Role of the secretome in manganese and carbon oxidation by filamentous ascomycete fungi
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CitationZeiner, Carolyn Alexandra. 2015. Role of the secretome in manganese and carbon oxidation by filamentous ascomycete fungi. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractFungi are the primary decomposers of recalcitrant plant and animal material in terrestrial environments, thereby serving as important drivers of global carbon cycling and climate dynamics and as mediators in renewable energy production. Degradation of organic litter is achieved through secretion of a large and diverse suite of extracellular enzymes and reactive metabolites, collectively referred to as the secretome. This thesis explores the secretomes of four filamentous Ascomycete fungi that were recently isolated from field sites and have the ability to oxidize manganese (Mn)(II) to Mn(III/IV) oxides: Alternaria alternata SRc1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a. Mn(II)-oxidizing fungi are of engineering and industrial interest due to their utility in the remediation of metal-contaminated waters and their ability to harness Mn(II) oxidation in the breakdown of lignocellulosic plant material. While the processes of Mn(II) oxidation and carbon oxidation are mediated by the secretome in white-rot Basidiomycete fungi, comparatively little is known about the oxidative capacity of the secretomes of Ascomycetes, particularly those of environmental isolates. Using a combination of microscopy and chemical assays, this thesis identifies extracellular superoxide as the oxidant of Mn(II) in Stagonospora sp. and Pyrenochaeta sp. during growth on solid substrate and suggests a role for secreted organic polymers in Mn(III) complexation and Mn oxide templation. Furthermore, through in-gel assays and bulk mass spectrometry, this work demonstrates that species-specific secreted enzymes confer Mn(II) oxidative capacity in the liquid, cell-free secretome of these two fungi and P. sporulosum and shows that Mn(II) oxidative capacity changes over time in a pattern unique to each organism. Through quantitative iTRAQ proteomics and custom bioinformatic analyses, the protein composition of the secretomes of all four filamentous Ascomycetes was fully characterized, revealing a rich and functionally diverse suite of extracellular enzymes that suggest the ability to oxidize carbon through both direct and indirect mechanisms. Although the functional diversity of the secretome was similar among the four organisms, the fungi exhibited striking differences in regulation of carbon-degrading enzymes over a three-week time course, illustrating species-specific and temporal shifts in carbon utilization strategies among the phylogenetically diverse species. Taken together, results of this thesis further our understanding of the mechanisms of Mn(II) oxidation by fungi, demonstrate the rich functional diversity and oxidative capacity of Ascomycete secretomes, and enhance our understanding of the role of filamentous Ascomycetes in recalcitrant carbon turnover in the environment.
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