The Genetic Manipulation of Staphylococci

This detailed book contains chapters using unbiased approaches such as chemical and transposon mutagenesis, as well as a protocol for allelic exchange, to make targeted mutants in Staphylococcus, as generating mutants of this bacteria can be accomplished in many ways, depending on the intended changes. Using these methods, the authors have been successful at making mutants that span individual single-nucleotide changes in the chromosome to whole genome mutant libraries. The latter strategy provides a useful tool for high-throughput screening, while single nucleotide changes are an elegant way of teasing apart the importance of single nucleotides in gene expression or to create specific amino acid substitutions to examine protein function. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Practical and authoritative, The Genetic Manipulation of Staphylococci: Methods and Protocols serves as a vital resource and guide to scientists in the Staphylococcus community as they pursue their studies on these bacteria.

Understanding Staphylococcal Nomenclature.- Restriction-Modification Systems as a Barrier for Genetic Manipulation of Staphylococcus aureus.- Complementation Plasmids, Inducible Gene-Expression Systems, and Reporters for Staphylococci.- De Novo Assembly of Plasmids Using Yeast Recombinational Cloning.- Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products.- Method for Preparation and Electroporation of S. aureus and S. epidermidis.- Rapid Isolation of DNA from Staphylococcus.- Bacteriophage Transduction in Staphylococcus aureus: Broth-Based Method.- Bacteriophage Transduction in Staphylococcus aureus.- Batch Transduction of Transposon Mutant Libraries for Rapid Phenotype Screening in Staphylococcus.- Conjugative Transfer in Staphylococcus aureus.- Allelic Exchange.- Creation of Staphylococcal Mutant Libraries Using Transposon Tn917.- Generation of a Transposon Mutant Library in Staphylococcus aureus and Staphylococcus epidermidis Using Bursa aurealis.- Chemical and UV Mutagenesis.- Pulsed Field Gel Electrophoresis.- RNA-Sequencing of Staphylococcus aureus Messenger RNA.- Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.- Electrophoretic Mobility Shift Assays.- Rapid Amplification of cDNA Ends for RNA transcript Sequencing in Staphylococcus.