Genomic insights into Penicillium chrysogenum adaptation to subseafloor sedimentary environments.

BACKGROUND: Penicillium chrysogenum is a filamentous fungal species with diverse habitats, yet little is known about its genetics in adapting to extreme subseafloor sedimental environments. RESULTS: Here, we report the discovery of P. chrysogenum strain 28R-6-F01, isolated from deep coal-bearing sediments 2306 m beneath the seafloor. This strain possesses exceptional characteristics, including the ability to thrive in extreme conditions such as high temperature (45 °C), high pressure (35 Mpa), and anaerobic environments, and exhibits broad-spectrum antimicrobial activity, producing the antibiotic penicillin at a concentration of 358 µg/mL. Genome sequencing and assembly revealed a genome size of 33.19 Mb with a GC content of 48.84%, containing 6959 coding genes. Comparative analysis with eight terrestrial strains identified 88 unique genes primarily associated with penicillin and aflatoxins biosynthesis, carbohydrate degradation, viral resistance, and three secondary metabolism gene clusters. Furthermore, significant expansions in gene families related to DNA repair were observed, likely linked to the strain's adaptation to its environmental niche. CONCLUSIONS: Our findings provide insights into the genomic and biological characteristics of P. chrysogenum adaptation to extreme anaerobic subseafloor sedimentary environments, such as high temperature and pressure.

Effect of oxygen concentrations and branched-chain amino acids on the growth and development of sub-seafloor fungus, Schizophyllum commune 20R-7-F01.

Fungi have been reported to be the dominant eukaryotic group in anoxic sub-seafloor sediments, but how fungi subsist in the anoxic sub-marine sedimental environment is rarely understood. Our previous study demonstrated that the fungus, Schizophyllum commune 20R-7-F01 isolated from a ~2 km sediment below the seafloor, can grow and produce primordia in the complete absence of oxygen with enhanced production of branched-chain amino acids (BCAAs), but the primordia cannot be developed into fruit bodies without oxygen. Here, we present the individual and synergistic effects of oxygen and BCAAs on the fruit-body development of this strain. It was found that the fungus required a minimum oxygen concentration of 0.5% pO2 to generate primordia and 1% pO2 to convert primordia into mature fruit body. However, if BCAAs (20 mM) were added to the medium, the primordium could be developed into fruit body at a lower oxygen concentration up to 0.5% pO2 where genes fst4 and c2h2 playing an important role in compensating oxygen deficiency. Moreover, under hypoxic conditions, the fungus showed an increase in mitochondrial number and initiation of auto-phagocytosis. These findings suggest that the fruit-body formation of S. commune may have multiple mechanisms, including energy and amino acid metabolism in response to oxygen concentrations.

The anaerobic survival mechanism of Schizophyllum commune 20R-7-F01, isolated from deep sediment 2 km below the seafloor.

Fungi dominated the eukaryotic group in the anaerobic sedimentary environment below the ocean floor where they play an essential ecological role. However, the adaptive mechanism of fungi to these anaerobic environments is still unclear. Here, we reported the anaerobic adaptive mechanism of Schizophyllum commune 20R-7-F01, isolated from deep coal-bearing sediment down to ~2 km below the seafloor, through biochemical, metabolomic and transcriptome analyses. The fungus grows well, but the morphology changes obviously and the fruit body develops incompletely under complete hypoxia. Compared with aerobic conditions, the fungus has enhanced branched-chain amino acid biosynthesis and ethanol fermentation under anaerobic conditions, and genes related to these metabolisms have been significantly up-regulated. Additionally, the fungus shows novel strategies for synthesizing ethanol by utilizing both glycolysis and ethanol fermentation pathways. These findings suggest that the subseafloor fungi may adopt multiple mechanisms to cope with lack of oxygen.

A Comparative Transcriptomic and Proteomic Analysis of Hexaploid Wheat's Responses to Colonization by Bacillus velezensis and Gaeumannomyces graminis, Both Separately and Combined.

