Publications by authors named "Rubén López-Mondéjar"

23 Publications

  • Page 1 of 1

Complementary Roles of Wood-Inhabiting Fungi and Bacteria Facilitate Deadwood Decomposition.

mSystems 2021 Jan 12;6(1). Epub 2021 Jan 12.

Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

Forests accumulate and store large amounts of carbon (C), and a substantial fraction of this stock is contained in deadwood. This transient pool is subject to decomposition by deadwood-associated organisms, and in this process it contributes to CO emissions. Although fungi and bacteria are known to colonize deadwood, little is known about the microbial processes that mediate carbon and nitrogen (N) cycling in deadwood. In this study, using a combination of metagenomics, metatranscriptomics, and nutrient flux measurements, we demonstrate that the decomposition of deadwood reflects the complementary roles played by fungi and bacteria. Fungi were found to dominate the decomposition of deadwood and particularly its recalcitrant fractions, while several bacterial taxa participate in N accumulation in deadwood through N fixation, being dependent on fungal activity with respect to deadwood colonization and C supply. Conversely, bacterial N fixation helps to decrease the constraints of deadwood decomposition for fungi. Both the CO efflux and N accumulation that are a result of a joint action of deadwood bacteria and fungi may be significant for nutrient cycling at ecosystem levels. Especially in boreal forests with low N stocks, deadwood retention may help to improve the nutritional status and fertility of soils. Wood represents a globally important stock of C, and its mineralization importantly contributes to the global C cycle. Microorganisms play a key role in deadwood decomposition, since they possess enzymatic tools for the degradation of recalcitrant plant polymers. The present paradigm is that fungi accomplish degradation while commensalist bacteria exploit the products of fungal extracellular enzymatic cleavage, but this assumption was never backed by the analysis of microbial roles in deadwood. This study clearly identifies the roles of fungi and bacteria in the microbiome and demonstrates the importance of bacteria and their N fixation for the nutrient balance in deadwood as well as fluxes at the ecosystem level. Deadwood decomposition is shown as a process where fungi and bacteria play defined, complementary roles.
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http://dx.doi.org/10.1128/mSystems.01078-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901482PMC
January 2021

Combined ozonation and solarization for the removal of pesticides from soil: Effects on soil microbial communities.

Sci Total Environ 2021 Mar 4;758:143950. Epub 2020 Dec 4.

CEBAS-CSIC, P.O. Box 164, 30100 Espinardo, Murcia, Spain.

Pesticides have been used extensively in agriculture to control pests and soil-borne diseases. Most of these pesticides can persist in soil in harmful concentrations due to their intrinsic characteristics and their interactions with soil. Soil solarization has been demonstrated to enhance pesticide degradation under field conditions. Recently, ozonation has been suggested as a feasible method for reducing the pesticide load in agricultural fields. However, the effects of ozonation in the soil microbial community have not been studied so far. Here, we evaluate the combined effects of solarization and ozonation on the microbial community of a Mediterranean soil. For this purpose, soil physico-chemical characteristics and enzyme activities and the biomass (through analysis of microbial fatty acids) and diversity (through 16S rRNA and ITS amplicon sequencing) of soil microbial communities were analyzed in a 50-day greenhouse experiment. The degradation of the pesticides was increased by 20%, 28%, and 33% in solarized soil (S), solarized soil with surface ozonation (SOS), and solarized soil with deep ozonation (SOD), respectively, in comparison to control (untreated) soil. Solarization and its combination with ozonation (SOS and SOD) increased the ammonium content as well as the electrical conductivity, while enzyme activities and soil microbial biomass were negatively affected. Despite the biocidal character of ozone, several microbial populations with demonstrated pesticide-degradation capacity showed increases in their relative abundance. Overall, the combination of solarization plus ozone did not exacerbate the effects of solarization on the soil chemistry and microbial communities, but did improve pesticide degradation.
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http://dx.doi.org/10.1016/j.scitotenv.2020.143950DOI Listing
March 2021

Feeding on fungi: genomic and proteomic analysis of the enzymatic machinery of bacteria decomposing fungal biomass.

Environ Microbiol 2020 11 31;22(11):4604-4619. Epub 2020 Aug 31.

Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 14220 Praha 4, Czech Republic.

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community.
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http://dx.doi.org/10.1111/1462-2920.15183DOI Listing
November 2020

Organic amendments exacerbate the effects of silver nanoparticles on microbial biomass and community composition of a semiarid soil.

