Pseudomonas aeruginosa: Methods and Protocols (Methods in Molecular Biology, 2721)

✍ Scribed by Giovanni Bertoni (editor), Silvia Ferrara (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 242
• Category: Library

✦ Synopsis

This book aims to provide methods, protocols, and discussion topics for those who wish to examine in depth the molecular mechanisms of adaptation and versality of bacteria and would like to envisage their evolution responses in the fast changing Antropocene.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Pseudomonas aeruginosa: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

✦ Table of Contents

Preface
Contents
Contributors
Part I: CRISPR-Based Genome Editing
Chapter 1: CRISPR/Cas9-based Genome Editing of Pseudomonas aeruginosa
1 Introduction
2 Materials
2.1 Construction of the Editing Plasmids
2.2 Preparation of P. aeruginosa Electrocompetent Cells
2.3 Genome Editing in P. aeruginosa
3 Methods
3.1 Construction of the Editing Plasmid
3.1.1 Insertion of the Suitable Spacer into the pACRISPR Plasmid
3.1.2 Assembly of the Repair Template into the pACRISPR-XX_spacer Plasmid
3.2 Genome Editing in P. aeruginosa Strains
3.2.1 Preparation of P. aeruginosa Electrocompetent Cells
3.2.2 Prepare P. aeruginosa Electrocompetent Cells Containing the pCasPA Plasmid
3.2.3 pCasPA/pACRISPR-mediated Genome Editing in P. aeruginosa
3.2.4 Plasmid Curing
3.3 pnCasPA-BEC-mediated Cytidine Base Editing in P. aeruginosa
4 Notes
References
Chapter 2: Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Plasmids
2.3 sgRNA Spacer Sequences
2.4 Bacterial Media and Additives
2.5 Equipment and Supplies for Microbiology and Molecular Biology
2.6 Nucleic Acid Purification Kits
2.7 Enzymatic Reactions
2.8 Equipment and Supplies for Mouse Infection and Analysis
2.8.1 General Laboratory Disposables
2.8.2 General Laboratory Equipment
2.8.3 General Mouse Facility Equipment
2.8.4 Surgical and Anesthesia Equipment
2.9 Reagents for Mouse Infection and Analysis
2.9.1 Reagents for Mouse Infection
2.9.2 Reagents for Mouse Anesthesia
2.10 Mice
3 Methods
3.1 Cloning sgRNA Spacers into Mobile-CRISPRi Vectors
3.1.1 Preparation of BsaI-digested Mobile-CRISPRi Plasmid
3.1.2 Annealing of sgRNA Spacer-encoding Oligonucleotides
3.1.3 Ligation of Annealed Oligonucleotides and BsaI-digested Vector
3.1.4 Transformation
3.1.5 Confirmation of Mobile-CRISPRi Plasmid Clones
3.2 Transferring Mobile-CRISPRi to Pseudomonas by Tn7 Transposition
3.2.1 Construction of Donor Strains for Tn7 Transposition
3.2.2 Tn7 Transposition of Mobile CRISPRi to the Pseudomonas aeruginosa Chromosome
3.3 Testing Gene Essentiality in a Murine Lung Infection Model
4 Notes
References
Part II: Genetic Tools and Biosensors
Chapter 3: Engineering Green-light-responsive Heterologous Gene Expression in Pseudomonas
1 Introduction
2 Materials
2.1 Strains and Plasmids
2.2 Media
2.3 Molecular Biology Reagents
2.4 Additional Materials
3 Methods
3.1 Construction of the Plasmid pGBF
3.2 Culture Conditions
3.3 Biofilm Staining
4 Notes
References
Chapter 4: Fluorescence-based Evaluation of Cyclic di-GMP Levels in Pseudomonas aeruginosa
1 Introduction
2 Materials
2.1 Bacterial Strains and Plasmids
2.2 Growth Media
2.3 Reagents
2.4 Other Equipment
3 Methods
3.1 Preparation of Competent P. aeruginosa Cells for Heat-shock Transformation
3.2 Transformation of pCdrA::gfpC Reporter Plasmid into P. aeruginosa Strains
3.3 Qualitative Analysis of c-di-GMP Levels
3.4 Quantitative Analysis of c-di-GMP Levels
4 Notes
References
Chapter 5: Whole-Cell Biosensors for Qualitative and Quantitative Analysis of Quorum Sensing Signal Molecules and the Investig...
