
		3 Methods
			3.1 Gliding Motility in Cell Suspension (Cell-Aggregate Formation)
			3.2 Gliding Motility on Solid Medium
			3.3 Gliding Motility in Solid Media
			3.4 Gliding Motility on Microscopic Glass Slide
			3.5 Cell-Surface Movements
		4 Notes
		References
Index