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Kenneth E. Hammel - One of the best experts on this subject based on the ideXlab platform.
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localizing gene regulation reveals a staggered wood decay mechanism for the Brown Rot fungus postia placenta
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Jiwei Zhang, Kenneth E. Hammel, Gerald N Presley, Jae San Ryu, Jon Menke, Melania Figueroa, Galya Orr, Jonathan S. SchillingAbstract:Wood-degrading Brown Rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that Brown Rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by Brown Rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for Brown Rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.
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evidence from serpula lacrymans that 2 5 dimethoxyhydroquinone is a lignocellulolytic agent of divergent Brown Rot basidiomycetes
Applied and Environmental Microbiology, 2013Co-Authors: Premsagar Korripally, Carl J Houtman, Vitaliy A I. Timokhin, Michael C D. Mozuch, Kenneth E. HammelAbstract:Basidiomycetes that cause Brown Rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of Brown Rot fungi have arisen independently from ligninolytic white Rot ancestors via loss of lignocellulolytic enzymes. Brown Rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe(3+) in colonized wood. Since there is little evidence that white Rot fungi produce these metabolites, one question is the extent to which independent lineages of Brown Rot fungi may have evolved different Fe(3+) reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry in Serpula lacrymans, a Brown Rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay by S. lacrymans. We found also that variegatic acid failed to reduce in vitro the Fe(3+) oxalate chelates that predominate in Brown-Rotting wood and that it did not drive Fenton chemistry in vitro under physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain Brown Rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white Rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.
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Evidence for cleavage of lignin by a Brown Rot basidiomycete
Environmental microbiology, 2008Co-Authors: Daniel J. Yelle, John Ralph, Kenneth E. HammelAbstract:Summary Biodegradation by Brown-Rot fungi is quantitatively one of the most important fates of lignocellulose in nature. It has long been thought that these basidi omycetes do not degrade lignin significantly, and that their activities on this abundant aromatic biopolymer are limited to minor oxidative modifications. Here we have applied a new technique for the complete solu bilization of lignocellulose to show, by one-bond 1 H- 13 C correlation nuclear magnetic resonance spec troscopy, that Brown Rot of spruce wood by Gloeo phyllum trabeum resulted in a marked, non-selective depletion of all intermonomer side-chain linkages in the lignin. The resulting polymer retained most of its original aromatic residues and was probably inter connected by new linkages that lack hydrogens and are consequently invisible in one-bond 1 H- 13 C corre
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fungal hydroquinones contribute to Brown Rot of wood
Environmental Microbiology, 2006Co-Authors: Melissa R Suzuki, Christopher G Hunt, Zachary D Dalebroux, Kenneth E. Hammel, Corine J HoutmanAbstract:Summary The fungi that cause Brown Rot of wood initiate ligno- cellulose breakdown with an extracellular Fenton system in which Fe 2+ and H2O2 react to produce hydroxyl radicals (·OH), which then oxidize and cleave the wood holocellulose. One such fungus, Gloeophyllum trabeum, drives Fenton chemistry on defined media by reducing Fe 3+ and O2 with two extra- cellular hydroquinones, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 4,5-dimethoxycatechol (4,5-DMC). However, it has never been shown that the hydro- quinones contribute to Brown Rot of wood. We grew G. trabeum on spruce blocks and found that 2,5- DMHQ and 4,5-DMC were each present in the aqueous phase at concentrations near 20 mM after 1 week. We determined rate constants for the reactions of 2,5- DMHQ and 4,5-DMC with the Fe 3+ -oxalate complexes that predominate in wood undergoing Brown Rot, finding them to be 43 l mol -1 s -1 and 65 l mol -1 s -1 respectively. Using these values, we estimated that the average amount of hydroquinone-driven ·OH production during the first week of decay was 11.5 mmol g -1 dry weight of wood. Viscometry of the degraded wood holocellulose coupled with computer modelling showed that a number of the same general magnitude, 41.2 mmol oxidations per gram, was required to account for the depolymerization that occurred in the first week. Moreover, the decrease in holocellulose viscosity was correlated with the mea- sured concentrations of hydroquinones. Therefore, hydroquinone-driven Fenton chemistry is one com- ponent of the biodegradative arsenal that G. trabeum expresses on wood.
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processive endoglucanase active in crystalline cellulose hydrolysis by the Brown Rot basidiomycete gloeophyllum trabeum
Applied and Environmental Microbiology, 2005Co-Authors: Roni Cohen, Melissa R Suzuki, Kenneth E. HammelAbstract:Brown Rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg pRotein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this Brown Rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.