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Growth Media
2.3 Reagents
2.4 Other Equipment
3 Methods
3.1 Overlay Assay for Qualitative Analysis of 3OC12-HSL Production
3.2 Quantification of 3OC12-HSL Levels in P. aeruginosa Supernatants
3.2.1 Growth of Bacterial Strain(s) of Interest and Recovery of the Supernatant(s)
3.2.2 Analysis of 3OC12-HSL Level in the Supernatant(s)
3.2.3 Data Analysis
3.3 Coculture Assay for Estimation of 3OC12-HSL Production During P. aeruginosa Growth
4 Notes
References
Chapter 6: A Pseudomonas aeruginosa-Suitable Fluorescent Reporter System for Analyzing Small RNA-Mediated Regulation of Target...
1 Introduction
2 Materials
2.1 Bacterial Strains and Plasmids
2.2 Oligonucleotides (5′3′)
2.3 Growth Media
2.4 Reagents
2.5 Other Equipment
3 Methods
3.1 Preparation of Competent E. coli DH5α Cells for Heat-Shock Transformation
3.2 Preparation of Competent P. aeruginosa Cells for Heat-Shock Transformation
3.3 Cloning into pXG10-SF Vector
3.4 Cloning into pBBR1-MCS5 Vector
3.5 Transformation of pBBR1-MCS5 Derivatives Expressing Translational Fusions into P. aeruginosa
3.6 Quantitative Evaluation of GFP Levels
3.7 Qualitative Analysis of GFP Levels
4 Notes
References
Chapter 7: The Pseudomonas aeruginosa Resistome: Permanent and Transient Antibiotic Resistance, an Overview
1 Introduction
2 Analysis of P. aeruginosa Intrinsic Resistome
3 Methods for the Study of the Mutation-Driven Acquisition of Antibiotic Resistance
4 Methods for Measuring Transient Antibiotic Resistance
5 In Silico Analysis of P. aeruginosa Resistome
References
Chapter 8: Biosensors for Inducers of Transient Antibiotic Resistance
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Plasmids
2.3 Oligonucleotides
2.4 Growth Media
2.5 Reagents
2.6 Equipment
3 Methods
3.1 Luminescence-Based Methods
3.1.1 Primer Design and Cloning of the Promoter Region of the mexCD-oprJ Operon (PmexCD)
3.1.2 Preparation of Chemocompetent E. coli S17-1λpir by CaCl2-Based Method
3.1.3 Construction of the luxCDABE-Based Plasmid Containing the Promoter Region of Interest
3.1.4 Integration of the Reporter luxCDABE-Based Plasmid into the Chromosome of P. aeruginosa Strains
3.1.5 Real-Time Monitoring of Luminescence for Promoter Inducers Screening
3.1.6 Normalization of Luminescence Measurements for Detection of Inducers
3.2 Fluorescence-Based Methods
3.2.1 Primer Design and Cloning of the Desired Promoter Region
3.2.2 Construction of the pSEVA237Y-Based Plasmid Containing the Promoter Region of Interest
3.2.3 Alternative Method for the Delivery of Reporter Plasmid into Pseudomonas aeruginosa by Conjugation
3.2.4 Real-Time Monitoring of Fluorescence for Promoter Inducers Screening
3.2.5 Normalization of Fluorescence Measurements for Detection of Inducers
3.2.6 Flow Cytometry Assay
4 Notes
References
Part III: Secreted Factors and Microscale Analysis of Biofilm
Chapter 9: Pseudomonas aeruginosa Soluble Pyocins as Antibacterial Weapons
1 Introduction
2 Materials
2.1 Bacterial Strains and Plasmids
2.2 Growth Media
2.3 Oligonucleotides (5′3′)
2.4 Reagents
2.5 Equipment
3 Methods
3.1 Plasmid Construction
3.2 Overexpression and Purification of Pyocins S4 and S5
3.3 Pyocin Sensitivity Assays
3.4 Identification of the S4 and S5 Pyocin Receptors on Sensitive Strains