Sci Total Environ 2020 Nov 15;744:140919. Epub 2020 Jul 15.

CEBAS-CSIC. Department of Soil and Water Conservation. Campus Universitario de Espinardo, 30100 Murcia, Spain.

Increased utilization of silver nanoparticles (AgNPs) can result in an accumulation of these particles in the environment. The potential detrimental effects of AgNPs in soil may be associated with the low fertility of soils in semiarid regions that are usually subjected to restoration through the application of organic amendments. Microbial communities are responsible for fundamental processes related to soil fertility, yet the potential impacts of low and realistic AgNPs concentrations on soil microorganisms are still unknown. We studied the effects of realistic citrate-stabilized AgNPs concentrations (0.015 and 1.5 μg kg) at two exposure times (7 and 30 days) on a sandy clay loam Mediterranean soil unamended (SU) and amended with compost (SA). We assessed soil microbial biomass (microbial fatty acids), soil enzyme activities (urease, β-glucosidase, and alkaline phosphatase), and composition of the microbial community (bacterial 16S rRNA gene and fungal ITS2 sequencing) in a microcosm experiment. In the SA, the two concentrations of AgNPs significantly decreased the bacterial biomass after 7 days of incubation. At 30 days of incubation, only a significant decrease in the Gram+ was observed at the highest AgNPs concentration. In contrast, in the SU, there was a significant increase in bacterial biomass after 30 days of incubation at the lowest AgNPs concentration. Overall, we found that fungal biomass was more resistant to AgNPs than bacterial biomass, in both SA and SU. Further, the AgNPs changed the composition of the soil bacterial community in SA, the relative abundance of some bacterial taxa in SA and SU, and fungal richness in SU at 30 days of incubation. However, AgNPs did not affect the activity of extracellular enzymes. This study demonstrates that the exposure time and organic amendments modulate the effects of realistic concentrations of AgNPs in the biomass and composition of the microbial community of a Mediterranean soil.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140919DOI Listing
November 2020

GlobalFungi, a global database of fungal occurrences from high-throughput-sequencing metabarcoding studies.

Sci Data 2020 07 13;7(1):228. Epub 2020 Jul 13.

Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 14220, Praha 4, Czech Republic.

Fungi are key players in vital ecosystem services, spanning carbon cycling, decomposition, symbiotic associations with cultivated and wild plants and pathogenicity. The high importance of fungi in ecosystem processes contrasts with the incompleteness of our understanding of the patterns of fungal biogeography and the environmental factors that drive those patterns. To reduce this gap of knowledge, we collected and validated data published on the composition of soil fungal communities in terrestrial environments including soil and plant-associated habitats and made them publicly accessible through a user interface at https://globalfungi.com . The GlobalFungi database contains over 600 million observations of fungal sequences across > 17 000 samples with geographical locations and additional metadata contained in 178 original studies with millions of unique nucleotide sequences (sequence variants) of the fungal internal transcribed spacers (ITS) 1 and 2 representing fungal species and genera. The study represents the most comprehensive atlas of global fungal distribution, and it is framed in such a way that third-party data addition is possible.
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http://dx.doi.org/10.1038/s41597-020-0567-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359306PMC
July 2020

Environmentally relevant concentrations of silver nanoparticles diminish soil microbial biomass but do not alter enzyme activities or microbial diversity.

J Hazard Mater 2020 06 4;391:122224. Epub 2020 Feb 4.

CEBAS-CSIC. Department of Soil and Water Conservation, Campus Universitario de Espinardo, 30100, Murcia, Spain.

The increasing use of silver nanoparticles (AgNPs) due to their well-known antimicrobial activity, has led to their accumulation in soil ecosystems. However, the impact of environmental realistic concentrations of AgNPs on the soil microbial community has been scarcely studied. In this work, we have assessed the impact of AgNPs, that mimic real concentrations in nature, on tropical soils cultivated with Coffea arabica under conventional and organic management systems. We evaluated the biomass, extracellular enzyme activities, and diversity of the soil microbial community, in a microcosm experiment as a function of time. After seven days of incubation, we found an increase in microbial biomass in an AgNPs-concentration-independent manner. In contrast, after 60-day-incubation, there was a decrease in Gram+ and actinobacterial biomass, in both soils and all AgNPs concentrations. Soil physico-chemical properties and enzyme activities were not affected overall by AgNPs. Regarding the microbial community composition, only some differences in the relative abundance at phylum and genus level in the fungal community were observed. Our results suggest that environmental concentrations of AgNPs affected microbial biomass but had little impact on microbial diversity and may have little effects on the soil biogeochemical cycles mediated by extracellular enzyme activities.
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http://dx.doi.org/10.1016/j.jhazmat.2020.122224DOI Listing
June 2020

A meta-analysis of global fungal distribution reveals climate-driven patterns.

