Bacterial Cell Wall Homeostasis: Methods and Protocols (Methods in Molecular Biology, 1440)

✍ Scribed by Hee-Jeon Hong (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 286
• Category: Library

✦ Synopsis

This volume brings together the most widely used and important protocols currently being employed in researching and understanding bacterial cell wall homeostasis. Chapters in Bacterial Cell Wall Homeostasis cover a variety of subjects, such as: modern microscopy techniques and other biophysical methods used to characterize the subcellular structure of the bacterial cell wall; high-throughput approaches that can be used to identify all the genes and proteins that participate in the correct functioning of an organism’s cell wall; protocols for assaying individual gene products for specific cell wall functions or identify chemicals with inhibitory activity against the cell wall; and methods for analyzing the non-protein components of the cell wall and the increasing use of computational approaches for predicting and modeling cell wall related functions and processes. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary material and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and cutting-edge, Bacterial Cell Wall Homeostasis: Methods and Protocols emphasizes the diversity of the research taking place in bacterial cell wall homeostasis, and explains how the integration of information from across multiple disciplines is going to be essential if a holistic understanding of this important process is to be obtained.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Analyzing Physical Properties of the Bacterial Cell Wall
Chapter 1: Atomic Force Microscopy Analysis of Bacterial Cell Wall Peptidoglycan Architecture
1 Introduction
2 Materials
2.1 Purification of Peptidoglycan Sacculi
2.1.1 Purification of Peptidoglycan Sacculi from Gram-­Positive Bacteria
2.1.2 Purification of Peptidoglycan Sacculi from Gram-­Negative Bacteria
2.2 Mounting of Peptidoglycan Sacculi for AFM Imaging
3 Methods
3.1 Purification of Peptidoglycan Sacculi from Gram-Positive Bacteria
3.2 Purification of Peptidoglycan Sacculi from Gram-Negative Bacteria
3.3 Breaking S. aureus
3.4 Breaking B. subtilis
3.5 Breaking Gram-Negative Bacteria
3.6 Preparation of Steel-Mica Stubs for AFM
3.7 Preparation of Sacculus Suspensions (Gram-Positive)
3.8 Preparation of Sacculus Suspensions (Gram-­Negative)
3.9 Mounting of Peptidoglycan Sacculi for AFM Imaging
4 Notes
References
Chapter 2: Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure
1 Introduction
2 Materials
2.1 Sacculi Preparation
2.2 Gram-Negative Bacteria Peptidoglycan Purification
2.3 Gram-Positive Bacteria Peptidoglycan Purification
2.4 Muramidase Digestion
2.5 Sample Reduction and Filtration
2.6 UPLC Separation
3 Methods
3.1 Sacculi Preparation
3.2 Gram-Negative Bacteria Peptidoglycan Isolation
3.3 Gram-Positive Bacteria Peptidoglycan Isolation
3.4 Muramidase Digestion
3.5 Sample Reduction and Filtration
3.6 UPLC Inorganic Separation
3.7 UPLC Organic Separation
3.8 UPLC Data Processing
4 Notes
References
Part II: Genome-Wide Approaches for the Identification of Gene Products with Roles in Cell Wall Homeostasis
Chapter 3: Microarray Analysis to Monitor Bacterial Cell Wall Homeostasis
1 Introduction
2 Materials
