Characterization of Essential Genes by Parasexual Genetics in the Human Fungal Pathogen Aspergillus fumigatus: Impact of Genomic Rearrangements Associated With Electroporation of DNA

Corresponding author: Christophe d'Enfert, CNRS URA 2172, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France., denfert{at}pasteur.fr (E-mail)

Communicating editor: M. S. SACHS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have evaluated the usefulness of parasexual genetics in the identification of genes essential for the growth of the human fungal pathogen Aspergillus fumigatus. First, essentiality of the A. fumigatus AfFKS1 gene, encoding the catalytic subunit of the ß-(1,3)-glucan synthase complex, was assessed by inactivating one allele of AfFKS1 in a diploid strain of A. fumigatus obtained using adequate selectable markers in spore color and nitrate utilization pathways and by performing haploidization under conditions that select for the occurrence of the disrupted allele. Haploid progeny could not be obtained, demonstrating that AfFKS1 and, hence, ß-(1,3)-glucan synthesis are essential in A. fumigatus. Second, random heterozygous insertional mutants were generated by electroporation of diploid conidia with a heterologous plasmid. A total of 4.5% of the transformants failed to produce haploid progeny on selective medium. Genomic analysis of these heterozygous diploids led in particular to the identification of an essential A. fumigatus gene encoding an SMC-like protein resembling one in Schizosacccharomyces pombe involved in chromosome condensation and cohesion. However, significant plasmid and genomic DNA rearrangements were observed at many of the identified genomic loci where plasmid integration had occurred, thus suggesting that the use of electroporation to build libraries of A. fumigatus insertional mutants has relatively limited value and cannot be used in an exhaustive search of essential genes.

ASPERGILLUS fumigatus is a ubiquitous filamentous fungus of human environments and an opportunist pathogen (for review see LATGE 1999 , LATGE 2001 ). Dissemination of A. fumigatus occurs by release of asexual spores, called conidia, into the atmosphere. Due to their small diameter (2–3 µm), conidia can reach the lung alveolae and cause invasive pulmonary aspergillosis (IPA) when innate immunity is deficient. With the rising number of immunocompromised hosts and the development of severe immunosuppressive therapies, the incidence of IPA has dramatically increased to become the most frequent airborne fungal infection in developed countries (MCNEIL et al. 2001 ). Because of a difficult diagnosis and the lack of efficient, nontoxic antifungal treatments, IPA is associated with a mortality rate as high as 85% (LIN et al. 2001 ).

A rational approach to develop new therapies relies on the identification of virulence factors (PERFECT 1996 ). Despite intensive efforts, no genuine virulence factor has been identified yet in A. fumigatus (reviewed in D'ENFERT 2000 ; LATGE 2001 ). This suggests that A. fumigatus virulence is a multifactorial process that depends mostly on the host immune system status, reflecting the opportunistic pathogenicity of this species (ODDS et al.. 2001 ). An alternative strategy for finding novel antifungal targets might be in the identification of genes that are essential for fungal growth under in vitro growth conditions (REICH 2000 ). Compendia of essential genes have been obtained for the yeast Saccharomyces cerevisiae through various approaches including identification and characterization of temperature-sensitive mutants, systematic gene inactivation, or insertional mutagenesis in a diploid background followed by the analysis of meiotic progeny (ROSS-MACDONALD et al. 1999 ; WINZELER et al.. 1999 ). More recently, a compendium of Candida albicans essential genes has been produced by expressing antisense RNA molecules (DE BACKER et al.. 2001 ).

Several of these approaches are not yet applicable to filamentous fungi, mainly because of the lack of complete genome sequences and of the low efficiency of homologous recombination (D'ENFERT et al.. 1999 ). In this regard, insertional mutagenesis in a diploid background appears as the most suitable method to identify essential genes in filamentous fungus. Aspergillus species are natural haploids that reproduce asexually, although a sexual cycle is performed by some species, in particular A. nidulans, when diploids are transiently formed. Alternatively, some fungi undergo a parasexual cycle, as first described by PONTECORVO et al. 1953 in A. nidulans and subsequently in other fungal species including A. fumigatus (STROMNAES and GARBER 1963 ). Stable diploids can be obtained using adequate genetic markers in the parent strains and appropriate selection procedures. These diploids do not enter meiosis but destabilizing reagents like benomyl or p-fluorophenylalanine can be used to induce the formation of haploid segregants because of mitotic chromosomal nondisjunction (HASTIE 1970 ; TIMBERLAKE and MARSHALL 1988 ; TIMBERLAKE 1991 ; CLUTTERBUCK 1992 ).

Parasexual genetics has been used to demonstrate gene essentiality in A. nidulans (TIMBERLAKE 1991 ; SOM and KOLAPARTHI 1994 ). In these experiments, one allele of the target gene is inactivated by homologous gene replacement in a stable diploid. The absence of the inactivated allele in all haploid segregants indicates that the gene is essential for fungal growth. This strategy has not yet been used to identify essential fungal genes without prior sequence information by random insertional mutagenesis of a stable diploid strain.