Nat Commun 2019 11 13;10(1):5142. Epub 2019 Nov 13.

Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 14220, Praha 4, Czech Republic.

The evolutionary and environmental factors that shape fungal biogeography are incompletely understood. Here, we assemble a large dataset consisting of previously generated mycobiome data linked to specific geographical locations across the world. We use this dataset to describe the distribution of fungal taxa and to look for correlations with different environmental factors such as climate, soil and vegetation variables. Our meta-study identifies climate as an important driver of different aspects of fungal biogeography, including the global distribution of common fungi as well as the composition and diversity of fungal communities. In our analysis, fungal diversity is concentrated at high latitudes, in contrast with the opposite pattern previously shown for plants and other organisms. Mycorrhizal fungi appear to have narrower climatic tolerances than pathogenic fungi. We speculate that climate change could affect ecosystem functioning because of the narrow climatic tolerances of key fungal taxa.
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http://dx.doi.org/10.1038/s41467-019-13164-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853883PMC
November 2019

Lignocellulolytic systems of soil bacteria: A vast and diverse toolbox for biotechnological conversion processes.

Biotechnol Adv 2019 11 22;37(6):107374. Epub 2019 Mar 22.

Laboratory of Environmental Microbiology, Institute of Microbiology of the CAS, Průmyslová 595, Vestec 252 50, Czech Republic. Electronic address:

Lignocellulose from plant biomass represents an abundant and renewable source for the production of environmentally friendly chemicals and biofuels. However, its recalcitrant nature entails the use of complex biochemical reactions that are still challenging. Since the degradation of lignocellulose is the current bottleneck of the conversion processes, the search for novel enzymes and microbial strains for degrading plant biomass is of high importance. Currently, bacteria are in the spotlight as promising candidates for novel conversion strategies due to their wide functional diversity and versatility. Here, we review the lines of evidence that show the high potential of bacterial strains from soil for biomass conversion ranging from strain characterization to metagenome and metatranscriptome analysis. Substantial and diverse fractions of soil bacteria are able to decompose the major lignocellulose components. To do that, bacteria evolved structurally variable and often highly complex lignocellulolytic systems composed of enzymes as well as proteins involved in efficient substrate binding. Both as individual components or in combination, bacterial enzymes, and accessory proteins appear to be promising tools in the biotechnological valorization of lignocellulose.
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http://dx.doi.org/10.1016/j.biotechadv.2019.03.013DOI Listing
November 2019

Land use shapes the resistance of the soil microbial community and the C cycling response to drought in a semi-arid area.

Sci Total Environ 2019 Jan 23;648:1018-1030. Epub 2018 Aug 23.

CEBAS-CSIC, Campus Universitario de Espinardo, PO Box 164, CP 30100 Murcia, Spain.

The aim of this study was to understand the responses of the microbial community of soil under different land uses to drought in a semi-arid Mediterranean area. In a laboratory incubation, soil samples from different land uses (natural forest, drip-irrigated orchard, rain-fed almond tree cultivation and abandoned area) were maintained at 20% and 60% of the WHC. The microbial biomass and potential enzyme activities were determined after four and fifty days of soil incubation. The diversity and composition of the microbial community were studied after 50 days of incubation. The total mineralisation of soil organic C (SOC), as well as, the mineralisation of fresh organic matter (FOM) and the "priming effect" were analysed after addition of C-enriched plant tissue. Both land use and drought had significant effects in the soil microbial community, but the effect of land use was stronger than that of drought. The PLFA content (microbial biomass) of the forests soil was greater under drought. After 50 days of soil incubation, the microbial biomass and most of potential enzyme activities of the almond tree and abandoned soil samples were not significantly affected by drought contrary to those in orchard soil. The total and FOM mineralisation were on average lower in soil under drought than under optimal moisture for all land uses. However, the responses of the priming effect to drought were dependent on the land use. Overall, we conclude that the resistance to drought of the soil microbial community from an agroecosystem having a semi-arid climate is strongly influenced by the previous land use.
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http://dx.doi.org/10.1016/j.scitotenv.2018.08.214DOI Listing
January 2019

Discovery of Phloeophagus Beetles as a Source of Strains That Produce Potentially New Bioactive Substances and Description of sp. nov.

