Genetic manipulation of S. aureus mutants

Genetic manipulation of Staphylococcus aureus 

This protocol describes a general workflow of genetic manipulation of S. aureus. Information on how to work with pKASBAR based vectors is also included.

Construction of an S. aureus mutants consists of the following steps:

1. Plasmid construction in E. coli.

2. Plasmid transformation into S. aureus RN4220 by electroporation: 

A. Preparation of electrocompetent S. aureus RN4220,

B. Transformation of S. aureus by electroporation.

3. Transduction of a mutation from a donor strain (RN4220 derivative) into a recipient S. aureus strain by phages:

C. Phage lysate preparation by phage propagation on a donor strain (RN4220 derivative),

D. Transduction into a recipient strain.

Protocols for steps 2 (A and B) and 3 (C and D) are detailed below.

A.   Preparation of electrocompetent S. aureus RN4220

Materials:

Growth medium: Brain Heart Infusion Broth (BHI) or Tryptic Soy Broth (TSB)

Deionised water (Milli-Q)

10% glycerol

Procedure:

1. Streak out frozen glycerol stock of RN4220 onto a BHI or TSB plate (no antibiotics). Grow cells overnight at 37℃ with 200 rpm shaking.

2. Inoculate 400 ml of growth medium with a single colony. Grow overnight for 10-12h (no more than 14) at 37℃ with 200 rpm shaking.

3. Inoculate 400 ml pre-warmed growth medium with the overnight culture to an OD₆₀₀ of 0.1. Grow cells at 37℃, 200 rpm to OD₆₀₀  0.4 – 0.6. 

Note: Check the OD₆₀₀ after 60 min and then after 30 min. If cells haven’t reached the desired OD₆₀₀ after 90 min, cells should not be used.

4. Split the culture into four sterile centrifuge bottles. Collect cells by centrifugation at 5,000 x g for 10 min at RT. Discard the supernatant.

5. Resuspend cells in 25 ml of RT dH₂O (do not vortex). 

Note: Resuspend each pellet gently in 1 ml dH₂O, add remaining 24 ml of dH₂O and mix by gently inverting the centrifuge bottle a few times.

6. Centrifuge at 5,000 x g for 10 min at RT. Discard the supernatant.

7. Repeat steps 5 – 6 two more times.

8. Resuspend each pellet in 5 ml of 10% glycerol. Combine all suspensions into one centrifuge bottle. 

9. Centrifuge at 5,000 x g for 10 min at RT. Discard the supernatant. 

10. Resuspend the pellet in 10 ml sterile 10% glycerol.  Incubate at room temperature for 30 min.

11. Centrifuge cells at 5,000 x g for 10 min at RT. Discard the supernatant.

12. Resuspend the pellet in 400 μl 10% glycerol.

13. Aliquot 50 μl into 1.5 ml microfuge tubes and snap freeze.

14. Store cells at -80℃.

B.   Transformation of S. aureus by electroporation

Materials:

Plasmid DNA

Growth medium: Brain Heart Infusion Broth (BHI) or Tryptic Soy Broth (TSB)

TSB or BHI agar plates with a selective antibiotic

Procedure:

1. Remove competent cells from -80℃.  Place cells on ice for 5 min and then at room temperature for additional 5 min.

2. Add from 1 to 5 μg of a purified plasmid to the cells, mix by gentle pipetting up and down.

3. Transfer the cells to a 1-mm electroporation cuvette at room temperature.

4. Pulse cells at 21 kV/cm (2.1 kV for a 1-mm cuvette), 100 Ω and 25 mF. Time constant is usually about 2.0 – 2.4 ms.

5. Immediately add 1 ml of TSB (or BHI).

6. Transfer cells to 30 ml universal tube.

7. Incubate cells at 37℃ (or 28℃ for temperature-sensitive plasmids) for 2 – 3 h.

8. Spread 5 x 200 μl aliquots on TSB (or BHI) agar plates with a selective antibiotic.

9. Incubate at 37℃ (or 28℃ for temperature-sensitive plasmids). Colonies usually appear after 20-48 h.

10. Re-streak several colonies onto TSB (or BHI) agar plates with a selective antibiotic and confirm mutation by an appropriative method.

C.   Preparation of phage lysate for transduction

Materials:

Growth medium: TSB or BHI

Stock of a serogroup B phage: φ11 or φ85α - It is a phage lysate amplified on a sensitive strain with no resistance marker

Phage buffer: 0.264 g/L MgSO₄, 0.588 g/L CaCl₂ • 2H₂O, 50 mM Tris-HCl pH 7.8, 5.99 g/L NaCl, 1 g/L gelatin

Procedure:

1. Inoculate 5 ml of growth medium containing a suitable antibiotic with a single colony of a donor strain. Grow cells overnight at 37℃ (or 28℃ for temperature-sensitive plasmids) with 200 rpm shaking.