Currently available procedures for random insertional mutagenesis in A. fumigatus rely on the integration of plasmid DNA into the fungal genome using transformation. Work by WEIDNER et al. 1998 and BROWN et al. 1998 has shown that electroporation of intact conidia with a nonhomologous plasmid is more appropriate than protoplast transformation for the generation of collections of insertional mutants since it provides (1) high transformation efficiencies, (2) integration of only one copy of the transforming DNA in the majority of transformants, and (3) an apparent random distribution of the plasmid in the genome, as judged from the various sizes of restriction fragments that have integrated the transforming DNA molecule. Addition of restriction enzymes during protoplast transformation (restriction enzyme-mediated integration) also appears to increase transformation efficiency and to favor single-site insertion of the transforming DNA (BROWN et al.. 1998 ). Although electroporation has been applied to generate a large collection of insertional mutants in a haploid background and subsequent screening of the mutants in an animal model of IPA, little is known about the consequence of plasmid integration on the genome structure in A. fumigatus (BROWN et al.. 2000 ).

Here, we have combined parasexual genetics and insertional mutagenesis for the systematic identification of essential genes in A. fumigatus at a genomic scale. First, we have validated this strategy by inactivating one allele of a presumed essential A. fumigatus gene by homologous recombination. ß-(1,3)-Glucan is a major component of the fungal cell wall synthesized by an enzymatic complex whose catalytic subunit is encoded by the A. fumigatus AfFKS1 gene (BEAUVAIS et al.. 2001 ). Glucan synthase is the target of the new antifungal drugs, candins (ONISHI et al.. 2000 ; TKACZ and DIDOMENICO 2001 ). In several species where it has been attempted (S. cerevisiae, Cryptococcus neoformans), inactivation of the homologous gene is lethal (MAZUR et al.. 1995 ; THOMPSON et al.. 1999 ) and repeated attempts to inactivate AfFKS1 in a haploid strain of A. fumigatus have failed (BEAUVAIS et al.. 2001 ). Here, we use parasexual analysis of heterozygous diploid transformants to demonstrate that the ß-(1,3)-glucan synthase enzyme is essential for the growth of A. fumigatus. We also generated and analyzed random insertions in diploid mutants of A. fumigatus. Our results show that, although the strategy has the potential to identify A. fumigatus genes that are essential for growth, insertional mutagenesis based on the electroporation of DNA is associated with numerous genomic rearrangements that limit the value of this approach.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A. fumigatus strains and culture conditions:
A. fumigatus strains used in this study are listed in Table 1. A. fumigatus strains were propagated at 37° on complete medium or minimal medium (MM) with 0.5 mM of one of the following nitrogen sources: sodium glutamate, ammonium tartrate, sodium nitrate, sodium nitrite, or hypoxanthine (COVE 1966 ). Uridine and uracil were added at a concentration of 5 mM when appropriate. Selection of A. fumigatus mutants unable to utilize nitrate as the sole nitrogen source was obtained by plating spores on MM containing ammonium tartrate and 100 mM sodium chlorate (COVE 1966 ). The nature of the mutation was assessed by growth on MM supplemented with different nitrogen sources, as previously described (COVE 1976 ). Selection of A. fumigatus mutants auxotrophic for uridine and uracil was achieved on MM containing 1 mg/ml 5-fluoroorotic acid plus uridine and uracil (D'ENFERT 1996 ). Liquid cultures used for DNA-mediated transformation and genomic DNA preparation were grown in YG (0.5% yeast extract, 2% glucose). Haploidization of A. fumigatus diploid strains was conducted on selective haploidization medium [SHM; complete medium containing 1.2 µg/ml benomyl (Aldrich, Milwaukee; 10 mg/ml in DMSO)] or on nonselective haploidization medium (NSHM; selective haploidization medium plus uridine and uracil).

Plasmid and DNA manipulation:
General recombinant DNA techniques and Southern-blot analyses were performed essentially according to SAMBROOK et al. 1989 and AUSUBEL et al. 1992 . Transformation of calcium-manganese-treated Escherichia coli was as described (HANAHAN et al.. 1991 ). Oligonucleotides used in this study were obtained from Genset (Paris) and are listed in Table 2. pCB::Fks is a derivative of pCB1004 (CAROLL et al.. 1994 ), which contains a hygromycin-resistance marker for selection of fungal transformants and in which a 2.3-kb KpnI internal fragment [nucleotides (nt) 1342–3672] of AfFKS1 has been subcloned (BEAUVAIS et al.. 2001 ). pF4 was obtained by subcloning an XbaI fragment containing the A. niger pyrG gene (VAN HARTINGSVELDT et al.. 1987 ) into pCB::Fks. ppyrG, a pUC18 derivative containing the A. nidulans pyrG gene (OAKLEY et al.. 1987 ) that efficiently complements the A. fumigatus pyrG1 mutation (WEIDNER et al.. 1998 ) was kindly provided by Claudio Scazzocchio (Université Paris-Sud, Orsay, France).