Front Microbiol 2018 8;9:913. Epub 2018 May 8.

Microbiology and Genetics Department, University of Salamanca, Salamanca, Spain.

Antimicrobial resistance is a worldwide problem that threatens the effectiveness of treatments for microbial infection. Consequently, it is essential to study unexplored niches that can serve for the isolation of new microbial strains able to produce antimicrobial compounds to develop new drugs. Bark beetles live in phloem of host trees and establish symbioses with microorganisms that provide them with nutrients. In addition, some of their associated bacteria play a role in the beetle protection by producing substances that inhibit antagonists. In this study the capacity of several bacterial strains, isolated from the bark beetles , and , to produce antimicrobial compounds was analyzed. Several isolates exhibited the capacity to inhibit Gram-positive and Gram-negative bacteria, as well as fungi. The genome sequence analysis of three isolates predicted the presence of several gene clusters implicated in the production of already described antimicrobials and moreover, the low similarity of some of these clusters with those previously described, suggests that they encode new undescribed substances, which may be useful for developing new antimicrobial agents. Moreover, these bacteria appear to have genetic machinery for producing antitumoral and antiviral substances. Finally, the strain IA19 showed to represent a new species of the genus . The 16S rRNA gene sequence analysis showed that its most closely related species include and 98.6, 98.5 98.4, and 98.4% identity, respectively. MLSA of the housekeeping genes B, B, and D confirmed that strain IA19 clearly separates from its closest related species. Average nucleotide identity between strains IA19 and ATCC 700689, DSM 11363, KL28 and DSM 17257 were 85.3, 80.2, 79.0, and 72.1%, respectively. Growth occurs at 4-37°C and pH 6.5-8. Optimal growth occurs at 28°C, pH 7-8 and up to 2.5% NaCl. Respiratory ubiquinones are Q9 (97%) and Q8 (3%). C16:0 and in summed feature 3 are the main fatty acids. Based on genotypic, phenotypic and chemotaxonomic characteristics, the description of sp. nov. has been proposed. The type strain is IA19 (=CECT 9403 = LMG 30182).
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http://dx.doi.org/10.3389/fmicb.2018.00913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953339PMC
May 2018

Drivers of microbial community structure in forest soils.

Appl Microbiol Biotechnol 2018 May 30;102(10):4331-4338. Epub 2018 Mar 30.

Institute of Microbiology of the CAS, Laboratory of Environmental Microbiology, Prague, Czech Republic.

Forests are essential biomes for global biogeochemical cycles, and belowground microorganisms have a key role in providing relevant ecosystem services. To predict the effects of environmental changes on these ecosystem services requires a comprehensive understanding of how biotic and abiotic factors drive the composition of microbial communities in soil. However, microorganisms are not homogeneously distributed in complex environments such as soil, with different features affecting microbes at different extent depending on the niche they occupy. Indeed, this spatial heterogeneity hampers the extrapolation of microbial diversity study results from particular habitats to the ecosystem level, even if the resolution of the more recent studies has increased significantly after the standardization of high-throughput sequencing techniques. The present work intends to give a comprehensive view of the knowledge accumulated until date defining the more important drivers determining the structure of forest soil microbial communities from fine to continental scales.
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http://dx.doi.org/10.1007/s00253-018-8950-4DOI Listing
May 2018

Decomposer food web in a deciduous forest shows high share of generalist microorganisms and importance of microbial biomass recycling.

ISME J 2018 06 28;12(7):1768-1778. Epub 2018 Feb 28.

Institute of Microbiology of the CAS, Průmyslová 595, 252 50, Vestec, Czech Republic.

