Transcriptome profiling of Paraburkholderia aromaticivorans AR20-38 during ferulic acid bioconversion.

The importance and need of renewable-based, sustainable feedstocks increased in recent years. Lignin-derived monomers have high potential, energetic and economic value in the microbial bioconversion to valuable biomolecules. The bacterium Paraburkholderia aromaticivorans AR20-38 produces a remarkable yield of vanillic acid from ferulic acid at moderate and low temperatures and is therefore a good candidate for biotechnological applications. To understand this bioconversion process on a molecular level, a transcriptomic study during the bioconversion process was conducted to elucidate gene expression patterns. Differentially expressed genes, cellular transporters as well as transcriptional factors involved in the bioconversion process could be described. Additional enzymes known for xenobiotic degradation were differentially expressed and a potential membrane vesicle mechanism was detected. The bioconversion mechanism on a transcriptional level of P. aromaticivorans could be elucidated and results can be used for strain optimization. Additionally, the transcriptome study showed the high potential of the strain for other degradation applications.

Biodegradation Potential and Putative Catabolic Genes of Culturable Bacteria from an Alpine Deciduous Forest Site.

Microbiota from Alpine forest soils are key players in carbon cycling, which can be greatly affected by climate change. The aim of this study was to evaluate the degradation potential of culturable bacterial strains isolated from an alpine deciduous forest site. Fifty-five strains were studied with regard to their phylogenetic position, growth temperature range and degradation potential for organic compounds (microtiter scale screening for lignin sulfonic acid, catechol, phenol, bisphenol A) at low (5 °C) and moderate (20 °C) temperature. Additionally, the presence of putative catabolic genes (catechol-1,2-dioxygenase, multicomponent phenol hydroxylase, protocatechuate-3,4-dioxygenase) involved in the degradation of these organic compounds was determined through PCR. The results show the importance of the Proteobacteria phylum as its representatives did show good capabilities for biodegradation and good growth at -5 °C. Overall, 82% of strains were able to use at least one of the tested organic compounds as their sole carbon source. The presence of putative catabolic genes could be shown over a broad range of strains and in relation to their degradation abilities. Subsequently performed gene sequencing indicated horizontal gene transfer for catechol-1,2-dioxygenase and protocatechuate-3,4-dioxygenase. The results show the great benefit of combining molecular and culture-based techniques.

Wildfire-Derived Pyrogenic Carbon Modulates Riverine Organic Matter and Biofilm Enzyme Activities in an In Situ Flume Experiment.

Wildfires produce large amounts of pyrogenic carbon (PyC), including charcoal, known for its chemical recalcitrance and sorption affinity for organic molecules. Wildfire-derived PyC can be transported to fluvial networks. Here it may alter the dissolved organic matter (DOM) concentration and composition as well as microbial biofilm functioning. Effects of PyC on carbon cycling in freshwater ecosystems remain poorly investigated. Employing in-stream flumes with a control versus treatment design (PyC pulse addition), we present evidence that field-aged PyC inputs to rivers can increase the dissolved organic carbon (DOC) concentration and alter the DOM composition. DOM fluorescence components were not affected by PyC. The in-stream DOM composition was altered due to leaching of pyrogenic DOM from PyC and possibly concurrent sorption of riverine DOM to PyC. Decreased DOM aromaticity indicated by a lower SUVA245 (-0.31 unit) and a higher pH (0.25 unit) was associated with changes in enzymatic activities in benthic biofilms, including a lower recalcitrance index (ß-glucosidase/phenol oxidase), suggesting preferential usage of recalcitrant over readily available DOM by biofilms. The deposition of particulate PyC onto biofilms may further modulate the impacts of PyC due to direct contact with the biofilm matrix. This study highlights the importance of PyC for in-stream biogeochemical organic matter cycling in fire-affected watersheds.

Biodegradation of lignin monomers and bioconversion of ferulic acid to vanillic acid by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest soil.

Lignin bio-valorization is an emerging field of applied biotechnology and has not yet been studied at low temperatures. Paraburkholderia aromaticivorans AR20-38 was examined for its potential to degrade six selected lignin monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, vanillic acid, benzoic acid) from different upper funneling aromatic pathways. The strain degraded four of these compounds at 10°C, 20°C, and 30°C; syringic acid and vanillic acid were not utilized as sole carbon source. The degradation of 5 mM and 10 mM ferulic acid was accompanied by the stable accumulation of high amounts of the value-added product vanillic acid (85-89% molar yield; 760 and 1540 mg l-1, respectively) over the whole temperature range tested. The presence of essential genes required for reactions in the upper funneling pathways was confirmed in the genome. This is the first report on biodegradation of lignin monomers and the stable vanillic acid production at low and moderate temperatures by P. aromaticivorans. KEY POINTS: • Paraburkholderia aromaticivorans AR20-38 successfully degrades four lignin monomers. • Successful degradation study at low (10°C) and moderate temperatures (20-30°C). • Biotechnological value: high yield of vanillic acid produced from ferulic acid.