Heterozygous A. fumigatus FKS1/fks1 strains construction:
Plasmid pF4 was introduced in A. fumigatus strain CEA136 by transformation of protoplasts as described previously (OSMANI et al.. 1987 ; D'ENFERT 1996 ). Genomic DNA of transformants was prepared according to GIRARDIN et al. 1993 . EcoRV- and HindIII-digested genomic DNA was probed with the 2.3-kb KpnI fragment of pCB::Fks that had been labeled using the Rediprime labeling kit (Amersham, Arlington Heights, IL) and [-³²P]dCTP. Integration of pF4 at one of the AfFKS1 loci in the diploid strain is expected to result in the appearance of two EcoRV fragments of 3.6 and 8.8 kb and two HindIII fragments of 1.7 and 7.4 kb in addition to the EcoRV fragment of 3.8 kb and two HindIII fragments of 1.25 and 1.75 kb that are also detected in genomic DNA of a wild-type diploid. Integration of pF4 at the AfFKS1 locus is confirmed by PCR analysis. Genomic DNA amplification with oligonucleotides fks1 and fks2 yields a 2.4-kb fragment in all diploid strains with at least one wild-type AfFKS1 allele. In strain CEA136 and in transformants with an ectopic integration of pF4, amplification with oligonucleotides T7 and fks2 does not yield any product. In contrast, amplification of genomic DNA of transformants with pF4 integrated at the AfFKS1 locus results in a 2.6-kb fragment corresponding to the disrupted fks1 allele when the T7 and fks2 oligonucleotides are used.

Production and characterization of A. fumigatus insertional mutants:
A. fumigatus insertional mutants were obtained by electroporation of intact conidia of strain CEA136 with EcoRI-digested ppyrG. On average, 0.5 µg of linearized DNA was used to transform 5 x 10⁷ conidia prepared according to WEIDNER et al. 1998 . Electroporation was carried out in a 0.2-cm electroporation cuvette (Bio-Rad, Richmond, CA) and cells were subjected to a 1-kV pulse using a Bio-Rad electroporation device (400 , 25 µF). Transformants were selected on complete medium. Following purification to single colonies on complete medium, transformants were scored on SHM and NSHM.

Genomic DNA of transformants was prepared as described above. ClaI-digested genomic DNA was subjected to Southern analysis using ³²P-labeled ppyrG as a probe. Genomic DNA regions flanking ppyrG were amplified using the method adapted from CHUN et al. 1997 . First, 100 ng of genomic DNA was amplified in 50 µl using oligonucleotides ppyr1 and PCRal1 or ppyr3 and PCRal1 (4 pmol/µl final) and the following amplification protocol: a denaturation step at 94° for 3 min followed by 5 cycles of the following steps: denaturation at 94° for 30 sec, annealing at 35° for 30 sec, extension at 72° for 1 min, and 30 cycles of the following steps: denaturation at 94° for 30 sec, annealing at 45° for 30 sec, extension at 72° for 1 min. A last elongation step was performed at 72° for 3 min. Final concentrations for MgCl₂ and dNTPs were 3 mM and 0.2 mM, respectively. One microliter of the PCR reaction was subjected to a second amplification using similar reaction conditions and oligonucleotides ppyr2 and PCRal2 (if ppyr1 and PCRal1 had been used in the first reaction) or ppyr4 and PCRal2 (if ppyr3 and PCRal1 had been used in the first reaction). The following amplification protocol was used: 30 cycles of the following steps: denaturation at 94° for 30 sec, annealing at 60° for 30 sec, extension at 72° for 1 min. A last elongation step was performed at 72° for 3 min. In some instances, oligonucleotides PCRal3, PCRal4, and PCRal5 were used in place of PCRal1. PCR products were separated by electrophoresis on a 2% agarose-TBE gel and major PCR products were purified with the Qiaquick gel purification kit (QIAGEN, Valencia, CA) according to the supplier's instructions. Purified PCR products were sequenced using ppyr2 or ppyr4 as primers (ESGS, Evry, France). Nucleotide sequences obtained in this manner and trimmed for ppyrG sequences were compared to protein databases and to the preliminary sequence data of the A. fumigatus genome project obtained from The Institute for Genomic Research (TIGR) website (6x shotgun sequencing publicly available on 14 November 2001 at http://www.tigr.org) using Blastx or Blastn (ALTSCHUL et al.. 1990 ).

