Wood Coloration and Decay Capabilities of Mycoparasite Scytalidium ganodermophthorum.

Scytalidium ganodermophthorum (telomorph: Xylogone ganodermopthora) Kang, Sigler, Lee & Yun is a destructive fungal pathogen that produces a yellow pigment that is used in sustainable product development. Similar pigmenting ascomycetes cause soft rot in woody substrates, however, the decay capabilities of S. ganodermophthorum have not been assessed or related to pigment production. A wood block decay test showed highly variable production of the expected bright yellow pigment and a secondary darker pigment when tested against multiple wood species and nutrient conditions. Microscopic examination showed cell wall erosion typical of type-2 soft rot in wood, although enzymatic analysis did not show detectible levels of endocellulase. Chitinase was detected in plate cultures but not wood cultures, indicating adaption of the fungus to a variety of environmental growth conditions. The high variability of pigmentation in wood cultures suggests that growth of S. ganodermophthorum on liquid media and use of extracted pigment is a superior method for obtaining consistent yellow coloration.

Sphingobium lignivorans sp. nov., isolated from river sediment downstream of a paper mill.

A bacterial isolate, B1D3AT, was isolated from river sediment collected from the Hiwassee River near Calhoun, TN, by enrichment culturing with a model 5-5' lignin dimer, dehydrodivanillate, as its sole carbon source. B1D3AT was also shown to utilize several model lignin-derived monomers and dimers as sole carbon sources in a variety of minimal media. Cells were Gram-stain-negative, aerobic, motile, rod-shaped and formed yellow/cream-coloured colonies on rich agar. Optimal growth occurred at 30 °C, pH 7-8, and in the absence of NaCl. The major fatty acids of B1D3AT were C18 : 1 ω7c and C17 : 1 ω6c. The predominant hydroxy fatty acids were C14 : 0 2-OH and C15 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine and sphingoglycolipid. B1D3AT contained spermidine as the only major polyamine. The major isoprenoid quinone was Q-10 with minor amounts of Q-9 and Q-11. The genomic DNA G+C content of B1D3AT was 65.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 49 core, universal genes defined by Clusters of Orthologous Groups gene families indicated that B1D3AT was a member of the genus Sphingobium. B1D3AT was most closely related to Sphingobium sp. SYK-6, with a 100 % 16S rRNA gene sequence similarity. B1D3AT showed 78.1-89.9 % average nucleotide identity and 19.5-22.2% digital DNA-DNA hybridization identity with other type strains from the genus Sphingobium. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain B1D3AT should be classified as representing a novel species of the genus Sphingobium, for which the name Sphingobium lignivorans sp. nov. is proposed. The type strain is strain B1D3AT (ATCC TSD-279T=DSM 111877T).

The Functional Biogeography of eDNA Metacommunities in the Post-Fire Landscape of the Angeles National Forest.

Wildfires have continued to increase in frequency and severity in Southern California due in part to climate change. To gain a further understanding of microbial soil communities' response to fire and functions that may enhance post-wildfire resilience, soil fungal and bacterial microbiomes were studied from different wildfire areas in the Gold Creek Preserve within the Angeles National Forest using 16S, FITS, 18S, 12S, PITS, and COI amplicon sequencing. Sequencing datasets from December 2020 and June 2021 samplings were analyzed using QIIME2, ranacapa, stats, vcd, EZBioCloud, and mixomics. Significant differences were found among bacterial and fungal taxa associated with different fire areas in the Gold Creek Preserve. There was evidence of seasonal shifts in the alpha diversity of the bacterial communities. In the sparse partial least squares analysis, there were strong associations (r > 0.8) between longitude, elevation, and a defined cluster of Amplicon Sequence Variants (ASVs). The Chi-square test revealed differences in fungi−bacteria (F:B) proportions between different trails (p = 2 × 10−16). sPLS results focused on a cluster of Green Trail samples with high elevation and longitude. Analysis revealed the cluster included the post-fire pioneer fungi Pyronema and Tremella. Chlorellales algae and possibly pathogenic Fusarium sequences were elevated. Bacterivorous Corallococcus, which secretes antimicrobials, and bacterivorous flagellate Spumella were associated with the cluster. There was functional redundancy in clusters that were differently composed but shared similar ecological functions. These results implied a set of traits for post-fire resiliency. These included photo-autotrophy, mineralization of pyrolyzed organic matter and aromatic/oily compounds, potential pathogenicity and parasitism, antimicrobials, and N-metabolism.