Tritrophic interactions involving a biocontrol agent, a pathogen, and a plant have been analyzed predominantly from the perspective of the biocontrol agent. To explore the adaptive strategies of wheat in response to beneficial, pathogenic, and combined microorganisms, we performed the first comprehensive transcriptomic, proteomic, and biochemical analysis in wheat roots after exposure to Bacillus velezensis CC09, Gaeumannomyces graminis var. tritici, and their combined colonization, respectively. The transcriptional or translational programming of wheat roots inoculated with beneficial B. velezensis showed mild alterations compared with that of pathogenic G. graminis var. tritici. However, the combination of B. velezensis and G. graminis var. tritici activated a larger transcriptional or translational program than for each single microorganism, although the gene expression pattern was similar to that of individual infection by G. graminis var. tritici, suggesting a prioritization of defense against G. graminis var. tritici infection. Surprisingly, pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity made wheat pretreated with B. velezensis more sensitive to subsequent G. graminis var. tritici infection. Additionally, B. velezensis triggered a salicylic acid (SA)-dependent mode of induced systemic resistance that resembles pathogen-induced systemic acquired resistance. Wheat plants mainly depend on SA-mediated resistance, and not that mediated by jasmonic acid (JA), against the necrotrophic pathogen G. graminis var. tritici. Moreover, SA-JA interactions resulted in antagonistic effects regardless of the type of microorganisms in wheat. Further enhancement of SA-dependent defense responses such as lignification to the combined infection was shown to reduce the level of induced JA-dependent defense against subsequent infection with G. graminis var. tritici. Altogether, our results demonstrate how the hexaploid monocot wheat responds to beneficial or pathogenic microorganisms and prolongs the onset of take-all disease through modulation of cell reprogramming and signaling events.

Deep-Sea Fungi Could Be the New Arsenal for Bioactive Molecules.

Growing microbial resistance to existing drugs and the search for new natural products of pharmaceutical importance have forced researchers to investigate unexplored environments, such as extreme ecosystems. The deep-sea (>1000 m below water surface) has a variety of extreme environments, such as deep-sea sediments, hydrothermal vents, and deep-sea cold region, which are considered to be new arsenals of natural products. Organisms living in the extreme environments of the deep-sea encounter harsh conditions, such as high salinity, extreme pH, absence of sun light, low temperature and oxygen, high hydrostatic pressure, and low availability of growth nutrients. The production of secondary metabolites is one of the strategies these organisms use to survive in such harsh conditions. Fungi growing in such extreme environments produce unique secondary metabolites for defense and communication, some of which also have clinical significance. Despite being the producer of many important bioactive molecules, deep-sea fungi have not been explored thoroughly. Here, we made a brief review of the structure, biological activity, and distribution of secondary metabolites produced by deep-sea fungi in the last five years.

Bacillus velezensis CC09: A Potential 'Vaccine' for Controlling Wheat Diseases.

Biocontrol bacteria that can act like a "vaccine", stimulating plant resistance to pathogenic diseases, are still not fully elucidated. In this study, an endophytic bacterium, Bacillus velezensis CC09, labeled with green fluorescent protein, was tested for its colonization, migration, and expression of genes encoding iturin A synthetase within wheat tissues and organs as well as for protective effects against wheat take-all and spot blotch diseases. The results showed that strain CC09 not only formed biofilm on the root surface but was also widely distributed in almost every tissue, including the epidermis, cortex, and xylem vessels, and even migrated to stems and leaves, resulting in 66.67% disease-control efficacy (DCE) of take-all and 21.64% DCE of spot blotch. Moreover, the gene cluster encoding iturin A synthase under the control of the pitu promoter is expressed in B. velezensis CC09 in wheat tissues, which indicates that iturin A might contribute to the in-vivo antifungal activity and leads to the disease control. All these data suggested that strain CC09 can act like a 'vaccine' in the control of wheat diseases, with a single treatment inoculated on roots through multiple mechanisms.