Genomic regions corresponding to the site of insertion of ppyrG in selected transformants were characterized as follows. Probes were prepared by PCR using appropriate primers based on the sequence obtained from the genomic DNA of transformants and labeled with [³²P]dCTP using the Rediprime kit. Probes were used to identify cosmids in a genomic library of A. fumigatus (BORGIA et al.. 1994 ). In the case of transformant 96, cosmid 25D9 was identified and subjected to shotgun sequencing according to standard procedures. Following assembly, the sequence of cosmid 25D9 was deposited at the GenBank nucleotide sequence database (accession no. AY080962). Oligonucleotides were designed from the sequence of cosmid 25D9 to deduce the genomic organization at the site of integration of ppyrG by PCR reactions on genomic DNA of transformant 96 and A. fumigatus CEA136.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Characterization of candidate A. fumigatus essential genes through parasexual genetics construction of A. fumigatus diploid strains:
Stable A. fumigatus pyrG diploids heterozygous for spore color markers and for genes involved in nitrate utilization were obtained using the following strategy. Spore color mutants CEA82 and CEA85 (Table 1) of the A. fumigatus pyrG strain CEA17 (D'ENFERT 1996 ) were identified by insertional mutagenesis with plasmid pAfpyrG2 (WEIDNER et al. 1998 ). Derivatives of these strains (CEA88 and CEA92; Table 1) that were unable to use nitrate as the sole nitrogen source were obtained by incubation in the presence of chlorate. Heterokaryons could be obtained by growing these complementary strains on MM nitrate and produced gray-green spores in addition to spores with the parental colors. Only these gray-green spores were able to use nitrate and, when incubated in the presence of benomyl, produced sectors of parental-colored spores, indicating that they correspond to A. fumigatus diploids. One of these diploids was designated CEA99 (Table 1). A derivative of CEA99 that had lost pAfpyrG2 sequences as indicated by Southern hybridization (data not shown) was obtained by selection on 5-fluoroorotic acid and designated CEA136 (w1/+, +/r7, cnx1/+, +/nirA4 pyrG1/pyrG1). Upon haploidization, strains CEA99 and CEA136 produced sectors of parental-colored spores only, suggesting that both color mutations lie on the same chromosome, as expected if these mutations affect the cluster of genes involved in the synthesis of the spore color pigment in A. fumigatus (TSAI et al.. 1999 ).

ß-(1,3)-Glucan synthase is essential for growth in A. fumigatus:
Plasmid pF4 (Fig 1A) was used to inactivate one allele of AfFKS1 in strain CEA136 by homologous recombination. pF4 contains an internal fragment (nt 1342–3672) of the 5813-bp AfFKS1 open reading frame cloned into a vector carrying two fungal markers: hph, which confers hygromycin resistance, and pyrG, which confers uridine/uracil prototrophy. Analysis of nine transformants of diploid strain CEA136 revealed that two resulted from the integration of pF4 at one of the two AfFKS1 loci (Fig 1A and Fig B; data not shown). Haploidization of these AfFKS1/Affks1 heterozygous diploids was obtained on nonselective haploidization medium, which would enable the growth of haploid strains containing AfFKS1 or Affks1, but not on a selective haploidization medium, which would enable the growth of only haploid strains containing Affks1, suggesting that inactivation of AfFKS1 is lethal in A. fumigatus (Fig 1C). AfFKS1/Affks1 heterozygous diploids form a cal on haploidization selective medium that might result from the production of poorly viable aneuploids in the presence of the mitotic spindle inhibitor benomyl, which, at low concentration, induces chromosomal nonmitotic disjunction in Aspergillus diploid strains (HASTIE 1970 ). Essentiality of AfFKS1 was confirmed by testing haploid conidia obtained following growth of the AfFKS1/Affks1 heterozygous diploid on NSHM for uridine/uracil prototrophy. None of these conidia had retained the transformation marker and, hence, Affks1 allele (data not shown). Taken together, these results suggested that AfFKS1 is essential for growth in A. fumigatus and that parasexual genetics might be used to test the essentiality of a cloned A. fumigatus gene. Interestingly, the conditions that we have tested did not permit haploidization on media containing hygromycin, suggesting that hph is not a useful marker for parasexual genetics studies in A. fumigatus (data not shown).

View larger version (13K): In this window In a new window Download PPT slide   Figure 1. Disruption of one allele of an A. fumigatus AfFKS1 gene in a diploid strain and parasexual analysis. (A) Plasmid pF4 contains a 2.3-kb KpnI internal fragment of the A. fumigatus AfFKS1 gene and two fungal transformation markers (pyrG and hph). Transformation of diploid CEA136 protoplasts leads to the inactivation of one allele of AfFKS1 by homologous recombination. The locations of primers fks1, fks2, and T7 used for PCR analysis of transformants are indicated by arrowheads. (B) PCR analysis of the AfFKS1 loci in A. fumigatus strain CEA136 and transformants pF4-8, pF4-9, and pF4-10. The occurrence of a wild-type allele is reflected by a 2.4-kb fragment when primers fks1 and fks2 are used (a, all strains). A disrupted Affks1 allele is visualized by the amplification of a 2.6-kb fragment when primers fks 2 and T7 are used (b, transformants pF4-9 and pF4-10). The absence of an amplification product in diploid transformants with these two primers reflects ectopic integration of pF4 (transformant pF4-8). (C) Haploidization of transformant pF4-9 on nonselective haploidization medium (NSHM) or selective haploidization medium (SHM). Inability to form haploid sectors is correlated with the formation of a cal on SHM, indicating that the FKS1 gene is essential for the viability of A. fumigatus.