Jonathan S. Schilling - One of the best experts on this subject based on the ideXlab platform.
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oxidative damage control during decay of wood by Brown Rot fungus using oxygen radicals
Applied and Environmental Microbiology, 2018Co-Authors: Jesus D Castano, Jiwei Zhang, Claire E Anderson, Jonathan S. SchillingAbstract:ABSTRACT Brown Rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that Brown Rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that Brown Rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grew Postia placenta directionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H 2 O 2 and Fe 2+ concentrations in the extracellular environment were conducive to HO˙ production in early (H 2 O 2 :Fe 2+ ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producer Trichoderma reesei. Collectively, this indicates that P. placenta uses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this Brown Rot fungus to use ROS to degrade wood. IMPORTANCE Wood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white Rot) or by using Fenton-generated reactive oxygen species (ROS) to “loosen” wood cell walls, enabling selective sugar extraction (Brown Rot). Although white Rot lignin-degrading pathways are well characterized, there are many uncertainties in Brown Rot fungal mechanisms. Our study addressed a key uncertainty in how Brown Rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H 2 O 2 , Fe 2+ ), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.
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distinct growth and secretome strategies for two taxonomically divergent Brown Rot fungi
Applied and Environmental Microbiology, 2017Co-Authors: Gerald N Presley, Jonathan S. SchillingAbstract:Brown Rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful non-selective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in Postia placenta to segregate these reactions, but it is unclear if this two-step mechanism varies in other Brown Rot fungi with different traits and life history strategies, and that occupy different niches in nature. We used pRoteomics to analyze a progression of wood decay on thin wafers, using Brown Rot fungi with significant taxonomic and niche distance - Serpula lacrymans (Boletales; ‘dry Rot9 lumber decay) and Gloeophyllum trabeum (order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus pRotein secretion (resource capture), with ergosterol/extracted pRotein ratio increased 7x with S. lacrymans than with G. trabeum . In line with their native substrate associations, hemicellulase specific activities were dominated by mannanase in S. lacrymans and by xylanase in G. trabeum . Consistent with previous observations, S. lacrymans did not produce GH 6 cellobiohydrolases (CBH) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among Brown Rot fungi by having CBH genes. This work suggests that distantly related Brown Rot fungi employ staggered mechanisms to degrade wood, but that the underlying strategies vary among taxa. Importance Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown Rot fungi are unique among these fungi in that they use a non-enzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has evolved multiple times, independently, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers, and we found similar two-step mechanisms in taxonomically divergent Brown Rot fungi. Results, however, revealed strikingly different growth strategies, with S. lacrymans investing more in biomass production than secretion of pRoteins, and G. trabeum showing the opposite pattern with a high diversity of uncharacterized pRoteins. The “simplified” S. lacrymans secretomic system could help narrow gene targets central to oxidative Brown Rot pretreatments, and comparing its distinctions with G. trabeum and other Brown Rot fungi (e.g. Postia placenta ) might offer similar traction in non-catabolic genes.
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localizing gene regulation reveals a staggered wood decay mechanism for the Brown Rot fungus postia placenta
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Jiwei Zhang, Kenneth E. Hammel, Gerald N Presley, Jae San Ryu, Jon Menke, Melania Figueroa, Galya Orr, Jonathan S. SchillingAbstract:Wood-degrading Brown Rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that Brown Rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by Brown Rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for Brown Rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.
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lignocellulose modifications by Brown Rot fungi and their effects as pretreatments on cellulolysis
Bioresource Technology, 2012Co-Authors: Jonathan S. Schilling, Robert A Blanchette, Shona M Duncan, Timothy R Filley, Ulrike W TschirnerAbstract:Brown Rot fungi Gloeophyllum trabeum and Postia placenta were used to degrade aspen, spruce, or corn stover over 16 weeks. Decayed residues were saccharified using commercial cellulases or Brown Rot fungal extracts, loaded at equal but low endoglucanase titers. Saccharification was then repeated for high-yield samples using full strength commercial cellulases. Overall, Brown Rot pretreatments enhanced yields up to threefold when using either cellulase preparation. In the best case, aspen degraded 2 weeks by G. trabeum yielded 72% glucose-from-cellulose, a 51% yield relative to original glucan. A follow-up trial with more frequent harvests showed similar patterns and demonstrated interplay between tissue modifications and saccharification. Hemicellulose and vanillic acid (G6) or vanillin (G4) lignin residues were good predictors of saccharification potential, the latter notable given lignin's potential active role in Brown Rot. Results show basic relationships over a Brown Rot time course and lend targets for controlling an applied bioconversion process.