Exploration of cultivable fungal communities in deep coal-bearing sediments from ∼1.3 to 2.5 km below the ocean floor.

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal-associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal-sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ∼20 million years of depositional history.

A Brief Review of Bioactive Metabolites Derived from Deep-Sea Fungi.

Deep-sea fungi, the fungi that inhabit the sea and the sediment at depths of over 1000 m below the surface, have become an important source of industrial, agricultural, and nutraceutical compounds based on their diversities in both structure and function. Since the first study of deep-sea fungi in the Atlantic Ocean at a depth of 4450 m was conducted approximately 50 years ago, hundreds of isolates of deep-sea fungi have been reported based on culture-dependent methods. To date more than 180 bioactive secondary metabolites derived from deep-sea fungi have been documented in the literature. These include compounds with anticancer, antimicrobial, antifungal, antiprotozoal, and antiviral activities. In this review, we summarize the structures and bioactivities of these metabolites to provide help for novel drug development.

Novel insights into dimethylsulfoniopropionate cleavage by deep subseafloor fungi.

Dimethylsulfoniopropionate (DMSP), a key organic sulfur compound in marine and subseafloor sediments, is degraded by phytoplankton and bacteria, resulting in the release of the climate-active volatile gas dimethylsulfide (DMS). However, it remains unclear if dominant eukaryotic fungi in subseafloor sediments possess specific abilities and metabolic mechanisms for DMSP degradation and DMS formation. Our study provides the first evidence that fungi from coal-bearing sediments ∼2 km below the seafloor, such as Aspergillus spp., Chaetomium globosum, Cladosporium sphaerospermum, and Penicillium funiculosum, can degrade DMSP and produce DMS. In Aspergillus sydowii 29R-4-F02, which exhibited the highest DMSP-dependent DMS production rate (16.95 pmol/µg protein/min), two DMSP lyase genes, dddP and dddW, were identified. Remarkably, the dddW gene, previously observed only in bacteria, was found to be crucial for fungal DMSP cleavage. These findings not only extend the list of fungi capable of degrading DMSP, but also enhance our understanding of DMSP lyase diversity and the role of fungi in DMSP decomposition in subseafloor sedimentary ecosystems.

Synthesis and antitumor activity of 1,3,4-oxadiazole possessing 1,4-benzodioxan moiety as a novel class of potent methionine aminopeptidase type II inhibitors.

A series of 1,3,4-oxadiazole derivatives containing 1,4-benzodioxan moiety (7a-7q) have been designed, synthesized and evaluated for their antitumor activity. Most of the synthesized compounds were proved to have potent antitumor activity and low toxicity. Among them, compound 7a showed the most potent biological activity against Human Umbilical Vein Endothelial cells, which was comparable to the positive control. The results of apoptosis and flow cytometry (FCM) demonstrated that compound 7a induce cell apoptosis by the inhibition of MetAP2 pathway. Molecular docking was performed to position compound 7a into MetAP2 binding site in order to explore the potential target.

Design, synthesis and antimicrobial activities evaluation of Schiff base derived from secnidazole derivatives as potential FabH inhibitors.

FabH, ß-ketoacyl-acyl carrier protein (ACP) synthase III, is critically important to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of novel secnidazole derivatives (1-20) were synthesized and fully characterized by spectroscopic methods and elemental analysis. Among these compounds, 6, 8, 11, 13, 14, 16-20 were reported for the first time. These compounds were tested for antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. The compounds inhibitory assay and docking simulation indicated that compound 20 (E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3,4,5-trimethylbenzylidene)acetohydrazide with MIC of 3.13-6.25 µg/mL against the tested bacterial strains was a potent inhibitor of Escherichia coli FabH.

