Tn7-based genome-wide random insertional mutagenesis of Candida glabrata

Abstract

We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7. Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of approximately 10(4) allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms.

Figure 1.

Maps of mini-Tn7 derivatives used. Tn7 UKR (URA3 Kan · R6Kγ ori) is a 3.108-kb derivative of mini-Tn7 SpeI-Km^R-NotI containing the conditional origin of replication R6Kγ and the Saccharomyces cerevisiae URA3 gene. Relevant restriction sites are shown. Tn7 UKR-H is a 5.118-kb element derived from Tn7 UKR that contains the Klebsiella pneumoniae hph gene (which confers resistance to hygromycin B) driven by the PGK1 promoter from S. cerevisiae. The arrows underneath each map indicate the direction of transcription.

Figure 2.

Two of the pathways of homologous recombination after transformation of linearized mutant fosmids. (A) Class 2 (partially stable, PS) transformants: A linearized genomic fragment containing a transposon insertion can recircularize before, or at the time of, homologous recombination, generating a duplication of the region as indicated. Primers 1 and 2 are oriented toward, and primers 3 and 4 oriented away from the Tn7 insertion. (B) Result of PCR amplification of genomic DNA from one Class 2 transformant. (genom.PS lane) Template DNA for PCR was genomic DNA; (plasm. lane) template DNA for PCR was pIC14. (C) Class 3 (stable, S) transformants: allele replacement. A linearized genomic fragment containing a transposon insertion recombines by double crossover and replaces the genomic locus. (D) Result of PCR amplification of genomic DNA from nine Class 3 (S) transformants. (Lane 1) Genomic DNA from a nonhomologous recombinant was used for the PCR and bands for both the wild type and Tn7-disrupted copies are amplified. (Lanes 2–9) Results for homologous recombinants. Controls: (wt) Cg14 genomic DNA was used as template for the PCR; (ins.) pIC14 was used as template.

Figure 3.

PCR amplification for 11 Class 3 transformants using three primer pairs. (A) Genomic primers flanking the Tn7 insertion site and oriented toward the insertion. (B) One genomic primer combined with a Tn7R primer. (C) Genomic primer combined with the Tn7L primer. Note that the three positive transformants for B and C are the only transformants positive for the longer PCR product in A corresponding to the Tn7-disrupted band. Control lanes: (wt) BG14 genomic DNA, (C) pIC14 DNA used as template for PCR.

Figure 4.

Southern analysis of Candida glabrata transformants derived from two mutagenized fosmids. The probes are the URA3 gene from Tn7 UKR (lanes 1–9). All DNAs were digested with BcgI and BamHI prior to agarose gel electrophoresis. (Lane 1) C.g., genomic DNA from a pool of 35 Ura⁺ transformants derived from transformation with mutagenized fosmid 1. (Lane 2) E. coli, genomic DNA from 150 pooled Tn7 insertions in fosmid 1. (Lane 3) U, DNA from unmutagenized fosmid 1. (Lane 4) C.g., genomic DNA from a pool of 44 Ura⁺ transformants derived from transformation with mutagenized fosmid 2. (Lane 5) E. coli, genomic DNA from 150 pooled Tn7 insertions in fosmid 2. (Lane 6) C.g., genomic DNA from a pool of 5 Ura⁺ transformants derived from transformation with Tn7-mutagenized pBAC-Bcg3. (Lane 7) DNA from unmutagenized fosmid 2. (Lane 8) E. coli, genomic DNA from >1000 pooled Tn7 insertions in plasmid pBAC-Bcg3. (Lane 9) Cont, control pIC31 digested with PstI.