Pathway discovery and engineering for cleavage of a ß-1 lignin-derived biaryl compound.

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial ß-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a ß-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

Complete Genome Sequences of Four Natural Pseudomonas Isolates That Catabolize a Wide Range of Aromatic Compounds Relevant to Lignin Valorization.

Many soil microorganisms have evolved catabolic strategies to utilize phenolic compounds arising from depolymerized lignin. We report the complete genome sequences of four Pseudomonas sp. isolates that demonstrated robust growth on a wide range of aromatic monomers and dimers that are relevant to the valorization of lignin into value-added chemicals.

Migration of creosote components from timbers treated with creosote and processed using Best Management Practices.

While creosote components have very low water solubility, concerns have been raised about potential environmental effects and stimulated research to minimize migration. Best management practices have been developed but there are few data quantifying their effects. The effects of post-treatment steaming on migration of polycyclic aromatic hydrocarbons (PAHs) from horizontally oriented creosote treated southern pine decking subjected to simulated rainfall were examined. Twelve of the 16 PAHs examined were detected in runoff during the rainfall exposure but at concentrations well below those predicted by water solubility. PAH levels declined with increasing rainfall time, although the differences were sometimes slight. Steaming for 1 h had minimal effect on PAH levels in runoff while 3 h of steaming produced more noticeable reductions ranging from ~20 to 80% for naphthalene, acenaphthylene, and acenaphthene. Longer post-treatment steaming times reduced initial losses of creosote components from treated wood.

Functional Genomics, Transcriptomics, and Proteomics Reveal Distinct Combat Strategies Between Lineages of Wood-Degrading Fungi With Redundant Wood Decay Mechanisms.

Wood-degrading fungi vary in their strategies for deconstructing wood, and their competitive successes shape the rate and fate of carbon released from wood, Earth's largest pool of aboveground terrestrial carbon. In this study, one-on-one interspecific interactions between two model brown rot (carbohydrate-selective) fungi, Gloeophyllum trabeum and Rhodonia (Postia) placenta, were studied on wood wafers where a clearly resolved interaction zone (IZ) could be generated, reproducibly. Comparative RNAseq and proteomics between the IZ and non-interacting hyphae of each species identified combative strategies for each fungus. Glycoside hydrolases were a relatively smaller portion of the interaction secretome compared to non-interacting hyphae. The interaction zone showed higher pectinase specific activity than all other sampling locations, and higher laminarinase specific activity (branched ß-glucan proxy) was seen in the IZ secretome relative to equivalent hyphae in single-species cultures. Our efforts also identified two distinct competitive strategies in these two fungi with a shared nutritional mode (brown rot) but polyphyletic ancestral lineages. Gloeophyllum trabeum (Gloeophyllum clade) upregulated more secondary metabolite (SM) synthesis genes in response to a competitor than did R. placenta. R. placenta (Antrodia clade) upregulated a larger variety of uncharacterized oxidoreductases in interacting hyphae, suggesting that these may play a role in mediating competitor response in this fungus. Both species produced several hypothetical proteins exclusively in the interaction zone, leaving questions as to the function of these proteins. This work supports the existence of multiple interaction strategies among brown rot fungi and highlights the functional diversity among wood decay fungi.

Coupling Secretomics with Enzyme Activities To Compare the Temporal Processes of Wood Metabolism among White and Brown Rot Fungi.

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.

Brown Rot-Type Fungal Decomposition of Sorghum Bagasse: Variable Success and Mechanistic Implications.

Sweet sorghum is a promising crop for a warming, drying African climate, and basic information is lacking on conversion pathways for its lignocellulosic residues (bagasse). Brown rot wood-decomposer fungi use carbohydrate-selective pathways that, when assessed on sorghum, a grass substrate, can yield information relevant to both plant biomass conversion and fungal biology. In testing sorghum decomposition by brown rot fungi (Gloeophyllum trabeum, Serpula lacrymans), we found that G. trabeum readily degraded sorghum, removing xylan prior to removing glucan. Serpula lacrymans, conversely, caused little decomposition. Ergosterol (fungal biomarker) and protein levels were similar for both fungi, but S. lacrymans produced nearly 4x lower polysaccharide-degrading enzyme specific activity on sorghum than G. trabeum, perhaps a symptom of starvation. Linking this information to genome comparisons including other brown rot fungi known to have a similar issue regarding decomposing grasses (Postia placenta, Fomitopsis pinicola) suggested that a lack of CE 1 feruloyl esterases as well as low xylanase activity in S. lacrymans (3x lower than in G. trabeum) may hinder S. lacrymans, P. placenta, and F. pinicola when degrading grass substrates. These results indicate variability in brown rot mechanisms, which may stem from a differing ability to degrade certain lignin-carbohydrate complexes.

