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.

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.

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.

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.

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.

Strong red-emitting gold nanoclusters protected by glutathione S-transferase.

Glutathione S-transferase (GST) is distributed widely in tissues and has been proven to be vital in the body. For example, it catalyzes reduced glutathione (GSH) to a variety of electrophilic substances and thus protects cells against many toxic chemicals. Therefore, GST-related investigations have always been significant for medical and/or life sciences. In the present study, a new material of gold nanoclusters (Au-NCs) protected by GST, Au-NCs@GST, was fabricated via an improved one-step heating method. The products were fully characterized by X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), and Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra. The results confirmed that around 10 gold atoms are encapsulated in one intact GST, forming Au-NCs@GST with strong (QY = 13.5%) red emission at 670 nm. Therefore, a new nanomaterial possessing both strong luminescence and bio-functions of GST was developed, and it has great potential in GST-related investigations. To prove the concept, Au-NCs@GST was successfully applied to detect metronidazole (MNZ) both in solution and in living cells. Therefore, in the present study, we report not only a new nanomaterial of Au-NCs@GST but also a feasible fluorescence probe for antibiotic detection. Both the improved synthetic method and the design concept can be extended to the fabrication of other kinds of metal nanoclusters using different functional proteins for various purposes.

Rifampicin-Resistance Mutations in the rpoB Gene in Bacillus velezensis CC09 have Pleiotropic Effects.

Rifampicin resistance (Rifr) mutations in the RNA polymerase ß subunit (rpoB) gene exhibit pleiotropic phenotypes as a result of their effects on the transcription machinery in prokaryotes. However, the differences in the effects of the mutations on the physiology and metabolism of the bacteria remain unknown. In this study, we isolated seven Rifr mutations in rpoB, including six single point mutations (H485Y, H485C, H485D, H485R, Q472R, and S490L) and one double point mutation (S490L/S617F) from vegetative cells of an endophytic strain, Bacillus velezensis CC09. Compared to the wild-type (WT) strain (CC09), the H485R and H485D mutants exhibited a higher degree of inhibition of Aspergillus niger spore germination, while the H485Y, S490L, Q472R, and S490L/S617F mutants exhibited a lower degree of inhibition due to their lower production of the antibiotic iturin A. These mutants all exhibited defective phenotypes in terms of pellicle formation, sporulation, and swarming motility. A hierarchical clustering analysis of the observed phenotypes indicated that the four mutations involving amino acid substitutions at H485 in RpoB belonged to the same cluster. In contrast, the S490L and Q472R mutations, as well as the WT strain, were in another cluster, indicating a functional connection between the mutations in B. velezensis and phenotypic changes. Our data suggest that Rifr mutations cannot only be used to study transcriptional regulation mechanisms, but can also serve as a tool to increase the production of bioactive metabolites in B. velezensis.

Genomic and metabolic traits endow Bacillus velezensis CC09 with a potential biocontrol agent in control of wheat powdery mildew disease.

Bacillus velezensis CC09, which was isolated from healthy leaves of Cinnamomum camphora and previously identified as Bacillus amyloliquefaciens CC09, shows great potential as a new biocontrol agent, in control of many phytopathogenic diseases. To extend our understanding of the potential antifungal capacities, we did a whole genome analysis of strain CC09. Result shows that strain CC09 has a relatively large genome size (4.17Mb) with an average GC content of 46.1%, and 4021 predicted genes. Thirteen secondary metabolites encoding clusters have been identified within the genome of B. velezensis CC09 using genome mining technique. Data of comparative genomic analysis indicated that 3 of the clusters are conserved by all strains of B. velezensis, B. amyloliquefaciens and B. subtilis 168, 9 by B. velezensis and B. amyloliquefaciens, and 2 by all strains of B. velezensis. Another 2 clusters encoding NRPS (Non-Ribosomal Peptide Synthetases) and NRPS-TransATPKS (NRPS and trans-Acyl Transferase Polyketide Synthetases) respectively are observed only in 15 B. velezensis strains, which might lead to the synthesis of novel bioactive compounds and could be explored as antimicrobial agents in the future. These clusters endow B. velezensis CC09 with strong and broad antimicrobial activities, for example, in control of wheat powdery mildew disease. Moreover, our data further confirmed the taxonomy of strain CC09 is a member of B. velezensis rather than a strain of B. amyloliquefaciens based on core genome sequence analysis using phylogenomic approach.

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.

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.

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.

[Stringent RNA polymerase of E. coli and its in vivo transcriptional activity].

Several spontaneous E. coli mutants with the similar phenotype as that in the condition of amino acid deficiency were obtained on the selective media. One of the mutants (LCH001) showing slow growth phenotype on LB agar plate and pink or white colonies on MacConkey agar plate was mapped at rpoC gene encoding the beta' subunit of RNA polymerase by phage P1 transduction and transformation assays and found to be a new site mutation from G to T at 3406bp in the rpoC gene, which resulted in the amino acid change from Glycine (GGT) to Cysteine (TGT). The effect of the mutation on transcriptional activity of both stringent and non-stringent controlled promoters in vivo was measured by determining the beta-galactolactase activity of the growing cells. Results showed that the transcriptional activity of the mutant LCH001 reduced greatly on the stringent promoter, but increased significantly on the non-stringent promoter. The beta-galactolactase activity of the mutant LCH001 transcribed on stringent promoter was 18% lower, but 5-fold higher on the non-stringent controlled promoter than that of the wild-type strain CLT5034. This finding may give insights into future studies of the structure-function relationship of RNA polymerase as well as its role in the stringent response of bacteria.