Culturable bacteria from an Alpine coniferous forest site: biodegradation potential of organic polymers and pollutants.

The potential of the culturable bacterial community from an Alpine coniferous forest site for the degradation of organic polymers and pollutants at low (5 °C) and moderate (20 °C) temperatures was evaluated. The majority of the 68 strains belonged to the phylum Proteobacteria (77%). Other strains were related to Bacteroidetes (12%), Alphaproteobacteria (4%), Actinobacteria (3%), and Firmicutes (3%). The strains were grouped into 42 different OTUs. The highest bacterial diversity was found within the phylum Bacteroidetes. All strains, except one, could grow at temperatures from 5 to 25 °C. The production of enzyme activities involved in the degradation of organic polymers present in plant litter (carboxymethyl cellulose, microgranular cellulose, xylan, polygalacturonic acid) was almost comparable at 5 °C (68%) and 20 °C (63%). Utilizers of lignin compounds (lignosulfonic acid, lignin alkali) as sole carbon source were found to a higher extent at 20 °C (57%) than at 5 °C (24%), but the relative fractions among positively tested strains utilizing these compounds were almost identical at the two temperatures. Similar results were noted for utilizers of organic pollutants (n-hexadecane, diesel oil, phenol, glyphosate) as sole carbon source. More than two-thirds showed constitutively expressed catechol-1,2-dioxygenase activity both at 5 °C (74%) and 20 °C (66%). Complete phenol (2.5 mmol/L) degradation by strain Paraburkholderia aromaticivorans AR20-38 was demonstrated at 0-30 °C, amounts up to 7.5 mmol/L phenol were fully degraded at 10-30 °C. These results are useful to better understand the effect of changing temperatures on microorganisms involved in litter degradation and nutrient turnover in Alpine forest soils.

Draft Genome Sequence of the Bacterium Paraburkholderia aromaticivorans AR20-38, a Gram-Negative, Cold-Adapted Degrader of Aromatic Compounds.

Here, we report the draft genome sequence of Paraburkholderia aromaticivorans strain AR20-38, a cold-adapted Gram-negative bacterium. It was isolated from Alpine forest soil and can degrade a range of aromatic compounds.

Back to the Salt Mines: Genome and Transcriptome Comparisons of the Halophilic Fungus Aspergillus salisburgensis and Its Halotolerant Relative Aspergillus sclerotialis.

Salt mines are among the most extreme environments as they combine darkness, low nutrient availability, and hypersaline conditions. Based on comparative genomics and transcriptomics, we describe in this work the adaptive strategies of the true halophilic fungus Aspergillus salisburgensis, found in a salt mine in Austria, and compare this strain to the ex-type halotolerant fungal strain Aspergillus sclerotialis. On a genomic level, A. salisburgensis exhibits a reduced genome size compared to A. sclerotialis, as well as a contraction of genes involved in transport processes. The proteome of A. sclerotialis exhibits an increased proportion of alanine, glycine, and proline compared to the proteome of non-halophilic species. Transcriptome analyses of both strains growing at 5% and 20% NaCl show that A. salisburgensis regulates three-times fewer genes than A. sclerotialis in order to adapt to the higher salt concentration. In A. sclerotialis, the increased osmotic stress impacted processes related to translation, transcription, transport, and energy. In contrast, membrane-related and lignolytic proteins were significantly affected in A. salisburgensis.

Transcriptome Study of an Exophiala dermatitidis PKS1 Mutant on an ex Vivo Skin Model: Is Melanin Important for Infection?

The black yeast Exophiala dermatitidis is a polyextremophilic human pathogen, especially known for growing in man-made extreme environments. Reported diseases caused by this fungus range from benign cutaneous to systemic infections with 40% fatality rate. While the number of cases steadily increases in both immunocompromised and immunocompetent people, detailed knowledge about infection mechanisms, virulence factors and host response are scarce. To understand the impact of the putative virulence factor melanin on the infection, we generated a polyketide synthase (PKS1) mutant using CRISPR/Cas9 resulting in a melanin deficient strain. The mutant and the wild-type fungus were inoculated onto skin explants using an ex vivo skin organ culture model to simulate in vivo cutaneous infection. The difference between the mutant and wild-type transcriptional landscapes, as assessed by whole RNA-sequencing, were small and were observed in pathways related to the copper homeostasis, cell wall genes and proteases. Seven days after inoculation the wild-type fungus completely colonized the stratum corneum, invaded the skin and infected keratinocytes while the mutant had only partially covered the skin and showed no invasiveness. Our results suggest that melanin dramatically improves the invasiveness and virulence of E. dermatitidis during the first days of the skin infection.

Influence of compost and biochar on microbial communities and the sorption/degradation of PAHs and NSO-substituted PAHs in contaminated soils.

