A rapid method for generating gene deletions in Aspergillus fumigatus

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). The entire cosmid (circular or linearised) is then used to generate a gene deletion in A. fumigatus. The large flanking sequences result in a high frequency of homologous recombination so that gene deletions occur at a relatively high frequency and very few transformants need to be screened. The method was adapted from Chaveroche et al., 2000.

Materials

E. coli KS272 (pKOBEG) - grow on LB Cm (25 µg/ml) at 30°C

The strain carries the pKOBEG plasmid (Cm^R) necessary for the recombination event and is used for generating recombined cosmids. The plasmid is compatible with ColE1-derived replicons. Expression of the red genes is induced by addition of 0.02-0.2 % arabinose. The plasmid pKOBEG is temperature sensitive so that all steps must be carried out at 30 °C.

E. coli BW19610 (pCDA21) - grow on LB Amp (50 µg/ml) at 37°C

This strain carries a bi-functional selectable marker – the Zeocin resistance gene (Zeo^R) and the A. fumigatus pyrG gene (pyrG). Other cassettes may be available (e.g., using the E. coli hph gene, conferring hygromycin B resistance on A. fumigatus) but were not used here. The plasmid pCDA21 is used as the template for a PCR reaction.

A. fumigatus CEA17 or other pyrG deletion strain

In this strain pyrG can be used as a selectable marker. If different selectable markers are used (e.g., the hph gene) then a wild-type strain can also be transformed.

Strains and plasmids needed for this protocol can be obtained upon request from Dr. Christophe d’Enfert, Unité Microbiologie et Environnement, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. (Tel. +33 1 40 61 32 57, fax. +33 1 45 68 89 38, e-mail: denfert@pasteur.fr).

Zeocin^TM can be obtained from e.g., Cayla, Toulouse, France (www.cayla.com), or Invitrogen, Groningen, The Netherlands (www.invitrogen.com).

LBLS  LB with low salt concentration (5 % NaCl, 5 % yeast extract, 10 % peptone) since Zeocin is only effective at low salt concentrations.

A genomic cosmid library of A. fumigatus carrying the hygromycin B resistance marker was used (Langfelder et al., 1998).

Method

Step 1: Introduction of target cosmid into KS272(pKOBEG)

The E. coli strain KS272(pKOBEG) is transformed with the relevant target cosmid according to standard E. coli procedures (i.e., either electro-competent or CaCl₂-competent E. coli cells can be used). It is advisable to use a selectable marker other than Cm^R or Zeo^R. Transformants must be selected at 30 °C and LB agar plates should include both chloramphenicol (25 µg/ml) and the relevant antibiotic for the target cosmid (e.g., ampicillin or kanamycin (50 µg/ml)). A transformant is selected which carries both the plasmid pKOBEG and the target cosmid. This transformant is used for making electrocompetent cells.

In our case we used a chromosomal A. fumigatus cosmid library (Langfelder et al., 1998) derived from the plasmid pANsCos1 (Osiewacz, 1994).

Step 2: Amplification of the bi-functional marker

PCR products are generated using the plasmid pCDA21 as template (other templates are possible). The primers used have two distinct regions.

The 5’ region (50-60 bp) corresponding to the sequence on the cosmid where the recombination event should take place (e.g., upstream of the relevant gene).

The 3’ region (approximately 20 bp) which is homologous to the peripheral region of the bi-functional marker (i.e., either to zeoR or pyrG) – see figure 1.

ZEO-primer:                5’-N₅₅X:GGAATTCTCAGTCCTGCTCC-3’

PYRG-primer:            5’-N₅₅Y:GAATTCGCCTCAAACAATGC-3’

These two primers are oriented in opposite directions and bind at the outside ends of the zeo^R and pyrG genes, respectively, so that a 2.7 kb PCR product results. This PCR product also includes the 50-60 bp of homologous sequence required for the recombination event. The flanking sequences (denoted by N₅₅) should be chosen to lie outside the region to be deleted and should also be oriented in opposite directions (i.e., pointing towards each other).

Proposed PCR program:

1. 93 °C              5’
2. 93 °C              30’’
3. 58 °C              2’
4. 72 °C              3’            repeat steps 2-4, 4x           
5. 93 °C              30’’
6. 60 °C              2’
7. 72 °C              3’            repeat steps 5-7, 24x
8. 72 °C              10’

In 100 µl: 200 ng of pCDA21, 25 nmol dNTPs, 100 pmol primers, 5 U Taq DNA polymerase.

Step 3: Recombination

The PCR product is dialysed twice on a Millipore 0.025 µm filter floated on distilled water. 10 µl of this solution are used to transform electrocompetent E. coli cells (KS272(pKOBEG + target cosmid)).

Method for making electrocompetent cells.