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Synergy between pretreatment lignocellulose modifications and saccharification efficiency in two Brown Rot fungal systems
Applied Microbiology and Biotechnology, 2009Co-Authors: Jonathan S. Schilling, Jacob P. Tewalt, Shona M DuncanAbstract:Brown Rot wood-degrading fungi distinctly modify lignocellulose and completely hydrolyze polysaccharides (saccharification), typically without secreting an exo-acting glucanase and without removing lignin. Although each step of this two-step approach evolved within the same organism, it is unknown if the early lignocellulose modifications are made to specifically facilitate their own abbreviated enzyme system or if enhancements are more general. Because commercial pretreatments are typically approached as an isolated step, answering this question has immense implication on bioprocessing. We pretreated spruce and pine blocks with one of two Brown Rot fungi, Gloeophyllum trabeum or Fomitopsis pinicola . Wood harvested at weeks 1, 2, 4, and 8 showed a progression of weight loss from time zero due to selective carbohydrate removal. Hemicellulose losses progressed faster than cellulose loss. This “pretreated” material was then saccharified with commercially relevant Trichoderma reesei cellulases or with cellulases from the Brown Rot fungi responsible for degrading the wood to test for synergy. With increased decay, a significant increase in saccharification efficiency was apparent but not limited to same-species enzyme sources. We also calculated total sugar yields, and calculations that compensate for sugars consumed by fungi suggest a shorter residence time for fungal colonization than calculations based solely on saccharification yields.
Frederick Green - One of the best experts on this subject based on the ideXlab platform.
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Copper tolerance of Brown-Rot fungi : Oxalic acid production in southern pine treated with arsenic-free preservatives
International Biodeterioration & Biodegradation, 2005Co-Authors: Frederick Green, Carol A ClausenAbstract:The voluntary withdrawal of chromated copper arsenate (CCA)-treated wood from most residential applications has increased the use of non-arsenical copper-based organic wood preservatives. Because the arsenic component of CCA controlled coppertolerant fungi, scientists have renewed interest in and concern about the decay capacity in the important copper-tolerant group of Brown-Rot fungi. We have demonstrated that the primary means of inactivating copper in preservatives is by excess production of oxalic acid (OA). Oxalic acid production is a key metabolic indicator of Brown-Rot decay, and our objective was to estimate the production of OA in five commercial or experimental arsenic-free preservatives. Ten aggressive Brown-Rot fungi, chosen from previous studies and representing the genera Antrodia, Coniophora, Gloeophyllum, Postia, Serpula, Tyromyces, and Wolfiporia, were tested against southern yellow pine (SYP) blocks that were vacuum-treated with ground contact retentions of copper naphthenate, amine copper azole, alkaline copper quat type D (ACQ-D), N,N -naphthaloylhydroxylamine (NHA), and copper borate in a 12week soil-block test. After determination of block weight loss, blocks were also tested for the presence of OA. Weight loss ranged from 0.3% to 8.3% for treated blocks and from 16.4% to 59.6% for untreated controls. We conclude that SYP treated with these five preservatives limited OA production and prevented decay, and thus confirmed the efficacy of the co-biocides against coppertolerant fungi. © 2005 Elsevier Ltd. All rights reserved.
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copper tolerance of Brown Rot fungi time course of oxalic acid production
International Biodeterioration & Biodegradation, 2003Co-Authors: Frederick Green, Carol A ClausenAbstract:Abstract The increase in the use of non-arsenical copper-based wood preservatives in response to environmental concerns has been accompanied by interest in copper-tolerant decay fungi. Oxalic acid production by Brown-Rot fungi has been proposed as one mechanism of copper tolerance. Fifteen Brown-Rot fungi representing the genera Postia , Wolfiporia , Meruliporia , Gloeophyllum , Laetiporus , Coniophora , Antrodia , Serpula , and Tyromyces were evaluated for oxalic acid production bi-weekly in southern yellow pine (SYP) blocks treated with 1.2% ammoniacal copper citrate (CC). Eleven fungi were designated copper-tolerant based upon weight loss in CC-treated blocks. After 2 weeks, these fungi produced 2–17 times more oxalic acid in CC-treated blocks than in untreated blocks. After 10 weeks, weight loss ranged from 32% to 57% in CC-treated SYP. Four fungi were copper sensitive, producing low levels of oxalic acid and minimal weight loss in CC-treated blocks. Rapid induction of oxalic acid appeared to correlate closely with copper tolerance. We conclude that the Brown-Rot fungi tested that were able to exceed and maintain an oxalic acid concentration of ⩾600 μmol / g effectively decayed SYP treated with CC.
