Validation of a Self-Excising Marker in the Human Pathogen Aspergillus fumigatus by Employing the β-Rec/six Site-Specific Recombination System

Abstract

Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing β serine recombinase acting on six recognition sequences (β-rec/six) in a fungal host, the human pathogen Aspergillus fumigatus, and its use in establishing a self-excising resistance marker cassette for serial gene replacement.

Functional studies of genes often rely on methods of their manipulation, most often by means of gene targeting, such as deletion. Usually, this is achieved by replacing any sequence of interest by that of a marker gene, the presence of which can be selected under specific conditions. One particular obstacle in doing so lies in the fact that specific cellular activities in higher eukaryotes are often encoded by multiple and sometimes redundant genes, making the generation of null mutants completely ablated for such activities a difficult task. The genome sequences of filamentous fungi have revealed high degrees of redundancy for a plethora of genes, and it is a challenge of functional genomics to address cellular functions of such gene families on a comprehensive scale. This is of special relevance, for instance, in Aspergillus fumigatus when assessing the role of gene products for pathogenicity (20). Before drawing any conclusions about the contribution of a specific cellular activity to the virulome of this human pathogen, all members of its possibly encoding gene family ought to be inactivated to result in a definite null mutant. Genetic backgrounds of specific A. fumigatus mutant strains that are impaired in illegitimate recombination assist in successful gene targeting by increasing the otherwise poor rate of homologous recombination (5, 24). One shortcoming, however, is based on the fact that the number of preferred resistance markers for gene replacement is limited, with only hygromycin, phleomycin, and pyrithiamine being applicable antifungal selective agents (26, 31). Recyclable marker modules allow repetitive rounds of gene deletion, followed by marker rescue, making the recurring use of resistance cassettes in gene targeting tasks possible, and they also avoid the potential risk of phenotypic effects caused by expression of additional heterologous genes. Marker recycling may be achieved via repetitive sequences that flank the genetic marker accompanied by counterselection after homologous recombination and marker excision (6, 22) or by more sophisticated systems that employ enzymes to catalyze site-specific recombination between short recognition sequences (9, 21). Such site-specific recombinases are classified into two categories, tyrosine and small or large serine recombinases, based on a key catalytic amino acid (33). In fungi, two recombination systems have been used primarily in genome manipulation approaches, the bacteriophage P1-derived Cre/loxP system (15) and the Saccharomyces cerevisiae Flp/FRT system (19, 38). Both act via a tyrosine recombinase, Cre or Flp, that binds to a DNA sequence, loxP or FRT, respectively, to catalyze recombination, resulting in excision and/or integration. Cre/loxP recombination has been extensively used for genetic engineering of the model eukaryote S. cerevisiae and other yeasts (13, 14, 17, 23, 45), while the Flp/FRT system is predominantly employed for functional studies of the fungal pathogen Candida albicans (29, 30, 32). For the latter, the system was streamlined by including the recombinase-encoding sequence within the marker module to be excised, thereby rendering introduction of the FLP gene after the gene replacement event unnecessary but allowing gene deletion and marker rescue based on one single transformation event. This so-called flipper cassette has proven itself extremely valuable and versatile for genome manipulations in this opportunistic pathogen.

In aspergilli, the Cre/loxP system was recently successfully established for repetitive gene targeting (9, 21). Expression of the Cre enzyme was achieved either by using a genetically altered recipient strain or by transiently expressing the cre gene from an autonomously replicating plasmid that is afterward lost under unselective culture conditions. In the pathogen A. fumigatus, the latter approach was followed, which requires two rounds of transformation per gene deletion/marker rescue event. In order to circumvent this experimental drawback, we sought to establish a self-excising marker cassette for this fungal host using the prokaryotic small β serine recombinase (β-rec) acting on six recognition sequences (β-rec/six) (4, 35, 37). This site-specific recombination system had been demonstrated to operate in various hosts, such as Bacillus subtilis, plants, or mammalian cells (2, 7, 12); yet, no proof of functionality of the β-rec/six system in fungi has been provided.