A genomics-informed study of oxalate and cellulase regulation by brown rot wood-degrading fungi.

Wood-degrading fungi that selectively remove carbohydrates (brown rot) combine Fenton-based oxidation and enzymatic hydrolysis to degrade wood. These two steps are incompatible in close proximity. To explain this, brown rot fungi may stagger oxidative reactions ahead of hydrolysis, but the scale and environmental controls for such a mechanism have not been resolved in solid wood. Here, we focused on one reaction control parameter, oxalate. In coordination with Fe3+-reducing compounds (e.g., 2,5-dimethoxyhydroquinone), oxalate can either promote Fenton chemistry by mobilizing Fe3+ as mono-oxalates (facilitative) or inhibit Fenton chemistry (protective) by restricting reducibility and the formation of Fenton's reagent as Fe3+/Fe2-(oxalate)2,3. Here, we sectioned wood wafers colonized directionally by Postia placenta and Gloeophyllum trabeum to map end-to-end the expression of oxalate synthesis genes and to overlay enzyme activities, metabolites, and wood modifications. Near advancing hyphal fronts, oxaloacetase expression was up upregulated for both fungi, while regulation patterns of paralogous of isocitrate lyases and glyoxylate dehydrogenases varied, suggesting different physiological roles. Oxalate decarboxylase (ODC) expression in G. trabeum was induced in more decayed wood behind the hyphal front, but was constitutively expressed in all P. placenta sections. Relative ODC activities increased and oxalate levels stabilized in more decayed wood behind the hyphal front. Endoglucanase (EG) activity, on the other hand, peaked for both fungi in later decay stages. These oxalate optimization patterns are in line with previous whole-block 'spiking' experiments tracking oxalate, but we provide here information on its genetic controls across a spatial gradient. As a complement, we also demonstrate in vitro the plausibility of a protective role for oxalate, to emphasize that these fungi might be optimizing oxalate at a given level to maximize Fenton reactions but to minimize oxidative damage.

Distinct Growth and Secretome Strategies for Two Taxonomically Divergent Brown Rot Fungi.

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 nonselective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in the segregation of these reactions in Postia placenta, but it is unclear if this two-step mechanism varies in other brown rot fungi with different traits and life history strategies that occupy different niches in nature. We employed proteomics to analyze a progression of wood decay on thin wafers, using brown rot fungi with significant taxonomic and niche distances: Serpula lacrymans (Boletales; "dry rot" 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 the ergosterol/extracted protein ratio being 7-fold higher with S. lacrymans than with G. trabeum In line with the native substrate associations of these fungi, 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 glycoside hydrolase 6 (GH6) cellobiohydrolases (CBHs) 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 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 nonenzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has independently evolved multiple times, 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. The 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 a comparison of its distinctions with G. trabeum and other brown rot fungi (e.g., Postia placenta) might offer similar traction in noncatabolic genes.

Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta.

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.

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus.

The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of ß-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated. All gluco-oligosaccharides tested (up to celloheptose) supported growth in M2 minimal media but not cellulose or CM-cellulose. The periplasmic and outer membrane fractions showed highest BGL activity, but no significant BGL activity was observed in the cytosol or extracellular medium. Cells grown in cellobiose showed expression of specific BGLs and TonB-dependent receptors (TBDRs). Carbonyl cyanide 3-chlorophenylhydrazone lowered the rate of cell growth in cellobiose but not in glucose, indicating potential cellobiose transport into the cell by a proton motive force-dependent process, such as TBDR-dependent transport, and facilitated diffusion of glucose across the outer membrane via specific porins. These results suggest that C. crescentus acquires carbon from cellulose-derived gluco-oligosaccharides found in the environment by extracellular and periplasmic BGL activity and TBDR-mediated transport. This report on extracellular degradation of gluco-oligosaccharides and methods of nutrient acquisition by C. crescentus supports a broader suite of carbohydrate metabolic capabilities suggested by the C. crescentus genome sequence that until now have not been reported.