Identification of A. fumigatus essential genes by insertional mutagenesis: Random insertional mutagenesis and parasexual screening:
Transformation with a linearized heterologous plasmid (ppyrG; OAKLEY et al.. 1987 ) was used to generate a collection of independent insertional mutants of strain CEA136. Electroporation of intact conidia (BROWN et al.. 1998 ; WEIDNER et al.. 1998 ) yielded 1000–2000 transformants per µg DNA. Southern analysis (Fig 2 and data not shown) of genomic DNA of 12 randomly chosen transformants revealed two fragments of varying sizes in transformants harboring a single copy of ppyrG, while an additional 4.6-kb fragment corresponding to ppyrG was observed when tandem integration of ppyrG had occurred, and additional fragments were observed when ppyrG has integrated at several locations. Eight transformants resulted from the integration of a single copy of ppyrG in their genome; 4 transformants had integration of tandem copies of ppyrG. Complex integration events (at least two copies of ppyrG at different genomic locations) appeared underrepresented. PCR analysis using primers ppyr1 and ppyr3 (Table 2) on 95 independent transformants confirmed that 34% (32/95) of the transformants had at least two copies of the plasmid inserted at the same genomic location. The occurrence of fragments of different sizes in the genomic DNA of transformants analyzed by Southern hybridization (Fig 2 and data not shown) is consistent with a random distribution of integration sites. However, it does not preclude the possibility of integration hotspots.

View larger version (32K): In this window In a new window Download PPT slide   Figure 2. Southern analysis of six derivatives of A. fumigatus strain CEA136 obtained by transformation with linearized ppyrG. (A) Schematic representation of expected integration events and hybridization data obtained using genomic DNA cleaved by a restriction enzyme (double arrow) that cuts once in the transforming DNA and the transforming plasmid (open box) as a probe. Integration of one copy of the plasmid (open box) into genomic DNA results in two restriction fragments of varying size (thick lines). Tandem integrations of the plasmid result in two hybridizing fragments of varying size plus a constant fragment of 4.6 kb. Complex integrations of the plasmid result in four or more fragments of varying size. (B) Southern analysis of six transformants of strain CEA136. Genomic DNA was digested with ClaI, which cleaves once in ppyrG, and probed with -32P-labeled ppyrG. Two ClaI fragments are observed in transformants 1–4, which have undergone a single integration event. Three ClaI fragments, including a 4.6-kb fragment (arrows), are observed in transformants 5 and 6, which have undergone a tandem integration event.

A collection of 544 independent transformants was screened for growth on SHM and NSHM. Two categories of transformants were obtained. A total of 519 (95.5%) transformants produced virtually indistinguishable colonies on the two media, suggesting that integration of ppyrG in these transformants did not prevent the expression of a gene critical for the growth of A. fumigatus (see Fig 3 for an example, transformants A, B, and C). In contrast, 25 (4.5%) transformants produced a cal on SHM similar to the AfFKS1/Affks1 heterozygous diploid (e.g., Fig 3, transformant D). Sixteen diploid transformants in the second category were further characterized by analyzing spores produced on NSHM for their phenotypes after plating on selective and nonselective complete media. Plating 10⁵ haploid spores of each yielded no haploid pyrG⁺ colonies (data not shown). This indicated that these 16 transformants contained a copy of ppyrG genetically linked to the inability to produce haploid spores in the presence of benomyl. That is, they might have a ppyrG insertion in a gene necessary for the growth of A. fumigatus.

View larger version (50K): In this window In a new window Download PPT slide   Figure 3. Parasexual genetic screening to identify an essential gene. Haploidization of four diploid pyrG+ transformants on nonselective (NSHM) and selective (SHM) haploidization media gave a cal growth phenotype on selective medium when the plasmid is integrated in an essential gene (transformant D). Random disjunction of chromosomes is visualized by the parental color of haploid conidia.

Characterization of genomic sequences at the site of plasmid integration:

Since each haploid-lethal mutation should be physically marked by the insertion of a plasmid, the characterization of the junctions between the genome and the plasmid in the 16 diploid transformants described above was achieved using the method of CHUN et al. 1997 to identify the corresponding A. fumigatus genes.

Identification of the flanking genomic sequences in eight transformants was hampered because of two types of plasmid rearrangements. First, tandem plasmid integrations were observed in four transformants and confirmed by PCR analysis. Their frequency (25%) is in the range of that observed in the overall diploid transformant population (34%; see above). Second, mosaic structures with one end of the plasmid followed by internal fragments of the plasmid in different orientations and of variable length and position were observed in four transformants (data not shown).