2. In a universal tube mix together 5 ml of phage buffer, 5 ml of growth medium, 100 μl of stock phage and 150 μl of the overnight culture (or until slightly cloudy).

3. Incubate stationary at 25℃ until mixture has totally cleared. It should have cleared after overnight.

4. Filter sterilise (0.2 μm pore size) the phage lysate. 

5. Store the phage lysate of donor strain at 4℃.

D.   Phage transduction

Materials and reagents:

LK: 10 g/L tryptone, 5 g/L yeast extract, 7 g/L KCl

LK agar plates with 0.5% (w/v) sodium citrate and a selective antibiotic: add 0.1 M sodium citrate solution to autoclaved LK agar

1 mM CaCl₂

0.1 M sodium citrate, pH 6.5

Phage lysate of a donor strain

Procedure:

1. Inoculate 50 ml LK with a recipient strain. Grow cells overnight at 37℃ with 200 rpm shaking.

2. Collect cells by centrifugation at 5,000 x g for 10 min at RT.

3. Resuspend the pellet in 3 ml LK.

4. In 50 ml centrifuge tubes prepare:

1) 500 µl recipient cells, 1 ml LK, 10 μl 1M CaCl₂, 500 µl phage lysate

2) Control: 500 µl recipient cells, 1 ml LK, 10 µl 1M CaCl₂

5. Incubate cells for 25 min at 37℃ without shaking.

In the meantime, prepare 0.02 M sodium citrate from the 0.1 M stock. Leave on ice.

6. Incubate cells at 37℃ for 15 min at 200 rpm. 

In the meantime, chill a centrifuge to 4℃.

7. Add 1 ml of ice-cold 0.02 M sodium citrate to cells. Incubate for 5 min on ice.

8. Collect cells by centrifugation at 5,000 x g for 10 min at 4℃.

9. Resuspend cells in 1 ml ice-cold sodium citrate.

10. Incubate cells on ice from 45 min to 1.5 h.

11. Spread 200 µl aliquots onto LK plates that contain a selective antibiotic and 0.5% sodium citrate.

12. Incubate at 37℃ (or 28℃ for temperature-sensitive plasmids). Colonies usually appear after 20-48 h.

13. Re-streak several colonies onto TSB (or BHI) agar plates with a selective antibiotic and confirm mutation by an appropriative method.

Construction of S. aureus mutants using pKASBAR based plasmids

pKASBAR is a shuttle vector that was constructed by cloning an attP phage attachment site and a tetracycline resistance cassette from pCL84 into pUC18 (Bottomley et al., 2014). pKASBAR cannot replicate in S. aureus as it lacks the S. aureus origin of replication, but it can integrate into S. aureus chromosome via the attP attachment site present in the plasmid and the bacterial attB attachment site present in the 3’ region of the lipase gene (geh). Integration of pKASBAR and its derivatives into the S. aureus chromosome is based on the site-specific recombination system of the bacteriophage L54a (Lee et al., 1991). Recombination occurs in the presence of bacteriophage integrase, whose gene is expressed constitutively from the plasmid pYL112Δ19 (Lee et al., 1991). Integration of the plasmid results in a loss of lipase activity (Lee and Iandolo, 1986). pKASBAR is useful in incorporating a chromosomal based mutation at a single copy. 

1. Prepare electrocompetent CYL316.

2. CYL316 is S. aureus RN4220 carrying pYL112Δ19. When preparing electrocompetent CYL316, grow it in the presence of 10 μg/ml chloramphenicol.

3. Transform CYL316 with a pKASBAR based vector.

4. Confirm plasmid integration at the lipase (geh) locus:

Streak several colonies onto Baird-Parker agar. Also streak a control: CYL316 or RN4220. Incubate at 37℃ overnight. The control will produce opaque haloes around colonies, while transformants will show a reduction in halo formation which indicates plasmid integration at geh and thus loss of lipase activity. Additionally, confirm the mutation by an additional method (e.g., PCR on extracted genomic DNA).

5. Prepare a phage lysate from a CYL316 derivative.

6. Transduce mutation into a recipient strain by phage transduction.

7. Confirm successful transformation by streaking cells onto Baird-Parker agar. Use the recipient strain as a control. Confirm the mutation by an additional method (e.g., PCR on extracted genomic DNA).

References

Bottomley, A. L. et al. Staphylococcus aureus DivIB is a peptidoglycan-binding protein that is required for a morphological checkpoint in cell division. Mol Microbiol (2014) 94, 1041–1064 (2014).

Lee, C. Y., Buranen, S. L. & Ye, Z. H. Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103, 101–105 (1991).

Lee, C. Y. & Iandolo, J. J. Integration of staphylococcal phage L54a occurs by site-specific recombination: structural analysis of the attachment sites. Proc Natl Acad Sci U S A 83, 5474–5478 (1986).

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