Forest soils represent important terrestrial carbon (C) pools where C is primarily fixed in the plant-derived biomass but it flows further through the biomass of fungi and bacteria before it is lost from the ecosystem as CO or immobilized in recalcitrant organic matter. Microorganisms are the main drivers of C flow in forests and play critical roles in the C balance through the decomposition of dead biomass of different origins. Here, we track the path of C that enters forest soil by following respiration, microbial biomass production, and C accumulation by individual microbial taxa in soil microcosms upon the addition of C-labeled biomass of plant, fungal, and bacterial origin. We demonstrate that both fungi and bacteria are involved in the assimilation and mineralization of C from the major complex sources existing in soil. Decomposer fungi are, however, better suited to utilize plant biomass compounds, whereas the ability to utilize fungal and bacterial biomass is more frequent among bacteria. Due to the ability of microorganisms to recycle microbial biomass, we suggest that the decomposer food web in forest soil displays a network structure with loops between and within individual pools. These results question the present paradigms describing food webs as hierarchical structures with unidirectional flow of C and assumptions about the dominance of fungi in the decomposition of complex organic matter.
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http://dx.doi.org/10.1038/s41396-018-0084-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018761PMC
June 2018

Differential sensitivity of total and active soil microbial communities to drought and forest management.

Glob Chang Biol 2017 10 6;23(10):4185-4203. Epub 2017 Jul 6.

Department of Molecular Systems Biology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany.

Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning-that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 6 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community-based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought-plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial community and climate change.
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http://dx.doi.org/10.1111/gcb.13790DOI Listing
October 2017

Forest Soil Bacteria: Diversity, Involvement in Ecosystem Processes, and Response to Global Change.

Microbiol Mol Biol Rev 2017 06 12;81(2). Epub 2017 Apr 12.

Laboratory of Environmental Microbiology, Institute of Microbiology of the CAS, Vestec, Czech Republic

The ecology of forest soils is an important field of research due to the role of forests as carbon sinks. Consequently, a significant amount of information has been accumulated concerning their ecology, especially for temperate and boreal forests. Although most studies have focused on fungi, forest soil bacteria also play important roles in this environment. In forest soils, bacteria inhabit multiple habitats with specific properties, including bulk soil, rhizosphere, litter, and deadwood habitats, where their communities are shaped by nutrient availability and biotic interactions. Bacteria contribute to a range of essential soil processes involved in the cycling of carbon, nitrogen, and phosphorus. They take part in the decomposition of dead plant biomass and are highly important for the decomposition of dead fungal mycelia. In rhizospheres of forest trees, bacteria interact with plant roots and mycorrhizal fungi as commensalists or mycorrhiza helpers. Bacteria also mediate multiple critical steps in the nitrogen cycle, including N fixation. Bacterial communities in forest soils respond to the effects of global change, such as climate warming, increased levels of carbon dioxide, or anthropogenic nitrogen deposition. This response, however, often reflects the specificities of each studied forest ecosystem, and it is still impossible to fully incorporate bacteria into predictive models. The understanding of bacterial ecology in forest soils has advanced dramatically in recent years, but it is still incomplete. The exact extent of the contribution of bacteria to forest ecosystem processes will be recognized only in the future, when the activities of all soil community members are studied simultaneously.
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http://dx.doi.org/10.1128/MMBR.00063-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485800PMC
June 2017

Decoding the complete arsenal for cellulose and hemicellulose deconstruction in the highly efficient cellulose decomposer Paenibacillus O199.

Biotechnol Biofuels 2016 14;9:104. Epub 2016 May 14.

Laboratory of Environmental Microbiology, Institute of Microbiology of the CAS, v. v. i., Průmyslová 595, 252 42 Vestec, Czech Republic.

Background: The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose.

Results: We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31 % of these CAZymes were expressed on cellulose and 29 % on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 β-glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 β-xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo-β-1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases, and other GHs.

Conclusions: Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins-GHs, CBMs, transporters, and SBPs-implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.
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http://dx.doi.org/10.1186/s13068-016-0518-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867992PMC
May 2016

Cellulose and hemicellulose decomposition by forest soil bacteria proceeds by the action of structurally variable enzymatic systems.

Sci Rep 2016 04 29;6:25279. Epub 2016 Apr 29.

Laboratory of Environmental Microbiology, Institute of Microbiology of the CAS, Vestec, 25242, Czech Republic.