Genomic sequences flanking plasmid DNA could be obtained for eight transformants with a single integrated copy of the plasmid (Table 3). In these, the plasmid ends have not undergone major DNA rearrangements (Table 3). In most cases (10/15), only the single-strand protruding ends (3'-TTAA-5') resulting from EcoRI cleavage prior to transformation were deleted in the integrated plasmid. Two minor deletions (5 and 6 bp) were observed at one end of the plasmid in two transformants (3' end of transformant 99 and 5' end of transformant 503; Table 3). In some instances, nucleotides that did not originate from the plasmid or genomic DNA were observed at their junction (3' end of transformants 96 and 516, 5' end of transformant 197; Table 3). Comparison of genomic sequences surrounding the plasmid in the eight transformants did not reveal microhomologies between plasmid and genomic DNA, consistent with the apparent random selection of the integration sites.

Identification of a member of the structural maintenance of chromosome protein family as necessary for efficient growth in A. fumigatus:

Characterization of the sequences flanking the site of integration in transformant 234 (Table 3) revealed that integration was in a gene encoding a structural maintenance of chromosome (SMC)-like protein (STRUNNIKOV and JESSBERGER 1999 ). The sequence of a corresponding 4339-bp genomic clone was determined (accession no. AY081008). This sequence overlaps with contig 432 (nt 6899–11240) of the A. fumigatus genome sequence (http://www.tigr.org/; release of November 14, 2001). The predicted polypeptide is encoded by an open reading frame (ORF) of 3561-bp nucleotides interrupted by five putative introns. The insertion of ppyrG is located at nt 996 (amino acid 332).

The deduced polypeptide sequence shows 30% identity and 50% similarity with the Schizosaccharomyces pombe Spr18 protein (SMC protein partner of Rad18), which is involved in the maintenance of chromosome structure and is essential for growth in this species (HIRANO 1999 ; FOUSTERI and LEHMANN 2000 ). It is also 28% identical and 48% similar to S. cerevisiae YOL034w, which is essential for growth (WINZELER et al.. 1999 ). Alignment of the A. fumigatus predicted protein with members of the SMC protein family shows two blocks of conserved residues located at the amino and carboxy termini of the protein (Fig 4). These results suggest that the gene interrupted by ppyrG in transformant 234 encodes a member of the SMC protein family that is essential for growth of A. fumigatus.

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. Domain conservation of the predicted A. fumigatus SmcA protein. Alignment of SmcA with S. pombe Spr18 (Swissprot: accession no. O13710) and S. cerevisiae YOL034w (accession no. NP_014608) is shown for the amino- (SMC_N; 50% identity, 70% similarity over 150 amino acids) and carboxy- (SMC_C; 42% identity, 61% similarity over 232 amino acids) terminal domains. Conserved amino acids between the three homologs are marked by a star while similar amino acids are indicated by dots on the bottom lines. The SGG and DE motifs that are conserved in all SMC proteins are in boldface type.

Genomic rearrangements in transformants with a haploid-lethal phenotype:

Analysis of the seven remaining transformants (96, 99, 197, 209, 503, 516, and 536) revealed that the 5'- and 3'-flanking regions in six were homologous to different contigs of the A. fumigatus genome sequence (Table 3). Furthermore, amplification of genomic DNA of A. fumigatus strain CEA136 with primers designed on the basis of the 5' and 3' sequences flanking ppyrG were all unsuccessful (data not shown). For transformant 536, where only the region located in 3' of ppyrG could be characterized, primers designed on the basis of the corresponding A. fumigatus contig (Contig856, http://www.tigr.org/) did not generate PCR products with primers corresponding to the 5' end of ppyrG (data not shown). These results suggested that these seven transformants had undergone significant genomic DNA rearrangements at the site of integration of ppyrG.

This result was confirmed by the detailed characterization of transformant 96. In this case, cosmid 25D9 containing the genomic sequences flanking both ends of ppyrG was sequenced (accession no. AY080692). Comparison of the cosmid sequence with the sequence of genomic DNA flanking ppyrG in transformant 96 revealed that integration of ppyrG is actually associated with the deletion of a 16,057-bp genomic DNA fragment and the inversion of a 587-bp fragment (Fig 5). Consequently, integration of ppyrG was associated with the partial or complete deletion of at least five ORFs (Fig 5). Two of these ORFs have no significant homolog in public databases (Fig 5; 25d9-9 and 25d9-10), while three resemble S. cerevisiae genes, namely Mvp1p (EKENA and STEVENS 1995 ), Nha1p (PRIOR et al.. 1996 ), and Ste50p (JANSEN et al.. 2001 ). Although the three S. cerevisiae genes identified are not essential for yeast growth (WINZELER et al.. 1999 ), our results indicate that at least one of the five genes inactivated in transformant 96 is essential for growth in A. fumigatus.

