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

Laura Alcazar-Fuoli,Sandra Pérez,Yasuhiro Igarashi,Paul Bowyer,Bozo Zonja,Elaine Bignell,Timothy Cairns,Jordi F Lopez,Damià Barceló,Robert A Cramer

doi:10.1371/journal.pone.0111875

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.

Highlights

In the genomic era of fungal molecular genetics, the context and/or spatial organisation of genes is emerging as an important regulatory determinant [1]
Lack of clearly defined biosynthetic pathways for many secondary metabolites means that the boundaries and number of genes comprising each gene cluster are often poorly defined, common features can be identified including the involvement of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), and hybrids thereof [10]
In order to commence a functional genomic analysis of gene clusters and virulence in the human fungal pathogen A. fumigatus we developed the means to manipulate gene content of complex genetic loci using bacterial artificial chromosome (BAC)-mediated recombineering

Summary

Introduction

In the genomic era of fungal molecular genetics, the context and/or spatial organisation of genes is emerging as an important regulatory determinant [1]. In some instances the mechanistic significance of such organisational structures remains unclear but it is widely accepted that genes involved in the biosynthesis of certain secondary metabolites are co-localised, in series, as gene clusters [2]. Noteworthy is the fact that the majority of known and putative SM gene clusters are located at subtelomeric regions of the chromosomes, [8] most likely facilitating their epigenetic regulation by chromatin-based mechanisms [13]. This epigenetic control of secondary metabolism might provide a means by which SM biosynthesis can be tailored to specific growth conditions while remaining otherwise silent

Objectives

Methods

Results

Conclusion