Evidence shows that bacteria contribute actively to the decomposition of cellulose and hemicellulose in forest soil; however, their role in this process is still unclear. Here we performed the screening and identification of bacteria showing potential cellulolytic activity from litter and organic soil of a temperate oak forest. The genomes of three cellulolytic isolates previously described as abundant in this ecosystem were sequenced and their proteomes were characterized during the growth on plant biomass and on microcrystalline cellulose. Pedobacter and Mucilaginibacter showed complex enzymatic systems containing highly diverse carbohydrate-active enzymes for the degradation of cellulose and hemicellulose, which were functionally redundant for endoglucanases, β-glucosidases, endoxylanases, β-xylosidases, mannosidases and carbohydrate-binding modules. Luteibacter did not express any glycosyl hydrolases traditionally recognized as cellulases. Instead, cellulose decomposition was likely performed by an expressed GH23 family protein containing a cellulose-binding domain. Interestingly, the presence of plant lignocellulose as well as crystalline cellulose both trigger the production of a wide set of hydrolytic proteins including cellulases, hemicellulases and other glycosyl hydrolases. Our findings highlight the extensive and unexplored structural diversity of enzymatic systems in cellulolytic soil bacteria and indicate the roles of multiple abundant bacterial taxa in the decomposition of cellulose and other plant polysaccharides.
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http://dx.doi.org/10.1038/srep25279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850484PMC
April 2016

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt.

Pestic Biochem Physiol 2013 Sep 12;107(1):112-9. Epub 2013 Jun 12.

Department of Soil and Water Conservation and Organic Waste Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus de Espinardo, P.O. Box 164, 30100 Espinardo, Murcia, Spain.

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.
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http://dx.doi.org/10.1016/j.pestbp.2013.06.001DOI Listing
September 2013

Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods.

Appl Microbiol Biotechnol 2014 Feb 3;98(4):1531-7. Epub 2014 Jan 3.

Laboratory of Environmental Microbiology, Institute of Microbiology of the ASCR, Vídeňská 1083, 14220, Prague 4, Czech Republic,

Molecular methods for the analysis of biomolecules have undergone rapid technological development in the last decade. The advent of next-generation sequencing methods and improvements in instrumental resolution enabled the analysis of complex transcriptome, proteome and metabolome data, as well as a detailed annotation of microbial genomes. The mechanisms of decomposition by model fungi have been described in unprecedented detail by the combination of genome sequencing, transcriptomics and proteomics. The increasing number of available genomes for fungi and bacteria shows that the genetic potential for decomposition of organic matter is widespread among taxonomically diverse microbial taxa, while expression studies document the importance of the regulation of expression in decomposition efficiency. Importantly, high-throughput methods of nucleic acid analysis used for the analysis of metagenomes and metatranscriptomes indicate the high diversity of decomposer communities in natural habitats and their taxonomic composition. Today, the metaproteomics of natural habitats is of interest. In combination with advanced analytical techniques to explore the products of decomposition and the accumulation of information on the genomes of environmentally relevant microorganisms, advanced methods in microbial ecophysiology should increase our understanding of the complex processes of organic matter transformation.
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http://dx.doi.org/10.1007/s00253-013-5457-xDOI Listing
February 2014

qRT-PCR quantification of the biological control agent Trichoderma harzianum in peat and compost-based growing media.

Bioresour Technol 2011 Feb 8;102(3):2793-8. Epub 2010 Oct 8.

Department of Soil Water Conservation and Organic Waste Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Espinardo, Murcia, Spain.

To ensure proper use of Trichoderma harzianum in agriculture, accurate data must be obtained in population monitoring. The effectiveness of qRT-PCR to quantify T. harzianum in different growing media was compared to the commonly used techniques of colony counting and qPCR. Results showed that plate counting and qPCR offered similar T. harzianum quantification patterns of an initial rapid increase in fungal population that decreased over time. However, data from qRT-PCR showed a population curve of active T. harzianum with a delayed onset of initial growth which then increased throughout the experiment. Results demonstrated that T. harzianum can successfully grow in these media and that qRT-PCR can offer a more distinct representation of active T. harzianum populations. Additionally, compost amended with T. harzianum exhibited a lower Fusarium oxysporum infection rate (67%) and lower percentage of fresh weight loss (11%) in comparison to amended peat (90% infection rate, 23% fresh weight loss).
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http://dx.doi.org/10.1016/j.biortech.2010.09.120DOI Listing
February 2011

Analysis of subgroup C of fungal chitinases containing chitin-binding and LysM modules in the mycoparasite Trichoderma atroviride.

Glycobiology 2011 Jan 14;21(1):122-33. Epub 2010 Sep 14.

Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, Vienna, Austria.

