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A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

Alcazar-Fuoli L, Cairns T, Lopez JF, Zonja B, Pérez S, Barceló D, Igarashi Y, Bowyer P, Bignell E - PLoS ONE (2014)

Bottom Line: To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes.By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates.We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

View Article: PubMed Central - PubMed

Affiliation: Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.

ABSTRACT
Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

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Related in: MedlinePlus

Deletion of the PsoA cluster in A. fumigatus CEA17_ΔakuBKU80.A) Schematic representation of PsoA cluster replacement by BSM-A/H cassette in A. fumigatus CEA17_ΔakuBKU80. B) Expected structure of the replacement locus and C) Southern blot analysis of PsoAcluster deleted mutant and wild type (WT) strains. Expected hybridization band pattern: (1) 11659 bp for WT, and (2, 3, 4) 10501 bp for ΔPsoAcluster mutants.
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pone-0111875-g003: Deletion of the PsoA cluster in A. fumigatus CEA17_ΔakuBKU80.A) Schematic representation of PsoA cluster replacement by BSM-A/H cassette in A. fumigatus CEA17_ΔakuBKU80. B) Expected structure of the replacement locus and C) Southern blot analysis of PsoAcluster deleted mutant and wild type (WT) strains. Expected hybridization band pattern: (1) 11659 bp for WT, and (2, 3, 4) 10501 bp for ΔPsoAcluster mutants.

Mentions: Allelic replacements were first tested by PCR and single integration was confirmed by Southern blot (Figure 3, S2 and S3). Based on the PCR results frequency of homologous recombination in the A. fumigatus CEA17_ΔakuBKU80 genetic background was high with more than 85% of tested transformants (n = 6–9) undergoing allelic replacement at the correct genomic locus. Although lower frequencies of gene and gene cluster deletions were obtained when the clinical isolate ATCC46645 was used (Table 4), we obtained relevant mutants within the first 8 transformants tested, indicating practically useful, if not heightened, frequencies of gene replacement in non-mutated clinical isolates.


A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

Alcazar-Fuoli L, Cairns T, Lopez JF, Zonja B, Pérez S, Barceló D, Igarashi Y, Bowyer P, Bignell E - PLoS ONE (2014)

Deletion of the PsoA cluster in A. fumigatus CEA17_ΔakuBKU80.A) Schematic representation of PsoA cluster replacement by BSM-A/H cassette in A. fumigatus CEA17_ΔakuBKU80. B) Expected structure of the replacement locus and C) Southern blot analysis of PsoAcluster deleted mutant and wild type (WT) strains. Expected hybridization band pattern: (1) 11659 bp for WT, and (2, 3, 4) 10501 bp for ΔPsoAcluster mutants.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221250&req=5

pone-0111875-g003: Deletion of the PsoA cluster in A. fumigatus CEA17_ΔakuBKU80.A) Schematic representation of PsoA cluster replacement by BSM-A/H cassette in A. fumigatus CEA17_ΔakuBKU80. B) Expected structure of the replacement locus and C) Southern blot analysis of PsoAcluster deleted mutant and wild type (WT) strains. Expected hybridization band pattern: (1) 11659 bp for WT, and (2, 3, 4) 10501 bp for ΔPsoAcluster mutants.
Mentions: Allelic replacements were first tested by PCR and single integration was confirmed by Southern blot (Figure 3, S2 and S3). Based on the PCR results frequency of homologous recombination in the A. fumigatus CEA17_ΔakuBKU80 genetic background was high with more than 85% of tested transformants (n = 6–9) undergoing allelic replacement at the correct genomic locus. Although lower frequencies of gene and gene cluster deletions were obtained when the clinical isolate ATCC46645 was used (Table 4), we obtained relevant mutants within the first 8 transformants tested, indicating practically useful, if not heightened, frequencies of gene replacement in non-mutated clinical isolates.

Bottom Line: To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes.By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates.We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

View Article: PubMed Central - PubMed

Affiliation: Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.

ABSTRACT
Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

Show MeSH
Related in: MedlinePlus