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oxalic acid overproduction by copper tolerant Brown Rot basidiomycetes on southern yellow pine treated with copper based preservatives
International Biodeterioration & Biodegradation, 2003Co-Authors: Carol A Clausen, Frederick GreenAbstract:Accumulation of oxalic acid (OA) by Brown-Rot fungi and precipitation of copper oxalate crystals in wood decayed by copper-tolerant decay fungi has implicated OA in the mechanism of copper tolerance. Understanding the role of OA in copper tolerance is important due to an increasing reliance on copper-based wood preservatives. In this study, four copper-tolerant Brown-Rot fungi were evaluated for decay capacity and OA production in early stages of exposure to four waterborne copper-based wood preservatives (ammonical copper quat type B and D, ammonical copper citrate, and chromated copper arsenate, type C) and one oilborne copper-based wood preservative (oxine copper) in southern yellow pine blocks. Weight losses were less than 14% during the 4-week incubation. The presence of copper in waterborne preservatives uniformly stimulated OA production by the test fungi within 2 weeks of exposure of the treated blocks to test fungi; 66% to 93% more OA was produced in treated blocks than untreated controls. Oxine copper, a nickel-containing oilborne preservative, prevented both weight loss and OA production in all fungi tested.
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Mechanism of Brown-Rot decay : Paradigm or paradox
International Biodeterioration & Biodegradation, 1997Co-Authors: Frederick Green, Terry L. HighleyAbstract:Interest in understanding how Brown-Rot fungi degrade wood has received increasing attention in recent years because of a need to identify novel targets that can be inhibited for the next generation of antifungal wood preservatives. Brown-Rot fungi are unique in that they can degrade holocellulose (cellulose and hemicellulose) in wood without first removing the lignin. Furthermore, they degrade holocellulose in an unusual manner, causing a rapid decrease in degree of polymerization at low weight loss. Despite increased research effort, the mechanism of Brown-Rot decay remains unclear. Furthermore, this research has not pointed to biochemical targets for inhibition and development of new wood preservatives. In reviewing the Brown-Rot literature, it became apparent that many beliefs about Brown-Rot decomposition of wood are based more on tradition or conjecture than on facts. In some cases, these misconceptions have become near dogma. They cloud our understanding of Brown-Rot decay and as a result may contribute to a misdirection of research efforts. The purpose of this paper is to attempt to identify and clarify some of these misconceptions.
Gerald N Presley - One of the best experts on this subject based on the ideXlab platform.
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coupling secretomics with enzyme activities to compare the temporal processes of wood metabolism among white and Brown Rot fungi
Applied and Environmental Microbiology, 2018Co-Authors: Gerald N Presley, Ellen A Panisko, Samuel O PurvineAbstract:ABSTRACT Wood-degrading fungi use a sequence of oxidative and hydrolytic mechanisms to loosen lignocellulose and then release and metabolize embedded sugars. These temporal sequences have recently been mapped at high resolution using directional growth on wood wafers, revealing previously obscured dynamics as fungi progressively colonize wood. Here, we applied secretomics in the same wafer design to track temporal trends on aspen decayed by fungi with distinct nutritional modes: two Brown Rot (BR) fungi (Postia placenta and Gloeophyllum trabeum) and two white Rot (WR) fungi (Stereum hirsutum and Trametes versicolor). We matched secretomic data from three zones of decay (early, middle, and late) with enzyme activities in these zones, and we included measures of total pRotein and ergosterol as measures of fungal biomass. In line with previous transcriptomics data, the fungi tested showed an initial investment in pectinases and a delayed investment in glycoside hydrolases (GHs). Brown Rot fungi also staggered the abundance of some oxidoreductases ahead of GHs to produce a familiar two-step mechanism. White Rot fungi, however, showed late-stage investment in pectinases as well, unlike Brown Rot fungi. Ligninolytic enzyme activities and abundances were also different between the two white Rot fungi. Specifically, S. hirsutum ligninolytic activity was delayed, which was explained almost entirely by the activity and abundance of five atypical manganese peroxidases, unlike more varied peroxidases and laccases in T. versicolor. These secretomic analyses support Brown Rot patterns generated via transcriptomics, they reveal distinct patterns among and within Rot types, and they link spectral counts with activities to help functionalize these multistrain secretomic data. IMPORTANCE Wood decay, driven primarily by wood-degrading basidiomycetes, is an essential component of global carbon cycles, and decay mechanisms are essential for understanding forest ecosystem function. These fungi efficiently consolidate pretreatment and saccharification of wood under mild conditions, making them promising templates for low-cost lignocellulose conversion. Species are categorized as ligninolytic white Rots and polysaccharide-selective Brown Rots, with considerable undescribed variability in decay mechanism that may manifest in the sequential variation in pRotein secretion over the progression of decay. Here we resolved spatially a temporal progression of decay on intact wood wafers and compared secretome dynamics in two white and two Brown Rot fungi. We identified several universal mechanistic components among decay types, including early pectinolytic “pretreatment” and later-stage glycoside hydrolase-mediated saccharification. Interspecific comparisons also identified considerable mechanistic diversity within Rot types, indicating that there are multiple avenues to facilitate white and Brown Rots.