3.4.1 Multiplex PCR Receptor Typing for the Identification of the S4 fpvA Receptor Type (See Note 3)
3.4.2 Allelic Replacement of fpvAI from an S4-Sensitive Strain
3.4.3 Selection of S5-Insensitive Mutants by Mini-Transposon Mutagenesis
3.5 Construction of S5-S2 Pyocin Chimers
4 Notes
References
Chapter 10: Assays for Studying Pseudomonas aeruginosa Secreted Proteases
1 Introduction
2 Materials
2.1 Skim Milk Agar Plate Assay
2.1.1 Bacterial Strains (See Notes 1 and 2)
2.1.2 Growth Media (See Note 3)
2.1.3 Protease Substrate
2.2 Sulfanilamide-Azocasein Assay for Total Proteolytic Activity Determination
2.2.1 Bacterial Strains (See Notes 1 and 2)
2.2.2 Growth Media
2.2.3 Protease Substrate
2.2.4 Reagents
2.3 Elastin Congo-Red Assay for Elastinolytic Activity Determination
2.3.1 Bacterial Strains (See Notes 1 and 2)
2.3.2 Growth Media
2.3.3 Protease Substrate
2.3.4 Reagents
3 Methods
3.1 Skim Milk Agar Plate Assay
3.2 Sulfanilamide-Azocasein Assay for Total Proteolytic Activity Determination
3.3 Elastin Congo-Red Assay for Elastinolytic Activity Determination
4 Notes
References
Chapter 11: Single Microcolony Diffusion Analysis in Pseudomonas aeruginosa Biofilms
1 Introduction
2 Materials
2.1 Bacterial Strains (see Note 1)
2.2 Growth Media
2.3 Chemicals
2.4 Equipment
2.4.1 Commercial Light Sheet Microscope
2.4.2 Custom-built SPIM Microscope
2.5 Software
2.5.1 Data Analysis Software and Fitting Model
3 Methods
3.1 Biofilm Growth Condition and Flow Setup
3.1.1 Preparation of Inoculum
3.1.2 Setting Up the Flow Chamber
3.1.3 Sample Preparation for Light Sheet-based FCS (see Note 2)
3.2 Setting Up the Image Acquisition
3.2.1 Determination of Light Sheet Thickness (see Note 6)
3.2.2 Setting Up the Image Acquisition (see Notes 7 and 8)
3.3 Imaging FCS Data Analysis
3.3.1 Installation of Imaging FCS 1.52 Plugin
3.3.2 Imaging FCS Data Analysis
3.3.3 Calibration of SPIM
3.4 Preparation and Imaging FCS for Alginate Beads
3.5 Generation of PAO1 eGFP pBAD yhjH Strain
3.5.1 Preparation and Transformation of PAO1 eGFP Competent Cells
3.5.2 Isolation of pBAD yhjH Plasmid from P. aeruginosa PAO1 pBAD yhjH
3.5.3 Generation of Gentamycin-sensitive PAO1 eGFP
3.5.4 Generation of P. aeruginosa PAO1 eGFP pBAD yhjH
4 Notes
References
Part IV: Genome-Scale Approaches
Chapter 12: Broad Genome Sequencing of Environmental and Clinical Strains and Genotyping
1 Introduction
2 Materials
2.1 Growth Media
2.2 DNA Extraction and Quality Check
2.3 Library Preparation
3 Methods
3.1 Genomic DNA Extraction
3.2 DNA Quality Check
3.3 NGS Library Preparation
3.3.1 Tagmentation
3.3.2 Amplification and Cleaning
3.3.3 Library Normalization
3.3.4 Library Loading
3.4 Assembly
3.5 Genotyping of P. aeruginosa
3.6 Graphical Representation of MLST Data (eBURST Minimum Spanning Tree)
4 Notes
References
Chapter 13: Genome-Scale Analysis of the Structure and Function of RNA Pathways and Networks in Pseudomonas aeruginosa
1 Introduction
2 Exploring the Transcription Level
2.1 RNA-Seq for Quantification and Annotation of Transcripts at Single-Nucleotide Resolution
2.2 Dissection of Specific Regulatory Networks and Their Interplay by Combining RNA-Seq and ChIP-Seq
2.3 Term-Seq: A Genome-Wide Evaluation of the Transcription Termination with the Potential to Discover New Regulation and Ribo...