Genome-wide characterization of laccase gene family in Schizophyllum commune 20R-7-F01, isolated from deep sediment 2 km below the seafloor.

Laccases are ligninolytic enzymes that play a crucial role in various biological processes of filamentous fungi, including fruiting-body formation and lignin degradation. Lignin degradation is a complex process and its degradation in Schizophyllum commune is greatly affected by the availability of oxygen. Here, a total of six putative laccase genes (ScLAC) were identified from the S. commune 20R-7-F01 genome. These genes, which include three typical Cu-oxidase domains, can be classified into three groups based on phylogenetic analysis. ScLAC showed distinct intron-exon structures and conserved motifs, suggesting the conservation and diversity of ScLAC in gene structures. Additionally, the number and type of cis-acting elements, such as substrate utilization-, stress-, cell division- and transcription activation-related cis-elements, varied between ScLAC genes, suggesting that the transcription of laccase genes in S. commune 20R-7-F01 could be induced by different substrates, stresses, or other factors. The SNP analysis of resequencing data demonstrated that the ScLAC of S. commune inhabiting deep subseafloor sediments were significantly different from those of S. commune inhabiting terrestrial environments. Similarly, the large variation of conserved motifs number and arrangement of laccase between subseafloor and terrestrial strains indicated that ScLAC had a diverse structure. The expression of ScLAC5 and ScLAC6 genes was significantly up-regulated in lignin/lignite medium, suggesting that these two laccase genes might be involved in fungal utilization and degradation of lignite and lignin under anaerobic conditions. These findings might help in understanding the function of laccase in white-rot fungi and could provide a scientific basis for further exploring the relationship between the LAC family and anaerobic degradation of lignin by S. commune.

Structure and bioactivity of polysaccharide from a subseafloor strain of Schizophyllum commune 20R-7-F01.

Fungal polysaccharide is a kind of biomacromolecule with multiple biological activities, which has a wide application prospect and may play an important role in organisms to cope with extreme environments. Herein, we reported an extracellular polysaccharide (EPS) produced by Schizophyllum commune 20R-7-F01 that was isolated from subseafloor sediments at ~2 km below the seafloor, obtained during expedition 337. The monosaccharide of EPS was glucose and its molecular weight was 608.8 kDa. Methylation and NMR analysis indicated that the backbone of the EPS was (1 â†’ 3)-ß-D-glucan with a side chain (1 â†’ 6) ß-D-glucan linking at every third residue. Bio-active assays revealed that the EPS had potent antioxidant activity and could promote RAW264.7 cells viability and phagocytosis. These results suggest that fungi derived from sediments below seafloor are important and new source of polysaccharides and may be involved in the adaptation of fungi to anoxic subseafloor extreme ecosystem.

A Visual Discrimination of Existing States of Virus Capsid Protein by a Giant Molybdate Cluster.

We report a unique phenomenon, the opposite color response of a giant polyoxometalate, (NH4)42[Mo132O372(CHCOO)30] (H2O)72 ([Mo132]), to the existing states of human papillomavirus (HPV) major capsid protein, L1-pentamer (L1-p), and virus-like particles (VLPs). The color responses originate from the different assembly forms between [Mo132] and the capsid protein. The latter were inspected and separated by using CsCl gradient centrifugation, and validated in detail by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and transmission electron microscopy (TEM) imaging. Furthermore, the intrinsic mechanisms were investigated in-depth by using XPS-based semi-quantitative analysis and well-designed peptides, revealing the critical points of L1 that determine the charge-transfer ratio between Mo(V) to Mo(VI), and consequently, the levels of [Mo132] hypochromic in different assemblies. Such a unique phenomenon is significant as it supplies a colorimetry approach to distinguish the existing states of the HPV capsid protein and would be significant in the quality assay of the HPV vaccine and existing states of other viruses in the future.

Methane Production by Facultative Anaerobic Wood-Rot Fungi via a New Halomethane-Dependent Pathway.