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distinct growth and secretome strategies for two taxonomically divergent Brown Rot fungi
Applied and Environmental Microbiology, 2017Co-Authors: Gerald N Presley, Jonathan S. SchillingAbstract:Brown Rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful non-selective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in Postia placenta to segregate these reactions, but it is unclear if this two-step mechanism varies in other Brown Rot fungi with different traits and life history strategies, and that occupy different niches in nature. We used pRoteomics to analyze a progression of wood decay on thin wafers, using Brown Rot fungi with significant taxonomic and niche distance - Serpula lacrymans (Boletales; ‘dry Rot9 lumber decay) and Gloeophyllum trabeum (order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus pRotein secretion (resource capture), with ergosterol/extracted pRotein ratio increased 7x with S. lacrymans than with G. trabeum . In line with their native substrate associations, hemicellulase specific activities were dominated by mannanase in S. lacrymans and by xylanase in G. trabeum . Consistent with previous observations, S. lacrymans did not produce GH 6 cellobiohydrolases (CBH) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among Brown Rot fungi by having CBH genes. This work suggests that distantly related Brown Rot fungi employ staggered mechanisms to degrade wood, but that the underlying strategies vary among taxa. Importance Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown Rot fungi are unique among these fungi in that they use a non-enzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has evolved multiple times, independently, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers, and we found similar two-step mechanisms in taxonomically divergent Brown Rot fungi. Results, however, revealed strikingly different growth strategies, with S. lacrymans investing more in biomass production than secretion of pRoteins, and G. trabeum showing the opposite pattern with a high diversity of uncharacterized pRoteins. The “simplified” S. lacrymans secretomic system could help narrow gene targets central to oxidative Brown Rot pretreatments, and comparing its distinctions with G. trabeum and other Brown Rot fungi (e.g. Postia placenta ) might offer similar traction in non-catabolic genes.
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localizing gene regulation reveals a staggered wood decay mechanism for the Brown Rot fungus postia placenta
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Jiwei Zhang, Kenneth E. Hammel, Gerald N Presley, Jae San Ryu, Jon Menke, Melania Figueroa, Galya Orr, Jonathan S. SchillingAbstract:Wood-degrading Brown Rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that Brown Rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by Brown Rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for Brown Rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.
Carol A Clausen - One of the best experts on this subject based on the ideXlab platform.
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Copper tolerance of Brown-Rot fungi : Oxalic acid production in southern pine treated with arsenic-free preservatives
International Biodeterioration & Biodegradation, 2005Co-Authors: Frederick Green, Carol A ClausenAbstract:The voluntary withdrawal of chromated copper arsenate (CCA)-treated wood from most residential applications has increased the use of non-arsenical copper-based organic wood preservatives. Because the arsenic component of CCA controlled coppertolerant fungi, scientists have renewed interest in and concern about the decay capacity in the important copper-tolerant group of Brown-Rot fungi. We have demonstrated that the primary means of inactivating copper in preservatives is by excess production of oxalic acid (OA). Oxalic acid production is a key metabolic indicator of Brown-Rot decay, and our objective was to estimate the production of OA in five commercial or experimental arsenic-free preservatives. Ten aggressive Brown-Rot fungi, chosen from previous studies and representing the genera Antrodia, Coniophora, Gloeophyllum, Postia, Serpula, Tyromyces, and Wolfiporia, were tested against southern yellow pine (SYP) blocks that were vacuum-treated with ground contact retentions of copper naphthenate, amine copper azole, alkaline copper quat type D (ACQ-D), N,N -naphthaloylhydroxylamine (NHA), and copper borate in a 12week soil-block test. After determination of block weight loss, blocks were also tested for the presence of OA. Weight loss ranged from 0.3% to 8.3% for treated blocks and from 16.4% to 59.6% for untreated controls. We conclude that SYP treated with these five preservatives limited OA production and prevented decay, and thus confirmed the efficacy of the co-biocides against coppertolerant fungi. © 2005 Elsevier Ltd. All rights reserved.