Fungi have a plethora of chitinases, which can be phylogenetically divided into three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially interesting due to their multiple carbohydrate-binding modules, but they have not been investigated in detail yet. In this study, we analyzed sgC chitinases in the mycoparasites Trichoderma atroviride and Trichoderma virens. The expression of sgC chitinase genes in T. atroviride was induced during mycoparasitism of the fungal prey Botrytis cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of the former. Interestingly, only few sgC chitinase genes were inducible by chitin, suggesting that non-chitinous cell wall components can act as inducers. In contrast, the transcriptional profile of the most abundantly expressed sgC chitinase gene tac6 indicated a role of the protein in hyphal network formation. This shows that sgC chitinases have diverse functions and are not only involved in the mycoparasitic attack. However, sequence analysis and 3D modelling revealed that TAC6 and also its ortholog in T. virens have potentially detrimental deletions in the substrate-binding site and are thus probably not catalytically active enzymes. Genomic analysis showed that the genes neighboring sgC chitinases often encode proteins that are solely composed of multiple LysM modules, which were induced by similar stimuli as their neighboring sgC chitinase genes. This study provides first insights into fungal sgC chitinases and their associated LysM proteins.
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http://dx.doi.org/10.1093/glycob/cwq142DOI Listing
January 2011

Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass.

Bioresour Technol 2010 Apr 7;101(8):2888-91. Epub 2009 Nov 7.

Department of Soil Water Conservation and Organic Waste Management, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Espinardo, Murcia, Spain.

The species of the genus Trichoderma are used successfully as biocontrol agents against a wide range of phytopathogenic fungi. Among them, Trichoderma harzianum is especially effective. However, to develop more effective fungal biocontrol strategies in organic substrates and soil, tools for monitoring the control agents are required. Real-time PCR is potentially an effective tool for the quantification of fungi in environmental samples. The aim of this study consisted of the development and application of a real-time PCR-based method to the quantification of T. harzianum, and the extrapolation of these data to fungal biomass values. A set of primers and a TaqMan probe for the ITS region of the fungal genome were designed and tested, and amplification was correlated to biomass measurements obtained with optical microscopy and image analysis, of the hyphal length of the mycelium of the colony. A correlation of 0.76 between ITS copies and biomass was obtained. The extrapolation of the quantity of ITS copies, calculated based on real-time PCR data, into quantities of fungal biomass provides potentially a more accurate value of the quantity of soil fungi.
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http://dx.doi.org/10.1016/j.biortech.2009.10.019DOI Listing
April 2010

The beta-N-acetylglucosaminidases NAG1 and NAG2 are essential for growth of Trichoderma atroviride on chitin.

FEBS J 2009 Sep 6;276(18):5137-48. Epub 2009 Aug 6.

Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Austria.

The chitinolytic enzyme machinery of fungi consists of chitinases and beta-N-acetylglucosaminidases. These enzymes are important during the fungal life cycle for degradation of exogenous chitin, which is the second most abundant biopolymer, as well as fungal cell-wall remodelling. In addition, involvement of chitinolytic enzymes in the lysis of the host cell wall in mycoparasitic Trichoderma spp. has been reported. In view of the fact that fungi have on average 15-20 chitinases, but only two beta-N-acetylglucosaminidases, the question arises how important the latter enzymes actually are for various aspects of chitin degradation. In this study, the role of two beta-N-acetylglucosaminidases, NAG1 and NAG2, was analysed in the mycoparasitic fungus Trichoderma atroviride. No beta-N-acetylglucosaminidase activity was detected in T. atrovirideDeltanag1Deltanag2 strains, suggesting that NAG1 and NAG2 are the only enzymes in T. atroviride that possess this activity. Deltanag1Deltanag2 strains were not able to grow on chitin and chitobiose, but the presence of either NAG1 or NAG2 was sufficient to restore growth on chitinous carbon sources in solid media. Our results demonstrated that T. atroviride cannot metabolize chitobiose but only the monomer N-acetylglucosamine, and that N-acetylglucosaminidases are therefore essential for the use of chitin as a nutrient source. NAG1 is predominantly secreted into the medium, whereas NAG2 mainly remains attached to the cell wall. No physiological changes or reduction of the mycoparasitic potential of T. atroviride was detected in the double knockout strains, suggesting that the use of chitin as carbon source is only of minor importance for these processes.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07211.xDOI Listing
September 2009