View larger version (26K): In this window In a new window Download PPT slide   Figure 5. Structure of genomic DNA at the site of integration of ppyrG in A. fumigatus transformant 96 of strain CEA136 and at the corresponding locus in a wild-type A. fumigatus diploid strain. (A) Map of the proposed organization of genomic DNA at the mutant locus in transformant 96 and at the wild-type locus. Plasmid ppyrG is indicated as an open box. The genomic organization of coding regions is indicated by arrows and numbers. The approximate location of the primers used to analyze the genomic structure in wild-type and mutant strains is indicated by arrowheads. (B) PCR analysis of wild-type diploid strain CEA136 and transformant 96 with primer 96-a in combination with appropriately spaced primers (96-b to 96-h). One wild-type copy of the locus is present in the diploid transformant 96, as shown by the amplification of fragments of identical size to those obtained with CEA136 genomic DNA when primers 96-b, -c, -d, and -e are used. The nature of the inversion is revealed by the occurrence of PCR products with primers 96-f, -g, and -h in transformant 96 only.

The comparison of the sequence of the regions flanking ppyrG in the six remaining transformants with the genomic data provided by the corresponding TIGR contig suggested that they had experienced significant genomic rearrangements (e.g., deletions of at least 30.9, 20.7, and 21.7 kb at the integration sites in transformants 99, 503, and 516, respectively). Analysis of transformants 197 and 209 showed that integration had been associated with shuffling of DNA originating from various regions of the genome (Table 3). The exact nature of these rearrangements could not be deduced from the available sequence data.

Electroporation-mediated insertional mutagenesis is associated with major genomic rearrangements:

To test whether the selection of transformants with an insertion of ppyrG in a gene essential for growth in A. fumigatus was associated with an increased frequency of chromosomal rearrangements, we analyzed the structure of the integration site in seven transformants with a single copy of ppyrG that could form haploid sectors on selective haploidization medium (data not shown). Three transformants (190, 226, and 333) displayed variable rearrangements of ppyrG that prevented analysis of flanking genomic DNA regions. Two others (328 and 340) had similar rearrangements of ppyrG but the length of the nucleotide sequences obtained by semirandom PCR was sufficient to reveal genomic sequences located at the junction with rearranged ppyrG. Comparison of these genomic sequences to the wild-type A. fumigatus genome sequence showed that the plasmid integration had led to minor genomic DNA deletions in these two transformants (42 and 7 bp, respectively). Finally, analysis of the two remaining transformants (176 and 336) showed that deletions of at least 12.7 and 36.9 kb, respectively, had occurred at the site of integration of ppyrG. We conclude that the frequency of chromosomal or plasmid rearrangements in transformants is independent of their phenotype on selective haploidization media.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The parasexual cycle of A. fumigatus:
The first description of a parasexual cycle in A. fumigatus was by Stromnaes and Garber in 1963. Few applications of parasexual genetics have been reported for this organism since (LEVADOUX et al.. 1981 ), although parasexual genetics has proved valuable for genetic mapping and evaluating whether cloned genes are essential in the model species A. nidulans (CLUTTERBUCK 1992 ; MOMANY and HAMER 1997 ; REOYO et al.. 1998 ). Here, we describe the establishment of A. fumigatus stable diploids that can be used in transformation experiments and for parasexual genetics. Using the parasexual cycle, we showed that the AfFKS1 gene encoding the catalytic subunit of the ß-(1,3)-glucan synthase is essential for the growth of A. fumigatus. By examining the formation of cals by heterozygous diploids plated on selective haploidization medium, we screened for mutations lying in genes essential for A. fumigatus growth. We analyzed a collection of 544 heterozygous diploids obtained by insertional mutagenesis. Of these mutants, 4.5% produced a cal similar to that observed for AfFKS1/Affks1 strains and, therefore, presumably harbor a mutation that inactivates an essential gene.

Recent results (BRADSHAW et al. 2001 ) using A. nidulans show that it is possible to recover poorly growing haploid progenies with a null mutation in cytochrome c through cleistothecia originating from a transformed heterokaryon, but not through parasexual genetics. This suggests that parasexual genetics prevents the appearance of haploid strains with a strong decrease in their fitness. Approximately 7% of the A. fumigatus mutants in our collection display an ambiguous phenotype (reduced growth, absence of sporulation) upon growth on selective haploidization media (data not shown). Further studies will be needed to understand the requirements to produce haploid mutant spores during the parasexual cycle.

DNA rearrangements following plasmid integration:
To generate a collection of insertional mutants of an A. fumigatus diploid strain, we used electroporation of a linearized heterologous plasmid. Although our results would suggest that electroporation is the method of choice to generate insertional mutants in A. fumigatus, as previously observed in haploid strains (BROWN et al.. 1998 ), our subsequent characterization of insertional mutants with an apparent single-copy integration showed that integration is associated with significant rearrangements of both plasmid and genomic DNA at the site of insertion. Such rearrangements are not easily detected by hybridization techniques and are a major limitation for the subsequent molecular characterization of tagged mutants.

