Lipopolysaccharide Transport Methods and Protocols

This volume provides the most widely used protocols and techniques employed in lipopolysaccharide( LPS) transport studies. Chapters detail identification and analysis of essential genes and crucial mutations in LPS transport-related genes, LPS profile determination, LPS chemical analysis, protein-LPS interaction, and LPS visualization. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Lipopolysaccharide Transport: Methods and Protocols aims to deliver an overview on the multidisciplinary study of LPS transport and to identify new and innovative antibacterial strategies.

PART I       Genetic Strategies to Study Essential LPS Biogenesis Genes

1.          Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport

Andrew Wilson, Carlos Iniguez, and Natividad Ruiz

2.          Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa

Alessandra Lo Sciuto, Maria Concetta Spinnato, Martina Pasqua, and Francesco Imperi

3.          Analysing the Function of Essential Genes by Plasmid Shuffling

          Federica Anna Falchi

PART II      Functional Analysis of Proteins Involved in LPS Transport

4.          Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia Coli

Elisabete C. Cardoso Mendes Moura, Alessandra Polissi, and Paola Sperandeo

5.          Exploring The Topology of Cytoplasmic Membrane Proteins Involved in Lipopolysaccharide Biosynthesis by in silico and Biochemical Analyses

Julia Monjarás Feria and Miguel A. Valvano

6.          Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein–LPS Interactions

Kathryn M. Schultz and Candice S. Klug

7.          A Photo-Crosslinking Approach to Monitor Assembly of an LptD Intermediate with LptE in a Living Cell

Ryoji Miyazaki, Hiroyuki Mori, and Yoshinori Akiyama

8.          Mass Spectrometry Analysis of Dynamics and Interactions of the LPS translocon LptDE

Francesco Fiorentino and Jani R. Bolla

9.          Affinity Purification and Co-Immunoprecipitation of Trans-envelope Protein Complexes in Gram-negative Bacteria

Alessandra M. Martorana, Carlo Santambrogio, and Alessandra Polissi

10.       A Bacterial Two-Hybrid System for in vivo assays of Protein-Protein Interactions and Drug Discovery

Daniel Ladant

11.       Detecting Lipoproteins Sneaking out of the Lipopolysaccharide Leaflet

Naemi Csoma, Didier Colau, and Jean-François Collet

PART III     Tools for Structural Analysis of LPS and LPS Transport Proteins

12.       Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry

13.       Probing the Functional Interaction Interface of Lipopolysaccharide and Antimicrobial Peptides: A Solution-State NMR Perspective

Karishma Biswas and Anirban Bhunia

14.       Cryo-EM Analysis of the Lipopolysaccharide Flippase MsbA

Michela Bollati and Louise J. Gourlay

PART IV    Microscopy-Based Approaches

16.       Metabolic Incorporation of Azido-Sugars into LPS to Enable Live Cell Fluorescence Imaging

Inga Nilsson and David A. Six

17.       Use of Atomic Force Microscopy to Characterize LPS Perturbations