The greenhouse gas methane (CH4) is of pivotal importance for Earth's climate system and as a human energy source. A significant fraction of this CH4 is produced by anaerobic Archaea. Here, we describe the first CH4 production by facultative anaerobic wood-rot fungi during growth on hydroxylated/carboxylated aromatic compounds, including lignin and lignite. The amount of CH4 produced by fungi is positively correlated with the amount of CH3Cl produced during the rapid growth period of the fungus. Biochemical, genetic, and stable isotopic tracer analyses reveal the existence of a novel halomethane-dependent fungal CH4 production pathway during the degradation of phenol and benzoic acid monomers and polymers and utilization of cyclic sugars. Even though this halomethane-dependent pathway may only play a side role in anaerobic fungal activity, it could represent a globally significant, previously overlooked source of biogenic CH4 in natural ecosystems. IMPORTANCE Here, we demonstrate that wood-rot fungi produce methane anaerobically without the involvement of methanogenic archaea via a new, halomethane-dependent pathway. These findings of an anaerobic fungal methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Genome, genetic evolution, and environmental adaptation mechanisms of Schizophyllum commune in deep subseafloor coal-bearing sediments.

To understand the genomic evolution and adaptation strategies of fungi to subseafloor sedimentary environments, we de novo assembled the genome of Schizophyllum commune strain 20R-7-F01 isolated from ∼2.0 km-deep, ∼20-millionyearsago (Mya) coal-bearing sediments. Phylogenomics study revealed a differentiation time of 28-73 Mya between this strain and the terrestrial type-strain H4-8, in line with sediment age records. Comparative genome analyses showed that FunK1 protein kinase, NmrA family, and transposons in this strain are significantly expanded, possibly linking to the environmental adaptation and persistence in sediment for over millions of years. Re-sequencing study of 14 S. commune strains sampled from different habitats revealed that subseafloor strains have much lower nucleotide diversity, substitution rate, and homologous recombination rate than other strains, reflecting that the growth and/or reproduction of subseafloor strains are extremely slow. Our data provide new insights into the adaptation and long-term survival of the fungi in the subseafloor sedimentary biosphere.

Anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungi isolated from anaerobic coal-associated sediments at 2.5 km below the seafloor.

Fungi represent the dominant eukaryotic group in the deep biosphere and well-populated in the anaerobic coal-bearing sediments up to ∼2.5 km below seafloor (kmbsf). But whether fungi are able to degrade and utilize coal to sustain growth in the anaerobic sub-seafloor environment remains unknown. Based on biodegradation investigation, we found that fungi isolated from sub-seafloor sediments at depths of ∼1.3-∼2.5 kmbsf showed a broad range of polycyclic aromatic hydrocarbons (PAHs) anaerobic degradation rates (3-25%). Among them, the white-rot fungus Schizophyllium commune 20R-7-F01 exhibited the highest degradation, 25%, 18% and 13%, of phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP); respectively, after 10 days of anaerobic incubation. Phe was utilized well and about 40.4% was degraded by the fungus, after 20 days of anaerobic incubation. Moreover, the ability of fungi to degrade PAHs was positively correlated with the anaerobic growth of fungi, indicating that fungi can use PAHs as a sole carbon source under anoxic conditions. In addition, fungal degradation of PAHs was found to be related to the activity of carboxylases, but little or nothing to do with the activity of lignin modifying enzymes such as laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP). These results suggest that sub-seafloor fungi possess a special mechanism to degrade and utilize PAHs as a carbon and energy source under anaerobic conditions. Furthermore, fungi living in sub-seafloor sediments may not only play an important role in carbon cycle in the anaerobic environments of the deep biosphere, but also be able to persist in deep sediment below seafloor for millions of years by using PAHs or related compounds as carbon and energy source. This anaerobic biodegradation ability could make these fungi suitable candidates for bioremediation of toxic pollutants such as PAHs from anoxic environments.

