scholarly journals Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Rafael R. da Costa ◽  
Haofu Hu ◽  
Bo Pilgaard ◽  
Sabine M. E. Vreeburg ◽  
Julia Schückel ◽  
...  
Keyword(s):  
Plant Material ◽  
Plant Biomass ◽  
Fungal Spores ◽  
Decomposition Process ◽  
Old World ◽  
Content Type ◽  
Substrate Conversion ◽  
Fungus Comb ◽  
Dead Plant ◽  
Plant Polymer

ABSTRACTFungus-growing termites rely on mutualistic fungi of the genusTermitomycesand gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes producedin situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.IMPORTANCEFungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.

scholarly journals Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

2016 ◽  
Vol 82 (18) ◽  
pp. 5698-5708 ◽  
Author(s):  
Sagar M. Utturkar ◽  
W. Nathan Cude ◽  
Michael S. Robeson ◽  
Zamin K. Yang ◽  
Dawn M. Klingeman ◽  
...  
Keyword(s):  
Single Cell ◽  
Endophytic Bacteria ◽  
Plant Material ◽  
Plant Biomass ◽  
Plant Root ◽  
Gradient Centrifugation ◽  
Substantial Reduction ◽  
Comparative Genomic ◽  
Single Cell Genomics ◽  
Content Type

ABSTRACTBacterial endophytes that colonizePopulustrees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria fromPopulusroots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phylaArmatimonadetes,Verrucomicrobia, andPlanctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.IMPORTANCEPlant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.

scholarly journals Symbiont-Mediated Digestion of Plant Biomass in Fungus-Farming Insects

2021 ◽  
Vol 66 (1) ◽  
pp. 297-316 ◽  
Author(s):  
Hongjie Li ◽  
Soleil E. Young ◽  
Michael Poulsen ◽  
Cameron R. Currie
Keyword(s):  
Plant Material ◽  
Plant Biomass ◽  
Herbivorous Insects ◽  
Bacterial Symbionts ◽  
Seminal Work ◽  
Carbohydrate Active Enzyme ◽  
Plant Feeding ◽  
Enzyme Discovery ◽  
Genomic Studies ◽  
Dead Plant

Feeding on living or dead plant material is widespread in insects. Seminal work on termites and aphids has provided profound insights into the critical nutritional role that microbes play in plant-feeding insects. Some ants, beetles, and termites, among others, have evolved the ability to use microbes to gain indirect access to plant substrate through the farming of a fungus on which they feed. Recent genomic studies, including studies of insect hosts and fungal and bacterial symbionts, as well as metagenomics and proteomics, have provided important insights into plant biomass digestion across insect–fungal mutualisms. Not only do advances in understanding of the divergent and complementary functions of complex symbionts reveal the mechanism of how these herbivorous insects catabolize plant biomass, but these symbionts also represent a promising reservoir for novel carbohydrate-active enzyme discovery, which is of considerable biotechnological interest.

Bioremediation of Acid Mine Drainage in an Uranium Deposit

2007 ◽  
Vol 20-21 ◽  
pp. 248-257 ◽  
Author(s):  
Stoyan N. Groudev ◽  
Plamen S. Georgiev ◽  
Irena Spasova ◽  
Marina Nicolova
Keyword(s):  
Mine Drainage ◽  
Plant Biomass ◽  
Uranium Deposit ◽  
Water Flow Rate ◽  
Ambient Temperatures ◽  
Zinc Cadmium ◽  
Copper Zinc ◽  
Cadmium Lead ◽  
Dead Plant ◽  
The Individual

Acid drainage waters generated in the uranium deposit Curilo, Bulgaria, were treated by means of different passive systems such as natural and constructed wetlands, alkalizing limestone drains, permeable reactive multibarriers and a rock filter, used separately or in different combinations. The waters had a pH in the range of about 2 – 4 and contained radionuclides (uranium, radium), heavy metals (copper, zinc, cadmium, lead, nickel, cobalt, iron, manganese), arsenic and sulphates in concentrations usually much higher than the relevant permissible levels for waters intended for use in agriculture and/or industry. The water flow rate through the individual systems was different and not stable, and varied in the range approximately from 0.02 to 1.5 l/s. Efficient removal of pollutants was achieved by means of these systems during the different climatic seasons, even during the cold winter months at water and ambient temperatures close to 0 oC. The removal was due to different mechanisms but microbial sulphate reduction, biosorption by living and dead plant biomass and chemical neutralization played the main roles.

scholarly journals Efficient Plant Biomass Degradation by Thermophilic Fungus Myceliophthora heterothallica

2012 ◽  
Vol 79 (4) ◽  
pp. 1316-1324 ◽  
Author(s):  
Joost van den Brink ◽  
Gonny C. J. van Muiswinkel ◽  
Bart Theelen ◽  
Sandra W. A. Hinz ◽  
Ronald P. de Vries
Keyword(s):  
Wheat Straw ◽  
Plant Biomass ◽  
Hydrolytic Enzymes ◽  
Reaction Times ◽  
Giant Reed ◽  
Thermophilic Fungi ◽  
Biomass Degradation ◽  
Thermostable Enzymes ◽  
Content Type

ABSTRACTRapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such asTrichodermaandAspergillusspecies. The genusMyceliophthoracontains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging toM. heterothallicawere recently separated from the well-described speciesM. thermophila. We evaluate here the potential ofM. heterothallicaisolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilicMyceliophthoraspecies, isolates belonging toM. heterothallicaandM. thermophilagrew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles andin vitroassays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly betweenM. thermophilaandM. heterothallicaisolates. Compared toM. thermophila,M. heterothallicaisolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures ofMyceliophthoraspecies lack sufficient β-xylosidase activity. Sexual crossing of twoM. heterothallicashowed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures ofM. heterothallica.

scholarly journals Complementary Contribution of Fungi and Bacteria to Lignocellulose Digestion in the Food Stored by a Neotropical Higher Termite

2021 ◽  
Vol 9 ◽  
Author(s):  
Edimar A. Moreira ◽  
Gabriela F. Persinoti ◽  
Letícia R. Menezes ◽  
Douglas A. A. Paixão ◽  
Thabata M. Alvarez ◽  
...  
Keyword(s):  
Plant Material ◽  
Plant Biomass ◽  
Carbohydrate Active Enzymes ◽  
Expression Of Genes ◽  
Its Sequence Analysis ◽  
Gut Symbionts ◽  
Cazy Genes ◽  
Complementary Set ◽  
New Evidence ◽  
Differential Expression Of Genes

Lignocellulose digestion in termites is achieved through the functional synergy between gut symbionts and host enzymes. However, some species have evolved additional associations with nest microorganisms that collaborate in the decomposition of plant biomass. In a previous study, we determined that plant material packed with feces inside the nests of Cornitermes cumulans (Syntermitinae) harbors a distinct microbial assemblage. These food nodules also showed a high hemicellulolytic activity, possibly acting as an external place for complementary lignocellulose digestion. In this study, we used a combination of ITS sequence analysis, metagenomics, and metatranscriptomics to investigate the presence and differential expression of genes coding for carbohydrate-active enzymes (CAZy) in the food nodules and the gut of workers and soldiers. Our results confirm that food nodules express a distinct set of CAZy genes suggesting that stored plant material is initially decomposed by enzymes that target the lignin and complex polysaccharides from fungi and bacteria before the passage through the gut, where it is further targeted by a complementary set of cellulases, xylanases, and esterases produced by the gut microbiota and the termite host. We also showed that the expression of CAZy transcripts associated to endoglucanases and xylanases was higher in the gut of termites than in the food nodules. An additional finding in this study was the presence of fungi in the termite gut that expressed CAZy genes. This study highlights the importance of externalization of digestion by nest microbes and provides new evidence of complementary digestion in the context of higher termite evolution.

scholarly journals Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Tristan Cerisy ◽  
Tiffany Souterre ◽  
Ismael Torres-Romero ◽  
Magali Boutard ◽  
Ivan Dubois ◽  
...  
Keyword(s):  
Ferulic Acid ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Gene Dosage ◽  
Content Type ◽  
Chemical Inhibitors ◽  
Growth Selection ◽  
Clostridium Phytofermentans

ABSTRACT Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors. IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.

scholarly journals Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis inPenicillium oxalicum

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Qi-Peng He ◽  
Shuai Zhao ◽  
Jiu-Xiang Wang ◽  
Cheng-Xi Li ◽  
Yu-Si Yan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Genetic Engineering ◽  
Filamentous Fungi ◽  
Soil Fungi ◽  
Plant Biomass ◽  
Penicillium Oxalicum ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
Pigment Biosynthesis

ABSTRACTSoil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor,Penicillium oxalicumNsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes inP. oxalicum. PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development inAspergillus. In the present study, the regulatory roles of PoxNsdD inP. oxalicumwere further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdDstrain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70parental strain. During cultivation, ΔPoxNsdDcultures changed color, from pale orange to brick red, while the ΔPoxKu70cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed thatPoxNsdDdynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover,in vitrobinding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering ofP.oxalicumfor potential industrial and medical applications.IMPORTANCEMost filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungusPenicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential ofP.oxalicumvia genetic engineering.

scholarly journals Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal PathogenRhizopus delemar

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Poppy C. S. Sephton-Clark ◽  
Jose F. Muñoz ◽  
Elizabeth R. Ballou ◽  
Christina A. Cuomo ◽  
Kerstin Voelz
Keyword(s):  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Developmental Process ◽  
Fungal Spores ◽  
Hyphal Growth ◽  
Large Shift ◽  
Rhizopus Delemar ◽  
Content Type ◽  
Transcriptional Changes ◽  
Dormant Spore

ABSTRACTRhizopus delemaris an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores ofRhizopus delemarcause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transformingRhizopus delemarfrom a single-cellular to multicellular organism.IMPORTANCEGermination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.

scholarly journals ABC Transporters Required for Hexose Uptake byClostridium phytofermentans

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Tristan Cerisy ◽  
Alba Iglesias ◽  
William Rostain ◽  
Magali Boutard ◽  
Christine Pelle ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Extracellular Enzymes ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Anaerobic Bacteria ◽  
Model Organism ◽  
Industrial Transformation ◽  
Content Type ◽  
Plant Matter

ABSTRACTThe mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal howClostridium(Lachnoclostridium)phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based onin vitroactivities. These mechanisms used byClostridiato uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCEPlant-fermentingClostridiaare anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine.Clostridiadegrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organismClostridium phytofermentans. We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

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