Diffusely contaminated soils often remain untreated as classical remediation approaches would be disproportionately expensive. Adding compost can accelerate the biodegradation of organic contaminants and adding biochar can immobilize contaminants through sorption. The combined use of compost and biochar to reduce polycyclic aromatic hydrocarbon (PAH) and NSO-substituted PAH contamination has, however, not previously been systematically investigated. We have therefore investigated the processes involved (i) through sorption batch experiments, (ii) by monitoring changes in bacterial, fungal and archaeal communities using denaturing gradient gel electrophoresis, and (iii) through degradation experiments with fluorene, phenanthrene, pyrene, carbazole, dibenzothiophene, and dibenzofuran. Sorption coefficients for organic contaminants in soils increased tenfold following 10% compost addition and up to a hundredfold with further addition of 5% biochar. The rate of PAH and NSO-PAH degradation increased up to twofold following compost addition despite increased sorption, probably due to the introduction of additional microbial species into the autochthonous soil communities. In contrast, degradation of PAHs and NSO-PAHs in soil-compost-biochar mixtures slowed down up to tenfold due to the additional sorption, although some degradation still occurred. The combined use of biochar and compost may therefore provide a strategy for immobilizing PAHs and NSO-PAHs and facilitating degradation of remaining accessible contaminant fractions.

Draft Genome Sequences of the Black Rock Fungus Knufia petricola and Its Spontaneous Nonmelanized Mutant.

The fungal genus Knufia mostly comprises extremotolerant species from environmental sources, especially rock surfaces. The draft genome sequence of the rock fungus Knufia petricola presented here is the first whole-genome sequence of the only species among black fungi known to have a nonmelanized spontaneous mutant.

Genomic and transcriptomic analysis of the toluene degrading black yeast Cladophialophora immunda.

Cladophialophora immunda is an ascomycotal species belonging to the group of the black yeasts. These fungi have a thick and melanized cell wall and other physiological adaptations that allows them to cope with several extreme physical and chemical conditions. Member of the group can colonize some of the most extremophilic environments on Earth. Cladophialophora immunda together with a few other species of the order Chaetothyriales show a special association with hydrocarbon polluted environments. The finding that the fungus is able to completely mineralize toluene makes it an interesting candidate for bioremediation purposes. The present study is the first transcriptomic investigation of a fungus grown in presence of toluene as sole carbon and energy source. We could observe the activation of genes involved in toluene degradatation and several stress response mechanisms which allowed the fungus to survive the toluene exposure. The thorough comparative genomics analysis allowed us to identify several events of horizontal gene transfer between bacteria and Cladophialophora immunda and unveil toluene degradation steps that were previously reported in bacteria. The work presented here aims to give new insights into the ecology of Cladophialophora immunda and its adaptation strategies to hydrocarbon polluted environments.

The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection.

The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors.

Pathogenic Yet Environmentally Friendly? Black Fungal Candidates for Bioremediation of Pollutants.

A collection of 163 strains of black yeast-like fungi from the CBS Fungal Biodiversity Center (Utrecht, The Netherlands), has been screened for the ability to grow on hexadecane, toluene and polychlorinated biphenyl 126 (PCB126) as the sole carbon and energy source. These compounds were chosen as representatives of relevant environmental pollutants. A microtiter plate-based culture assay was set up in order to screen the fungal strains for growth on the selected xenobiotics versus glucose, as a positive control. Growth was observed in 25 strains on at least two of the tested substrates. Confirmation of substrate assimilation was performed by cultivation on closed vials and analysis of the headspace composition with regard to the added volatile substrates and the generated carbon dioxide. Exophiala mesophila (CBS 120910) and Cladophialophora immunda (CBS 110551), both of the order Chaetothyriales and isolated from a patient with chronic sinusitis and a polluted soil sample, respectively, showed the ability to grow on toluene as the sole carbon and energy source. Toluene assimilation has previously been described for C. immunda but this is the first account for E. mesophila. Also, this is the first time that the capacity to grow on alkylbenzenes has been demonstrated for a clinical isolate. Assimilation of toluene could not be demonstrated for the human opportunistic pathogen Pseudoallescheria boydii (CBS 115.59, Microascales), but the results from microtiter plate assays suggest that strains of this species are promising candidates for further studies. The outstanding abilities of black yeast-like fungi to thrive in extreme environments makes them ideal agents for the bioremediation of polluted soils, and for the treatment of contaminated gas streams in biofilters. However, interrelations between hydrocarbonoclastic and potentially pathogenic strains need to be elucidated in order to avoid the possibility of biohazards occurring.

Draft Genome Sequence of Exophiala mesophila, a Black Yeast with High Bioremediation Potential.

The fungal genus Exophiala comprises both pathogen species, which cause severe infections in humans, and environmental species, which are able to degrade alkylbenzene compounds. The draft genome sequence of Exophiala mesophila presented here is the first genome assembly of an alkylbenzene-degrading organism belonging to the genus Exophiala.