Natural genomic rearrangements are common in fungi and are the main cause of karyotype variability in populations, playing an important role in adaptation to environment changes (FIERRO and MARTIN 1999 ). Mutagenesis and DNA-mediated transformation enhance the appearance of genomic rearrangements, as seen in Neurospora crassa (ASCH et al.. 1992 ; PERKINS et al.. 1993 ), A. nidulans (XUEI and SKATRUD 1994 ), Nectria haematococca (KISTLER and BENNY 1992 ), and C. albicans (BARTON and SCHERER 1994 ). Nevertheless, insertional mutagenesis with heterologous plasmids has been used extensively in fungi (reviewed in BROWN and HOLDEN 1998 ). To identify A. fumigatus virulence factors, random insertional transformants were obtained mainly by electroporation of a haploid strain by BROWN et al. 2000 . The only characterized mutant with severely attenuated virulence has an integration of two copies of the heterologous plasmid in a tail-to-tail configuration without genomic rearrangements (BROWN et al.. 2000 ). Our characterization of a larger set of transformants indicates that DNA rearrangements following transformation with a heterologous plasmid are common in A. fumigatus and were probably underestimated in previous studies. Similar rearrangements also appear associated with the ectopic integration of a homologous plasmid introduced by electroporation (A. FIRON and C. D'ENFERT, unpublished data).

Analysis of the junctions between ppyrG and A. fumigatus genomic DNA suggests common features for illegitimate recombination in C. glabrata (CORMACK and FALKOW 1999 ) and A. fumigatus: absence of microhomologies between plasmid ends and genomic targets, absence of a topoisomerase I consensus next to the genomic target, variable deletions of the plasmid 5' protruding end, and addition of some bases at the junction that do not originate from plasmid or genomic DNA. This suggests that a nonhomologous end joining process is involved in the integration of heterologous DNA in A. fumigatus as proposed in C. glabrata. However, illegitimate recombination in C. glabrata and in S. cerevisiae is not associated with major genomic rearrangements, although genomic duplications and insertions of mitochondrial DNA or small deletions/insertions have been observed at the site of plasmid integration in these species (SCHIESTL et al.. 1993 ; CORMACK and FALKOW 1999 ). Further studies on the mechanisms of recombination and DNA repair in A. fumigatus and filamentous fungi will be needed to explain the increased frequency and extent of DNA rearrangements that occur in these species.

Identification of essential A. fumigatus genes:
A collection of 546 insertional mutants of a diploid A. fumigatus strain yielded 25 transformants for which insertion of the transforming DNA seemed to have occurred in a gene essential for A. fumigatus growth. As mentioned above, DNA-mediated transformation results in major plasmid and genomic rearrangements. An unambiguous correlation between the observed phenotype and a gene interrupted by the transforming DNA molecule could be obtained for only one transformant. In this transformant, the mutation results in the inactivation of a gene that encodes a member of the SMC protein family that we designate SmcA. Other members of this protein family have been described in lower and higher eukaryotes (STRUNNIKOV and JESSBERGER 1999 ). In yeasts, SMC proteins are also essential for cell viability (LEHMANN et al.. 1995 ; WINZELER et al.. 1999 ; FOUSTERI and LEHMANN 2000 ).

In summary, we have shown that insertional mutagenesis of a diploid A. fumigatus strain can be used as a means to identify genes that are required for efficient growth of this fungus. However, electroporation-mediated transformation does not appear to be an appropriate method to generate a collection of A. fumigatus insertional mutants that is amenable to rapid molecular characterization. Insertional mutagenesis using Agrobacterium tumefaciens T-DNA (DE GROOT et al.. 1998 ) or using heterologous transposons (DABOUSSI 1996 ; KEMPKEN and KUCK 1998 ; HUA-VAN et al.. 2001 ) may represent suitable alternative strategies to produce A. fumigatus insertional mutants. In this regard, we have recently shown that the Fusarium oxysporum class II transposable element impala (LANGIN et al.. 1995 ; HUA-VAN et al.. 2001 ) transposes efficiently in A. fumigatus without any associated genomic rearrangements and can be used in combination with parasexual genetics to establish a compendium of A. fumigatus essential genes (A. FIRON and C. D'ENFERT, unpublished data).

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY081008 and AY080962.

	ACKNOWLEDGMENTS

We thank Maxime Schwartz and the members of the laboratory for their constant support. We also acknowledge Marc-Henri Lebrun, Francois Villalba, Rolland Beffa, and Tony Pugsley for their critical discussion of the work and reading of the manuscript. Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org. Sequencing of A. fumigatus was funded by the National Institute of Allergy and Infectious Diseases U01 AI 48830 to David Denning and William Nierman. This work was supported by a Ph.D. fellowship to A. Firon from Association Nationale de la Recherche Technique (CIFRE-Ministère de la Recherche et de la Technologie) and by grants from Institut Pasteur and Aventis CropScience to C. d'Enfert.

Manuscript received February 22, 2002; Accepted for publication April 23, 2002.

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