2.4 Transcriptomics at Single-Cell Level
2.5 Transcriptome Analysis of P. aeruginosa During Infection: RNA-Seq on Ex Vivo Samples and Dual RNA-Seq
2.6 Combination of Transcription and Translation Profiling
3 Exploring the Post-Transcriptional Regulatory Networks
3.1 Genome-Wide Detection of RNA-RNA Interactions
3.2 High-Throughput Assays for RNA Binding Protein Activity
3.3 Probing the Dynamics of RNA Structure at a Global Scale
References
Chapter 14: In-Depth Quantitative Proteomics Analysis of the Pseudomonas aeruginosa Secretome
1 Introduction
2 Materials
2.1 Induction of the T3SS of Pseudomonas aeruginosa
2.2 TCA Precipitation
2.3 Solubilization of Protein Pellet Followed by Reduction and Alkylation
2.4 Single-Pot, Solid-Phase-Enhanced Sample Preparation (SP3) and Digestion
2.5 Solid Phase Extraction (C18 Purification) of Peptides
2.6 LC-MS/MS Measurement
2.7 Label-Free Quantification Using MaxQuant
3 Methods
3.1 Induction of the T3SS of Pseudomonas aeruginosa
3.2 TCA Precipitation of Supernatant
3.3 Solubilization of Protein Pellet Followed by Reduction, Alkylation, and Digestion
3.4 Single-Pot, Solid-Phase-Enhanced Sample Preparation (SP3) and Digestion
3.5 Solid Phase Extraction of Peptides (C18 Purification)
3.6 LC-MS/MS Measurement
3.7 Label-Free Quantification Using MaxQuant
3.8 Data Analysis Using Perseus
4 Notes
References
Part V: Host-Pathogen Interaction
Chapter 15: Improving the Predictive Value of Preclinical Mouse Models of Pseudomonas aeruginosa Respiratory Infection to Eval...
1 Introduction
2 Materials
2.1 Solutions
2.2 Mice
2.3 Infection of Mice
2.4 Drug Administration in Mice
2.4.1 Aerosol Treatment by Penn-Century MicroSprayer Aerosolizer
2.4.2 Intranasal Administration
2.4.3 Subcutaneous Administration and Intraperitoneal Injection
2.5 Mouse Health Monitoring
2.6 Evaluation of Infection and Inflammation in the Murine Airways
2.7 Determination of Protein Amounts in Murine Samples
3 Methods
3.1 Infection of Mice
3.2 Different Routes of Drug Administration
3.2.1 Aerosol Treatment by Penn-Century MicroSprayer Aerosolizer
3.2.2 Intranasal Administration
3.2.3 Subcutaneous Administration
3.2.4 Intraperitoneal Injection
3.3 Murine Health Monitoring
3.3.1 Monitoring Murine Health After Acute Infection
3.3.2 Monitoring Murine Health During Chronic Infection
3.4 Evaluation of Infection and Inflammation in Murine Airways
3.4.1 Bacterial Load and Inflammation in Bronchoalveolar Lavage Fluid (BALF)
3.4.2 Bacterial Load and Inflammation in the Lungs
3.5 Determination of Protein Amounts in Murine Samples
3.6 Normalization of Readout Levels and Presentation of Data
4 Notes
References
Chapter 16: Emerging In Vitro Models for the Study of Infection and Pathogenesis of Pseudomonas aeruginosa and Testing of Anti...
1 Introduction
2 Organoid-Based Models of P. aeruginosa Infections
3 Modeling P. aeruginosa Infection in Human Lung Airway-on-a-Chip
References
Index