Genetic Manipulation of Staphylococcus aureus

Olaf Schneewind1, Dominique Missiakas1

1 Department of Microbiology, University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9C.3
DOI:  10.1002/9780471729259.mc09c03s32
Online Posting Date:  February, 2014
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Staphylococcus aureus is a facultative anaerobic Gram‐positive coccus and a member of the normal skin flora, as well as that of the nasal passages of humans. However, S. aureus can also gain entry into the host and cause life‐threatening infections or persist as disease foci that develop into suppurative abscesses. While genetically tractable, the manipulation of S. aureus remains challenging. This unit describes methods developed in our laboratory for gene disruption by allelic replacement and transposition. We also provide a protocol for bacteriophage‐mediated transduction of mutants marked with selectable alleles and describe plasmid utilization for complementation studies. Curr. Protoc. Microbiol. 32:9C.3.1‐9C.3.19. © 2014 by John Wiley & Sons, Inc.

Keywords: Staphylococcus aureus; plasmid; bursa aurealis; transduction; allelic replacement

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Competent Cells of S. aureus
  • Basic Protocol 2: Electroporation of Plasmid DNA and Plating for Selection of Transformants
  • Basic Protocol 3: Transposon Mutagenesis Using bursa aurealis
  • Basic Protocol 4: Mapping bursa aurealis Insertions by Inverse PCR
  • Basic Protocol 5: Allelic Replacement Using pKOR1
  • Basic Protocol 6: Preparing S. aureus Lysates for Extraction of DNA
  • Basic Protocol 7: Transduction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Competent Cells of S. aureus

  • S. aureus strain from a frozen stock
  • Tryptic soy agar (TSA) plates (see recipe for plate preparation)
  • Tryptic soy broth (TSB; see recipe)
  • Ice‐cold sterile 0.5 M sucrose in deionized water
  • Ice
  • 37°C shaking incubator
  • Sterile loop
  • 15‐ml culture tubes
  • 1‐liter flasks
  • Spectrophotometer and plastic cuvette to measure cell density at 600 nm (A 600)
  • 50‐ml sterile centrifuge tubes (that can be centrifuged at up to 5,000 × g), sterile
  • Centrifuge
  • Microcentrifuge tubes, sterile
  • Dry ice‐ethanol bath
  • −80°C freezer

Basic Protocol 2: Electroporation of Plasmid DNA and Plating for Selection of Transformants

  • Frozen competent cells (see protocol 1)
  • Ice
  • Plasmid DNA (see protocol 6 and Troubleshooting section)
  • Tryptic soy broth (TSB; see recipe)
  • TSA plates (see recipe for plate preparation) containing either chloramphenicol at 5 or 10 µg/ml or tetracycline at 2.5 µg/ml (alone or in combination)
  • 0.1‐cm electroporation cuvettes (e.g., Bio‐Rad)
  • Electroporator
  • 1.5‐ml microcentrifuge tubes, sterile
  • 30°C incubator
  • Sterile tips and pipetman
  • Sterile spreader
  • 37°C incubator, optional

Basic Protocol 3: Transposon Mutagenesis Using bursa aurealis

  • A target S. aureus strain carrying plasmids pBursa and pFA545, prepared as described in Basic Protocols protocol 11 and protocol 22
  • TSA plates (see recipe for plate preparation) containing antibiotics (see below)
  • Antibiotics: chloramphenicol (Chl), erythromycin (Erm), and tetracycline (Tet) antibiotics used at the following final concentrations 5, 10, and 2.5 µg/ml, respectively
  • Sterile water
  • Tryptic soy broth (TSB; see recipe)
  • Tryptic soy agar (TSA; see recipe)
  • 2× cryopreservation solution (see recipe)
  • Flasks, sterile
  • 43°C shaking incubator
  • 1.5‐ml microcentrifuge tubes
  • Sterile loop
  • Vortex mixer
  • Sterile glass beads
  • 2‐ml cryotubes

Basic Protocol 4: Mapping bursa aurealis Insertions by Inverse PCR

  • Isolated cultures (see protocol 3)
  • 10× buffer for AciI (New England Biolabs or preferred commercial source)
  • Restriction enzyme AciI (New England Biolabs or preferred commercial source)
  • Chromosomal DNA (see protocol 3)
  • T4 DNA ligase and 10× buffer for T4 DNA ligase (New England Biolabs or preferred commercial source)
  • Qiagen MinElute 96 UF PCR purification kit (or preferred kit for purification of DNA fragments)
  • Oligonucleotide primers:
  • 1% agarose gel
  • 2× PCR Master Mix (Promega)
  • 96‐well plates
  • 37°C incubator
  • 200‐µl PCR tubes
  • Thermal cycler
  • Gel electrophoresis equipment
  • Additional reagents and equipment for lysing cells to extract genomic DNA (see protocol 6)

Basic Protocol 5: Allelic Replacement Using pKOR1

  • AccuPrime Pfx SuperMix (Invitrogen; for PCR amplification)
  • Cloning primers
  • Agarose gel
  • Preferred kit for extraction of DNA from agarose gel
  • T4 DNA ligase and buffer (NEB)
  • BP Clonase enzyme mix (Invitrogen)
  • Competent cells of E. coli DH5α and appropriate medium for growth
  • Ampicillin (Amp) and chloramphenicol (Chl) antibiotics used at the final concentrations of 100 and 10 µg/ml, for selection of E. coli and S. aureus carrying pKOR1, respectively
  • Tryptic soy broth (TSB; see recipe) and tryptic soy agar (TSA; see recipe) to grow S. aureus
  • Sterile water
  • TSA plates (see recipe for plate preparation) containing anhydrotetracycline at 100 ng/ml
  • TSA plates (see recipe for plate preparation) without anhydrotetracycline
  • Gateway manual downloaded from (consult as needed if encountering technical difficulties, and see additional comments in the Commentary section)
  • Gel electrophoresis equipment
  • 30°C, 37°C, and 43°C incubators
  • 15‐ml culture tubes
  • Additional reagents and equipment for amplifying the target DNA (see protocol 6)

Basic Protocol 6: Preparing S. aureus Lysates for Extraction of DNA

  • Tryptic soy broth (TSB; see recipe)
  • TSM buffer (see recipe)
  • Lysostaphin (see recipe)
  • 15‐ml culture tubes
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 37°C incubator
  • Additional reagents and equipment for growing the target strain (see protocol 1 in unit 9.1)

Basic Protocol 7: Transduction

  • The donor strain (Donor) that carries a mutation in a target gene marked with a selectable marker (such as bursa aurealis insertion)
  • Tryptic soy broth (TSB; see recipe)
  • 5 mM CaCl 2
  • Phage lysate amplified on a sensitive strain with no resistance marker [phage titer should be ∼109‐1010 plaque‐forming units (pfu)]
  • Recipient strain, preferentially the parent strain used for bursa aurealis mutagenesis (Recipient)
  • 40 mM Na‐citrate (citrate buffer; see recipe)
  • Tryptic soy agar (TSA; see recipe)
  • TSA plates containing erythromycin at 10 µg/ml and Na citrate at 40 mM
  • 15‐ml culture tubes
  • 250‐ml flasks
  • Spreader
  • 30°C, 37°C incubators
  • 50‐ml centrifuge tubes
  • Centrifuges (for 50‐ml tubes, such as Sorvall, and for microcentrifuge tubes)
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