Main description:

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.

Contents:

1. Understanding Staphylococcal Nomenclature

Christina N. Krute and Jeffrey L. Bose

2. Restriction-Modification Systems as a Barrier for Genetic Manipulation of Staphylococcus aureus

Marat R. Sadykov

3. Complementation Plasmids, Inducible Gene-Expression Systems, and Reporters for Staphylococci

Ralph Bertram

4. De Novo Assembly of Plasmids Using Yeast Recombinational Cloning

Ameya Mashruwala and Jeffrey M. Boyd

5. Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products

Justin A. Thornton

6. Method for Preparation and Electroporation of S. aureus and S. epidermidis

Melinda R. Grosser and Anthony R. Richardson

7. Rapid Isolation of DNA from Staphylococcus

Kelsey L. Krausz and Jeffrey L. Bose

8. Bacteriophage Transduction in Staphylococcus aureus: Broth-Based Method

Kelsey L. Krausz and Jeffrey L. Bose

9. Bacteriophage Transduction in Staphylococcus aureus

Michael E. Olson

10. Batch Transduction of Transposon Mutant Libraries for Rapid Phenotype Screening in Staphylococcus

Katherine L. Maliszewski

11. Conjugative Transfer in Staphylococcus aureus

Cortney R. Halsey and Paul D. Fey

12. Allelic Exchange

McKenzie K. Lehman, Jeffrey L. Bose, and Kenneth W. Bayles

13. Creation of Staphylococcal Mutant Libraries Using Transposon Tn917

Kelly C. Rice

14. Generation of a Transposon Mutant Library in Staphylococcus aureus and Staphylococcus epidermidis Using Bursa aurealis

Vijaya Kumar Yajjala, Todd J. Widhelm, Jennifer L. Endres, Paul D. Fey, and Kenneth W. Bayles

15. Chemical and UV Mutagenesis

Jeffrey L. Bose

16. Pulsed Field Gel Electrophoresis

Batu K. Sharma-Kuinkel, Thomas H. Rude, and Vance G. Fowler, Jr

17. RNA-Sequencing of Staphylococcus aureus Messenger RNA

Ronan K. Carroll, Andy Weiss, and Lindsey N. Shaw

18. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression

April M. Lewis and Kelly C. Rice

19. Electrophoretic Mobility Shift Assays

Sarah E. Rowe and James P. O'Gara

20. Rapid Amplification of cDNA Ends for RNA transcript Sequencing in Staphylococcus

Eric Miller