In order to validate a self-excising marker cassette for straightforward gene targeting purposes in A. fumigatus, we designed a suitable marker module consisting of several functional parts (Fig. 1). As the dominant selectable marker, the pyrithiamine resistance-conferring ptrA gene from Aspergillus oryzae was chosen (25). This was linked to an expression cassette for a synthetic, codon-optimized allele of the β recombinase-encoding gene, placed between the Penicillium chrysogenum endoxylanase-encoding xylP promoter sequence (46) and the Aspergillus nidulans trpC transcriptional termination region. The whole module is flanked by two identical, asymmetric β recombinase binding sites at 90 bp in size, termed six sites (4). Assembly of the plasmid construct was achieved by inserting the PCR amplified ptrA marker via BglII sites into the plasmid pSK479 (acquired custom-made by purchase from GeneArt, Germany), which contains the synthetic, codon-optimized β-rec allele and the trpC terminator between two six sites in the basic cloning vector pMA-RQ. The 1.7-kb xylP promoter region was inserted in the resulting plasmid as a PCR amplicon via PstI to yield the final construct pSK485. Samples of this plasmid (GenBank accession no. HM640258) and the A. fumigatus akuA::loxP strain AfS77 have been deposited at the Fungal Genetics Stock Center (Kansas) (28) to assist in further distribution. A 5.3-kb fragment can be isolated from pSK485 by SfiI digestion to generate incompatible overhangs that assist in further cloning steps (18). Accordingly, the marker module was ligated in a next step to the SfiI backbone of plasmid pSK346, which contains a gene replacement cassette for the A. fumigatus abr2 gene (24). From the resulting plasmid pSK509, a 8.9-kb HindIII fragment was isolated to target the abr2 coding sequence in the recipient strain AfS77, which is an akuA::loxP derivative (24) of the A. fumigatus clinical isolate ATCC 46645 (16). After transformation, strains carrying the respective abr2Δ gene deletion were easily distinguished from ectopic integrants by their brown conidia (44). Several brownish transformants were colony purified on minimal culture medium, with glucose as the source of carbon, and three clonal isolates were then allowed to germinate on xylose-containing minimal medium, from which two single colonies were chosen for further analyses. The resulting strains and their progenitors were screened for the presence or absence of the marker module by diagnostic PCR with oligonucleotides Sv300 (5′-CAACTCTAAATTCTACTGTTGTTTC-3′), Sv301 (5′-GTTCGAGCCATCCATAGTTCCAGTC-3′), Sv302 (5′-ATTCTGGACTGTACCTTATGTAAG-3′), and Sv630 (5′-TCGGAGAACACCTTGCTGACG-3′) and Southern hybridization (39), as well as by their sensitivity toward pyrithiamine (Fig. 2). A uniform pattern could be detected for all series of strains descending from a clonal transformant, as follows: the primary abr2Δ mutants stably maintained the six-pxylP::β-rec::trpCt-ptrA-six marker module, and after passage in the presence of xylose, the marker had been excised to leave an abr2::six lesion. The identity of the recombined six site was confirmed by sequencing a corresponding PCR fragment amplified from the genomic locus. Moreover, growth phenotypes of the examined strains on pyrithiamine-containing medium were in agreement with these genotypes, demonstrating successful marker rescue and suitability of the self-excising β-rec/six blaster cassette for repeated gene targeting.

FIG. 1.

Elements of the self-excising β-rec/six blaster cassette. A schematic presentation of the plasmid pSK485, which harbors the novel marker module, is given. The functional elements are as follows: (i) an expression cassette for the synthetic, codon-optimized β recombinase-encoding gene (β-rec), in which transcription is driven in dependency of the carbon source by the controllable xylP promoter from P. chrysogenum; (ii) the ptrA gene from A. oryzae as the dominant, selectable marker conferring resistance toward pyrithiamine; and (iii) flanking binding targets for the β recombinase, termed six sites. Incompatible restriction sites (SfiI-1 and SfiI-2) assist in the assembly of gene replacement cassettes.

FIG. 2.

Validation of site-specific recombination by the β-rec/six blaster cassette in A. fumigatus, resulting in marker recycling. (A) Schematic presentation of genotypes after abr2 gene replacement and subsequent marker excision. Positions of priming oligonucleotides, the hybridizing probe (black bar), and restriction sites are schematically indicated. Also, the recombined six site at the abr2::six locus, as determined by sequencing of the genomic locus, is presented, with subsites I and II denoting binding sites for the β recombinase (4) and convergent arrows specifying inverted repeat regions. wt, wild type. (B) Diagnostic PCR (left) and Southern analysis (right) of genomic DNA isolated from corresponding strains. The calculated sizes of DNA fragments or hybridization signals are specified. (C) Growth phenotypes of the validated isolates confirm marker rescue accompanied by restoration of pyrithiamine sensitivity. Shown are the parental abr2⁺ strain AfS77, one abr2Δ deletant, and its descendant after induction of β-rec expression via passage on xylose-containing culture medium. Strains were point inoculated on Aspergillus minimal medium (AMM) in the presence (+) or absence (−) of pyrithiamine.