2.1 Strain and Culture Media
2.2 RNA Extraction and Quality Control (See Note 4)
2.3 cDNA Synthesis and Cleanup
2.4 cDNA Fragmentation, Terminal Labeling, and Quality Control
2.5 Affymetrix GeneChip Hybridization, Washing, and Staining (See Note 6)
2.6 Affymetrix GeneChip Data Analysis (See Note 15)
3 Methods
3.1 Culture and Induction with Antibiotics
3.2 RNA Isolation (See Note 18)
3.3 cDNA Synthesis
3.4 cDNA Fragmentation and Terminal Labeling
3.5 Quality Control of Labeled and Fragmented cDNA Using a Gel Shift Assay (See Note 22)
3.6 Hybridization, Washing, Staining, and Scanning of Arrays
3.7 Data Analysis (See Note 25)
3.8 Database Submission
4 Notes
References
Chapter 4: Cell Shaving and False-Positive Control Strategies Coupled to Novel Statistical Tools to Profile Gram-Positive Bacterial Surface Proteomes
1 Introduction
1.1 Bacterial Surface Topography
1.2 Cell Shaving Proteomics
2 Materials
2.1 Growth of Microorganisms
2.2 Sample Preparation for Cell Shaving
2.3 Mass Spectrometry and Data Analysis
3 Methods
3.1 Cell Shaving and False-­Positive Control
3.2 LC-MS/MS (See Note 8)
3.3 Data Analysis (See Notes 11 and 12)
4 Notes
References
Chapter 5: Differential Proteomics Based on Multidimensional Protein Identification Technology to Understand the Biogenesis of Outer Membrane of Escherichia coli
1 Introduction
2 Materials
2.1 Membrane Purification and Fractionation
2.2 Resolution of Inner and Outer Membranes
2.3 MudPIT Analysis
2.4 MS Analysis
2.5 Computational Analysis
3 Methods
3.1 Bacterial Growth
3.2 Whole Membrane Purification
3.3 Membrane Fractionation
3.4 Resolution of Inner and Outer Membranes from Sucrose Gradient
3.4.1 Total Protein Profile: Bradford Assay
3.4.2 Inner Membrane Profile: NADH Activity Assay
3.4.3 Outer Membrane Profile: SDS-­Polyacrylamide Gel Electrophoresis and Coomassie Blue Staining
3.5 Sample Preparation for MudPIT Analysis
3.6 MS Analysis
3.7 Computational Analysis
4 Notes
References
Chapter 6: Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection
1 Introduction
2 Materials
2.1 Strains, Plasmids, and Phages
2.2 Bacterial Media
2.3 Buffers
2.4 Antibiotics and Inductor
2.5 Oligonucleotides
2.6 DNA-­Related Kits
2.7 Enzymes
2.8 Other Reagents
3 Methods
3.1 Random Transposon Mutagenesis
3.2 Selection of Host Mutants Resistant to mEp213
3.3 Electroporation of Phage-­Resistant Host Mutants with Viral DNA
3.3.1 Phage DNA Extraction
3.3.2 Preparation of Electro-­Competent Cells
3.3.3 Electroporation
3.3.4 Mutants Related to FhuA Receptor
3.4 Sequencing the Host Chromosome Regions Adjacent to the Transposon
3.4.1 Plasmid Constructions
4 Notes
References
Part III: Functional Analysis of Cell-Wall Associated Proteins
Chapter 7: Zymographic Techniques for the Analysis of Bacterial Cell Wall in Bacillus
1 Introduction
2 Materials
2.1 SDS-
2.2 Zymography
2.3 Protein Staining
2.4 Extraction of Cell Surface Proteins
3 Methods
3.1 Purification of B. subtilis Cell Wall (Peptidoglycan Containing [Lipo/Wall] Teichoic Acid)
3.2 Purification of  B. subtilis Peptidoglycan Without Lipo/Wall Teichoic Acids
3.3 Confirmation of Quality of B. subtilis Cell Wall and Peptidoglycan
3.4 Zymographic Analysis
3.5 Preparation of Cell Surface Proteins for Zymography
3.6 Preparation of Proteins Secreted to Culture on Zymography
4 Notes
References