Materials:

500 ml LB medium in 1 l flask
2 l of ice-cold, sterile dH₂O
50 ml of ice-cold, sterile 10 % [v/v] glycerol solution
sterile Eppendorf cups

Inoculate a 5 ml overnight culture of the strain containing both plasmid pKOBEG and the target cosmid at 30 °C, Cm (25µg/ml) + Amp (50µg/ml), 180 rpm. Use 2.5 ml of this culture to inoculate 500 ml of LB medium containing Cm (25µg/ml) + Amp (50µg/ml) + 0.2 % [w/v] arabinose. Shake at 30 °C and 180 rpm until the culture reaches OD₆₀₀ 0.5-0.8.

1. Centrifuge cells in a cold rotor at 4000 x g for 10 minutes. Remove supernatant.
2. Resuspend cells in 500 ml cold dH₂O and centrifuge again (4000 x g, 10 minutes). Repeat 3 times. Keep cells on ice at all times.
3. Remove supernatant and resuspend cells in 40 ml cold dH₂O. Centrifuge as above (in 50 ml corning tube).
4. Remove supernatant and gently resuspend cells in 1.5 ml 10 % [v/v] ice-cold glycerol solution (final cell concentration should be around 10¹⁰ cells/ml).
5. Freeze 50 µl aliquots in liquid nitrogen. Store at –70 °C for up to 6 months.

One aliquot can be used for electroporation using 10 µl of the dialysed PCR product. The conditions for electroporation are as follows: 200 Ohm, 25 µF and 2.5 kV (the time constant should be around 4.5 ms). After electroporation cells should be incubated in 1 ml LB medium for 30 minutes (30 °C) before plating on relevant agar plates (LBLS zeocin (50 µg/ml) + relevant antibiotic, ampicillin (50 µg/ml) in our case). Plates are incubated at 30 °C. Transformants appear within 24 hours.

Transformants can be grown at 37 °C (LBLS zeocin + e.g., ampicillin) in order to cure them of the plasmid pKOBEG (which is temperature sensitive). They should contain the target cosmid in which the relevant gene/region has been replaced by the bi-functional marker. This can be verified by restriction digest (sometimes inconclusive because of the large number of fragments), PCR or sequencing.

Two primers which are directed outwards from the bi-functional marker can be used in conjunction with primers which lie on the cosmid to generate PCR products (these can also be sequenced in order to check for correct integration).

ZEOinternal                 5’-GTGACCCTGTTCATCAGC-3’

PYRinternal                 5’-TCCACGGAACTTTTAACG-3’

Once a correct cosmid (i.e., one in which the relevant gene has been replaced by the bi-functional marker) has been identified, this can be used for transformation of A. fumigatus. The circular cosmid can be used or it can be digested with an appropriate restriction enzyme, followed by dephosphorylation. This will increase the probability of a gene deletion occurring and decrease the number of single recombination events – see figure 2.

In our case the circular cosmid was used to transform the A. fumigatus CEA17 strain. 20 transformants resulted from the first transformation. Of these 8 were analysed by Southern blot analysis and 3 were found to have a deletion of the relevant gene. It is possible to preselect transformants resulting from homologous recombination since these should still be sensitive to hygromycin B. In the case of an ectopic integration of the cosmid (which carries the hph gene and hence confers hygromycin B resistance) the transformants should be resistant to hygromycin B.

If a cosmid containing the relevant gene/region is available then this method can be used to generate deletion strains very rapidly and without much DNA manipulation. The large flanking regions result in a high percentage of A. fumigatus transformants with the relevant gene deletion. The critical step in this procedure is the quality of electrocompetent cells. Varying the arabinose concentration can increase the recombination efficiency as can an increase in the amount of PCR product used in the transformation.

Some cosmids may not be suited to this recombination system although it is not yet clear why this should be the case. Cosmids derived from pWE15 appear to work while LORIST2 cosmids have not done so to date.

References

1.                  Osiewacz H. D. (1994). A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes. Curr. Genet. 26:87-90.

2.                  Zhang Y., Muyrers J. P., Testa G., Stewart A. F. (2000). DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18:1314-1317.

3.                  Chaveroche M. K., Ghigo J. M., d'Enfert C. (2000). A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 28:E97.

4.                  Muyrers J. P., Zhang Y., Stewart A. F. (2001). Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA. Trends Biochem Sci. 26:325-331.  Review

5.                  Langfelder K., Jahn B., Gehringer H., Schmidt A., Wanner G., Brakhage A. A. (1998). Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol (Berl) 187:79-89.

Prepared by:

Kim Langfelder
Johannes Gutenberg-Universität Mainz,
D-55101 Mainz,
Institut für Mikrobiologie und Genetik,
Technische Universität Darmstadt,
D-64287 Darmstadt, Germany

Stephanie Gattung
Institut für Mikrobiologie,
Universität Hannover,
Am Schneiderberg 50,
30167 Hannover, Germany

Prof. Axel A. Brakhage
Department of Molecular and Applied Microbiology,
Leibniz Institute for Natural Product Research and Infection Biology
Hans-Knoell-Institute (HKI), and Friedrich Schiller University Jena,
Jena, Germany

and

Christophe d’Enfert
Associate Professor and Head of the Fungal Biology and Pathogenicity Unit
Institut Pasteur,
Paris, France

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