Lipopolysaccharide Transport: Methods and Protocols

Preface
Contents
Contributors
Part I: Genetic Strategies to Study Essential LPS Biogenesis Genes
	Chapter 1: Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport
		1 Introduction
		2 Materials
			2.1 Site-Directed Mutagenesis
			2.2 Agarose Gel and Gel Electrophoresis
			2.3 DpnI Digestion of Template DNA and Plasmid Purification
			2.4 Electroporation
			2.5 Plasmid Extraction and Sequencing
			2.6 Chemical Transformation
			2.7 Disk Diffusion Assay
		3 Methods
			3.1 Site-Directed Mutagenesis and Agarose Gel Electrophoresis
			3.2 DpnI Digestion of Template Plasmid and Purification
			3.3 Electroporation into Competent Cells
			3.4 Plasmid Extraction and Sequencing
			3.5 Chemical Transformation of Mutagenized Plasmid
			3.6 Blue/White Screening
			3.7 Disk-Diffusion Assay
		4 Notes
		References
	Chapter 2: Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa
		1 Introduction
		2 Materials
			2.1 Culture Media, Supplements, and Solutions
			2.2 Bacterial Strains and Plasmids
			2.3 DNA and Cloning Procedures
		3 Methods
			3.1 Generation of the Plasmid Construct for Arabinose-Dependent Inducible Expression
			3.2 Generation of the Plasmid Construct for the Deletion of the CDS of Interest
			3.3 Generation of the Conditional Mutant in the Gene of Interest
			3.4 Confirmation and Investigation of the Conditional Mutant
		4 Notes
		References
	Chapter 3: Analyzing the Function of Essential Genes by Plasmid Shuffling
		1 Introduction
		2 Materials
			2.1 Strains and Plasmids
			2.2 Culture Media
			2.3 Vector Construction
			2.4 Preparation and Transformation of E. coli Electrocompetent Cells
			2.5 Small-Scale LPS Extraction
		3 Methods
			3.1 Vector Construction
			3.2 Thermosensitive and Streptomycin-Resistant Host Strain Generation
				3.2.1 Preparation of Recipient E. coli Strain Electrocompetent Cells
				3.2.2 Cell Transformation and Selection Procedure
				3.2.3 Screening of pMBM-GOI1GOI2 Transformants
			3.3 Plasmid Shuffling
				3.3.1 Electrocompetent Host Cell Preparation
				3.3.2 Host Cell Transformation and Double Selection Against Cells Carrying pMBM-GOI1GOI2
				3.3.3 Screening of Transformants and Identification of Suppressor Mutation(s)
			3.4 LPS Extraction from Suppressor Mutants
		4 Notes
		References
Part II: Functional Analysis of Proteins Involved in LPS Transport
	Chapter 4: Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Esch...
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Bacterial Growth
			2.3 N-Acetyl-[3H]-Glucosamine Pulse Labeling
			2.4 Plasmolysis and Whole-Membrane Fraction Purification
			2.5 Membrane Fractionation by Sucrose Density Gradient Ultracentrifugation
			2.6 Bradford Assay
			2.7 NADH Oxidase Assay
			2.8 Outer Membrane Protein (OMP) Profile on Sodium-Dodecyl-Sulfate (SDS) Polyacrylamide Gel (PAGE)
			2.9 Tricine-SDS-PAGE
			2.10 LPS-Specific Silver Staining
		3 Methods
			3.1 Bacterial Growth
			3.2 Bacterial Growth for the Radiolabeling of Newly Synthesized LPS
			3.3 Total Membrane Fraction Purification
			3.4 Membrane Fractionation
			3.5 Analysis of [3H]-Glucosamine Incorporation (Applicable Only When Bacterial Growth Has Been Performed as Described in Subhe...
			3.6 Identification of Inner and Outer Membrane Fractions from Sucrose Gradient
				3.6.1 Determination of Protein Content Profile by Bradford Assay
				3.6.2 Identification of Inner Membrane Fractions by NADH Activity Assay
				3.6.3 Identification of Outer Membrane Fractions Through the Analysis of OMP Profile by SDS-PAGE
			3.7 Analysis of LPS Profile in Membrane Fractions
				3.7.1 Sample Preparation
				3.7.2 LPS Fractionation in Tricine-SDS-PAGE
				3.7.3 Gel Fixing and Staining
		4 Notes
		References
	Chapter 5: Exploring the Topology of Cytoplasmic Membrane Proteins Involved in Lipopolysaccharide Biosynthesis by in Silico an...
