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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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Deletion of the pacC gene in A. fumigatus CEA17_ΔakuBKU80 (referred as akuBKU80).A) Schematic view of pacC gene deletion. B) Primers used to check gene replacement at the pacC locus by PCR. C) Phenotypic analysis of ΔpacC mutants compared with the wild type. 2.5×104 spores were point inoculated onto MM pH 6.5 and MM pH 8. Plates were incubated at 37°C for 48 hours.
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pone-0111875-g002: Deletion of the pacC gene in A. fumigatus CEA17_ΔakuBKU80 (referred as akuBKU80).A) Schematic view of pacC gene deletion. B) Primers used to check gene replacement at the pacC locus by PCR. C) Phenotypic analysis of ΔpacC mutants compared with the wild type. 2.5×104 spores were point inoculated onto MM pH 6.5 and MM pH 8. Plates were incubated at 37°C for 48 hours.

Mentions: In order to establish and optimise the methodology we first elected to replace single genes, selecting two, physically unlinked, individual genes AFUA_1G17640 and AFUA_3G11970. AFUA_3G11970 is a telomere distal gene encoding the transcription factor, PacC which is involved in alkaline signal transduction [18]. AFUA_1G17640 is a telomere-proximal gene encoding a putative transcription factor, RegA, which resides in a cluster of genes upregulated during murine infection and has a possible role in melanin biosynthesis [44], [45]. For our initial experiments we exploited the well-characterised alkaline sensitivity of PacC mutants to permit rapid assessment of homologous gene replacements amongst transformants, and utilised two newly constructed biselectable marker plasmids (pBSM-Z/P and pBSM-A/H) to permit comparative assessment of achievable gene replacement frequencies. Schematic overviews of recombinant BAC construction in E. coli and BAC-mediated pacC gene deletion are provided in Figures 1 and 2A, respectively. A BAC clone (AfB28-mq1_36C04) having appropriate coverage of the pacC AFUA_3G11970 genomic locus was retrieved from the library (Table S1). The BAC insert spanned the entire AFUA_3G11970 gene incorporating 24 kb and 74 kb of 5′ and 3′ flanking regions respectively. Appropriately recombined BAC clones were identified by PCR with primers AmpR-F and CF5R (Figure 2B) or PtR-F and CF5R. Recombinant BACs were denoted as BAC36C4-Z/P and BAC36C4-A/H where the AFUA_3G11970 gene had been replaced with an ampicillin/hygromycin or zeocin/pyrithiamine biselectable marker respectively.


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 pacC gene in A. fumigatus CEA17_ΔakuBKU80 (referred as akuBKU80).A) Schematic view of pacC gene deletion. B) Primers used to check gene replacement at the pacC locus by PCR. C) Phenotypic analysis of ΔpacC mutants compared with the wild type. 2.5×104 spores were point inoculated onto MM pH 6.5 and MM pH 8. Plates were incubated at 37°C for 48 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111875-g002: Deletion of the pacC gene in A. fumigatus CEA17_ΔakuBKU80 (referred as akuBKU80).A) Schematic view of pacC gene deletion. B) Primers used to check gene replacement at the pacC locus by PCR. C) Phenotypic analysis of ΔpacC mutants compared with the wild type. 2.5×104 spores were point inoculated onto MM pH 6.5 and MM pH 8. Plates were incubated at 37°C for 48 hours.
Mentions: In order to establish and optimise the methodology we first elected to replace single genes, selecting two, physically unlinked, individual genes AFUA_1G17640 and AFUA_3G11970. AFUA_3G11970 is a telomere distal gene encoding the transcription factor, PacC which is involved in alkaline signal transduction [18]. AFUA_1G17640 is a telomere-proximal gene encoding a putative transcription factor, RegA, which resides in a cluster of genes upregulated during murine infection and has a possible role in melanin biosynthesis [44], [45]. For our initial experiments we exploited the well-characterised alkaline sensitivity of PacC mutants to permit rapid assessment of homologous gene replacements amongst transformants, and utilised two newly constructed biselectable marker plasmids (pBSM-Z/P and pBSM-A/H) to permit comparative assessment of achievable gene replacement frequencies. Schematic overviews of recombinant BAC construction in E. coli and BAC-mediated pacC gene deletion are provided in Figures 1 and 2A, respectively. A BAC clone (AfB28-mq1_36C04) having appropriate coverage of the pacC AFUA_3G11970 genomic locus was retrieved from the library (Table S1). The BAC insert spanned the entire AFUA_3G11970 gene incorporating 24 kb and 74 kb of 5′ and 3′ flanking regions respectively. Appropriately recombined BAC clones were identified by PCR with primers AmpR-F and CF5R (Figure 2B) or PtR-F and CF5R. Recombinant BACs were denoted as BAC36C4-Z/P and BAC36C4-A/H where the AFUA_3G11970 gene had been replaced with an ampicillin/hygromycin or zeocin/pyrithiamine biselectable marker respectively.

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