In order to validate the suitability of our system for multiple gene targeting events, a second region of the pigment-encoding gene cluster was targeted, the polyketide synthase-encoding pksP/alb1 gene (27, 43). Using a fusion PCR approach (42), a pksP replacement cassette containing the marker module flanked by 5′ and 3′ homologous regions of 1.8 kb and 1.6 kb in size, respectively, was generated. The corresponding replacement cassette of 8.65 kb was released from the resulting plasmid pSK510 via FspI to be transformed in the abr2::six recipient strain. Several primary transformants with white conidia could be retrieved and were purified on minimal medium containing glucose. Diagnostic PCR with genomic DNA extracted from 12 independent clonal transformants confirmed the proper integration of the pksP replacement cassette at the target locus (not shown), which is in accordance with their albino phenotype. Finally, conidia from six pksPΔ isolates were further propagated on xylose-containing medium, from which three single colonies per deletant were analyzed for proper marker excision by diagnostic PCR with priming oligonucleotides Sv712 (5′-ACAACGCCCAAACCTGTTATCC-3′) and Sv713 (5′-AAGGCTGTGAACAAGCCAAGCC-3′). The resulting 495-bp amplicons were in agreement with β-mediated marker excision leaving behind one six recombination site at the pksP locus, resulting in an abr2::six pksP::six genotype. Moreover, all the strains had regained sensitivity toward pyrithiamine, as demonstrated by the absence of growth when inoculated on pyrithiamine-containing medium (Fig. 3). Extended diagnostic PCR with the primer combination Sv302 and Sv713 did not yield a product for any of the analyzed strains, indicating that marker excision by the β recombinase was restricted to the six acceptor sites associated with the deletion marker and did not extend to the abr2::six one, which is located 8.1 kb upstream.

FIG. 3.

Validation of sequential gene targeting and marker rescue by deletion of the pksP gene. (A) Schematic representation of the abr2::six pksP::six locus after repeated gene deletion and marker excision. The positions of priming oligonucleotides for diagnostic PCR are indicated, and the distance between both six sites is specified for illustration. chr., chromosome. (B) Diagnostic PCR with genomic DNA isolated from a representative clonal strain after induction of β-rec expression by propagation on xylose-containing culture medium, interrogating the abr2::six and pksP::six loci. The calculated sizes of DNA fragments are indicated. (C) Pyrithiamine sensitivity of the abr2::six pksP::six isolate confirms marker excision. Shown are the parental abr2::six strain, one abr2::six pksPΔ isolate, and one of its abr2::six pksP::six descendants inoculated on Aspergillus minimal medium in the presence (+) or absence (−) of pyrithiamine.

We here describe the first validated use of the prokaryotic β-rec/six site-specific recombination system in the fungal host A. fumigatus, therefore introducing a novel tool for genetic manipulation of fungal genomes. From previous studies of bacteria, plants, and mammalian cells, it is known that β recombinase relies on supercoiled substrates and a protein factor associated to chromatin, such as bacterial HBsu or eukaryotic HMG1 protein (1, 34, 41). The successful recombination in A. fumigatus demonstrates that this fungal host apparently provides for such a host factor assisting in β recombination, and it can be speculated that other aspergilli or even fungal species in general are capable of supporting this recombination system. Successful establishment of the β-rec/six system includes several benefits, most important of which is the strict cis action of the β recombinase. While other recombinases, such as Cre or Flp, are able to act on unlinked recombination sites, the β recombinase is restricted exclusively to intramolecular recombination events (1, 35, 36). This is advantageous when several rounds of recombination are needed, as intermolecular recombination between residual acceptor sites and therefore undesired and uncontrolled chromosomal rearrangements are prevented. We have utilized the recombination system to compose a novel, self-excising marker for gene targeting purposes in A. fumigatus, which allows marker rescue in a convenient manner. One particular crucial component of this marker module is the promoter sequence regulating expression of the recombinase gene. A complete of shutoff transcription in Aspergillus, as is essential in this particular setting, is rarely achieved for controllable promoters. The employed heterologous xylP promoter, however, appears to be sufficiently repressed in the presence of glucose, while adequate levels of expression are in fact generated upon induction by the alternative carbon source xylose. Another feature of the module lies in the synthetic, fully codon-optimized sequence that encodes the β recombinase, which was used to avoid potential obstacles from poor expression of this heterologous gene due to negative codon bias. This strategy has proven useful in several instances by enhancing the levels of gene expression or making it at all possible (3, 11). Deletion of two genes of the pigment-encoding cluster proved the feasibility of the marker system for sequential gene targeting approaches. At least for the targeted abr2 and pksP genes, no site-specific effects acting on expression of the recombinase became evident. However, such obstacles, which might interfere with controlled marker excision and therefore with selection, cannot be ruled out in general and may depend on the locus at which the marker is integrated.

By establishing the functionality of the β-rec/six system in A. fumigatus, we have extended the range of recombination systems available for this fungal species. This might assist not only in gene targeting procedures but also in recombination-based screening procedures, such as RIVET (recombination-based in vivo expression technology) or RAGE (recombination-activated gene expression) systems (8, 10, 40), which could be of use in comprehensive approaches to identify virulence-related characteristics of this saprophyte.