		1 Introduction
		2 Materials
			2.1 Construction of Cysteine-Less Proteins
			2.2 Growth Media and Strains
				2.2.1 Media
				2.2.2 Strains
			2.3 Sulfhydryl Labeling
			2.4 Bradford Assay
			2.5 Protein Purification and Cross-Linking
			2.6 SDS-Polyacrylamide Gel Electrophoresis
			2.7 Western Blotting
			2.8 Protein Oligomerization
		3 Methods
			3.1 In Silico Transmembrane Protein Topology Prediction
			3.2 Construction of Cysteine-Less Proteins
			3.3 Sulfhydryl Labeling EDTA-Permeabilized Intact Cell Preparation
			3.4 Sulfhydryl Labeling of Total Membrane Fraction
			3.5 Thiol-Specific Chemical Blocking with N-Ethylmaleimide (NEM)
			3.6 Detection of Sulfhydryl Labeling Proteins and Total Membrane Preparation by Western Blotting
			3.7 Protein Oligomerization
		4 Notes
		References
	Chapter 6: Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions
		1 Introduction
		2 Materials
		3 Methods
			3.1 Protein Preparation
			3.2 Spin Labeling
			3.3 Sample Preparation
			3.4 Sample Tube Preparation
			3.5 Data Collection
			3.6 Data Visualization
		4 Notes
		References
	Chapter 7: A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell
		1 Introduction
		2 Materials
			2.1 Strain and Plasmids
			2.2 Media
			2.3 PiXie (Pulse-Chase and In Vivo Photo-Crosslinking Experiment)
			2.4 Purification of Crosslinked Products
			2.5 1 M MgSO4
		3 Methods
			3.1 Culture of Cells
			3.2 Radioisotope (RI) Label and Photo-Crosslinking (See Fig. 3)
			3.3 Purification of Crosslinked Products
		4 Notes
		References
	Chapter 8: Mass Spectrometry Analysis of Dynamics and Interactions of the LPS Translocon LptDE
		1 Introduction
		2 Materials
			2.1 Native Mass Spectrometry (nMS)
				2.1.1 Reagents
				2.1.2 Equipment
				2.1.3 Software
			2.2 Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)
				2.2.1 Reagents
				2.2.2 Equipment
				2.2.3 Software
		3 Methods
			3.1 Native Mass Spectrometry (nMS)
				3.1.1 Sample Preparation
				3.1.2 nMS Experimental Procedure: Obtaining the LptDE Spectrum
				3.1.3 nMS Experimental Procedure: Lipid-Binding Experiments
				3.1.4 nMS Experimental Procedure: Thanatin-Binding Experiments
				3.1.5 nMS Spectra Interpretation and Data Analysis
			3.2 HDX-MS
				3.2.1 Liquid Chromatography-Mass Spectrometry (LC-MS) Setup
				3.2.2 HDX-MS Experimental Procedure
				3.2.3 HDX-MS Data Analysis
		4 Notes
		References
	Chapter 9: Affinity Purification and Coimmunoprecipitation of Transenvelope Protein Complexes in Gram-Negative Bacteria
		1 Introduction
		2 Materials
			2.1 Cell Growth
			2.2 Preparation of Cell Lysate
			2.3 Affinity Purification and Coimmunoprecipitation (Co-IP)
			2.4 Resolution of Protein Complexes by SDS-Polyacrylamide Gel (PAGE)
			2.5 Western Blotting
			2.6 In-Gel Digestion and Protein Identification by Mass Spectrometry
		3 Methods
			3.1 Bacterial Growth
			3.2 Affinity Purification
			3.3 Affinity Purification Verification by SDS-PAGE and Western Immunoblotting
			3.4 Tandem Affinity Purification - Coimmunoprecipitation
			3.5 Resolution of Protein Co-IP Complexes
			3.6 In-Gel Digestion and Protein Identification by HPLC-MS
		4 Notes
		References
	Chapter 10: A Bacterial Two-Hybrid System for In Vivo Assays of Protein-Protein Interactions and Drug Discovery
		1 Introduction
			1.1 Principle of the BACTH System
			1.2 Overview of the Procedure
		2 Materials
			2.1 Solutions and Buffers
			2.2 Bacterial Media
			2.3 Bacterial Strains and Plasmids
			2.4 Equipment
		3 Methods
			3.1 Construction of Recombinant Plasmids Expressing the Two Proteins of Interest (Say X and Y) as Fusions with T25 and T18
			3.2 Assay of Interaction Between Hybrid Proteins in E. coli cya Cells
			3.3 Quantification of Functional Complementation Between Hybrid Proteins by β-Galactosidase Assays
			3.4 β-Galactosidase Data Analysis
			3.5 Further Characterization of Hybrid Proteins
			3.6 Applications of BACTH Assays
		4 Notes
		References
	Chapter 11: Detecting Lipoproteins Sneaking Out of the Lipopolysaccharide Leaflet
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Bacterial Strains and Growth Medium
			2.3 Solutions
		3 Methods
			3.1 Sample Preparation
			3.2 Dotblot
		4 Notes
		References
Part III: Tools for Structural Analysis of LPS and LPS Transport Proteins
	Chapter 12: Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry
		1 Introduction
			1.1 General Overview
			1.2 GC-MS
			1.3 MALDI-TOF MS
		2 Materials
			2.1 GC-MS
			2.2 MALDI
		3 Methods
			3.1 GC-MS
				3.1.1 Acetylated Methyl Glycosides (AMG)
				3.1.2 Acetylated Alditols (AA)
				3.1.3 Acetylated 2-Octyl Glycosides (AOG)
				3.1.4 Partially Methylated Acetylated Alditols (PMAA)
				3.1.5 Fatty Acid Methyl Esters (FAME)
			3.2 MALDI-TOF MS
				3.2.1 Analysis of the Lipid A from LPS or LOS Molecules
				3.2.2 Determination of the LOS Structure
		4 Notes
		References
	Chapter 13: Probing the Functional Interaction Interface of Lipopolysaccharide and Antimicrobial Peptides: A Solution-State NM...