Chapter 8: Liquid Chromatography-Tandem Mass Spectrometry to Define Sortase Cleavage Products
1 Introduction
2 Materials
2.1 Enzymes and Substrates
2.2 Enzyme Concentration Determination
2.3 Cleavage Assay Components
2.4 LC/MS Analysis
3 Methods
3.1 Sortase Substrate Cleavage
3.2 Cleavage Product Analysis
4 Notes
References
Chapter 9: Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant
1 Introduction
1.1 Allelic Exchange in A. oris
1.2 Generation of Conditional Deletion Mutants in A. oris
1.3 Random gene Disruptions by Tn5-Based Transposition
2 Materials
2.1 Plasmids (Fig. 1)
2.2 Preparation of  A. oris Competent Cells
2.3 Electroporation
2.4 Allelic Replacement
2.5 Conditional srtA Deletion in A. oris
2.6 Tn5 Transposition
2.7 Thin Section and Electron Microscopy
3 Methods
3.1 Construction of Deletion Plasmids
3.2 Preparation of  A. oris Competent Cells
3.3 Electroporation of A. oris with pCWU2 or pCWU3
3.4 Allelic Exchange Using galK (pCWU2) as a Counterselection Marker
3.5 Allelic Exchange Using mCherry (pCWU3) as a Counterselection Marker
3.6 Generation of the A. oris Conditional srtA Deletion Mutant
3.7 Construction of the Tn5 Transposon Plasmid pMOD-­2/ Kan215
3.8 Production of the Tn5 Transposome
3.9 Characterization of Tn5 Insertion by TAIL-PCR
3.10 Preparation of A. oris Thin Sections
3.11 Transmission Electron Microscopy (TEM)
4 Notes
References
Part IV: Reporter Assays for Cell Wall Stress
Chapter 10: Construction of a Bioassay System to Identify Extracellular Agents Targeting Bacterial Cell Envelope
1 Introduction
2 Materials
2.1 Bacterial Strains and Culture (See Note 1)
2.2 Construction and Preparation of Plasmid DNA
2.3 Preparation of Streptomyces Protoplasts and DNA Transformation
2.4 Agarose Gel Electrophoresis
2.5 Antibiotics
3 Methods
3.1 Preparation of Protoplasts
3.2 Construction and Preparation of sigEp-neo Reporter Plasmid
3.3 Construction of sigEp-neo Bioassay System in S. coelicolor M600
3.4 Bioassay for Inducers of the sigEp-neo Reporter System
4 Notes
References
Chapter 11: Luciferase Reporter Gene System to Detect Cell Wall Stress Stimulon Induction in Staphylococcus aureus
1 Introduction
2 Materials
2.1 Luciferase Fusion Construct Components
2.2 Luciferase Assay Components
3 Methods
3.1 Construction of Promoter–Luciferase Reporter Gene Fusion Plasmids
3.2 Luciferase Assay
4 Notes
References
Part V: Analysis of the Non-Protein Components of the Cell Wall
Chapter 12: Extraction and Analysis of Peptidoglycan Cell Wall Precursors
1 Introduction
2 Materials
2.1 Strains
2.2 Cultivation Media
2.3 Antibiotics and Solution Preparation
2.4 Instruments
3 Methods
3.1 Cultivation of Microbes and Extraction of PG Precursors
3.1.1 Cultivation of B. megaterium and Extraction of PG Precursors
3.1.2 Cultivation of A. teichomyceticus and Nonomuraea sp. and Extraction of PG Precursors
3.2 LC and MS-MS Analysis
3.3 Data Analysis
3.3.1 Effect of Different Antibiotics on the PG Precursor Synthesis in B. megaterium
3.3.2 PG Precursor Modification in Glycopeptide Producing Actinomycetes
4 Notes1
References
Chapter 13: Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases
1 Introduction
2 Materials
2.1 Purification of the Peptidoglycan Synthase PBP1B
2.1.1 Overproduction of PBP1B and Its Solubilization from Cell Membranes
2.1.2 Ni-NTA Affinity Chromatography