A hybrid HPV capsid protein L1 with giant Mo-containing polyoxometalate improves the stability of virus-like particles and the anti-tumor effect of [Mo154].

We report a bio-inorganic hybrid system, [Mo154]@VLPs, constructed from the virus-like particles (VLPs) of the HPV capsid protein L1 and a giant disc-shaped, molybdenum-containing polyoxometalate of [Mo154]. The hybrid was purified by CsCl gradient centrifugation and further validated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and transmission electron microscopy (TEM). An assembly with [Mo154] improved the tolerance of VLPs to pH, temperature, and storage time, thereby defining an opportunity to reduce the cost of HPV vaccines. Moreover, the ability of [Mo154] to kill cancer cells was improved by 6% after being encapsulated inside the VLPs, which is mainly attributed to the enhanced biocompatibility of [Mo154]. The irradiation of both [Mo154] and [Mo154]@VLPs with an infrared light of 808 nm further enhanced their ability to destroy cancer cells by 3- and 2-fold, respectively, confirming that [Mo154] is an effective anti-tumor photo-thermal agent. Therefore, the successful hybrid of L1-p and [Mo154] improves the stability of VLPs and simultaneously paves the way to enhance the anti-tumor ability of [Mo154] and further extends its application prospects as a future anti-tumor drug.

Co-assembly of HPV capsid proteins and aggregation-induced emission fluorogens for improved cell imaging.

In order to improve the cell-imaging ability, and particularly, to extend the bio-application of AIEgen, human papillomavirus (HPV) capsid protein L1 was assembled with the complex of DNA and aggregation-induced emission fluorogen 9,10-distyrylhydrazine (DSAI), where the virus-like particles (VLPs) of HPV encapsulate the complex via electrostatic interaction. The co-assembled nanoparticles, DSAI-DNA@VLPs, showed homogeneous size (∼53 nm), enhanced fluorescence (8 × 2.5-fold), considerable stability (anti-DNase digestion), improved biocompatibility and commendable protection for the DSAI-DNA complex, ensuring virtual brighter imaging in live cells, both for HeLa and normal 293T cell lines.

Comparative Transcriptome Profiling of Gaeumannomyces graminis var. tritici in Wheat Roots in the Absence and Presence of Biocontrol Bacillus velezensis CC09.

This study aimed to explore potential biocontrol mechanisms involved in the interference of antagonistic bacteria with fungal pathogenicity in planta. To do this, we conducted a comparative transcriptomic analysis of the "take-all" pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) by examining Ggt-infected wheat roots in the presence or absence of the biocontrol agent Bacillus velezensis CC09 (Bv) compared with Ggt grown on potato dextrose agar (PDA) plates. A total of 4,134 differentially expressed genes (DEGs) were identified in Ggt-infected wheat roots, while 2,011 DEGs were detected in Bv+Ggt-infected roots, relative to the Ggt grown on PDA plates. Moreover, 31 DEGs were identified between wheat roots, respectively infected with Ggt and Bv+Ggt, consisting of 29 downregulated genes coding for potential Ggt pathogenicity factors - e.g., para-nitrobenzyl esterase, cutinase 1 and catalase-3, and two upregulated genes coding for tyrosinase and a hypothetical protein in the Bv+Ggt-infected roots when compared with the Ggt-infected roots. In particular, the expression of one gene, encoding the ABA3 involved in the production of Ggt's hormone abscisic acid, was 4.11-fold lower in Ggt-infected roots with Bv than without Bv. This is the first experimental study to analyze the activity of Ggt transcriptomes in wheat roots exposed or not to a biocontrol bacterium. Our results therefore suggest the presence of Bv directly and/or indirectly impairs the pathogenicity of Ggt in wheat roots through complex regulatory mechanisms, such as hyphopodia formation, cell wall hydrolase, and expression of a papain inhibitor, among others, all which merit further investigation.