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copper tolerance of Brown Rot fungi time course of oxalic acid production
International Biodeterioration & Biodegradation, 2003Co-Authors: Frederick Green, Carol A ClausenAbstract:Abstract The increase in the use of non-arsenical copper-based wood preservatives in response to environmental concerns has been accompanied by interest in copper-tolerant decay fungi. Oxalic acid production by Brown-Rot fungi has been proposed as one mechanism of copper tolerance. Fifteen Brown-Rot fungi representing the genera Postia , Wolfiporia , Meruliporia , Gloeophyllum , Laetiporus , Coniophora , Antrodia , Serpula , and Tyromyces were evaluated for oxalic acid production bi-weekly in southern yellow pine (SYP) blocks treated with 1.2% ammoniacal copper citrate (CC). Eleven fungi were designated copper-tolerant based upon weight loss in CC-treated blocks. After 2 weeks, these fungi produced 2–17 times more oxalic acid in CC-treated blocks than in untreated blocks. After 10 weeks, weight loss ranged from 32% to 57% in CC-treated SYP. Four fungi were copper sensitive, producing low levels of oxalic acid and minimal weight loss in CC-treated blocks. Rapid induction of oxalic acid appeared to correlate closely with copper tolerance. We conclude that the Brown-Rot fungi tested that were able to exceed and maintain an oxalic acid concentration of ⩾600 μmol / g effectively decayed SYP treated with CC.
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oxalic acid overproduction by copper tolerant Brown Rot basidiomycetes on southern yellow pine treated with copper based preservatives
International Biodeterioration & Biodegradation, 2003Co-Authors: Carol A Clausen, Frederick GreenAbstract:Accumulation of oxalic acid (OA) by Brown-Rot fungi and precipitation of copper oxalate crystals in wood decayed by copper-tolerant decay fungi has implicated OA in the mechanism of copper tolerance. Understanding the role of OA in copper tolerance is important due to an increasing reliance on copper-based wood preservatives. In this study, four copper-tolerant Brown-Rot fungi were evaluated for decay capacity and OA production in early stages of exposure to four waterborne copper-based wood preservatives (ammonical copper quat type B and D, ammonical copper citrate, and chromated copper arsenate, type C) and one oilborne copper-based wood preservative (oxine copper) in southern yellow pine blocks. Weight losses were less than 14% during the 4-week incubation. The presence of copper in waterborne preservatives uniformly stimulated OA production by the test fungi within 2 weeks of exposure of the treated blocks to test fungi; 66% to 93% more OA was produced in treated blocks than untreated controls. Oxine copper, a nickel-containing oilborne preservative, prevented both weight loss and OA production in all fungi tested.
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relationships between mechanical properties weight loss and chemical composition of wood during incipient Brown Rot decay
Forest Products Journal, 2002Co-Authors: Simon Curling, Carol A Clausen, Jerrold E WinandyAbstract:Incipient decay of wood by Brown-Rot fungi causes measurable strength losses in wood before measurable weight loss occurs. Previous studies have shown that the high levels of strength loss that occur during incipient Brown-Rot decay may be related to loss in hemicellulose. This study investigates the effect of decay on hemicellulose composition and the relationship of decay to the mechanical properties of the wood. An in vitro test method was used to allow progressive sampling of southern pine exposed to monocultures of Brown-Rot fungi. The wood was subsequently analyzed by mechanical testing and chemical analysis. The results demonstrated a ratio of strength to weight loss of approximately 4:1. The chemical data indicated that early strength loss (up to 40%) was associated with loss of arabinan and galactan components. Subsequent strength loss (greater than 40%) was associated with the loss of the mannan and xylan components. Significant loss of glucan (representing cellulose) was only detected at greater than 75 percent modulus of rupture loss.