2.1.3 Ion Exchange Chromatography
2.2 Glycosyltrans
3 Methods
3.1 Purification of the Peptidoglycan Synthase PBP1B
3.1.1 Overproduction of PBP1B and Its Solubilization from Cell Membranes
3.1.2 Ni-NTA Affinity Chromatography
3.1.3 Ion Exchange Chromatography
3.2 Glycosyltrans
3.3 Data Analysis
4 Notes
References
Chapter 14: Analysis of Peptidoglycan Fragment Release
1 Introduction
2 Materials
2.1 [3H]-Glucosa
2.2 Size-Exclusion Chromatography
2.3 Reversed-­Phase HPLC
3 Methods
3.1 Quantitative Metabolic Radiolabeling of Bacteria and Collection of Released Peptidoglycan Fragments
3.2 Analysis of Peptidoglycan Turnover
3.3 Size-Exclusion Chromatography
3.3.1 Pouring New Size-Exclusion Columns
3.3.2 Running Samples on Size-­Exclusion Columns
3.3.3 Concentration and Desalting of Collected Fractions for HPLC
3.4 Analysis of Fragments by Reversed-­Phase HPLC
4 Notes
References
Chapter 15: Analysis of Cell Wall Teichoic Acids in Staphylococcus aureus
1 Introduction
2 Materials
2.1 Extraction of WTA
2.2 WTA Phosphate Quantification
2.3 Analysis of WTA Monosaccharides
2.4 Analysis of WTA Extracts
3 Methods
3.1 Extraction of WTA
3.2 WTA Phosphate Quantification
3.3 Analysis of WTA Monosaccharides
3.4 Analysis of WTA Extracts
4 Notes
References
Chapter 16: Analysis of Bacterial Cell Surface Chemical Composition Using Cryogenic X-Ray Photoelectron Spectroscopy
1 Introduction
2 Materials
3 Methods
3.1 Cleaning of Sample Holder
3.2 Sample Preparation
3.3 Spectrometer Preparation
3.4 Loading of Sample into XPS Instrument
3.5 Measurement Procedure
3.6 Data Analysis
4 Notes
References
Part VI: Bioinformatics and Computational Biology Based Approaches
Chapter 17: Biophysical Measurements of Bacterial Cell Shape
1 Introduction
1.1 Active Contours and Meshes
1.1.1 Active Contours in 2D
1.1.2 3D Active Meshes
1.1.3 Adaptive Meshes
1.2 External Energy
1.2.1 Surface-Labeled Objects
1.2.2 Filled Objects
1.3 Fitting Polymers on Surfaces
1.4 Results
1.4.1 Fitting Accuracy
1.5 Localization and Curvature
2 Materials
3 Methods
3.1 PSF Measurements
3.2 Image Acquisition
3.3 Shape Initialization
3.4 Shape Fitting
3.5 Polymer Fitting
3.6 Benchmarks
4 Notes
References
Chapter 18: Coarse-Grained Molecular Dynamics Simulations of the Bacterial Cell Wall
1 Introduction
2 Materials
3 Method
3.1 Coarse-Grained Peptidoglycan Model
3.1.1 Glycan Mechanical Properties
3.1.2 Peptide Crosslink Mechanical Properties
3.1.3 Initial Sacculus
3.2 Turgor Pressure
3.3 Coarse-Grained Model of Enzymes
3.3.1 Transglycosylation
3.3.2 Transpeptidation
3.3.3 Endopeptidation
3.3.4 Enzyme Diffusion
3.3.5 Periplasmic Confinement
3.3.6 Interaction with LpoA and LpoB
3.3.7 Enzyme-Enzyme Tethering
3.3.8 Enzyme-PG Interaction
3.4 Relaxation
3.5 Shape Characterization
3.5.1 Hole Size
3.5.2 Central Line
3.5.3 Bulges
3.5.4 Straightness
3.5.5 Roughness
3.6 Visualization of Sacculus Growth
4 Notes
References
Chapter 19: Structural Comparison and Simulation of Pneumococcal Peptidoglycan Hydrolase LytB
1 Introduction
2 Materials
2.1 The Atomic Coordinates
2.2 Websites and Programs
2.3 Hydrolytic Activity Assays
3 Methods
3.1 Structural Comparison of the C-Terminal α-Helix Module
3.2 Homology Search of the Two All-β Modules
3.3 Simulation of LytBCAT Against the Putative Substrate
3.4 Hydrolytic Activity Assays
4 Notes
References
Index