		1 Introduction
			1.1 General Overview
			1.2 Probing the Peptide-LPS Interaction Interface: Contributions of Nuclear Magnetic Resonance (NMR)
				1.2.1 Bioactive Conformation: Applications of Transferred NOESY (trNOESY)
				1.2.2 Defining the Epitope of Hetero-molecular Interactions: Applications of Saturation Transfer Difference NMR Spectroscopy
		2 Materials
		3 Methods
			3.1 General Procedure for Loading Sample in NMR Tubes
			3.2 Solvent Suppression
			3.3 Setting a trNOESY Experiment
				3.3.1 Outline of Structure Calculation from NOESY Spectra
			3.4 Setting a STD NMR Experiment
				3.4.1 Analysis of STD Spectrum
		4 Notes
		References
	Chapter 14: Cryo-EM Analysis of the Lipopolysaccharide Flippase MsbA
		1 Introduction
		2 Materials
			2.1 Cloning and Expression
			2.2 Protein Purification
			2.3 Nanodisc Reconstitution
			2.4 Cryo-EM Sample Preparation
			2.5 Cryo-EM Data Processing
		3 Methods
			3.1 Construct and Expression
			3.2 Protein Purification
			3.3 Nanodisc Reconstitution
			3.4 Cryo-EM Sample Preparation
			3.5 Cryo-EM 3D Data Processing
		4 Notes
		References
	Chapter 15: Protein Crystallization of Two Recombinant Lpt Proteins
		1 Introduction
		2 Materials
			2.1 Recombinant Lpt* Protein Production in E. coli Cells (*Refers to Both Pa-LptH and EcLptC24-191G153R)
			2.2 Recombinant Lpt* Protein Purification
			2.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.4 Dynamic Light Scattering (DLS)
			2.5 Crystallization
		3 Methods
			3.1 Recombinant Lpt* Protein Production in E. coli Cells
			3.2 Recombinant Lpt* Protein Purification
			3.3 SDS-PAGE
			3.4 Dynamic Light Scattering (DLS)
			3.5 Protein Crystallization
		4 Notes
		References
Part IV: Microscopy-Based Approaches
	Chapter 16: Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging
		1 Introduction
		2 Materials
			2.1 Kdo-N3 Incorporation into Bacterial Cells
			2.2 Fluorescent SPAAC Labeling of Kdo-N3-Containing Bacterial Cells
			2.3 Fluorescence Microscopy and Plasmolysis of Intact Bacterial Cells
			2.4 SDS-PAGE Gel (LPS Gel)
			2.5 Fluorescence Flow Cytometry
		3 Methods
			3.1 Kdo-N3 Incorporation into Bacterial Cells
			3.2 Fluorescent SPAAC Labeling (Click Labeling) of Kdo-N3
			3.3 Fluorescence Microscopy and Plasmolysis of Labeled Bacteria
			3.4 Visualization of Fluorescent LPS by SDS-PAGE
			3.5 Fluorescence Flow Cytometry Analysis of Fluorescently Labeled Bacteria
		4 Notes
		References
	Chapter 17: Use of Atomic Force Microscopy to Characterize LPS Perturbations
		1 Introduction
		2 Materials
			2.1 LPS and Antibiotic Conjugation
			2.2 Bacterial Cell Culture
		3 Methods
			3.1 LPS Conjugation on the Mica Surface
			3.2 Antibiotic Treatment
			3.3 Bacterial Culture
			3.4 Bacterial Sample Preparation for Imaging in Ambient Environment
			3.5 AFM Setup for Imaging
			3.6 Imaging LPS Layer in Physiological Environment
			3.7 Measure Height Changes (Nano-shaving)
			3.8 Imaging Effects of PMB on Bacterial Surface
			3.9 Data Analysis
		4 Notes
		References
Index